CN116814700A - Application of ACSM5-P425T in constructing a drug detection model for the treatment of Xuanwei lung cancer - Google Patents

Abstract

The invention belongs to the field of biomedicine technology and specifically discloses the application of ACSM5-P425T in constructing a drug detection model for treating Xuanwei lung cancer. The ACSM5-P425T sequence is a C-base missense mutation at the chr16:20442608 position in the gene ACSM5. For the formation of single nucleotide polymorphisms caused by the A base, the model constructed by the present invention can be used for precise treatment of Xuanwei lung cancer, screening more effective drugs, and from in vitro cell experiments and in vivo animal tumorigenesis experiments level, reveal the impact of drugs on the occurrence and development of Xuanwei lung cancer, and provide theoretical basis for the treatment of Xuanwei lung cancer.

Description

Application of ACSM5-P425T in constructing a drug detection model for the treatment of Xuanwei lung cancer

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to the application of ACSM5-P425T in constructing a drug detection model for treating Xuanwei lung cancer.

Background technique

Xuanwei lung cancer is a unique endemic disease in Yunnan Province. It has the characteristics of high incidence and mortality of lung adenocarcinoma in non-smoking women, younger age of onset, familial aggregation, poor sensitivity to radiotherapy and chemotherapy, and high drug resistance. Because of its distinctive onset characteristics , attracted worldwide attention. In recent years, a variety of targeted therapeutic drugs such as TK inhibitors (Tyrosine Kinase inhibitors, TKIs) gefitinib have been developed, which has brought lung cancer to the forefront of precision treatment. However, only a small number of patients with epidermal growth factor receptor (EGFR) mutations (such as L858R, G719C, etc.) are effective in gefitinib treatment. Cases without EGFR mutations are resistant to gefitinib targeted therapy. It is almost ineffective, which reminds us that the prerequisite for precise treatment needs to be based on accurate diagnosis. Although many lung cancer-related gene mutation sites have been identified for targeted therapy with TKIs, Xuanwei lung cancer has unique onset characteristics and the therapeutic effect of TKIs is poor. It may have different characteristics from non-Xuanwei lung cancer. driver mutation spectrum.

Intrinsic factors in the occurrence and development of tumors include activation of oncogenes and inactivation of tumor suppressor genes. By searching for oncogenes and tumor suppressor genes related to Xuanwei lung cancer, and by constructing related cell and animal models, we can screen more effective drugs for the precise treatment of Xuanwei lung cancer, and use in vitro cell experiments and in vivo animal tumorigenesis At the experimental level, the impact of drugs on the development of Xuanwei lung cancer is revealed.

Contents of the invention

The main purpose of the present invention is to provide a method for constructing a drug detection model for treating Xuanwei lung cancer. The prepared model can be used for precise treatment of Xuanwei lung cancer and screening of more effective drugs.

To achieve the above objectives, the present invention provides the following technical solutions:

The application of ACSM5-P425T in constructing a drug detection model for the treatment of Xuanwei lung cancer. The ACSM5-P425T sequence is a single nucleotide polypeptide caused by a missense mutation of the C base at the chr16:20442608 position in the gene ACSM5 to an A base. Morphological formation.

Further, the model overexpresses the ACSM5-P425T gene sequence.

Further, the model is a Xuanwei lung cancer cell line model or an animal model.

Further, the Xuanwei lung cancer cell line model construction method is: cell culture, construction of the target gene plasmid containing the ACSM5-P425T gene sequence, lentivirus packaging of the target gene plasmid, cell infection, and detection of the lentivirus packaged target gene plasmid. Titer.

Furthermore, the method for constructing the animal model is as follows: injecting cells overexpressing the ACSM5-P425T gene sequence into mice, and culturing them to obtain a mouse model.

The invention also provides a recombinant plasmid. The ACSM5-P425T sequence is a single nucleotide polymorphism caused by a missense mutation of the C base at the chr16:20442608 site in the gene ACSM5 to an A base.

The present invention also provides an application of knocking out or inhibiting the expression of the ACSM5 gene and/or its encoded protein in constructing a drug detection model for treating Xuanwei lung cancer.

Technical effects achieved by this invention:

1. Through preliminary testing and screening, the present invention believes that ACSM5-P425T is an oncogene related to Xuanwei lung cancer and ACSM5 is a tumor suppressor gene. Through further research and construction of relevant cell and animal models, it can provide precise treatment and screening for Xuanwei lung cancer. More effective drugs, and reveal the impact of drugs on the development of Xuanwei lung cancer from in vitro cell experiments and in vivo animal tumorigenesis experiments

2. The stably expressed wild-type ACSM5, A549 and JT cells successfully constructed by the method of the present invention are significantly inhibited in their proliferation, plate colony formation ability, migration and invasion. The cell cycle is mainly blocked in the G0/G1 phase, and early apoptotic cells increase, in contrast, mutant ACSM5-P425T obtained the opposite result. It is worth noting that ACSM5 wild type and ACSM5-P425T mutant type have a greater impact on JT cell proliferation than A549 cells, while the plate colony formation ability is opposite, indicating that A549 cells are more malignant than JT cells; compared with ACSM5 wild type, paclitaxel In ACSM5-P425T mutant A549 cells and JT cells, the drug concentrations CC50 that caused half of the cell death were 3.507 μg/ml and 75.95 μg/m respectively. The difference between the groups was statistically significant (P<0.05), indicating that paclitaxel has a significant effect on JT cells are more susceptible to drug resistance, further demonstrating the application of ACSM5-P425T mutant cells in screening drugs for the treatment of Xuanwei lung cancer.

3. The results of the in vivo nude mouse tumor-bearing experiment showed that compared with the stably expressing wild-type ACSM5 group, the transplanted tumors in the ACSM5-P425T mutant group grew faster, increased in weight, increased ki-67 expression, and decreased Tunel apoptotic bodies. The difference between groups was statistically significant (P<0.05).

Description of the drawings

Figure 1 ACSM5 wild-type sequence and mutant sequence (base 1273 in the ORF region is replaced from C to A) and schematic diagram of the PReceiver-Lv201 vector;

Figure 2 Western Blot analysis results of ACSM5 total protein expression in Xuanfei lung cancer JT cells and different NSCLC lines;

Figure 3 Sanger reverse sequencing of plasmids of ACSM5 wild strain and ACSM5-P425T mutant strain (A) and forward sequencing of A549 cells and JT cells (B);

Figure 4 qPCR detection results after lentivirus infection of 293T cells; (including titer amplification CT curve (A), standard curve (B) and titer (C))

Figure 5 A549 and JT cells construct the control empty strain, ACSM5 wild strain and ACSM5-1 mutant strain and the verification results; ((A) A549 cells construct the stability of the empty strain, ACSM5 wild strain and ACSM5-1 mutant strain Vectors and verification results, (B) Stable vectors and verification results of JT cells constructing empty strain, ACSM5 wild strain and ACSM5-1 mutant strain, (C) A549 and JT cells constructing stable vectors of empty strain, ACSM5 wild strain and ACSM5-1 Western Blot verification in mutant strains)

Figure 6 Effect diagram of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the proliferation ability of A549 and JT cells;

Figure 7 Effects of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the clonogenic ability of A549 and JT cells (40×);

Figure 8 Effect diagram of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the cycle of A549 cells (A) and JT cells (B);

Figure 9 The effects of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the apoptosis of A549 cells (A) and JT cells (B);

Figure 10 Scratch experiment to detect the effects of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the migration ability of A549 cells (A) and JT cells (B) (4×);

Figure 11 Through Transwell experiment, detect the effects of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the migration ability of A549 cells (A) and JT cells (B) (10×);

Figure 12 Through Transwell experiment, the effects of NC, ACSM5-W wild type (ACSM5) and ACSM5-T mutant (ACSM5-1) on the invasion ability of A549 cells (A) and JT cells (B) are detected (10×);

Figure 13 Western blot experiment detects the expression levels of EMT-related proteins in each experimental group in A549 cells and JT cells;

Figure 14 The CCK-8 method detects the CC50 (unit: μg/ml) effect of paclitaxel in different treatment groups;

Figure 15: Establishment of nude mouse tumor-bearing model (A) and comparison of the growth rate of transplanted tumors in each cell group (B);

Figure 16 A. The cells of each group were inoculated subcutaneously at a single point in the anterior axillary limb and the tumors were taken out 15 days later to compare the size of the cell transplanted tumors in each group; B. Statistical analysis of the weight of the cell transplanted tumors in each group;

Figure 17 A and C. Ki-67 staining of transplanted tumor tissue and statistical analysis of cells in each group (10×); B and D. TUNEL staining of transplanted tumor tissue of cells in each group and statistical analysis (10×);

In the above figure, comparisons between groups are involved: *: P＜0.05; **; P＜0.01; ***; P＜0.005; ****P＜0.001;

Detailed ways

The concept of the present application and the technical effects produced will be clearly and completely described below in conjunction with the embodiments and drawings to fully understand the purpose, features and effects of the present application. Unless otherwise defined herein, scientific and technical terms used in conjunction with the disclosure of the present invention shall have meanings commonly understood by those of ordinary skill in the art. Products purchased in test methods, if specific conditions are not specified, shall be manufactured in accordance with conventional conditions or It is carried out under the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they can all be purchased from commercially available conventional products.

