A siRNA that targets and inhibits FAM195A gene expression and its application

Abstract

This invention relates to a siRNA that targets and inhibits the expression of the FAM195A gene and its applications, belonging to the field of biomedical technology. This invention designs and synthesizes siRNA that inhibits the expression of the FAM195A gene. The siRNA is siFAM195A, comprising a sense strand and an antisense strand; wherein the sense strand is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2. This invention, by transfecting siFAM195A into cervical cancer cells, can effectively inhibit the expression of the FAM195A gene, effectively inhibit the growth and proliferation of cervical cancer cells, and promote apoptosis of cervical cancer cells. This invention provides a new potential drug for the treatment of cervical cancer, possessing high clinical application prospects and value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a siRNA that targets and inhibits the expression of the FAM195A gene and its applications. Background Technology

[0002] Cervical cancer ( Cervical Carcinoma, CC Cervical cancer is a significant public health issue for women worldwide, ranking fourth among female cancers and second among malignant tumors of the female reproductive system. Due to the often subtle nature of early symptoms, 75%-90% of patients are diagnosed at an intermediate or advanced stage when typical symptoms appear. Early-stage cervical cancer is primarily treated surgically, while the standard treatment for intermediate and advanced-stage patients is concurrent chemoradiotherapy. Despite significant advancements in surgery, chemoradiotherapy, and immunotherapy, approximately 30%-40% of cervical cancer patients experience recurrence and metastasis. Patients with metastasis have a 5-year survival rate of only about 17%, indicating a poor prognosis and high mortality rate, which is a major cause of treatment failure. Therefore, in-depth research into the specific mechanisms of cervical cancer pathogenesis and progression, and the identification of highly sensitive diagnostic biomarkers and important pathogenic targets, is beneficial for improving the early detection rate of cervical cancer and has important guiding significance for the development of new therapeutic drugs.

[0003] FAM195A ( Family with Sequence Similarity 195, Member A FAM195A is a protein-coding gene (Gene ID: 84331) in the human genome. It is located in the 16p13.3 region of chromosome 16 and encodes a small protein. The FAM195A protein is primarily located in cytoplasmic stress granules (…). Stress Granules FAM195A is a type of RNA-binding protein found in the cell nucleus and other structures. Recent studies have revealed its important role in metabolic regulation and neuropsychiatric processes. However, there are no published reports on the role of FAM195A in the development and progression of cervical cancer or its application in the preparation of anti-cervical cancer drugs. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a siRNA that targets and inhibits FAM195A gene expression and its application. This invention, through transfection of siFAM195A into cervical cancer cells, effectively inhibits FAM195A gene expression, thereby effectively suppressing cervical cancer cell growth and proliferation and promoting apoptosis. This invention provides a novel potential drug for the treatment of cervical cancer, possessing high clinical application prospects and value.

[0005] Based on the above technical solution, the present invention can be further improved as follows.

[0006] The first objective of this invention is to provide a siRNA that targets and inhibits the expression of the FAM195A gene, wherein the siRNA is siFAM195A and comprises a sense strand and an antisense strand; The justice chain is shown in SEQ ID NO.1, and the antisense chain is shown in SEQ ID NO.2.

[0007] The beneficial effects of this invention are as follows: Currently, there are no reports on the specific role of FAM195A in the occurrence and development of cervical cancer or its use in the preparation of anti-tumor drugs. This invention synthesizes siRNA targeting FAM195A and demonstrates that this siRNA can effectively regulate and interfere with the expression of the FAM195A gene in human cervical cancer cells Siha and Hela, thereby reducing the growth and proliferation of human cervical cancer cells Siha and Hela and promoting apoptosis of cervical cancer cells Siha and Hela. This proves that the siRNA of this invention can be used in the preparation of drugs for the treatment of cervical cancer and has great application prospects.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] A second objective of this invention is to provide an application of siRNA that targets and inhibits the expression of the FAM195A gene, wherein the siRNA that targets and inhibits the expression of the FAM195A gene is used in the preparation of a pharmaceutical composition for treating tumor diseases.

