CN111944812A - siRNA molecule of targeting Fascin gene and application thereof - Google Patents

Abstract

The invention discloses a group of siRNA molecules targeting Fascin gene and application thereof, and belongs to the technical field of biomedicine. The siRNA molecule is composed of a sense strand and an antisense strand. In vitro experiments have shown that the antisense strand of the siRNA molecule of the present invention can specifically bind to the mRNA that inhibits the Fascin gene to degrade the mRNA, thereby interfering with the post-transcriptional translation process and inducing tumor cells. Apoptosis, inhibition of tumor cell metastasis and invasion, to achieve the purpose of tumor treatment.

Description

siRNA molecule targeting Fascin gene and its application

technical field

The invention belongs to the technical field of biomedicine, and particularly relates to a group of siRNA molecules targeting Fascin gene and applications thereof.

Background technique

Primary liver cancer, especially hepatocellular carcinoma (HCC), is the most common primary liver tumor, and its morbidity and mortality are gradually increasing. At present, its global incidence ranks fifth among all malignant tumors, and the cancer mortality ranks third, and about 800,000 people die of hepatocellular carcinoma every year. About half of the new cases of liver cancer in the world each year occur in my country, ranking the second cause of cancer death in my country, seriously endangering national health. Hepatocellular carcinoma has a high degree of malignancy, insidious onset, difficult early diagnosis, and rapid progression. When diagnosed, most patients have reached the middle and advanced stages with extensive invasion or distant metastasis, and cannot be surgically removed. An important reason for the poor prognosis of liver cancer is the lack of effective treatment. Therefore, in addition to emphasizing and attaching importance to early detection and early treatment, it is of great significance to explore effective treatment methods to improve the prognosis of patients with liver cancer.

The human Fascin gene belongs to the Fascin family, and its protein-encoded product is a cytoskeletal protein with a molecular weight of 55kDa, which binds to F-actin and is located in the core muscle of the cytoplasmic tension fibers and the edge of the cell membrane ruffles and microspins. in actin bundles. Fascin plays an important role in cell migration, cell adhesion, and intercellular information exchange. Studies have shown that Fascin protein is significantly increased in a variety of tumors, participates in tumor proliferation, growth, invasion and other malignant processes, and is closely related to tumor prognosis.

RNA interference (RNAi) is a form of post-transcriptional gene silencing in which small interfering RNA (siRNA) triggers the degradation of its cognate messenger RNA (mRNA) (Nature.1998,391:806 -811). It has been confirmed that it has great potential in the treatment of various viral infectious diseases and tumors, and is an ideal treatment method for blocking gene expression. RNAi technology has opened up a whole new field of treatment. At present, dozens of siRNA drugs have entered the clinical stage in the world.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a group of siRNA molecules targeting Fascin gene and its application. The antisense strand of the siRNA molecule can specifically bind to the mRNA that inhibits the Fascin gene, degrade the mRNA, thereby interfering with the post-transcriptional translation process and inducing tumors. Cell apoptosis, inhibition of tumor cell metastasis and invasion, to achieve the purpose of tumor treatment.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

The siRNA molecule targeting the Fascin gene consists of a sense strand and an antisense strand, and its sequence is:

Sense strand: 5'-CGUUCGGGUUCAAGGUGAAdTdT-3' (SEQ ID NO: 1),

Antisense strand: 5'-UUCACCUUGAACCCGAACGdTdT-3' (SEQ ID NO: 2).

The application of the above siRNA molecule in the preparation of a medicine for inhibiting the function of Fascin gene in cells.

The application of the above-mentioned siRNA molecules in the preparation of medicines for preventing and/or treating liver cancer.

Further, the siRNA molecule can induce apoptosis of liver cancer cells.

Further, the siRNA molecule can inhibit the metastasis and invasion of liver cancer cells.

In vitro experiments have proved that the antisense strand of the siRNA molecule of the present invention can specifically bind to the mRNA that inhibits the Fascin gene, degrade the mRNA, thereby interfering with the post-transcriptional translation process, inducing tumor cell apoptosis, inhibiting tumor cell metastasis and invasion, and achieving therapeutic effects. tumor purpose.