The ACSM5 gene in the present invention is a public sequence, and the NCBI sequence number is: NM_017888.2.

The ACSM5-T, ACSM5-1 and ACSM5-P425T mutations in the drawings and instructions of the present invention all refer to the single nucleotide caused by the missense mutation of the C base at the chr16:20442608 position in the gene ACSM5 to the A base. Polymorphism.

Preliminary research of the present invention shows that ACSM5 plays the role of a tumor suppressor gene in Xuanwei lung cancer patients. When the C base at position 1273 of the SNP site chr16:20442608 of ACSM5 is missense-mutated into an A base, the encoded hydrophobic proline After the acid is converted into hydrophilic threonine, its mutant ACSM5-P425T may function as an oncogene. qPCR, Westernblot, immunohistochemistry and other experiments have further confirmed that high and low expression of ACSM5 are related to clinically relevant information such as tumor size and stage. In summary, we believe that the ACSM5-P425T mutant may be involved in the occurrence and development of Xuanfei lung cancer. Therefore, the construction of cells or animal models containing the ACSM5-P425T mutant, or cells or animal models with low expression of ACSM5, can be targeted. Screening of drugs for the treatment of Xuanwei lung cancer.

As a specific embodiment, ACSM5-P425T can be used in the construction of a drug detection model for the treatment of Xuanwei lung cancer. The ACSM5-P425T sequence is caused by a missense mutation of the C base at the chr16:20442608 position in the gene ACSM5 to the A base. The single nucleotide polymorphism is formed in the model overexpressing the ACSM5-P425T gene sequence.

As some embodiments, optional models are Xuanwei lung cancer cell line models or animal models.

As a more specific implementation, the Xuanwei lung cancer cell line model construction method is: cell culture, construction of the target gene plasmid containing the ACSM5-P425T gene sequence, lentivirus packaging of the target gene plasmid, cell infection, and detection of lentivirus packaging. The titer of the target gene plasmid.

As another more specific embodiment, the method for constructing an animal model is to inject cells overexpressing the ACSM5-P425T gene sequence into mice, and then culture the cells to obtain a mouse model.

As another embodiment, the present invention also provides a recombinant plasmid, the recombinant plasmid contains the ACSM5-P425T base sequence, and the ACSM5-P425T sequence is the C base missense mutation at the chr16:20442608 site in the gene ACSM5 It is the formation of single nucleotide polymorphism caused by the A base.

As another embodiment, knocking out or inhibiting the expression of the ACSM5 gene and/or its encoded protein can be used to construct a drug testing model for treating Xuanwei lung cancer.

As described above, preferred embodiments are shown for ease of understanding. The scope of the invention is not limited to the embodiments and examples specifically described herein, and is limited only by the scope of the claims. The embodiments of the present invention are specifically shown below based on the test process.

Example 1

1 ACSM5 protein expression in Xuanwei lung cancer JT cell lines and NSCLC cell lines

Compared with HBE cells, the ACSM5 protein expression level of A549 cells in NSCLC cell lines (A549, H460, H1299, H1975) is slightly lower than HBE, and the ACSM5 protein expression level of Xuanwei lung cancer JT cells is slightly higher than HBE. There is no difference in the above. Statistical significance (P>0.05). However, the expression of ACSM5 in NSCLC lines H1299 and H1975 was significantly higher than that in HBE, and that in H460 was significantly lower than that in HBE. There was a statistical difference between the groups (P<0.05) (Figure 2). Therefore, A549 cells and JT cells will be used in subsequent in vivo and in vitro cell experiments.

2 Plasmid construction and verification of ACSM5 wild strain and ACSM5-P425T mutant strain

According to the plasmids of the ACSM5 wild strain (ACSM5-W) and the ACSM5-P425T mutant strain (ACSM5-T) constructed in step 2.3 of the present invention, before transfection and screening of cell lines stably expressing the target gene, Sanger sequencing is performed to ensure that the corresponding plasmids are constructed. Successful (the specific process was entrusted to Beijing Qingke Biological Company to complete). The reverse sequencing results of the plasmids showed that base 1273 of the ACSM5-W plasmid was C, and base 1273 of the ACSM5-T plasmid was A, indicating that the plasmid for the target gene was successfully constructed (see Figure 3-A).

At the same time, the candidate A549 cells and JT cells were sequenced by Sanger to ensure that before corresponding plasmid transfection and cell line screening, the candidate A549 cells and JT cells did not have the natural mutation of the C base to base A at position 1273. Sequencing The results were consistent with the wild-type plasmid sequencing results, indicating that there was no naturally mutated A base at base 1273 in A549 cells and JT cells (see Figure 3-B).

The above results show that the plasmids of the ACSM5 wild strain and the ACSM5-P425T mutant strain were successfully constructed. At the same time, there is no natural mutation of the A base at position 1273 of ACSM5 in A549 cells and JT cells, which is convenient for subsequent plasmid transfection and stable expression. Gene-based cell line screening provides accuracy assurance.

3 Lentivirus titer detection status

According to step 2.3.3 of the invention, the target gene plasmid undergoes a series of processes such as lentivirus packaging, infection of 293T cells, concentration and purification, extraction of lentiviral RNA in the supernatant, and reversal, and then the lentivirus-packaged target gene plasmid is detected by qPCR method Titer to ensure the success rate of subsequent infection and screening of cells A549 and JT that stably express the target gene plasmid. Figure 4 shows that the titers of the target genes ACSM5 (wild type) and ACSM5-1 (mutated type) and the empty PReceiver are both higher than the medium concentration standard value of 16.13. This result shows that the titer of the target gene is consistent with the concentration value for subsequent infection and selection of stably transfected A549 and JT cells.

4Construction of wild-type ACSM5-W and mutant ACSM5-T stable vectors

JT cells and A549 cells were infected with the PReceiver-ACSM5-W wild-type lentiviral vector, PReceiver-ACSM5-T mutant lentiviral vector, and the control vector PReceiver-GFP constructed by the present invention, expressing green fluorescent GFP, and treated with 1.5ug Puromycin at a concentration of /ml was used to screen out infection-positive cells until the infection positivity rate reached more than 80%. The selected positive cells were expanded and cultured, and the cells were harvested for subsequent experiments. The construction effect of stable cell lines was verified through qPCR and WesternBlot experiments (Figure 5).

The above results show that A549 and JT cell lines stably expressing control empty, ACSM5-W wild type and ACSM5-T mutant were successfully constructed.

Example 2

Effects on the in vitro biological behavior of wild-type ACSM5-W and mutant ACSM5-T cell lines 1. Effect on cell proliferation ability

NC cells (ngetivecontrol), wild-type A549 cells and JT cells, and mutant A549 cells and JT cells were seeded in 96-well plates respectively. The CCK-8 method was used to detect cell proliferation ability every 24 hours, with time as the horizontal axis and relative cell changes. The absorbance is the ordinate to draw the growth curve (Figure 6). The results showed: ① The proliferation ability of JT cells in each group was stronger than that of A549 cells, which was especially obvious starting from 72 hours; ② The growth of ACSM5-W wild-type A549 cells and JT cell group was significantly inhibited compared with the other two groups (P＜0.05), suggesting that ACSM5- The P425T mutant can restore the proliferation ability of ACSM5-W wild-type A549 cells and JT cells; ③ There is no significant difference between the NC group and the mutant A549 cells and JT cell groups (P<0.05), suggesting that the lentiviral expression vector has no effect on cell proliferation.