[0010] The beneficial effects of this invention are as follows: RT-qPCR experiments were used to detect that siFAM195A specifically inhibits FAM195A gene expression. CCK-8 staining, EdU staining, colony formation assays, and apoptosis assays revealed that siFAM195A, designed and synthesized in this invention, effectively regulates and interferes with FAM195A gene expression after transfection into human cervical cancer Siha and HeLa cells, thereby reducing the growth and proliferation of human cervical cancer cells and promoting their apoptosis. Therefore, this FAM195A-targeted siFAM195A has the ability to inhibit FAM195A expression, effectively suppress cervical cancer cell growth and proliferation, and promote apoptosis in human cervical cancer cells. This invention provides a novel gene target and drug for cervical cancer treatment, possessing high clinical application value.

[0011] A third object of the present invention is to provide a pharmaceutical composition for treating neoplastic diseases, wherein the active ingredient of the pharmaceutical composition comprises the siRNA that targets and inhibits the expression of the FAM195A gene, and wherein the final concentration of siFAM195A in the pharmaceutical composition is 25 nM.

[0012] The beneficial effects of this invention are: the siRNA provided by this invention inhibits the proliferation and growth of tumor cells and promotes tumor cell apoptosis by knocking down the expression of the FAM195A gene, thereby treating tumor diseases.

[0013] Furthermore, the dosage form of the pharmaceutical composition is at least one of the following: aerosol, tablet, capsule, drop pill, pill, powder, solution, suspension, emulsion, granule, lipid preparation, transdermal preparation, lozenge, suppository, and lyophilized powder for injection.

[0014] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable adjuvant ingredients.

[0015] Furthermore, the pharmaceutical composition further includes at least one of the following: filler, lubricant, dispersant, wetting agent, binder, regulator, solubilizer, antioxidant, antibacterial agent, emulsifier, and disintegrant.

[0016] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0017] Furthermore, the vector is a transfection vector.

[0018] Furthermore, the neoplastic disease is a neoplastic disease related to the expression and function of the FAM195A gene.

[0019] Furthermore, the neoplastic disease is cervical cancer.

[0020] The beneficial effect of adopting the above-mentioned further approach is that siRNA treats cervical cancer by inhibiting the expression of the FAM195A gene. Attached Figure Description

[0021] Figure 1 The images show the reduction effect of FAM195A gene mRNA in Siha cells and HeLa cells after transfection with siRNA in Example 1 of the present invention; where (A) is the reduction effect of FAM195A gene mRNA in Siha cells after transfection with siRNA; and (B) is the reduction effect of FAM195A gene mRNA in HeLa cells after transfection with siRNA. Figure 2 The figures are cell growth curves of Siha cells and HeLa cells after inhibiting FAM195A gene expression in Example 2 of the present invention; wherein (A) is the cell growth curve of Siha cells after inhibiting FAM195A gene expression; and (B) is the cell growth curve of HeLa cells after inhibiting FAM195A gene expression. Figure 3 The diagram shows the cell clone formation of Siha cells and HeLa cells after inhibiting FAM195A gene expression in Example 2 of the present invention; wherein (A) is the cell clone formation of Siha cells after inhibiting FAM195A gene expression; and (B) is the cell clone formation of HeLa cells after inhibiting FAM195A gene expression. Figure 4The images show cell proliferation of Siha cells and HeLa cells after inhibiting FAM195A gene expression in Example 2 of this invention; where (A) is a cell proliferation image of Siha cells after inhibiting FAM195A gene expression; and (B) is a cell proliferation image of HeLa cells after inhibiting FAM195A gene expression. Figure 5 The images show apoptosis in Siha cells and Hela cells after inhibiting FAM195A gene expression in Example 2 of this invention; where (A) is apoptosis in Siha cells after inhibiting FAM195A gene expression; and (B) is apoptosis in Hela cells after inhibiting FAM195A gene expression. Detailed Implementation

[0022] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] The experimental materials and instruments used in the embodiments of this invention are shown in Tables 1 and 2: Table 1 Table 2 Example 1: Design and Synthesis of siRNA Sequences The FAM195A mRNA sequence (NM_001331229.2) was obtained by directly searching the Genebank database (https: / / www.ncbi.nlm.nih.gov / genbank / ) using bioinformatics methods. The nucleotide sequence of the FAM195A coding region (nucleotides 165-545, the CDS region of the FAM195A gene, the nucleotide sequence directly encoding the protein) was selected, as shown in SEQ ID NO.3. Using the DSIR design website, siRNA sequences were designed according to the optimal siRNA sequences recommended by DSIR. Furthermore, BLAST software provided by NCBI GenBank (compiled by the National Library of Medicine and the National Institutes of Health) was used to perform homology analysis on the selected target sequence to rule out the possibility of non-specific inhibition of other gene fragments by the siRNA. The selected target gene sequence for siFAM195A was GGCTCGGCGAGCTCCTGAA. In addition, dTdT dangling bases were designed at the end of the sequence to increase the stability of the siRNA. The designed sequence was sent to Shanghai Sangon Biotech Co., Ltd. for synthesis, and the company provided the negative control siNC.