The siRNA molecule of the present invention can be applied to prepare a drug that regulates the function of inhibiting Fascin gene in cells to play the role of RNA interference, induce tumor cell apoptosis, inhibit tumor cell metastasis and invasion, and achieve the purpose of treating tumors.

Description of drawings

Figure 1 shows the mRNA expression levels of Fascin in hepatoma cells (HepG2 and Huh7) and normal hepatocytes LO2 detected by real-time quantitative PCR in Example 1.

Figure 2 shows the protein expression levels of Fascin in hepatoma cells (HepG2 and Huh7) and normal hepatocytes LO2 detected by Western Blot in Example 1.

FIG. 3 is the real-time quantitative PCR detection of siRNA in Example 1 to down-regulate the mRNA expression level of Fascin in hepatoma cells HepG2.

FIG. 4 is the real-time quantitative PCR detection of siRNA in Example 1 to down-regulate the protein expression level of Fascin in HepG2 hepatoma cells.

FIG. 5 shows the Western Blot detection of siRNA in Example 1 to down-regulate the mRNA expression level of Fascin in liver cancer cells Huh7.

FIG. 6 is the real-time quantitative PCR detection of siRNA in Example 1 to down-regulate the protein expression level of Fascin in liver cancer cells Huh7.

Figure 7 shows the effect of siRNA down-regulating the expression level of Fascin in hepatoma cells HepG2 by MTT assay in Example 1 on the cell proliferation ability.

Figure 8 shows the effect of siRNA down-regulating the expression level of Fascin in liver cancer cells Huh7 detected by MTT method in Example 1 on the cell proliferation ability.

FIG. 9 shows the effect of siRNA on the cell migration ability after down-regulating the expression level of Fascin in hepatoma cells HepG2 by cell scratch assay in Example 1. FIG.

FIG. 10 shows the effect of siRNA down-regulating the expression level of Fascin in liver cancer cells Huh7 on cell migration ability detected by cell scratch assay in Example 1. FIG.

Figure 11 shows the effect of siRNA down-regulating the expression level of Fascin in hepatoma cells HepG2 on the cell invasion ability detected by Transwell cell invasion assay in Example 1.

Figure 12 shows the effect of siRNA down-regulating the expression level of Fascin in liver cancer cells Huh7 on the cell invasion ability detected by Transwell cell invasion assay in Example 1.

Detailed ways

For convenience, in the following, the terms "siRNA", "siRNA sequence" or "siRNA molecule" are used interchangeably and have the same meaning and scope.

Among them, siRNA is a double-stranded structure formed by the annealing of the sense strand and the antisense strand.

The siRNA molecule of the present invention is designed from the functional conserved region of the open reading frame of Fascin gene.

Various methods can be used to prepare siRNA, such as chemical synthesis, in vitro transcription, enzymatic cleavage of long-chain dsRNA, vector expression of siRNA, and PCR synthesis of siRNA expression elements. Better access to gene silencing efficiency.

The siRNA molecule of the present invention can be used as an effective ingredient for preparing a drug for regulating the function of Fascin gene in cells, especially an effective ingredient for an antitumor drug.

For application purposes, siRNA molecules can be directly administered as a drug to a specific part of the recipient's body, such as tumor tissue.

The dosage form of the medicament of the present invention can be in various forms as long as it is suitable for administration of the corresponding disease and the activity of the siRNA molecule is properly maintained. For example, for injectable drug delivery systems, the dosage form can be a lyophilized powder.

Optionally, any pharmaceutically acceptable carrier and adjuvant may be included in the above-mentioned pharmaceutical dosage form, as long as it is suitable for the corresponding administration system and properly maintains the activity of the siRNA molecule.

In order to make the present invention more obvious and comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

The following examples are only used to illustrate the present invention, not to limit the present invention.

Embodiment 1

1. Experimental method

Step 1, Cell Culture

Hepatocellular carcinoma cell lines HepG2 and Huh7 were purchased from the Institute of Cell Research, Chinese Academy of Sciences, Shanghai, and were cultured in DMEM medium (Thermo Fisher Company) containing 10% FBS (Thermo Company), and penicillin and streptomycin (Thermo Company) were added to the medium. The final concentrations of penicillin and streptomycin were 100 U/mL and 100 μg/mL, respectively, and were cultured in a carbon dioxide incubator at 37°C.