The above results show that the ACSM5 wild strain can inhibit cell proliferation (JT cells > A549 cells); the ACSM5-P425T mutant strain can promote the above biological processes (JT cells > A549 cells).

2 Effects of cell colony formation

Cell proliferation experiments indicate that the ACSM5-P425T mutant can restore the proliferation ability of ACSM5-W wild-type A549 cells and JT cells. In order to further verify that the ACSM5-P425T mutant has a higher colony-forming ability than the ACSM5-W wild type, NC cells and Wild-type A549 cells and JT cells, and mutant A549 cells and JT cells were verified by plate colony formation experiments (Figure 7). The results show that: ① In A549 cells and JT cells, the ACSM5-P425T mutant has stronger clonogenic ability than the ACSM5-W wild type (P＜0.05), but weaker than the NC group (P＜0.05); ② Although JT cells have stronger proliferation ability than A549 cells (5.1), A549 cells have stronger clonogenic ability than JT cells.

The above results show that the ACSM5 wild strain can inhibit cell clone formation (malignant degree A549 cells > JT cells); the ACSM5-P425T mutant strain can promote the above biological processes.

3 Effects of Cell Cycle

In the A549 cell group, compared with the ACSM5-P425T mutant group and NC control group (Figure 8-A), A549 cells stably transfected with ACSM5-W wild type: ① The proportion of cells in the G0-G1 phase increased (P<0.05) , and was significantly lower than that of JT cells (53.85%<70.71%, A549VSJT); ②The proportion of cells in the G2-M phase was between the ACSM5-P425T mutant group and the NC control group, and was different from the NC group (P<0.05). There was no difference from the ACSM5-P425T mutant group (P＞0.05); ③The proportion of S phase cells decreased (P＜0.05).

In the JT cell group, compared with the ACSM5-P425T mutant group and NC control group (Figure 8-B), JT cells stably transfected with ACSM5-W wild type: ① The proportion of cells in the G0-G1 phase was significantly increased (P<0.05 ); ② The proportion of cells in G2-M phase decreased, and there was no statistical difference between groups (P>0.05); ③ The proportion of cells in S phase decreased (P<0.05).

The above results show that the ACSM5 wild strain can promote cell arrest in the G0-G1 phase; the ACSM5-P425T mutant strain can inhibit the above biological processes.

4 Effects of Apoptosis

Annexin-V/7-ADD flow cytometry was used to detect the proportion of early apoptotic cells in A549 cells and JT cells in NC, ACSM5-W wild type and ACSM5-T mutant. As shown in Figure 9, early stage of flow cytometry analysis Apoptotic cells, namely Q4 group, were statistically analyzed for the ratio of early apoptotic cells. The results showed that compared with the ACSM5-P425T mutant group and NC control group, the rates of early apoptotic cells in A549 and JT cells stably transfected with ACSM5-W wild type were significantly increased (P<0.05), among which ACSM5-T The early apoptotic cell rate of mutant JT cells was higher than that of the corresponding A549 cells, and the difference was statistically significant (P<0.05).

The above results show that the ACSM5 wild strain can promote early apoptosis of cells; the ACSM5-P425T mutant strain can inhibit early apoptosis of A549 cells and JT cells.

5. Effect of cell migration ability

5.1 Scratch test

In the A549 cell group (Figure 10-A), the number of migrating cells under the scratched area was almost not affected by time in the ACSM5-W wild-type group. On the contrary, in the ACSM5-P425T mutant group and NC control group, the number of migrating cells under the scratch area increased in a time-dependent manner. The number of migrating cells at 48h was significantly higher than that at 0h and 24h (P<0.05), and the number of migrating cells in the NC control group was significantly higher than that at 0h and 24h (P<0.05). The number of migrating cells under the scar area was more than that in the ACSM5-P425T mutant group, and there was a statistical difference between the groups (P<0.05).

In the JT cell group (Figure 10-B), the trend of the number of migrating cells under the scratch area is basically the same as that of A549 cells. However, the number of migrating cells is significantly less than that of the A549 cell group.

The above results show that the ACSM5 wild strain has the ability to inhibit cell migration; the ACSM5-P425T mutant strain has the ability to promote cell migration (A549 cells > JT cells).

5.2 Transwell migration experiment

In the A549 cell group (Figure 11-A) and JT cell group (Figure 11-B), the number of cells entering the lower chamber in the ACSM5-W wild-type group was approximately ((443 cells/field of view) and (385 cells/field of view)) It was less than the ACSM5-P425T mutant group ((583/field) and (523/field)) and the NC control group ((655/field) and (575/field)) (P<0.05). This shows that the ACSM5-W wild strain significantly affects the migration ability of A549 cells and JT cells, and the conclusion is consistent with the scratch experiment in 5.5.1.

The above 5.5.1+5.5.2 results show that: the ACSM5 wild strain has the ability to inhibit cell migration; the ACSM5-P425T mutant strain has the ability to promote cell migration.

6. Influence of cell invasion ability

In the A549 cell group (Figure 12-A) and JT cell group (Figure 12-B), the number of cells in the ACSM5-W wild-type group that penetrated Matrigel and entered the lower chamber was approximately ((408/field of view) and (363 individuals/field)) are less than the ACSM5-P425T mutant group ((487/field) and (408/field)) and the NC control group ((624/field) and (558/field)) ( P<0.05). This shows that the ACSM5-W wild strain significantly affects the invasion ability of A549 cells and JT cells.

7 Epithelial to mesenchymal transition

Epithelial-Mesenchymal Transition (EMT) is a process in which epithelial cells lose the characteristics of epithelial cells and acquire the characteristics of mesenchymal cells. It has been judged by relevant literature to be the key to invasion and metastasis. The expression level of its related epithelial protein E-Cadherin decrease, and the expression levels of related interstitial proteins N-Cadherin and Vimentin increase. The changing trends in the expression levels of the above related proteins often indicate the occurrence of EMT.

Therefore, the ACSM5-W wild type has the ability to inhibit the invasion of A549 and JT cells, while the ACSM5-T mutant type, on the contrary, can promote the invasion of A549 and JT cells. We used Western-blot experiments to verify the changes in the expression levels of the above three EMT-related proteins at the molecular level to verify whether A549 and JT cells stably expressing ACSM5-W wild type and ACSM5-T mutant inhibit or promote the EMT process. .

The results in Figure 13 show: ① In A549 cells, compared with the ACSM5-W wild-type group, the expression levels of interstitial cell-related proteins N-Cadherin and Vimentin increased to varying degrees in the NC group and ACSM5-T mutant group. There was a statistical difference between the three groups (P<0.05), while the expression levels of the epithelial cell-related protein E-Cadherin were significantly weaker (not statistically significant); ② In JT cells, only the expression level of Vimentin protein was compared among the three groups. There is a statistical difference (P<0.05).

The above results cannot yet clarify whether the ACSM5 wild strain and the ACSM5-P425T mutant strain can inhibit or promote the epithelial-to-mesenchymal transition of cells.

8Paclitaxel resistance index detection

Paclitaxel is one of the anti-tumor drugs that is often used in combination with cisplatin in chemotherapy for advanced lung cancer. However, after being used for a period of time, it can easily cause drug resistance and affect the effect of chemotherapy. Therefore, in this study, different concentrations of paclitaxel (0μg/ml, 5μg/ml, 10μg/ml, 20μg/ml, 40μg/ml, 80μg/ml and 160μg/ml) were used to treat NC, ACSM5-W wild type and ACSM5-T. For mutant A549 and JT cells, the CCK-8 method was used to calculate the drug concentration at which half of the cells died (50% cytoxicity concentration, CC50). The results in Figure 14 show that compared with the NC group and ACSM5-W wild-type group, the ACSM5-T mutant group is less sensitive to paclitaxel (P<0.05), that is, the drug resistance is higher (JT cells>A549 cells) .