[0024] The positive chain sequence of siNC is UUCUCCGAACGUGUCACGUTT, and the negative chain sequence is ACGUGACACGUUCGGAGAATT. The positive chain sequence of siFAM195A is shown in SEQ ID NO.1, and the negative chain sequence is shown in SEQ ID NO.2.

[0025] Example 2: (1) Cell culture One day before transfection, 2 mL of Siha and Hela cell suspensions in the first growth phase were seeded into 6-well cell culture plates. After mixing, the 6-well cell culture plates were placed in a cell culture incubator at 37°C and 5% CO2. The next day, when the cells reached 30%-50% confluence, Siha and Hela cells were transfected with siNC and siFAM195A, respectively.

[0026] (2) Cell transfection The transfection methods for siNC and siFAM195A were performed according to the Lipofectamine RNAiMAX Transfection Reagent instructions. The Lipofectamine RNAiMAX Transfection Reagent includes the Lipofectamine RNAiMAX transfection reagent. The specific steps are as follows: Dilute 5 μL of Lipofectamine RNAiMAX transfection reagent with 125 μL of Opti-MEM medium; dilute siNC and siFAM195A with 125 μL of Opti-MEM medium (final concentration 25 nM). Mix the diluted siNC and siFAM195A with the diluted Lipofectamine RNAiMAX, gently mix, and incubate at room temperature for 10-20 min to obtain siNC-Lipofectamine RNAiMAX complex and siFAM195A-Lipofectamine RNAiMAX complex, respectively. Add 250 μL of siNC-Lipofectamine RNAiMAX complex and siFAM195A-Lipofectamine RNAiMAX complex to Siha cells and HeLa cells, respectively, mix well, and incubate in a cell culture incubator for 24-48 h. Transfection was successful.

[0027] (3) RT-qPCR detection RNA was extracted from Siha and HeLa cells 48 hours after transfection with siNC and siFAM195A, respectively. Total RNA was extracted from the cells using Trizol reagent, and the specific extraction method is as follows: a. Add 1 mL of TRIzol reagent to the Siha and HeLa cell pellets, mix by pipetting, transfer the cell lysis buffer to a 1.5 mL centrifuge tube, add 200 μL of chloroform, shake well for 20 s, let stand on ice for 5 min, and then place in a pre-cooled centrifuge at 4 °C and centrifuge at 12000 rpm for 15 min. b. Carefully transfer 500 μL of the upper aqueous phase containing RNA to a new centrifuge tube, add 500 μL of isopropanol, vortex thoroughly to mix, let stand at room temperature for 5 min, then place in a small refrigerated centrifuge at 4°C and centrifuge at 12000 rpm for 10 min. After centrifugation, discard the supernatant, retain the RNA precipitate, wash twice with 1 mL of 75% ethanol, centrifuge at 12000 rpm for 2 min at 4°C, discard the supernatant, air dry the precipitate, and dissolve the RNA in 30 μL of enzyme-free water; c. RNA concentration was detected using a NanoDrop micro-spectrophotometer. 1 μg of RNA was taken and processed according to the PrimeScript quantitative PCR reverse transcription kit. TM cDNA was synthesized via reverse transcription according to the instructions of the Rreagent Kit with gDNA Eraser. The resulting cDNA product was diluted 10-fold and then processed according to Power SYBR. TM The qPCR detection and analysis were performed using the Green PCR premix solution according to the instructions. The PCR reaction program was as follows: 95℃ pre-denaturation for 10 min, 1 cycle; 95℃ denaturation for 15 s, 60℃ annealing / extension for 1 min, for a total of 40 cycles. The specific PCR reaction system is shown in Table 3. Table 3 Primer sequences are shown in Table 4: Table 4 The relative expression level was calculated using the ΔΔCt algorithm. The formula for calculating the expression level of the target gene relative to the internal reference gene is as follows: RNA expression level was normalized using ACTB mRNA, and the relative expression level was compared by setting the control to 1. Relative expression level = 2 -ΔΔCt Where ΔCt=(Ct) 目的基因 -Ct 内参基因 Experimental group - (Ct) 目的基因 -Ct 内参基因 Control group. Results are expressed as mean ± standard deviation of three independent replicates, where *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0028] The results are as follows Figure 1As shown: Using ACTB as an internal reference gene, the RT-qPCR experiment was used to detect the inhibitory effect of FAM195A. The results showed that siRNA targeting FAM195A in Siha cells and HeLa cells could significantly reduce the expression level of FAM195A mRNA.