Step 2. In vitro siRNA transfection

The siRNA sequence Fascin siRNA targeting the Fascin (NCBI number: NM_003088) gene was designed. A negative control sequence (NC) was designed as a control. See Table 1 for the sequence.

Table 1 siRNA sequences and control sequences

2000(Thermo Fisher公司)转染到细胞内，使siRNA终浓度为50nM，37℃培养4h后，培养液换成含10％FBS的DMEM培养基。Take the cells in the logarithmic growth phase cultured in step 1, the seeding density of 96-well plate is 5×10 ⁴ /well, 24-well plate is 1.5×10 ⁵ /well, 6-well plate is 1×10 ⁶ /well Cells were seeded and divided into Fascin siRNA transfected group and untreated group without siRNA transfection, and NC transfected group served as a negative control. Each experimental group used the corresponding siRNA and liposome according to the operation instructions.

2000 (Thermo Fisher Company) was transfected into cells to make the final concentration of siRNA 50nM, after culturing at 37°C for 4h, the culture medium was changed to DMEM medium containing 10% FBS.

All experiments were repeated three times, and the results were expressed as mean ± SD. SPSS19.0 was used for statistical analysis. Statistical differences were analyzed by one-way ANOVA and two-sided t-test. P<0.05 indicates a significant difference. In all graphs, * indicates a significant difference compared to the untreated group.

2. Detection of mRNA expression levels by real-time quantitative PCR (RT-qPCR)

Cell culture and in vitro transfection of siRNA were performed using the methods described above. The in vitro transfection was carried out in a 96-well plate.

RNA提取试剂(Thermo Fisher公司)提取，并按操作说明用RT-qPCR试剂盒(Biomics公司)进行检测。引物序列见表2，PCR反应条件为：95℃预变性5min，45个循环为95℃变性20s、58℃退火30s、72℃延伸30s。48h after transfection, total cell RNA was

RNA extraction reagent (Thermo Fisher Company) was extracted and detected by RT-qPCR kit (Biomics Company) according to the operating instructions. The primer sequences are shown in Table 2. The PCR reaction conditions are: 95°C pre-denaturation for 5 min, 45 cycles of 95°C denaturation for 20s, 58°C annealing for 30s, and 72°C extension for 30s.

Table 2 Primers for RT-QPCR detection

As shown in Figure 1, the mRNA expression levels of Fascin in hepatoma cell lines HepG2 and Huh7 were significantly higher than those in normal hepatocyte LO2 (P<0.05); as shown in Figures 3 and 4, Fascin siRNA effectively inhibited the expression of Fascin in HepG2 and Huh7 cells. mRNA expression of the Fascin gene. Compared with the untreated group, the inhibition rates of Fascin siRNA reached 65% and 75%, respectively (P<0.05).

3. Western Blot to detect the protein expression level of the gene

Cell culture and in vitro transfection of siRNA were performed using the methods described above.

The cells were seeded in 6-well plates and cultured in a 37°C/5% CO ₂ incubator for 24 hours. When the cell confluency reached 70-80%, siRNA was transfected; after 24 hours, the culture medium was discarded and washed with PBS for 2 hours. The operation was carried out on ice, 50 μL of SDS protein lysis solution was added to each well to lyse the cells, and the cells were fully mixed until viscous, scraped off with a scraper, transferred to a 1.5 mL centrifuge tube, and placed on ice after a boiling water bath for 10 min. 4°C, 12000rpm×15min, extract the supernatant; after electrophoresis by SDS-PAGE (5% stacking gel, 8% separating gel), use a wet transfer instrument to transfer at a constant current of 200mA for 2h, and transfer the proteins in the gel to On PVDF membrane; immerse PVDF membrane in blocking solution (5% nonfat milk) and shake slowly on a shaker for 2 hours; add primary antibody (1:1000) and incubate at 37°C for 2 hours; rinse membrane three times with TBST for 10 minutes each time; Secondary antibodies (goat anti-mouse IgG-HRP, 1:1000) were added respectively, and incubated at 37°C for 2 h; after the secondary antibody incubation, the membrane was rinsed three times with TBST for 5-10 min each time; the washed membrane was subjected to ECL luminescence development; the results Analysis: Using the internal reference gene as the internal control, use Image J software to analyze the gray value of the target band, and calculate the relative expression level of the target gene=the gray value of the target band/the gray value of the internal reference in the same sample.