The above results show that the stably expressing ACSM5-P425T mutant strain is more likely to be resistant to paclitaxel than the wild strain ACSM5 (JT cells > A549 cells).

Example 3

Effects of tumor-forming ability of wild-type ACSM5-W and mutant ACSM5-T cell lines in vivo

1. Influence of tumor volume and mass in vivo:

Animal models of non-Xuanwei lung cancer and Xuanwei lung cancer were established through NC, A549 cells and JT cells stably expressing ACSM5-W wild type and ACSM5-T mutant type (Figure 15-A). Since the tumor-bearing model of A549 cells in nude mice is significantly better than that of JT cells (the tumor formation of JT cells in nude mice is not good and is not shown in this article), the A549 cell model was selected for the subsequent in vivo tumor formation experiments in nude mice.

The growth curves of transplanted tumors in each group of cells (NC, A549 cells stably expressing ACSM5-W wild type and ACSM5-T mutant) are shown (Figure 15-B and C). The growth rate of the group stably expressing ACSM5-W wild type was significantly lower than NC and stably expressing ACSM5-T mutant group (P<0.05), indicating that stably expressing ACSM5-W wild type significantly inhibits the growth rate of tumor cells, while ACSM5-T mutant type can promote the growth rate of tumor cells.

After 15 days, the tumors were peeled off and weighed, and the tumor weights in each group were compared. The results showed (Figure 16): The transplanted tumor weight of the wild-type group stably expressing ACSM5-W was lighter than that of the NC group and the mutant group stably expressing ACSM5-T (P<0.05). This shows that the ACSM5-W wild type can inhibit the tumor formation ability of tumor cells, while the ACSM5-T mutant type can promote the tumor formation of tumor cells.

2 Effects on proliferation and apoptosis of transplanted tumor cells:

The nude mice were sacrificed 15 days later, and the transplanted tumors were embedded in paraffin and sectioned. The cell proliferation activity of each group was observed by Ki-67 immunohistochemical staining. As shown in the figure (Figure 17-A): Compared with the wild-type group stably expressing ACSM5-W, the cells in the tissue sections of the NC and stably expressing ACSM5-T mutant groups were significantly stained for Ki-67 (green fluorescence, indicated by red arrows). increased, and the difference between groups was statistically significant (P<0.05), indicating that ACSM5-W wild type can inhibit tumor cell proliferation, while ACSM5-T mutant type can promote tumor cell proliferation. This is consistent with the results of previous in vitro cell proliferation experiments.

The TUNEL apoptosis detection method was used to observe the early apoptosis of transplanted tumor tissue cells. The results showed (Figure 17-B): Compared with the NC and stably expressing ACSM5-T mutant groups, the tissue sections of the stably expressing ACSM5-W wild-type group had significantly more cells stained by TUNEL (green fluorescence, indicated by red arrows). The results suggest that ACSM5-W wild type can significantly promote NSCLC cell apoptosis, while ACSM5-T mutant type can inhibit NSCLC cell apoptosis. This is consistent with the results of previous in vitro AnnexinV/7-AAD flow cytometry analysis.

The above results show that the ACSM5 wild strain can inhibit the proliferation of NSCLC tumor cells and promote their apoptosis. On the contrary, the ACSM5-P425T mutant strain promotes the proliferation of NSCLC tumor cells and inhibits their apoptosis.

In summary, the present invention constructs A549 and JT cells that stably express ACSM5 wild type and ACSM5-P425T mutant type, and observes the proliferation, plate colony formation, cell cycle, apoptosis, and migration of the above two cell lines at the cell level in vitro. and changes in invasion, epithelial-to-mesenchymal transition and paclitaxel resistance. The results prove that ACSM5 can inhibit Xuanwei lung cancer cell proliferation, plate colony formation, migration and invasion, block cells in the G0/G1 phase, and promote early apoptosis. Playing the role of a tumor suppressor gene, its mutant ACSM5-P425T can promote the changes in the above-mentioned cell biological behaviors and play the function of "driver mutation", thereby affecting the prognosis of Xuanwei lung cancer patients; at the level of nude mouse transplant tumors in vivo, Conclusions consistent with in vitro cell experiments were also obtained; the paclitaxel resistance index experiment also showed that JT cells are more susceptible to drug resistance than A549 cells.

Research Materials and Methods

1Experimental materials

1.1 Experimental cell lines

The human Xuanwei lung cancer cell line JT was preserved by the laboratory of the Medical Laboratory Department of the First Affiliated Hospital of Kunming Medical University. For cell properties, please refer to (Ma Liju, Wang Hongzhi, Bian Li, et al. Establishment and characteristics of Xuanwei lung adenocarcinoma cell line XLA-07 [J]. Chinese Journal of Pathology, 2012, 41(5): 335-339. The JT cells in the present invention are passage cells of the cell line XLA-07); 4 types of non-small cell lung cancer NSCLC cell lines (A549, H460 , H1299, H1975) and normal human bronchial epithelial cell line HBE were purchased from the cell bank of Kunming Institute of Zoology, Chinese Academy of Sciences.

1.2 Experimental animals

4-week-old BALB/C-nude female nude mice (N0.430727210100378365) were purchased from Hunan Slack Jingda Experimental Co., Ltd. and weighed approximately 120 mg when purchased. The living environment is maintained at a suitable temperature and humidity, and water and feed are added regularly.

1.3 Main reagents and consumables

1.4 Preparation of routine laboratory reagents

(1) PBS phosphate buffer for cell culture (1L, needs to be sterilized by high temperature and high pressure before use): NaCl (8.0g), KCl (0.2g), Na2HPO4·12H2O (2.86g), KH2PO4 (0.27g). (2) SDS electrophoresis buffer (1mL): Glycine (14.4g), Tris (3g), SDS (1g).

(3) SDS transfer buffer (1mL): Glycine (14.4g), Tris (3g), 200ML methanol.

(4) 6×SDS loading buffer: 4×Tris-HCl/SDS (pH6.8) (7mL), Glycerol (3.0mL), DTT (0.933g), Bromophenolblue (1.2mg).

2Experimental methods

2.1 Cell culture

The culture medium of JT and A549 cells is RPMI1640, 10% FBS; the culture medium of other cells is DMEM, 10% FBS. Screen the JT and A549 cells that stably express the target gene. Add 100 U/mL Penicyllin and 100 μg/mL Streptomycin to the culture medium.

2.2 ACSM5 protein expression in Xuanwei lung cancer and various lung cancer cell lines and screening of candidate cell lines

(1) Collect the target protein: JT cells in the logarithmic growth phase and cells of each NSCLC cell line (A549, H460, H1299, H1975) and normal human cells in a 6-well plate at a density of 5×10 ⁵ cells/mL/well Bronchial epithelial cell line HBE, after culturing in RPMI1640 or DMEM serum-free medium for 24 hours, remove the culture medium and wash the cells three times with PBS buffer. After removing the PBS buffer, add 200 μL of RIPA lysis buffer + protease inhibitor to each well of the 6-well plate. Add 2 μL of agent + 2 μL of phosphatase inhibitor, place on ice for lysis for 30 minutes, centrifuge at 12,000 rpm for 5 minutes, and draw the supernatant to obtain the total protein of each cell line. (2) Use BCA method to quantitatively measure the target total protein. (3) Conduct electrophoresis test by SDS-PAGE method. (4) Analysis results: Analyze the gray value of each cell line according to Image software, and compare the differences in the total protein expression of ACSM5 between each cell line. (5) Select candidate cell lines for subsequent in vivo and in vitro experiments according to step (4).

2.3 Construction of plasmids for ACSM5 wild strain and ACSM5-P425T mutant strain

Query the ORF sequence of the ACSM5 gene from the NCBI website, and load its wild-type sequence and mutant sequence (the 1273rd base of the ORF region is replaced from C to A) on the PReceiver-Lv201 empty vector, using E. coli amplification and ampicillin A large number of target vectors were screened, and this process was entrusted to Guangzhou Funeng Gene Co., Ltd. (Figure 1).