[0029] Example 3: Knocking down FAM195A gene expression inhibits the growth and proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells. (1) Cell viability detection The effects of siRNA on the growth of Siha and Hela cells were detected using the CCK-8 assay.

[0030] Siha and HeLa cell suspensions were seeded in 6-well plates. The cells were transfected with siNC and siFAM195A (as described in Example 1), following the same transfection method and steps as in Example 2. After trypsin digestion, the cells were observed to be rounded under an inverted fluorescence microscope. Digestion was terminated by adding 2 mL of fresh culture medium containing 10% FBS and 1% penicillin-streptomycin, and cell counting was performed. 100 μL of cell suspension was seeded into 96-well plates (3 × 10⁶ cells per well). 3 (1 cell), 4 replicates per group.

[0031] Cell viability was assessed on days 1, 2, 3, 4, and 5 post-inoculation using the CCK-8 assay kit. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C with 5% CO2 for 1 hour. The absorbance (OD) at 450 nm was measured using a continuous wavelength multi-mode microplate reader. Blank culture medium readings were used as background signals and subtracted from the measured values. The absorbance values ​​of siNC cells on day 1 were normalized, and the growth changes of Siha and HeLa cells were calculated. Results are expressed as mean ± standard deviation of three independent replicates, where *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0032] The results are as follows Figure 2 As shown, knockdown of FAM195A reduced the viability of Siha and Hela cells, indicating that downregulation of FAM195A by siFAM195A significantly inhibited the growth of Siha and Hela cells.

[0033] (2) Cell clone formation experiment 2 mL of MEM medium containing 10% FBS and 1% penicillin-streptomycin was added to each well of a 6-well plate. Transfected Siha and HeLa cells were seeded evenly at a density of 800 cells / well. The medium was replaced with fresh medium every 3 days. After 2 weeks of culture, the cells were fixed with 1 mL of 4% paraformaldehyde at room temperature for 10 min. After washing twice with PBS, the cells were stained with 1% crystal violet for 15 min. The staining solution was discarded, and the cells were washed three times with PBS buffer. After air-drying at room temperature, photographs were taken, and the number of colonies formed was statistically analyzed. Results are expressed as mean ± standard deviation of three independent replicates, where *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0034] The results are as follows Figure 3 As shown, knocking down FAM195A significantly reduced the clonogenic ability of Siha and Hela cells, indicating that downregulation of FAM195A by siFAM195A significantly inhibited the continuous growth of Siha and Hela cells.

[0035] (3) Cell proliferation experiment The effect of siRNA on the proliferation of Siha and HeLa cells was detected by EdU staining assay. The BeyoClick™ EdU-594 cell proliferation assay kit was used, and its components include EdU working solution, Triton-X permeable membrane, and Hoechst-33342 solution.

[0036] Siha and HeLa cell suspensions were seeded in 6-well plates. The cells were transfected with siNC and siFAM195A, following the same transfection methods and procedures as in Example 1. Forty-eight hours after transfection, EdU working solution was added as per the manufacturer's instructions, and the cells were incubated at 37°C for 2 hours. Fixation was performed with 4% paraformaldehyde for 15 min, followed by Triton-X permeation membrane incubation for 15 min, click reaction incubation for 30 min, and nucleus staining with Hoechst-33342 solution for 15 min. Images were taken using an inverted fluorescence microscope. Finally, the images were analyzed using ImageJ software. Results are expressed as the mean ± standard deviation of three independent replicates, where *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0037] The results are as follows Figure 4 As shown, knocking down FAM195A significantly reduced the proliferation capacity of Siha and Hela cells, indicating that downregulation of FAM195A by siFAM195A significantly inhibited the proliferation of Siha and Hela cells.

