CN116004817A - Gastric cancer circRNA marker and application thereof - Google Patents

Abstract

The invention discloses a gastric cancer circRNA marker and application thereof, and belongs to the technical field of biology. The circRNA marker is at least one of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_ 0043438. The invention discovers that the four kinds of annular RNA are related to the occurrence of gastric cancer for the first time, the expression of the four kinds of annular RNA in serum of a gastric cancer patient is obviously reduced, the occurrence of gastric cancer can be well predicted, and the four kinds of annular RNA can be used as a gastric cancer diagnosis marker. In addition, after the four markers are over-expressed, the proliferation capacity of gastric cancer cells can be obviously inhibited, and the four markers can be used as potential targets for gastric cancer treatment.

Description

Gastric cancer circRNA markers and their application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a gastric cancer circRNA marker and its application.

Background technique

Gastric cancer is a multi-stage, slow-going and multi-factorial pathological process. Helicobacter pylori infection, obesity, and excess salt and nitrate intake are associated with increased gastric cancer. In addition, gene mutations, epigenetic changes, and abnormal molecular signaling pathways are also involved in the occurrence and metastasis of gastric cancer. Therefore, it is crucial to determine the molecular patterns of gastric cancer and their specific biomarkers for the development of therapeutics targeting specific tumor behaviors.

Contents of the invention

The purpose of the present invention is to provide a gastric cancer circRNA marker and its application. The gastric cancer circRNA marker can well predict the occurrence of tumors, and can also inhibit the proliferation of gastric cancer cells after overexpression, thus suggesting that it can be used as a diagnosis of gastric cancer. It can also be used as a potential way for the treatment of gastric cancer.

Invention concept of the present invention:

A large number of studies have confirmed that many noncoding RNAs (ncRNAs) such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are related to the carcinogenesis process of gastric cancer and can be used as biomarkers for early risk assessment, clinical treatment and survival assessment. In addition, circRNAs are important members of the non-coding RNA family discovered in recent years. Increasing evidence indicates that circRNAs are involved in the occurrence and development of various diseases, including gastric cancer. Studies have found that most circRNAs are involved in the occurrence and development of some tumors, and are abnormally expressed in a tissue-specific manner under pathological conditions. These characteristics make circRNA an important target and potential marker for tumor diagnosis and treatment.

DNAJC6 (DnaJ Heat Shock Protein Family (Hsp40) Member C6, DnaJ Heat Shock Protein Family (Hsp40) Member C6), which belongs to the evolutionarily conserved DNAJ/HSP40 protein family, regulates molecular chaperone activity by stimulating ATPase activity. DNAJ proteins may have as many as 3 distinct domains: a conserved 70 amino acid J domain, usually at the N-terminus, a glycine/phenylalanine (G/F)-rich region, and a Domain of cysteine, containing 4 motif domains similar to zinc. Studies have reported that DNAJC6 promotes hepatocellular carcinoma progression by inducing epithelial-mesenchymal transition (Yang T, 2014).

TDRD3 (Tudor Domain Containing 3, Tudor Domain Containing 3) specifically recognizes and binds to the scaffolding protein of dimethylarginine-containing proteins. In the nucleus, TDRD3 acts as a coactivator: recognizes and binds asymmetric dimethylation on core histone tails (H3R17me2a and H4R3me2a) associated with transcriptional activation and recruits proteins at these arginine methylation loci . In the cytoplasm, TDRD3 may play a role in the assembly and/or disassembly of mRNA stress granules and the regulation of translation of target mRNAs by binding to the Arg/Gly-rich motif (GAR) in dimethylarginine-containing proteins . TDRD3 promotes DHX9 chromatin recruitment and R-loop release (Wei Yuan, 2021). TDRD3 promotes tumorigenesis and invasive ability of breast cancer cells (Alan Morettin, 2017).

GON4L (Gon-4 Like, Gon-4-like protein) is predicted to have transcriptional co-regulator activity, and GON4L drives cancer growth through the YY1-androgen receptor-CD24 axis (Neeraj Agarwal, 2016), but the role of its CircRNA in gastric cancer unclear.

