Application of small interfering RNA for knocking down TMEM14B in treatment of hepatocellular carcinoma

Abstract

The invention relates to the field of liver cancer treatment, in particular to application of small interfering RNA (Ribonucleic Acid) for knocking down TMEM14B in preparation of a medicine for treating hepatocellular carcinoma. In the invention, the TMEM14B is found to be highly expressed in HCC tissues and cell lines. The small interfering RNA is used for reducing expression of TMEM14B in LM3 and HepG2 cells for the first time, it is found that knock-down TMEM14B can remarkably inhibit proliferation of hepatocellular carcinoma cells and inhibit the migration ability of the hepatocellular carcinoma cells in a two-dimensional plane and a three-dimensional space, and it is confirmed that the small interfering RNA of the knock-down TMEM14B has the definite effect of inhibiting proliferation of the hepatocellular carcinoma cells; the small interfering RNA for knocking down the TMEM14B has a definite effect of inhibiting migration and invasion of hepatocellular carcinoma cells. Therefore, the small interfering RNA for knocking down the TMEM14B can be used for preparing the medicine for treating the hepatocellular carcinoma. The small interfering RNA for knocking down the TMEM14B can be used for preparing medicines for treating metastasis and relapse of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of tumor diagnosis and treatment, and specifically refers to the application of small interfering RNA for knocking down TMEM14B in the treatment of hepatocellular carcinoma. Background Art

[0002] Hepatocellular carcinoma has become one of the digestive tract tumors with the highest number of deaths in the world due to its hidden onset and poor prognosis. The 2023 Global Tumor Epidemiology Statistics (GLOBOCAN) show that the proportion of deaths from hepatocellular carcinoma in the world ranks fifth among men and seventh among women. After liver cells become cancerous, they mainly metastasize to distant sites through the blood or lymphatic pathways, and can also invade other organs through direct contact. Due to the rich blood supply to the liver, hepatocellular carcinoma can invade the intrahepatic arteries and veins, and then return to the pulmonary capillary network through systemic venous return, followed by systemic circulation, and can also spread retrogradely through the portal vein system. Due to the hidden characteristics of hepatocellular carcinoma, most patients have lost the opportunity for surgery when they are diagnosed with hepatocellular carcinoma. Although chemotherapy and radiotherapy have certain effects on the treatment of hepatocellular carcinoma, they often cannot help patients achieve continuous treatment progress. The 5-year survival rate of hepatocellular carcinoma is only 21%, and the 5-year survival rate of hepatocellular carcinoma that has metastasized is only 3%. In summary, the grim status of HCC treatment cannot be ignored. The high metastatic characteristics of HCC are the main problems facing the current treatment of HCC. Improving early screening methods and developing more efficient treatment methods can effectively improve the survival rate of HCC. Therefore, the top priority of HCC clinical research is to deeply analyze the potential mechanisms that affect the occurrence, development and prognosis of HCC and to find effective diagnostic and therapeutic targets.

[0003] Transmembrane Protein 14B (TMEM14B) is a protein-coding gene. It is a primate-specific protein that participates in the expansion and folding of the developing neocortex. Currently, there are no reports on the phenotypic regulation and regulatory mechanism of the TMEM14B gene in the development of hepatocellular carcinoma.

[0004] Based on this, this study will explore the effects of small interfering RNA knocking down TMEM14B on the proliferation and migration of hepatocellular carcinoma through an in vitro model, providing ideas for the development of new applications and new treatment strategies for hepatocellular carcinoma in clinical practice. Summary of the invention

[0005] The purpose of the present invention is to retrospectively analyze the existing data to reveal the role of TMEM14B in judging the prognosis of hepatocellular carcinoma, and at the same time reveal the specific role of TMEM14B in hepatocellular carcinoma cells through in vitro experimental studies, and use small interfering RNA to knock down TMEM14B to reduce the vitality and proliferation ability of hepatocellular carcinoma cells and inhibit the migration of hepatocellular carcinoma cells.

[0006] Therefore, the main contents of the present invention are summarized as follows: high TMEM14B expression level indicates poor prognosis of hepatocellular carcinoma, and reducing TMEM14 expression by using small interfering RNA to knock down TMEM14B can reduce the vitality and proliferation ability of hepatocellular carcinoma cells and inhibit the migration of hepatocellular carcinoma, and small interfering RNA for knocking down TMEM14B can be used to treat hepatocellular carcinoma.