2.3.1 Vector sequencing identification

2.3.1.1 Vector amplification

(1) Add 10 μL of the lentiviral plasmid synthesized by Fu Neng Company into 100 μL of Escherichia coli DH5a competent cells, flick the tube wall to mix, and place on ice for 30 minutes. Place it in a circulating water bath preheated to 42°C for about 90 seconds, then quickly move it to an ice water bath and incubate for 2 minutes. Then 500 μL LLB medium was added and placed on a shaking table at 37°C for 1 h. Take an appropriate amount of bacterial solution and apply it evenly on a plate containing ampicillin, place it in a constant temperature incubator and incubate it upside down for 16 hours.

(2) Inoculate a single colony into LB liquid medium containing ampicillin and culture it at 37°C for 12-16 hours. Then use the EndoFreemidiPlasmidKit kit for plasmid extraction.

(3) Collect the above-mentioned overnight bacterial culture in a 5mL centrifuge tube, centrifuge at 12000 rpm for 2 minutes and collect the bacterial liquid. Discard the supernatant and add 250 μL of cell resuspension solution. Shake thoroughly to make the bacterial liquid evenly suspended, then add 250 μL of cell lysis solution, and then Add 10 μL of proteinase K, invert 6 times to mix, and then let stand for 1-2 minutes to lyse the bacteria until they are clear. Add 350 μL of neutralizing solution and mix again by inverting upside down to precipitate the protein. Place it in an ice bath for 5 minutes. Centrifuge at 10,000 rpm for 10 minutes and then discard the protein. Collect the supernatant into a sterile E tube. Centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to Centrifuge again at 12,000 rpm for 1 min in the recovery column and discard the lower waste liquid. Add 600 μL of the pre-configured rinse solution and centrifuge at 12,000 rpm for 1 min. Discard the waste liquid in the lower layer. Centrifuge at 12,000 rpm for 2 min and then remove the residual rinse solution again. Transfer the recovery column to a new 1.5 mL EP tube on the ultra-clean bench and let it stand for 10- 20mmin, wait for it to dry naturally, add 95μL Nuclease-freeWater into the recovery column, let it stand for 2min, centrifuge at 2000rpm for 2min, collect the sample and perform numbering, electrophoresis, concentration determination and quality inspection.

2.3.1.2 Electrophoresis identification

(1) Gel preparation: Weigh 0.5g agarose with an electronic balance, boil it in a microwave oven for 1 minute and dissolve it in 50ml TAE buffer (1×). After cooling to 50-60°C, add 5μL nucleic acid dye, mix gently, and carefully pour in the previously prepared Insert the comb into the gel making tank, and be gentle to prevent the generation of a large number of bubbles. If bubbles are accidentally generated, you can use a sterile pipette tip to puncture them and let them sit for 40 minutes. After natural solidification, the 1% agarose will be solidified. glue.

(2) Sample loading: Before loading the sample, place the prepared gel into an electrophoresis tank containing TAE buffer and drain the air bubbles in the gel holes. When loading the sample, add 2 μL of sample mixed with 1 μL Loading Buffer at a ratio of 15,000 bp DNAmarker of the same volume in the leftmost hole or the rightmost hole. (3) Electrophoresis: During electrophoresis, the voltage is set to 150V and the electrophoresis time is 15-20 minutes. (4) Judgment: After electrophoresis, turn off the power, take out the gel block and put it into the gel imager, turn on the ultraviolet transmission light to observe the electrophoresis bands, judge the molecular length, and select samples with bright, single bands and consistent molecular weights to send for sequencing. .

2.3.1.3 Sequencing

Sequencing and comparison were performed near the mutation site in the ORF region of the ACSM5 gene carried by the vector. The specific process was entrusted to Beijing Qingke Biotechnology Company to complete.

2.3.2 Cell sequencing identification

After infecting 293T cells with the lentiviral vector, DNA was extracted and sequenced to compare whether ACSM5 had mutated. The specific sequencing process was completed by Beijing Qingke Biotechnology Company.

2.3.3 Lentivirus titer detection

2.3.3.1 Lentivirus packaging

(1) Prepare lentivirus packaging helper cells 293T

Resuscitate the 293T cells. When the 293T cells grow to 90%, discard the old culture medium, add PBS solution, shake gently, wash the cell growth surface, then discard the PBS solution, add an appropriate amount of EDTA-trypsin, and digest at 37°C for 1 ~2 minutes, observe under an inverted microscope. When the cells separate from each other and become round, pour out the trypsin solution, add fresh complete culture medium, mix the cells, and inoculate them into a 10cm cell culture plate. When the cells increase to 80%, lentivirus packaging is carried out.

(2) The virus is packaged in a sterile centrifuge tube, dilute 2.5ug of the vector to be transfected and 5μL of Lenti-PacHIV with 200μL of Opti-MEM medium, and culture it in another centrifuge tube with 200μL of Opti-MEM Dilute 15 μL of EndoFectin Lenti with base and incubate at room temperature for 5 minutes. Add the diluted EndoFectinLenti dropwise to the diluted carrier, gently shake the centrifuge tube containing the mixed solution, and incubate at room temperature for 20 minutes. Take the cells inoculated in a 10cm culture plate. When the cells grow to 80% confluence, discard the old culture medium, wash twice with PBS solution, and use new culture medium (containing 10% inactivated fetal bovine serum, without double antibodies). DMEM medium) to replace the old medium in the culture plate. Add the incubated carrier-EndoFectinLenti complex to the 293T cell culture plate, mix well, and place it in a cell culture incubator for 8-14 hours. Replace the old medium with DMEM medium containing 5% inactivated fetal bovine serum and 1% double antibody, and add 1/500 volume of TiterBoost. Collect the 293T cell culture medium 48 hours and 72 hours after transfection, centrifuge at 500g for 10 minutes, collect the supernatant, concentrate or aliquot and freeze at -80°C for later use.

(3) Lentivirus concentration and purification

After completing the lentivirus packaging operation, collect the supernatant from the tool cell culture plate or culture bottle. The supernatant contains lentivirus particles. The supernatant can be centrifuged at 2000g for 10 minutes at 4°C to remove cell debris, or you can choose to filter the cell debris with a 0.45μm filter membrane; mix the lentivirus supernatant and concentrate at a ratio of lentiviral liquid volume:concentrated reagent volume=5:1 Reagent (concentrated reagent can be added directly to 6X stock solution), and incubate at 0 to 4°C for 2 hours or more (it can also be incubated overnight). After completing the incubation, centrifuge the mixture at 3500g for 25 minutes at 4°C. After centrifugation, carefully aspirate and discard the supernatant; measure 1/10-1/100 volume of DMEM or PBS of the original solution, and re-suspend by pipetting. The lentivirus is precipitated; the resuspended lentivirus liquid has been concentrated and can be aliquoted and stored at -80°C. At the same time, a small amount is taken to measure the concentration of the lentivirus titer.

2.3.3.2 Lentivirus titer detection

(1) Viral RNA extraction:

Add 0.25ml RNAzol to 50 or 100 μL virus stock solution or 10 μL virus concentrate, invert 10 times to dissolve the virus, and incubate at room temperature for more than 15 minutes. Add 50 μL of pure water to the tube containing 50 μL of virus stock solution or 10 μL of virus concentrate. In short, the sum of the volume of virus liquid and the total volume of water is 100 μL. 20℃, 18000g, centrifuge for 10 minutes. Transfer the supernatant to a new 1.5 ml centrifuge tube, and add 1 μL of 1.5 mg/ml linear polyacrylamide per 100 μL of supernatant. Add an equal volume of isopropyl alcohol or three times the volume of absolute ethanol, mix well, and place in a -20°C refrigerator for 4 hours or overnight. 10℃, 18000g, centrifuge for 20min, discard the supernatant. Wash the RNA twice with 0.5 ml of 75% ethanol, each time at 18000g, 10°C, and centrifuge for 5 minutes. After drying in the air for 3 minutes, add 50 μL of water to dissolve the RNA.