[0038] (4) Apoptosis experiment The effect of siRNA on the apoptotic capacity of Siha and HeLa cells was detected by flow cytometry. The Annexin V-FITC / PI apoptosis double staining kit was used for detection. The Annexin V-FITC / PI apoptosis double staining kit consists of 1× Binding Buffer, Annexin V-FITC, and PI.

[0039] Siha and HeLa cell suspensions were seeded in 6-well plates. The cells were transfected with siNC and siFAM195A, following the same transfection methods and procedures as in Example 2. Forty-eight hours after transfection, the cells were digested with EDTA-free trypsin and centrifuged at 2000 rpm for 5 min at room temperature to collect the cells. The cells were resuspended in pre-chilled 1×PBS, centrifuged at 2000 rpm for 5 min, and washed twice. 100 μL of 1×Binding Buffer was added to resuspend the cells. 5 μL of Annexin V-FITC was added, mixed well, and incubated at room temperature for 15 min in the dark. 5 μL of PI staining was added 5 min before flow cytometry. 400 μL of 1×Binding Buffer was added before flow cytometry to detect apoptosis.

[0040] The results are as follows Figure 5 As shown, knockdown of FAM195A significantly increased apoptosis in Siha and Hela cells, indicating that downregulation of FAM195A by siFAM195A promoted apoptosis in Siha and Hela cells.

[0041] In summary, the siFAM195A designed and synthesized in this invention, after transfection into human cervical cancer cells Siha and Hela, can effectively regulate and interfere with the expression of the FAM195A gene, thereby reducing the growth and proliferation of human cervical cancer cells Siha and Hela, and effectively promoting the apoptosis of human cervical cancer cells Siha and Hela. Therefore, the FAM195A gene-targeted siFAM195A has the function of knocking down the expression of FAM195A, effectively inhibiting the growth and proliferation of cervical cancer cells and promoting the apoptosis of cervical cancer cells.

[0042] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A siRNA that targets and inhibits the expression of the FAM195A gene, characterized in that, The siRNA is siFAM195A, which includes a sense strand and an antisense strand; The justice chain is shown in SEQ ID NO.1, and the antisense chain is shown in SEQ ID NO.

2.

2. The application of a siRNA that targets and inhibits the expression of the FAM195A gene, characterized in that, The siRNA that targets and inhibits the expression of the FAM195A gene as described in claim 1 is used in the preparation of a pharmaceutical composition for treating tumor diseases.

3. A pharmaceutical composition for treating neoplastic diseases, characterized in that, The active ingredient of the pharmaceutical composition comprises the siRNA that targets and inhibits the expression of the FAM195A gene as described in claim 1, and its final concentration in the pharmaceutical composition is 25 nM.

4. The pharmaceutical composition for treating neoplastic diseases according to claim 3, characterized in that, The dosage form of the pharmaceutical composition is at least one of the following: aerosol, tablet, capsule, drop pill, pill, powder, solution, suspension, emulsion, granule, lipid preparation, transdermal preparation, lozenge, suppository, and lyophilized powder for injection.

5. The pharmaceutical composition for treating neoplastic diseases according to claim 3, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable adjuvant ingredients.

6. The pharmaceutical composition for treating neoplastic diseases according to claim 3, characterized in that, The pharmaceutical composition further includes at least one of the following: filler, lubricant, dispersant, wetting agent, binder, regulator, solubilizer, antioxidant, antibacterial agent, emulsifier, and disintegrant.

7. The pharmaceutical composition for treating neoplastic diseases according to claim 3, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

8. The pharmaceutical composition for treating neoplastic diseases according to claim 7, characterized in that, The carrier is a transfection carrier.

9. A pharmaceutical composition for treating neoplastic diseases according to any one of claims 3 to 8, characterized in that, The neoplastic disease is a neoplastic disease related to the expression and function of the FAM195A gene.

10. A pharmaceutical composition for treating neoplastic diseases according to claim 9, characterized in that, The neoplastic disease mentioned is cervical cancer.

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