[0007] The details of the present invention are as follows:

[0008] 1. We first revealed that the gene expression level of TMEM14B was significantly upregulated in hepatocellular carcinoma tissues compared with adjacent tissues, and that the expression level of TMEM14B in tumor tissues was negatively correlated with the overall survival and disease-specific survival of hepatocellular carcinoma patients;

[0009] 2. We first revealed that the gene expression level of TMEM14B in hepatocellular carcinoma epithelial cells was significantly upregulated compared with adjacent tissues;

[0010] 3. We observed that oxidative phosphorylation, MYC target V1 and DNA repair-related pathways were activated in epithelial cells of hepatocellular carcinoma compared with those in epithelial cells of adjacent tissues;

[0011] 4. We first revealed that TMEM14B is positively correlated with oxidative phosphorylation, MYC target V1 and DNA repair-related genes in tumor tissues;

[0012] 5. We first revealed that knocking down TMEM14B inhibits cell viability and proliferation in hepatocellular carcinoma cells;

[0013] 6. We revealed for the first time that knocking down TMEM14B can inhibit the migration and invasion of hepatocellular carcinoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 .Expression of TMEM14B in hepatocellular carcinoma tissue samples and adjacent tissues: Based on the hepatocellular carcinoma sample data provided by the TCGA database and the normal human sample data provided by GTEx (TCGA-LIHC), the gene expression level of TMEM14B in hepatocellular carcinoma tumor tissues (369 cases) and normal tissues (160 cases) of hepatocellular carcinoma patients was analyzed.

[0015] Figure 2.Correlation between the expression of TMEM14B in hepatocellular carcinoma and the overall survival and disease-specific survival of patients: (A) Kaplan-Meier survival curves of hepatocellular carcinoma patients (n=369) obtained from the TCGA-LIHC dataset were used to analyze the correlation between the expression level of TMEM14B and the overall survival of hepatocellular carcinoma patients. *P<0.05. (B) Kaplan-Meier survival curves of hepatocellular carcinoma patients (n=369) obtained from the TCGA-LIHC dataset were used to analyze the correlation between the expression level of TMEM14B and the disease-specific survival of hepatocellular carcinoma patients. *P<0.05; **P<0.01; ***P<0.001.

[0016] Figure 3 .TMEM14B expression in hepatocellular carcinoma epithelial cells and adjacent tissue epithelial cells: (A) tSNE diagram of 9 cell types. (B) tSNE diagram of hepatocytes. (C) Based on the hepatocellular carcinoma samples (GSE149614) provided by the GEO database, the gene expression level of TMEM14B in hepatocellular carcinoma tumor epithelial cells and adjacent tissue epithelial cells was analyzed.

[0017] Figure 4 .KEGG and hallmarker analysis results of differential genes between hepatocellular carcinoma epithelial cells and adjacent tissue epithelial cells: (A) Hallmarker analysis of differential genes between hepatocellular carcinoma epithelial cells and adjacent tissue epithelial cells was performed using the clusterProfiler software package. Different colors in p.adjust indicate different significance, and the size of Count indicates the number of genes. (B) KEGG analysis of differential genes between hepatocellular carcinoma epithelial cells and adjacent tissue epithelial cells was performed using the clusterProfiler software package. Different colors in p.adjust indicate different significance, and the size of Count indicates the number of genes.

[0018] Figure 5.Relationship between the expression of TMEM14B in hepatocellular carcinoma epithelial cells and genes related to oxidative phosphorylation, MYC target V1, and DNA repair: (A) Hallmarker analysis of genes related to TMEM14B using the clusterProfiler software package. Different colors in p.adjust indicate different significance, and the size of Count indicates the number of genes. (B) Regulatory network of TMEM14B and genes related to DNA repair. (C) Regulatory network of TMEM14B and genes related to MYC target V1. (D) Regulatory network of TMEM14B and genes related to oxidative phosphorylation. (E) Pearson correlation coefficient between TMEM14B and genes related to DNA repair. (F) Pearson correlation coefficient between TMEM14B and genes related to MYC target V1. (G) Pearson correlation coefficient between TMEM14B and genes related to oxidative phosphorylation.

[0019] Figure 6 .Changes in proliferation ability of hepatocellular carcinoma cells after knockdown of TMEM14B: (A) LM3 cells with low TMEM14B expression were obtained after transfection with two siRNAs (siTMEM14B#1, siTMEM14B#2) for 48 hours, and the cell proliferation level was detected by CCK8 assay. (B) HepG2 cells with low TMEM14B expression were obtained after transfection with two siRNAs (siTMEM14B#1, siTMEM14B#2) for 48 hours, and the cell proliferation level was detected by CCK8 assay. *P<0.05; **P<0.01; ***P<0.001.