The protein encoded by MED1 (Mediator Complex Subunit 1) is a subunit of the CRSP (cofactor required for SP1 activation) complex, which together with TFIID is necessary for the efficient activation of SP1, and MED1 is also a component of other multi-subunit complexes Some, such as thyroid hormone receptor (TR)-associated protein, interact with TR and together with initiation factors and cofactors promote TR function on DNA templates, it also regulates p53-dependent apoptosis and is critical for adipogenesis . Studies have found that MED1 phosphorylated MED1 is a targetable driver of Pol II recycling dysregulation in cancer (ZhongChen, 2022), and MED1 is also involved in the process of CDK7 inhibitors inhibiting castration-resistant prostate cancer progression (Reyaz UrRasool, 2019), These results indicate that MED1 plays a key role in tumors, but the role of its circRNA in gastric cancer is unclear.

In the study of circRNAs derived from the above four genes, we found that four circRNAs (hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438) were significantly low-expressed in gastric cancer, and overexpression significantly inhibited the proliferation, invasion and Migration phenotype. This suggests that the significantly low expression of four circRNAs in gastric cancer makes them useful as diagnostic markers for gastric cancer, and the effect of inhibiting the progression of gastric cancer after overexpression suggests that they may be used as nucleic acid drugs for the treatment of gastric cancer.

The present invention is realized through the following technical solutions:

In a first aspect, the present invention provides a gastric cancer circRNA marker, the circRNA marker being at least one of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438;

The nucleotide sequence of the hsa_circ_0002454 is shown in SEQ ID No.1;

The nucleotide sequence of the hsa_circ_0003441 is shown in SEQ ID No. 2;

The nucleotide sequence of the hsa_circ_0014624 is shown in SEQ ID No. 3;

The nucleotide sequence of the hsa_circ_0043438 is shown in SEQ ID No.4.

In a second aspect, the present invention provides an application of a reagent for detecting the above-mentioned gastric cancer circRNA markers in the preparation of a kit for diagnosing gastric cancer.

Further, in a preferred embodiment of the present invention, the above-mentioned reagents include a primer set for specifically amplifying any one of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438.

Further, in a preferred embodiment of the present invention, the above primer set of hsa_circ_0002454 includes a forward primer and a reverse primer;

The nucleotide sequence of the forward primer is shown in SEQ ID NO.5: TGACATTCGAAGCTTTTTGG;

The nucleotide sequence of the reverse primer is shown in SEQ ID NO.6: ATAGCTGGGCTCCATGTCTG.

Further, in a preferred embodiment of the present invention, the above primer set of hsa_circ_0003441 includes a forward primer and a reverse primer;

The nucleotide sequence of the forward primer is shown in SEQ ID NO.7: TGGATTCCTGCTCTTGAATG.

The nucleotide sequence of the reverse primer is shown in SEQ ID NO.8: TTTGTCTGGAGAGCTTGTGC.

Further, in a preferred embodiment of the present invention, the above primer set of hsa_circ_0014624 includes a forward primer and a reverse primer;

The nucleotide sequence of the forward primer is shown in SEQ ID NO.9: CCTCACCCAAGATGAGGAAG.

The nucleotide sequence of the reverse primer is shown in SEQ ID NO.10: CTAGCACCATCAGCCATGTG.

Further, in a preferred embodiment of the present invention, the above primer set of hsa_circ_0043438 includes a forward primer and a reverse primer;

The nucleotide sequence of the forward primer is shown in SEQ ID NO.11: TAATGGTCATTTAA.

The nucleotide sequence of the reverse primer is shown in SEQ ID NO.12: TTCTCATGCAAAATGATG.

In specific applications, the forward primer and reverse primer of the internal reference GAPDH are also included:

Internal reference GAPDH Primer F: GGAGCGAGATCCCTCCAAAAT (SEQ ID NO.13);

Internal reference GAPDH Primer R: GGCTGTTGTCATACTTCTCATGG (SEQ ID NO.14).

Further, in a preferred embodiment of the present invention, the above reagents also include: reverse transcription PCR reagents and fluorescent quantitative PCR reagents;

Preferably, the reverse transcription PCR reagents include: 2X RT Mix, RT Enzyme Mix and RNA free ddH ₂ O;

Preferably, the reverse transcription of RNA into cDNA reaction system and conditions are as follows:

The reaction conditions are: 37°C for 10 minutes, 42°C for 20 minutes, 85°C for 5 minutes, and 4°C for 2 minutes.

Preferably, the fluorescent quantitative PCR reagents include: 2X PCR Master Mix and RNA free ddH ₂ O.