(2) DNase I treatment: Add reagents according to the following system: DEPCwater--1.5μL, LentiviralRNA--20μL, DNaseIbuffer (10×)--2.5μL, DNaseI--1.0μL, incubate at 37°C for 30-60min, then 75 °C for 10 minutes to inactivate DNaseI.

(3) Reverse transcription: Add reagents according to the following system:

(DNaseItreated) RNA--10μL, 4.0umcDNAsynthesisprimer--5μL, incubate at 70°C for 5 minutes, then cool the sample on ice.

Add reagents according to the following system:

10×ReverseTranscriptionBuffer--2.0μL, 25mMDNTP--1.0μL, RNaseInhibitor--1.0μL, ReverseTranscriptase--1.0μL, incubate at 37℃ for 60min, 90℃ for 10min, the product can be used directly for subsequent qPCR or stored in a -20℃ refrigerator .

(4) qPCR detection of virus titer: Add reagents according to the following system: Water--6.0μL, 2XAll-in-OneqPCRMIX--10.0μLqPCRprimermix (2.5uMeach)--2.0μL, DNAstrandardorcDNAsampleorwater--2.0μL.

Follow the following procedure to react

Temperature Interval Duration Read 72--95℃ 0.5℃ 6sec/each On 30℃ 30sec off

(5)Data analysis

CT value corresponds to the copy number of the standard curve = CT value corresponds to the titer × PCR system standard (sample) volume, virus titer = CT value corresponds to the copy number of the standard curve × dilution factor (copies/ml)

Dilution factor = (RNA volume/virus sample volume) × (Total volume of DNaseI treatment/Volume of RNA used for DNaseI treatment) × (Total volume of reverse transcription/Volume of RNA used for reverse transcription) × (1000 μL/ml/for qPCR volume of).

2.4 Construction of stably transfected cell lines

2.4.1 Cell culture

(1) Cell recovery: Adjust the water bath temperature to 37°C. According to the records, take out the frozen A549 and JT cells from the liquid nitrogen irrigation, shake them quickly in a 37°C water bath, wait for them to dissolve, quickly transfer them to a 15ml centrifuge tube containing PBS, and centrifuge at 1000r/min for 5 minutes. Pour off the supernatant, add culture medium to resuspend the cells, mix by pipetting, transfer the cell suspension to a T25 cell culture flask, and place it in a 37°C, 5% CO2 incubator.

(2) Cell passage: When the cells grow to 80%, aspirate the culture medium in the cell culture flask, rinse the cells twice with PBS, add an appropriate amount of 0.25% trypsin for digestion, and wait until most of the cells become round and separated from each other. , add an appropriate amount of RPMI1640 complete culture medium containing fetal bovine serum to terminate digestion, pipe gently to make a single cell suspension, centrifuge at 1000r/min for 5min, and passage 1/3.

(3) Sequencing identification: DNA was extracted from some JT and A549 cells after passage and sequenced to compare whether ACSM5 had natural mutations. The specific sequencing process was completed by Beijing Qingke Biotechnology Company.

2.4.2 Infection of A549 and JT cells with wild-type and mutant ACSM5 lentivirus

The above lentivirus and culture medium were used to infect A549 and JT cells (106) with a growth density of 75% at a ratio of 1:1, and Polybrene with a final concentration of 8ug/ml was added to facilitate virus infection. 8 hours after infection, replace the old medium with fresh medium containing 5% FBS (inactivated) and 1% double antibody, and continue to culture in a CO2 incubator. After 72 hours, fresh culture medium containing puromycin at a concentration of 1.5ug/ml was added to screen positive cells for infection. The medium was changed every 2 days, and the optimal concentration of puromycin was used to screen positive cells for infection. Until the infection positive rate reaches more than 80%, the selected positive cells will be expanded and cultured, and the cells will be harvested for subsequent experiments. Wild-type ACSM5 and mutant ACSM51 cells were taken from A549 and JT cells respectively, and normal cultured A549 and JT cells were used as blank strains and A549 and JT cell PReceiver-Lv201 were used as empty strains for qPCR, proliferation, migration, Detection of invasion, cycle and apoptosis.

2.4.3 Detection of changes in mRNA levels of ACSM5 gene related to stably transformed cell lines

2.4.3.1 Extraction of total cellular RNA

(1) After the adherent cells to be tested are cultured, carefully pour out the culture medium, gently add PBS prepared in advance, wash twice, centrifuge at 4000 rpm for 10 minutes, discard the supernatant and set aside. (2) Add 1ml Trizol reagent to the culture flask, shake the culture flask left and right to fully contact the cells, and let it stand on ice for 10 minutes to allow the cells to fully lyse. (3) After the cells are lysed, transfer them to a labeled and cooled 1.5 ml RNaes-free EP tube. (4) Add 200 μL of chloroform to 1 ml of Trizol reagent, shake vigorously manually for 15 seconds, and let stand at room temperature for 2-3 minutes. (5) Centrifuge at 12000g and 4°C for 15 minutes. It can be seen that the liquid is divided into three layers. The lower layer is pink phenol and chloroform, the middle layer is white, and RNA remains in the colorless upper aqueous phase. (6) Carefully absorb the upper aqueous phase and put it into another EP tube. Be careful not to absorb the middle layer and lower layer (if you accidentally absorb it, you must squeeze it out gently), then add 0.5ml of 100% isopropyl alcohol and mix upside down. Mix well and let stand for 10 minutes (when the sample amount is small, it can be placed in a -20 degree refrigerator overnight to improve the RNA extraction rate). (7) Centrifuge at 12000g and 4°C for 10 minutes, and it will be seen that white RNA precipitate is formed at the bottom of the tube (the precipitate is not visible to the naked eye when the sample amount is small, and does not affect normal operation). (8) Gently tilt the tube orifice to discard the supernatant. Be careful not to pour out the precipitate. Dry the tube orifice with absorbent paper. Add 1ml of 75% ethanol to the precipitate. Invert the tube several times to make the precipitate float and let it stand for 2 minutes. (9) Centrifuge at 7500g and 4°C for 5 minutes to allow the precipitate to adhere to the bottom of the tube again. If necessary, repeat this step twice to clean out the impurities. (10) Discard the supernatant, place the centrifuge tube upside down on absorbent paper, and dry naturally for 10 minutes or place it on a clean workbench to blow dry to evaporate the ethanol and water as much as possible but not too dry, otherwise it will affect the subsequent dissolution of RNA. . (11) Add 30 to 50 μL of RNase water to the dried RNA pellet and let it stand for 15 minutes to completely dissolve the RNA. Take 2 μL of RNA for electrophoresis detection. Dilute 3 μL of RNA to 3 ml with TAE buffer and put it on the spectrophotometer. Detect OD260 and OD280, calculate RNA purity/concentration and the loading amount in the following steps. (12) The remaining RNA is reverse transcribed immediately or frozen in a -80°C refrigerator.

2.4.3.2 Electrophoresis detection

(1) Gel preparation: Weigh 0.5g agarose with an electronic balance, boil it in a microwave oven for 1 minute and dissolve it in 50ml TAE buffer (1×). After cooling to 50~60℃, add 5μL nucleic acid dye, mix gently, and carefully pour in the previously prepared Insert the comb into the gel making tank, and be gentle to prevent the generation of a large number of bubbles. If bubbles are accidentally generated, you can use a sterile pipette tip to puncture them and let them sit for 40 minutes. After natural solidification, the 1% agarose will be solidified. glue. (2) Sample loading: Before loading the sample, place the prepared gel into an electrophoresis tank containing TAE buffer and drain the air bubbles in the gel holes. When loading the sample, add 2 μL of sample mixed with 1 μL LoadingBuffer, and place the same volume of DNAmarker in the leftmost hole or the rightmost hole. (3) Electrophoresis: During electrophoresis, the voltage is set to 150V and the electrophoresis time is 15 to 20 minutes. (4) Judgment: After electrophoresis, turn off the power, take out the gel block and put it into the gel imager, turn on the UV transmission light to observe the electrophoresis bands, judge the RNA quality (18s band, 28s band, brightness, etc.), take pictures and save them.