[0020] Figure 7 .Effect of knockdown of TMEM14B in hepatocellular carcinoma cells on 2D cell migration: (A) Scratch healing of LM3 cells transfected with TMEM14B-specific siTMEM14B#1 and siTMEM14B#2 at 0, 24 and 48 hours. (B) Scratch healing of HepG2 cells transfected with TMEM14B-specific siTMEM14B#1 and siTMEM14B#2 at 0, 24 and 48 hours.

[0021] Figure 8 The effect of knocking down TMEM14B in hepatocellular carcinoma cells on their three-dimensional spatial migration: (A) LM3 cells transfected with TMEM14B-specific siTMEM14B passing through the Transwell pores in a 24-hour Transwell experiment. (B) HepG2 cells transfected with TMEM14B-specific siTMEM14B passing through the Transwell pores in a 24-hour Transwell experiment. DETAILED DESCRIPTION

[0022] All experimental materials and methods mentioned in the Examples section

[0023] 1. Cell Recovery

[0024] 1) Preheat the water bath to 37℃, quickly take out the cryotube from the liquid nitrogen tank, and place it in a 37℃ water bath for 2 minutes until it is completely thawed. The thawed cell suspension is quickly transferred to a culture dish containing culture medium and transferred to a constant temperature cell culture incubator.

[0025] 2) Centrifuge the cell suspension at 300g for 4-5 minutes in a benchtop centrifuge; discard the supernatant and use

[0026] Resuspend the pellet evenly in 2 mL of complete culture medium and transfer to a cell culture dish, which is then placed in a 5% CO2, 37°C constant temperature cell culture incubator for culture.

[0027] 3) After 6 hours of cell culture, observe the cell adhesion status, discard the upper floating cells, replace with fresh culture medium and continue culturing.

[0028] 2. Cell Culture

[0029] LM3 cells and HepG2 cells were cultured in DMEM medium. 10% FBS and 1% penicillin-streptomycin solution were added to the medium. The cells were cultured in a 5% CO2, 37℃ constant temperature cell culture incubator.

[0030] Observe the cell growth status, replace fresh culture medium from time to time, and subculture when the cell density in the culture dish reaches 80-90%.

[0031] 3. Cell Passaging

[0032] 1) Cells can be subcultured when the cell growth density is about 80-90%.

[0033] 2) After removing the upper culture medium, add sterile PBS to wash away a small amount of residual culture medium, repeat twice; digest the cells with 2ml of trypsin for 2-3min, tap the culture dish gently to see that the cells have a large range of cell wall detachment, and add 2ml of complete culture medium to terminate the digestion.

[0034] 3) Use a pipette to blow the cells off the dish wall to completely remove them, and centrifuge the cell suspension at 300g for 4-5 minutes.

[0035] 4) Discard the supernatant and add an appropriate amount of culture medium to resuspend the cell pellet, add culture medium according to the subculture ratio and transfer it to a cell culture dish; place the culture dish in a 5% CO2, 37°C constant temperature incubator.

[0036] 4. Cell Cryopreservation

[0037] 1) After removing the upper culture medium, add sterile PBS to wash away a small amount of residual culture medium, repeat twice; digest the frozen cells with 2ml trypsin for 2-3min. After a large range of cell wall detachment occurs, add 2ml complete culture medium to terminate the digestion.

[0038] 2) Use a pipette to gently blow against the dish wall to completely detach the cells, and centrifuge the cell suspension at 300g in a desktop centrifuge for 4-5 minutes.

[0039] 3) After centrifugation, discard the supernatant, aspirate 1-2 ml of complete medium to evenly resuspend the cells and transfer them to a cryopreservation tube, add 10% volume of DMSO and pipette evenly; the cryopreservation tube is placed in a 4°C and -20°C refrigerator for 20 min, then placed in a -80°C refrigerator overnight, and finally placed in liquid nitrogen for long-term storage.

[0040] 5. siRNA Construction and Transfection

[0041] siRNA was synthesized by GenScript. According to the design principle of RNA interference, two sequences siTMEM14B#1 and siTMEM14B#2 were constructed as TMEM14B gene interference fragments, and a non-functional fragment siControl was used as a negative control.

[0042] siRNA transfection steps:

[0043] 1) Cell culture: Before transfection, target cells were cultured in serum-containing medium in 12-well plates, with 1×10 cells per well. 4 When the cell confluence reached 30-40%, the medium was replaced with serum-free medium.