Preferably, the reaction system and reaction conditions of fluorescent quantitative PCR amplification detection are as follows:

The fluorescent quantitative PCR reaction conditions are: denaturation at 95°C for 5 minutes; 10 seconds at 95°C, 35 seconds at 60°C; 40 cycles.

In the third aspect, the present invention also provides a gastric cancer diagnostic kit, which is characterized in that it includes a specific primer set for the circRNA marker described in claim 1.

In a fourth aspect, the present invention also provides an application of the above-mentioned expression promoter of gastric cancer circRNA markers in the preparation of a drug for treating gastric cancer.

Compared with the prior art, the present invention has at least the following technical effects:

The present invention first discovered that four circular RNAs, hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438, are related to the occurrence of gastric cancer, and their expression in the serum of patients with gastric cancer is significantly reduced, which shows that these four circular RNAs can well predict gastric cancer It can be used as a diagnostic marker for gastric cancer. The fluorescent quantitative PCR method can be used to quantitatively detect this circRNA marker to realize the diagnosis of gastric cancer. Using this circRNA marker to diagnose gastric cancer has strong specificity, high sensitivity, stable results, and no false positives, and has a good clinical application prospect.

In addition, the overexpression of these four markers hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438 can significantly inhibit the proliferation ability of gastric cancer cells, thus indicating that these four circular RNA genes can be used as potential targets for gastric cancer treatment, that is, using their Expression promoters are used to develop drugs for the treatment of gastric cancer, which is beneficial to the effective treatment of gastric cancer.

Description of drawings

Figure 1 is an identification diagram of circular RNAs hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, and hsa_circ_0043438 in Example 1 of the present invention.

Figure 2 is a graph showing the results of fluorescent quantitative PCR detection of the decreased expression of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, and hsa_circ_0043438 in gastric cancer serum in Example 2 of the present invention;

Fig. 3 is a graph showing that hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, and hsa_circ_0043438 have good marker performance in gastric cancer serum in Example 2 of the present invention;

Fig. 4 is a graph showing the experimental results of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438 in Example 3 of the present invention for inhibiting the proliferation of gastric cancer cells.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention, and are not indicated in the examples The specific conditions were carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1

Identification of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_004348

1. RNA extraction (Trizol method)

(1) Add 1 ml trizol to the plasma of patients with gastric cancer and healthy people;

(2) Add 200 μL of chloroform, shake vigorously for 10 seconds, and let stand at room temperature for 10 minutes;

(3) Centrifuge at 12,000g for 10min at 4°C, the solution is divided into three layers, the RNA is dissolved in the water phase, and the water phase is transferred to another new RNase free EP tube;

(4) Add 1 volume of isopropanol, vortex and mix thoroughly;

(5) Centrifuge at 12,000g for 15min at 4°C. After centrifugation, RNA precipitates at the bottom of the tube, discard the supernatant;

(6) Add 1ml of 75% ethanol, invert gently by hand, centrifuge at 12,000g for 5min, and discard the supernatant;

(7) Dry at room temperature, add 20 μL DEPC water to dissolve the precipitate.

2. Genomic DNA Removal

To remove the residue of genomic DNA in the total RNA, use DNA digestion enzyme. The specific reaction system and conditions are as follows. The total volume of the reaction solution is 10 μL, and the composition is as follows:

After digestion in the reaction solution at 37°C for 40 minutes, DNA digestion enzymes were inactivated at 85°C for 3 minutes.

3. Reverse transcription of RNA into cDNA

The reaction system and conditions for reverse transcription of RNA into cDNA are as follows:

The reaction conditions are: 37°C for 10 minutes, 42°C for 20 minutes, 85°C for 5 minutes, and 4°C for 2 minutes.

4. Design reverse amplification PCR primers to amplify the circRNA interface and flanking sequence DNA sequencing verification

According to the partial reference sequences provided in the Circbase database, design primers to recognize reverse PCR amplification, amplify hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 interface and flanking sequence primer sequences are shown in Table 1:

Table 1. Primer sequences of gastric cancer circRNA markers

Primers amplify the partial sequence of circular RNA hsa_circ_0002454 with a size of 350bp; using cDNA as a template, PCR amplifies the partial sequence on both sides of the circular interface of circular RNA hsa_circ_0002454, and separates by 1.2% nucleic acid agarose electrophoresis, and the PCR product is purified Verified by DNA sequencing. The results showed that the circular RNA hsa_circ_0002454 molecule is a circular RNA molecule composed of the first positions of the 2nd, 3rd and 4th exons of the DNAJC6 gene. After amplification, the circular sequence of hsa_circ_0002454 was confirmed by first-generation Sanger sequencing (Figure 1A). The full length of hsa_circ_0002454 is 350 bases, and the full-length sequence is:

GTGCCTCATCTCCAGACATGGAGCCCAGCTATGGGGGAGGTCTCTTTGACATGGTAAAAGGAGGTGCAGGGAGGCTCTTTAGTAACCTAAAGGACAACTTGAAAGACACCCCAAAGACACATCTTCTAGAGTGATACAATCTGTGACCAGCTACACAAAGGGAGATTTAGACTTCACTTATGTTACTCCAGAATTATTGTGATGTCCTTTCCTCTGGACAAT GTTGACATAGGATTCAGGAATCAGGTTGATGACATTCGAAGCTTTTTGGATTCCAGACATCTTGACCACTACACAGTATACAATCTGTCACCTAAGTCTTATCGAACTGCCAAGTTTCACAGCCGG (SEQ ID NO. 1).

The primers amplify the partial sequence of circular RNA hsa_circ_0003441 with a size of 450bp; using cDNA as a template, PCR amplifies the partial sequence on both sides of the circular interface of circular RNA hsa_circ_0003441, and separates by 1.2% nucleic acid agarose electrophoresis, and the PCR product is purified Verified by DNA sequencing. The results showed that the circular RNA hsa_circ_0003441 molecule is a circular RNA molecule formed by connecting the first positions of exons 2, 3, 4 and 4 of TDRD3 gene. After amplification, the circular sequence of hsa_circ_0003441 was confirmed by first-generation Sanger sequencing (Figure 1B). The full length of hsa_circ_0003441 is 450 bases, and the full-length sequence is:

GTATTCTTTCAGATGAAGGCATTGAAGCTTGCACAAGCTCTCCAGACAAAGTCAATGTAAATGACATCATCCCTGATTGCTCTCCAATACAGATCTGAGAACAATTGGCAAGAAATTCCTCCCAGTGACATCAATAGTGGAAAGGTAGAAAAGCTCGAAGGTCCATGTGTTTTGCAAATTCAAAAAAATTCGCAATGTTGCTGCACCAAAGGATAATGAAGAATCTCAGGCT GCACCAAGGATGCTGCGATTACAGATGACTGATGGTCATATAAGTTGCACAGCAGTAGAATTTAGTTATATGTCAAAAATAAGCCTGAACACACCCACCTGGAACTAAAGTTAAGCTCTCAGGCATTGTTGACATAAAAAATGGATTCCTGCTCTTGAATGACTCTAACACCAGTTCTTGGTGGTGAAGTGGAACACCTTATTGAGAAATGGGAGTTACAGAGA (SEQ ID NO. 2).

The primers amplified the partial sequence of circular RNA hsa_circ_0014624 with a size of 1710bp; using cDNA as a template, PCR amplified the partial sequence on both sides of the circular interface of circular RNA hsa_circ_0014624, separated by 1.2% nucleic acid agarose electrophoresis, and purified the PCR product Verified by DNA sequencing. The results showed that the circular RNA hsa_circ_0014624 molecule is a circular RNA molecule formed by connecting the first position of exon 21 of GON4L gene. After amplification, the circular sequence of hsa_circ_0014624 was confirmed by first-generation Sanger sequencing (Figure 1C). The full length of hsa_circ_0014624 is 1710 bases, and the full-length sequence is:

GCCAGTCTGCCATCCATCCAGGAAGAACTGCGGCACATGGCTGATGGTGCTAGAGAGGTAGGAAATATGACTGGAACCACTGAGATCAACTCAGATCGAAGCCTAGAAAAAGACAATTTGGAGTTGGGGAGTGAATCTCGGTACCCCACTGCTATTGCCTAAGGGTGTAGTCCTGAAACTGAAGCCAGTTGCCACCCGTTTCCCCAGGAAGGCTTGGAGACA GAAGCGTTCATCAGTCCTGAAGCCCCTCCTTATCCAACCCAGCCCCTCTCCAGCCCAGCTTCAACCCTGGGAAAACACCAGCCCGATCAACTCATTCAGAAGCCCCTCCGAGCAAAATGGTGCTCCGGATTCCTCACCCAATACAGCCAGCCACTGTTTTCAGACAGTTCCAGGTGTCCCTCACTGGGGGTCAGTGGAGGTGAGAGTTTTGAGTCTCCTGCAG CACTGCCTGCTGTGCCCCCTGAGGCCAGGACAAGCTTCCCCTGTCTGAGTCCCAGACTTTGCTCTCTTCTGCCCCTGTGCCCAAGGTAATGCTGCCCTCCCTTGCCCCTTCTAAGTTTCGAAAGCCATATGTGAGACGGAGACCCTCAAAGAGAAGAGGAGTCCAAGGCCTCTCCCTGTATGAAACCTGCCCCTGTTATCCACCACCCTGCATCTGTTACCTTCACTGTTCCT GCTACCACTGTGAAGATTGTGAGCCTTGGCGGTGGCTGTAACATGATCCAGCCTGTCAATGCGGCTGTGGCCCAGAGTCCCCAGACTATTCCCATCACTACCCTCTTGGTTAACCCTACTTCCTTCCCCTGTCCATTGAACCAGTCCCTTGTGGCCTCCTCTGTCTCACCCTTAATTGTTTCTGGCAATTCTGTGAATCTTCCTATACCATCCACCCCTGAAGATA AGGCCCACGTGAATGTGGACATTGCTTGTGCTGTGGCTGATGGGGAAAATGCCTTTCAGGGCCTAGAACCCAAATTAGAGCCCCAGGAACTATTCTCCTCTCTCTGCTACTGTTTTTCCCGAAAGTGGAACATAGCCCAGGGCCTCCACTAGCCAGATGCAGAGTGCCAAGAAGGATTGTCAGAGAATAGTGCCTGTCGCTGGACCGTTGTGAAAACAGAGGAGGG GAGGCAAGCTCTGGAGCCGCTCCCTCAGGGCATCCAGGAGTCTCTAAAACAACCCTACCCCTGGGGATTTAGAGGAAATTGTCAAGATGGAACCTGAAGAAGCTAGAGAGGAAATCAGTGGATCCCCTGAGCGTGATATTGTGATGACATCAAAGTGGAACATGCTGTGGAATTGGACACTGGTGCCCCAAGCGAGGAGTTGAGCAGTGCTGGAGAAG TAACGAAACAGACAGTCTTACAGAAGGAAGAGGAGAGGAGTCAGCCAACTAAAAACCCCTTCATCTTCTCAAAGAGCCCCCTGATGAAGGAACCTCAGGGACAGATGTGAACAAAGGATCATCAAAGAATGCTTTGTCCTCAATGGATCCTGAAGTGAGGCTTAGTAGCCCCCAGGGAAGCCAGAAGATTCATCAGTGTTGATGGTCAGTCAGTGGGGACTCCAGTT GGGCCAGAAACTGGAGGAGAGAAGAATGGGCCAGAAGAAGAGGAAGAAGAGGACTTTGATGACCTCACCCCAAGATGAGGAAGATGAAATGTCATCAGCTTCTGAGGAATCTGTGCTTTCTGTCCCAGAACTCCAG (SEQ ID NO. 3).

The primers amplify the partial sequence of circular RNA hsa_circ_0043438 with a size of 90bp; using cDNA as a template, PCR amplifies the partial sequence on both sides of the circular interface of circular RNA hsa_circ_0043438, separates by 1.2% nucleic acid agarose electrophoresis, and purifies the PCR product Verified by DNA sequencing. The results showed that the circular RNA hsa_circ_0043438 molecule is a circular RNA molecule formed by connecting the first position of exon 10 of MED1 gene. After amplification, the circular sequence of hsa_circ_0043438 was confirmed by first-generation Sanger sequencing (Figure 1D). The full length of hsa_circ_0043438 is 90 bases, and the full-length sequence is:

GTCATTTAATGAACCTGAAGTACTATGTCCTCCTTCTGACCTACTGGATGACAAGACTGCATCTCCCATCATTTTGCATGAGAATAATG (SEQ ID NO. 4).

Example 2

Fluorescent quantitative PCR detection of circRNA markers

Fluorescent quantitative PCR amplification detection method of circular hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 molecules in the plasma expression of gastric cancer patients and healthy people is as follows:

The total RNA was extracted according to the method described in Example 1, and the remaining genomic DNA in the extracted RNA was removed with DNase, and the RNA was reverse-transcribed into cDNA; finally, the fluorescent quantitative PCR amplification was used for detection, and the primer sequences of the fluorescent quantitative PCR were shown in Table 1 Shown in SEQ ID NO.5-14 among. Fluorescent quantitative PCR amplification detection of circular RNA hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 molecules in the plasma expression of gastric cancer patients and healthy people The reaction system and reaction conditions are as follows:

The fluorescent quantitative PCR reaction conditions are: denaturation at 95°C for 5 minutes; 10 seconds at 95°C, 35 seconds at 60°C; 40 cycles.