2.4.3.3 Concentration determination

Turn on the spectrophotometer in advance, then take 3 μL of the extracted RNA sample, dilute it with TAE to a volume of 3 ml, and after zeroing with TAE buffer, replace the cuvette with the sample to be tested, measure OD260 and OD280 respectively, and calculate both. If the ratio is between 1.8 and 2.0, it indicates that the RNA purity is better. Then calculate the RNA concentration,

The formula is RNA concentration (ug/ml) = OD260 × dilution factor × 0.04.

2.4.3.4 Detection of mRNA expression level of AMSC5 gene

(1) Reverse transcription of mRNA into cDNA

Use Guangzhou Genecopoeia company's

SureScript-First-strand-cDNA-synthesis-kit kit, follow the instructions, specifically: take out the components of the reverse transcription kit, melt them at room temperature, centrifuge briefly, place on ice, and add each component in the following table in sequence on ice Reagents and solutions:

After a brief centrifugation, perform reverse transcription on an ordinary PCR machine according to the conditions in the table below:

Note that since the loading amounts of SureScriptRTaseMix (20×) and SureScriptRTReactionBuffer (5×) are very small, you can mix them with an appropriate amount of ddH2O (RNase/DNasefree) in advance, and then mix them with RNA respectively. This operation can Improve sample loading accuracy and reduce unnecessary errors. All sample loading operations can refer to this method.

(2) RT-PCR (real-time fluorescence quantitative PCR) detection

First dilute the above reverse transcription product cDNA 5 to 20 times (all samples must be diluted according to the same multiple. Too high or too low concentration will affect the detection results. Gradient verification can be done first to determine the optimal dilution ratio) and then perform the following Q- PCR reaction. RT-PCR detection of mRNA used BlazeTaq ^TM from Guangzhou Genecopoeia Company GreenqPCRMix2.0 kit, add the reagents in the following table in order:

You can mix 5×BlazeTaqqPCRMix, PCRforwardprimer (2μM), PCRreverseprimer (2μM) and ddH2O (RNase/DNasefree) according to the calculated ratio in advance, and then mix them with cDNA according to their total volume. This operation can improve the accuracy of loading.

During the sample loading operation, you should check whether there are bubbles in the tip of the tip every time you draw a liquid. After blowing the liquid into the hole, you should also check whether there is any liquid residue in the tip. If there is any residue, the residual liquid should be blown into the instrument. in the corresponding wells, and the sample loading operation must be completed in one go. Other experimental operations or unrelated things cannot be performed at the same time to ensure the consistency of repeated wells.

After brief centrifugation, perform 40 cycles of reaction on the CFX96 real-time quantitative fluorescence PCR instrument under the following conditions:

Collect and record fluorescence, create amplification curves and dissolution curves, read Ct values, and analyze the relative expression of ACSM5. Note: The ACSM5 primer sequence is as follows:

The upstream primer is: 5′-TTGTGGATGATGAGGGCAACG-3′

The downstream primer is: 5′-AGAAACAGAAGGGCCGAGTGG-3′

2.4.3.5 Detection of AMSC5 protein expression level: This step is the same as 2.2 of the invention.

2.5 Functional experiment of stably transformed cell lines

2.5.1 Cell proliferation detection

A549 and JT cells in the logarithmic growth phase (empty control cells, cells transfected with ACSM5 wild type or mutant type) were digested and resuspended respectively and then seeded in 96-well plates, with 100 μL (2000 cells) in each well, respectively. After culturing for 24h, 48h, 72h, 96h and 120h, add 10 μL CCK8 solution to each well, and add corresponding amounts of cell culture medium and CCK8 solution but no cells to the blank control. Incubate the culture plate in the incubator for 4 hours. Use a microplate reader to measure the absorbance at 450 nm, and analyze the differences in cell proliferation in each group.

2.5.2 Apoptosis detection

(1) After digesting A549 and JT cells (empty control cells, cells transfected with wild-type or mutant ACSM5), transfer the cell solution to an EP tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. (2) Add 1 ml of pre-cooled PBS, rinse twice, discard the supernatant, and resuspend the cells in 1× binding buffer (10×, diluted with double-distilled water) to 1×106 cells/ml. (3) Transfer 100 μL of cell suspension (105 cells) to a centrifuge tube, add 5 μL of AnnexinV-PE and 5 μL of 7-AAD, mix and incubate at room temperature in the dark for 15 minutes. (4) Add 300 μL of 1×bindingbuffer and run it on the flow cytometer for detection within one hour. (5) Take the sum of the second quadrant (late apoptosis) and the fourth quadrant (early apoptosis) to compare the apoptosis changes after transfection with lentivirus.

2.5.3 Cell cycle detection

(1) Configuration of staining solution: Configure the corresponding PI staining solution according to the number of samples.

(2) Collect sample cells: Carefully aspirate the cell culture medium, digest the cells with trypsin, and prepare a single cell suspension. Centrifuge at 1000g for 3-5 minutes to pellet the cells, discard the supernatant, and rinse the cells once with 1 ml of ice-cooled PBS. Collect the cells by centrifugation, add about 1 ml of pre-cooled PBS again, resuspend the cells, centrifuge to pellet the cells, carefully remove the supernatant, add 1 ml of ice-cold PBS, and resuspend the cells. The number of cells is 1×10 ⁶ /ml. (3) Cell fixation: Take 4 ml of pre-cooled 95% ethanol, vortex at low speed, and add 1 ml of cell suspension drop by drop. Maintain the final concentration of ethanol between 70 and 75%. Mix well and immediately place on ice. Fix cells at 4°C for 2 hours or longer, and fix cells for 12-24 hours for better results. Fixed cells can be stored at 4°C for 2 days and -20°C for 1 week. (4) Cell resuspension: Centrifuge at 1000g for 3 to 5 minutes to precipitate the cells. If the precipitation of specific cells is insufficient, the centrifugation time can be appropriately extended or the centrifugal force can be slightly increased. Carefully aspirate the supernatant, leaving about 50 μL of ethanol to avoid aspirating the cells. Add about 5 ml of ice-cooled PBS, resuspend the cells, collect the cells by centrifugation, and carefully aspirate the supernatant. You can reserve 50 μL of PBS to avoid aspirating the cells. Gently tap the bottom of the centrifuge tube to properly disperse the cells and avoid clumping. (5) Cell staining: Add 500 μL of prepared staining solution to each tube of sample, and slowly and fully resuspend the cells. Incubate at 37°C in the dark for 30 minutes. After completion, it can be stored briefly at 4°C in the dark, but the sample needs to be tested within 24 hours, and the flow cytometry test must be completed on the same day. (6) Flow cytometry detection and analysis: Use a flow cytometer for detection, detect at the excitation wavelength of 488nm, and detect light scattering at the same time. Cellular DNA content analysis and light scattering analysis were performed using appropriate analysis software modfit-1.

2.5.4 Cell invasion detection

(1) The aliquoted Matrige gel is diluted to 300ug/ml with culture medium in advance, and 200 μL is added to the bottom polycarbonate membrane of the Transwell chamber with a pore size of 8um, so that all pores on the membrane can be covered by Mattige Matrigel. Dry at 37°C overnight. (2) Add 500 μL of five-serum culture medium to the 24-well plate until it just touches the bottom of the Transwell chamber. (3) A549 and JT cells (empty control cells, cells transfected with wild-type or mutant ACSM5) were taken from each group and 3.5×104 cells/well were suspended into 200 μL (complete medium containing 10% FBS) and inoculated in Transwell In the upper chamber, place it in an incubator and culture it for 5 hours. When the cells adhere, replace the medium. Wash the upper chamber with PBS and then add serum-free medium. Add complete medium containing 10% FBS to the lower chamber and culture for 24 hours. (4) 24h Then take it out and fix it with methanol for 10 minutes, discard the methanol, and dry it. (5) Staining: Stain with 0.5% crystal violet solution (prepared with formaldehyde) for 20 minutes, wipe off the cells on the top of the PET membrane with a cotton swab, and rinse again with PBS until the background is clear. ( 6) Decolorize with acetic acid and read the OD value. (7) Repeat step 4. (8) Cover the slide and count the cells.