As shown in Figure 2, the protein expression level of Fascin in hepatoma cell lines HepG2 and Huh7 was significantly higher than that in normal hepatocyte LO2 (P<0.05). As shown in Figures 5 and 6, Fascin siRNA effectively inhibited the expression of Fascin in HepG2 and Huh7 cells. Protein expression of the Fascin gene. Compared with the untreated group, the inhibition rates of Fascin siRNA reached 68% and 75%, respectively (P<0.05).

4. Detection of cell proliferation by MTT assay

Cell culture and siRNA in vitro transfection were performed using the methods described above using 96-well plates. When the cell confluence reaches about 75% before transfection, the cells in the logarithmic growth phase are plated into a 96-well cell culture plate at a seeding density of 5×10 ⁴ /well, repeating 3 wells.

Measure the OD values of Fascin siRNA transfection group, NC transfection group and untreated group samples at 24h, 48h, 72h, and 96h of transfection, as well as the above-mentioned untransfected samples. 10 μl MTT, placed in a 37 °C incubator for 4 h in the dark; add 150 μl DMSO to each well, and place in a 37 °C incubator for 10 min; after pipetting and mixing, take 120 μl in another clean 96-well plate, and take 120 μl DMSO as a blank control for zero adjustment. OD was measured on a microplate reader (Bio-Rad Company) with a wavelength of 490 nm; data processing was performed to draw a cell growth curve.

As shown in Figures 7 and 8, Fascin siRNA treated HepG2 and Huh7 cells for 48h, 72h and 96h had a significant cell growth inhibitory effect compared with the untreated group and the NC group (P<0.05).

5. Cell scratch assay to detect cell migration

Cell culture and in vitro transfection of siRNA were performed using the methods described above.

HepG2 and Huh7 cells at a density of ^1.5 x 105/well were plated into 24-well plates and transfected. After 48 h, the confluent cells were scratched with a pipette tip, then washed with PBS buffer pH 7.4, and serum-free DMEM medium (Thermo Fisher) was added. 24h and 48h after scratching, photos were taken to observe cell migration. The experiment was performed in 3 parallel groups, with 4 fields of view per plate.

As shown in Figure 9 and Figure 10, compared with the untreated group and the negative control group, Fascin siRNA treatment of HepG2 and Huh7 cells for 24 and 48 h could effectively inhibit the migration of HepG2 and Huh7 (P<0.05).

6. Transwell cell invasion assay

Cell culture and in vitro transfection of siRNA were performed using the methods described above. The in vitro transfection was performed in a 24-well plate.

Cell migration was detected by 24-well membrane filters (Corning Bioscience, USA) 72h after transfection. 1.5×10 ⁵ cells were plated into the upper chamber to migrate into serum-containing DMEM medium (Gibco) for 24 h. Cells remaining in the upper chamber were removed with a cotton swab, and cells that migrated into the lower chamber were fixed with 10% formaldehyde for 30 s. Finally, cells were stained with 0.1% crystal violet for 4 min, followed by 3 washes with PBS buffer pH 7.4. Cells were counted in 200x magnification fields, 5 fields per condition.

As shown in Figures 11 and 12, Fascin siRNA could significantly inhibit the invasion of HepG2 and Huh7 cells compared with the untreated group and the NC group (P<0.05).

sequence listing

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Claims (5)

1. the siRNA molecule of targeting Fascin gene, is characterized in that: be made up of sense strand and antisense strand, and its sequence is: Sense strand: 5'-CGUUCGGGUUCAAGGUGAAdTdT-3', Antisense strand: 5'-UUCACCUUGAACCCGAACGdTdT-3'. 2. The application of the siRNA molecule of claim 1 in the preparation of a medicine for inhibiting the function of Fascin gene in cells. 3. The application of the siRNA molecule of claim 1 in the preparation of a drug for preventing and/or treating liver cancer. 4. The application according to claim 3, wherein the siRNA molecule can induce apoptosis of liver cancer cells. 5. The application according to claim 3, wherein the siRNA molecule can inhibit the metastasis and invasion of liver cancer cells.

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