Real-time quantitative PCR detected the expression of circular RNA hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 in the plasma of gastric cancer patients and healthy people, and the results are shown in Figure 2 and Figure 3:

Among them, Figure 2A shows the expression level of hsa_circ_0002454 in the plasma of gastric cancer patients and healthy people. It can be seen from the figure that the expression of hsa_circ_0002454 in gastric cancer plasma is significantly reduced;

Figure 2B shows the expression level of hsa_circ_0003441 in the plasma of gastric cancer patients and healthy people. It can be seen from the figure that the expression of hsa_circ_0003441 in gastric cancer plasma is significantly reduced;

Figure 2C shows the expression level of hsa_circ_0014624 in the plasma of gastric cancer patients and healthy people. It can be seen from the figure that the expression of hsa_circ_0014624 in gastric cancer plasma is significantly reduced;

Figure 2D shows the expression level of hsa_circ_0043438 in the plasma of gastric cancer patients and healthy people. It can be seen from the figure that the expression of hsa_circ_0043438 in gastric cancer plasma is significantly reduced;

Figure 3A shows that hsa_circ_0002454 has good marker performance in gastric cancer serum, AUC=0.7598;

Figure 3B shows that hsa_circ_0003441 has good marker performance in gastric cancer serum, AUC=0.8408;

Figure 3C shows that hsa_circ_0014624 has good marker performance in gastric cancer serum, AUC=0.8288;

Figure 3D shows that hsa_circ_0043438 has good marker performance in gastric cancer serum, AUC=0.7788;

Summarized in the table below:

This shows that hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 can be used as tumor diagnostic markers, hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 in Gastric cancer serum significantly reduced, and can well predict the occurrence of tumors.

The fluorescent quantitative PCR detection method of the present invention can ideally detect the expression of circular RNAs hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438 in organisms.

Example 3

CCK8 assay detects the effect of circRNAs on cell proliferation

In order to study the effect of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 on cell proliferation, first construct hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 Overexpression vectors to efficiently upregulate hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 in gastric cancer cell lines BGC-823 and SGC -Expression in 7901. Control refers to the group transfected with empty plasmids when transfecting cells, that is to say, the control group; hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 refer to the transfection of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014 respectively 624, hsa_circ_0043438 overexpression vector set. Gastric cancer cells BGC-823 and SGC-7901 in the logarithmic growth phase were inoculated into six-well plates at a cell suspension of 2×104 cells/mL, and the culture plates were cultured in an incubator for 24 hours (37°C, 5% CO2 ). Take out the six-well plate, discard the old medium, and replace it with 1.5mL serum-free medium to prepare the cell plate for liposome transfection. The hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 overexpression vector groups were transfected respectively. Take out the gastric cancer cell lines BGC-823 and SGC-7901 in the logarithmic growth phase that have been transfected for 24 hours in the six-well plate, discard the old medium, wash the cells with PBS, add an appropriate amount of trypsin to digest the cells, and add 10 Resuspend cells in 1640 medium with %FBS to stop digestion. Count the number of cells with a hemocytometer, add 100 uL of cell suspension to a 96-well plate, and inoculate about 1,000 cells in the 96-well plate. Set the orifice plate and the control group, and set 3-5 duplicate holes for each group. Place in an incubator to continue culturing. Take out a 96-well plate for detection at 0 hour after plating (6 hours after plating, the cells are completely adhered to the wall and positioned at 0 hour after plating), 24 hours, 48 hours and 72 hours. Add 10uL of CCK-8 reagent to each well, shake gently and place in a 37°C incubator. Clock an hour. Measure the absorbance at a wavelength of 450 nm with a microplate reader. Analyze the data according to the obtained absorbance value, and draw the cell growth curve to evaluate the effect of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, hsa_circ_0043438 overexpression on cell proliferation.