2.5.5 Cell migration detection

(1) Add 500 μL of serum-free medium to the 24-well plate until it just touches the bottom of the Transwell chamber.

(2) For A549 and JT cells (empty control cells, cells transfected with ACSM5 wild type or mutant type), 3.5×10 ⁴ cells/well were taken from each group and suspended in 200 μL (containing 10% FBS complete medium, Inoculate the upper chamber of the Transwell and place it in an incubator for 5 hours. When the cells adhere, replace the culture medium. The upper chamber is washed with PBS and serum-free medium is added. The lower chamber is added with complete medium containing 10% FBS and cultured for 24 hours. ( 3) After 24 hours, remove and fix with methanol for 10 minutes, discard the methanol, and dry. (4) Staining: Stain with 0.5% crystal violet solution (prepared with formaldehyde) for 20 minutes, wipe off the cells on the upper part of the PET membrane with a cotton swab, and rinse again with PBS until the background Clear.

2.5.6 Scratch test

(1) Cell preparation: Take out A549 and JT cells in the logarithmic growth phase (empty control cells, cells transfected with ACSM5 wild-type or mutant cells), aspirate the original culture medium, and wash the cells with sterile PBS. Add 1 ml of 0.25% trypsin digestion solution to digest the cells. Observe under a microscope that all cells have completely shrunk and become round. Then add complete culture medium to terminate digestion. Collect the cell suspension in a 15 ml centrifuge tube and centrifuge at 800 rpm/min at room temperature for 5 min. Use a high-precision flow cytometer FILPLUS to count cells, discard the supernatant, and add culture medium to resuspend the cells. The cells were seeded into a 6-well culture plate at a density of 3.5 to 4 × 10 ⁴ cells per well, and cultured in RPMI1640 medium containing 10% fetal bovine serum for one day. It is better to cover it overnight. (2) Linear scratching: Take out the cells covering the six-well plate, use a 200 μL pipette tip perpendicular to the cell surface, and scratch from one end of the well to the other end. Try to make the scratch perpendicular to the horizontal line on the back. The tip of the gun should be vertical and not tilted. At this time, #-shaped scratches on the surface of the cells can be clearly seen on the surface of the culture dish. (3) Wash cells and culture with PBS: Aspirate off the old culture medium, wash the cell surface of each group three times with sterile PBS, remove the scratched cells, and add culture medium containing 1% fetal bovine serum. Gently shake the six-well plate so that the cell culture medium covers the entire 6-well plate, and place it in a 37°C, 5% CO2 incubator for culture. (4) Observation of results: Observe the scratch track widths of different treatment groups after scratching at 0 hours, 24hr, and 48hr respectively. The results can be seen: 48 hours after cell scratching, it was observed that the scratch width of the cells in the control group recovered by about 90%, and that in the experimental group recovered by about 60%, indicating that the cell migration in the experimental group was inhibited, while the cells in the control group maintained their original migration ability. The scratches are then covered up by migration.

2.5.7 Plate colony formation experiment

(1) Cell preparation: Trypsinize the A549 and JT cells of each experimental group (empty control cells, cells transfected with ACSM5 wild type or mutant type) in the logarithmic growth phase, and resuspend them in complete culture medium to prepare cells. Suspension, count. (2) Cell seeding: Inoculate about 100 cells/well of each experimental group in a 6-well culture plate (the normal proliferation rate is 1:5 ~ 1:10 passage, the cells will be full in 3 days, and 50 ~ 200 cells can be inoculated, and the rest For slow-proliferating cells, 800 to 1,000 can be inoculated; when inoculating, pay attention to the gradient dilution of the cell suspension and observe the cell density to avoid bias in the experimental results due to inaccurate counting). Each experimental group has 3 duplicate wells, and the culture medium contains 10% FBS complete medium. (3) Shake the inoculated cells well and place them gently in the incubator to continue culturing. Change the medium every 3 days and observe the cell status, and observe the clone size under a microscope. (4) Culture for about 10 to 14 days. Stop the culture when the number of cells in most single clones in the wells is about 50. Discard the supernatant and wash the cells once with PBS.

(5) Add 1 mL of 4% paraformaldehyde (toxic, please operate in a safety cabinet) to each well, fix the cells in a refrigerator at 4°C for 60 minutes, and wash the cells once with PBS. (6) Add 1000 μL of clean, impurity-free crystal violet dyeing solution to each well and stain the cells for 2 minutes. (Just dilute it from 0.5% to 0.1%, use methanol as solvent and PBS for dilution), (Add 10ml methanol to 50mg, and then dilute 5 times with PBS). (7) Wash the cells several times with ddH20, dry them, take pictures with a digital camera, and count the clones.

2.5.8 ACSM5 and ACSM5-P425T are resistant to paclitaxel

(1) Dilute paclitaxel according to the following proportions: 0, 5, 10, 20, 40, 80 and 160 μg/ml.

(2) After the cells of each experimental group are passaged and adhered to the wall, add the paclitaxel diluted in the above concentration gradient to continue culturing the cells. The remaining steps are the same as in 2.5.1 of the invention.

2.6 In vitro animal experiments

(1) Experimental grouping: BALB/c nude mice were randomly divided into three groups, namely A549-NC group,

A549-ACSM5-1 group (mutant type) and A549-ACSM5 group (wild type), 6 animals in each group, 18 animals in total. (2) Before the experiment, the cells in the A549-NC group, A549-ACSM5-1 (mutant type) and A549-ACSM5 group (wild type) in the logarithmic growth phase were resuspended to 1×106, and then mixed with Matrigel Prepare according to the ratio of 1:1 and mix thoroughly. (3) Aseptically inject 100 μL of the above three groups of cell suspensions into the forelimb axilla of each nude mouse randomly grouped in step (1). (4) Parameter recording: From the beginning of tumor formation, the size of the tumor (long diameter and short diameter) is measured every other day. After two weeks, the nude mice are sacrificed, and the complete tumor is taken. After measurement and photography, it is divided into two parts, and the part is fixed on Paraformaldehyde (for subsequent pathological sectioning), part of which is frozen in a -80°C refrigerator (can be used for PCR and other related tests).

2.6 Statistical analysis

Each experiment was repeated at least three times. SPSS21.0 software was used for analysis. Before analyzing the measurement data, a normality test was performed on the data. Normal data are expressed as X±SD, and skewed data are expressed as median (P25-P75). Comparison between two groups of normally distributed data uses the t test of two independent samples, and comparison between multiple groups uses ANOVA one-way analysis of variance; comparison between two groups of skewed distribution data uses the Mann-Whitney U test, and comparison between multiple groups uses Kruskal-Wallis. Rank sum test. Count data were expressed as rate (%), and chi-square test was used for comparison between groups. P<0.05 means the difference is statistically significant.

Claims (7)

1. The application of ACSM5-P425T in constructing a drug detection model for treating Xuanwei lung cancer is characterized in that the ACSM5-P425T sequence is formed by single nucleotide polymorphism caused by missense mutation of C base at chr16:20442608 site in gene ACSM5 into A base.
2. 2. The use according to claim 1, wherein the model overexpresses the ACSM5-P425T gene sequence.
3. 3. The use according to claim 2, wherein the model is an xuwei lung cancer cell line model or an animal model.
4. 4. The use according to claim 3, wherein the method for constructing the lung cancer cell line model comprises the following steps: cell culture, construction of a target gene plasmid containing ACSM5-P425T gene sequence, carrying out slow virus packaging on the target gene plasmid, carrying out cell infection and detecting the target gene plasmid titer of slow virus packaging.
5. 5. The use according to claim 3, wherein the animal model is constructed by the following steps: and injecting cells which over-express ACSM5-P425T gene sequence into mice, and culturing to obtain the mice model.
6. 6. A recombinant plasmid is characterized in that the recombinant plasmid contains an ACSM5-P425T base sequence, wherein the ACSM5-P425T base sequence is formed by single nucleotide polymorphism caused by missense mutation of C base at chr16:20442608 locus in a gene ACSM5 into A base.
7. 7. The application of knocking out or inhibiting the expression of ACSM5 gene and/or coded protein thereof in constructing a drug detection model for treating Xuanwei lung cancer.