After overexpressing hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, and hsa_circ_0043438 in gastric cancer cell lines BGC-823 and SGC-7901, the cell proliferation ability was detected by CCK8 assay, and the results are shown in Figure 4:

Among them, Figure 4A is the test results of cell proliferation ability after hsa_circ_0002454 was overexpressed in gastric cancer cell lines BGC-823 and SGC-7901. It can be seen from the figure that gastric cancer cells were significantly inhibited, and the inhibition rates were 29% and 30%, respectively.

Figure 4B is the detection results of cell proliferation ability after hsa_circ_0003441 was overexpressed in gastric cancer cell lines BGC-823 and SGC-7901. It can be seen from the figure that gastric cancer cells were significantly inhibited, and the inhibition rates were 26% and 38%, respectively.

Figure 4C is the detection results of cell proliferation ability after hsa_circ_0014624 was overexpressed in gastric cancer cell lines BGC-823 and SGC-7901. It can be seen from the figure that gastric cancer cells were significantly inhibited, and the inhibition rates were 41% and 43%, respectively.

Figure 4D shows the test results of the cell proliferation ability of hsa_circ_0043438 after the overexpression of gastric cancer cell lines BGC-823 and SGC-7901. It can be seen from the figure that gastric cancer cells were significantly inhibited, and the inhibition rates were 51% and 47%, respectively.

In summary, the present invention provides a circRNA marker for the diagnosis and treatment of gastric cancer, namely hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438 circular RNA genes. The inventor detected by fluorescent quantitative PCR that the expression level of the circular RNA gene in the serum of patients with gastric cancer was significantly lower than that of healthy people, thus indicating that these four circRNA markers can predict the occurrence of gastric cancer and can be used as a diagnosis of gastric cancer landmark. Further, the effects of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624, and hsa_circ_0043438 on cell proliferation were detected by CCK8 experiments. It was found that the growth of gastric cancer cells was significantly inhibited after these four markers were overexpressed in gastric cancer cell lines BGC-823 and SGC-7901. inhibition. This shows that the four markers hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438 can be used not only as markers for the diagnosis of gastric cancer, but also as potential targets for the treatment of gastric cancer. The reagents for detecting the four markers hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_0043438 can be used in products for diagnosing or treating cancer, such as chips, kits or nucleic acid membrane strips.

Finally, it should be noted that: the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A gastric cancer circRNA marker, wherein the circRNA marker is at least one of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_ 0043438;

the nucleotide sequence of hsa_circ_0002454 is shown in SEQ ID No. 1;

the nucleotide sequence of hsa_circ_0003441 is shown in SEQ ID No. 2;

the nucleotide sequence of hsa_circ_0014624 is shown in SEQ ID No. 3;

the nucleotide sequence of hsa_circ_0043438 is shown in SEQ ID No. 4.

2. Use of a reagent for detecting the gastric cancer circRNA marker of claim 1 in the preparation of a kit for diagnosing gastric cancer.

3. The use according to claim 2, wherein the reagent comprises a primer set that specifically amplifies any one of hsa_circ_0002454, hsa_circ_0003441, hsa_circ_0014624 and hsa_circ_ 0043438.

4. The use according to claim 3, wherein the primer set of hsa_circ_0002454 comprises a forward primer and a reverse primer;

the nucleotide sequence of the forward primer is shown as SEQ ID NO. 5;

the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 6.

5. The use according to claim 3, wherein the primer set of hsa_circ_0003441 comprises a forward primer and a reverse primer;

the nucleotide sequence of the forward primer is shown as SEQ ID NO. 7;

the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 8.

6. The use according to claim 3, wherein the primer set of hsa_circ_0014624 comprises a forward primer and a reverse primer;

the nucleotide sequence of the forward primer is shown as SEQ ID NO. 9;

the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 10.

7. The use according to claim 3, wherein the primer set of hsa_circ_0043438 comprises a forward primer and a reverse primer;

the nucleotide sequence of the forward primer is shown as SEQ ID NO. 11;

the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 12.

8. The use according to claim 3, wherein the agent further comprises: reverse transcription PCR reagent and fluorescent quantitative PCR reagent;

preferably, the reverse transcription PCR reagent comprises: 2X RT Mix, RT Enzyme Mix and RNA free ddH2O;

preferably, the fluorescent quantitative PCR reagent comprises: 2X PCR Master Mix and RNA free ddH2O.

9. A gastric cancer diagnostic kit comprising a specific primer set for the circRNA marker of claim 1.

10. Use of the expression promoter of gastric cancer circRNA marker of claim 1 in the preparation of a medicament for treating gastric cancer.

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