[0044] 2) Prepare transfection solution: according to the instructions of the plasmid manual, take an appropriate amount of lyophilized powder and add 50 μl Opti-MEM medium to mix thoroughly to obtain siRNA dilution solution, and let it stand at room temperature for 5 minutes; according to the instructions of Lipofectamine 3000 manual, pipette an appropriate amount of Lipofectamine RNAi-MAX into an EP tube (shake gently before taking), add an equal volume of Opti-MEM medium to the siRNA dilution solution to dilute Lipofectamine RNAi MAX, and let it stand at room temperature for 5 minutes.

[0045] 3) siRNA transfection: Use a pipette to repeatedly pipette and mix the siRNA and Lipofectamine RNAiMAX dilution, and let stand at room temperature for 15 minutes; add all the mixed solution to the cell culture plate in 2).

[0046] 4) Cell medium replacement: Incubate the cells in a 5% CO2 37°C incubator for 6 hours and then replace with fresh complete medium; after continuing to incubate for 24-48 hours, qPCR and Western blot experiments can be performed to identify the expression level of the target gene.

[0047] 6. Cell Counting

[0048] The digested cells were diluted evenly in an appropriate ratio, and 20 μl of the cell suspension was mixed evenly with an equal volume of trypan blue solution; 20 μl of the mixture was added to a cell counting plate, and cells in three fields of view were randomly selected for counting using Count Star software to obtain the average cell concentration.

[0049] 7. Cell activity detection (CCK8 method)

[0050] 1) Each group of cells was inoculated in a 96-well plate, with 100 μl of cell suspension in each well. The cell concentration was

[0051] 1×10 5 / ml, set up 6 replicate wells and incubate at 37℃.

[0052] 2) Cell activity was detected 24h, 48h, and 72h after cell attachment.

[0053] 3) According to the instructions of the CCK8 kit, slowly add 10% (10 μl) CCK8 reagent to each well along the well wall and incubate at 37°C for 1-2 hours.

[0054] 4) Remove the culture medium and measure the cell absorbance at a wavelength of 450 nm using an ELISA reader; draw a survival curve.

[0055] 8. Real-time quantitative polymerase chain reaction (Realtime PCR)

[0056] Template RNA extraction

[0057] 1) Digest the cells to be tested in the logarithmic growth phase and take 5×10 6 cells;

[0058] The cell suspension was centrifuged at 350 g for 3 minutes and washed 1-2 times with PBS to obtain a cell pellet; the operating table was sprayed with alcohol to maintain an enzyme-free environment.

[0059] 2) Use a pipette to absorb 1 ml of Trizol and gently blow on the cells. Transfer the cell suspension to a 1.5 ml EP tube and place it on ice for 5 minutes to allow the cells to fully lyse.

[0060] 3) Add 200 μl of chloroform and shake up and down for 15 seconds to mix, then let stand at room temperature for 5 minutes; precool the high-speed centrifuge to 4°C, centrifuge at 12,000 g for 15 minutes; transfer the upper aqueous phase (about 500 μl) to an enzyme-free EP tube and discard the lower phenol phase.

[0061] 4) Add an equal volume of isopropanol (about 500 μl) and mix thoroughly by inversion. Let stand at room temperature for 5-10 minutes. Centrifuge at 4°C, 12,000 g for 15 minutes, carefully remove the supernatant, and the RNA precipitate is at the bottom of the tube. Invert the tube to drain.

[0062] 5) Add about 500 μl of 75% ethanol and gently shake the EP tube. Centrifuge at 8000 g for 5 min at 4°C and carefully remove the supernatant.

[0063] 6) Repeat step 5) 1-2 times to purify RNA, turn the tube upside down to dry at room temperature or place it in a clean bench to air dry (avoid over-drying).

[0064] 7) Add 50 μl of enzyme-free DEPC water to the EP tube, place it in a 65°C drying oven and heat for 5-10 minutes to fully dissolve the RNA; store it in a -80°C refrigerator until use.

[0065] RNA reverse transcription

[0066] 1) Heat the RNA in 2.2.11.1 7) at 65°C for 5 min and measure the RNA concentration using a spectrophotometer.

[0067] 2) Prepare the following reverse transcription system (20 μl):

[0068]

[0069] 3) Reverse transcription of the system in 2) using a PCR instrument (remix the system before reverse transcription), the reaction conditions are:

[0070]

[0071] 4) The reverse transcription product was used for qPCR immediately or stored at -80°C for a long time.

[0072] RT qPCR

[0073] 1) Prepare the following system (20 μl) in an eight-tube strip in a dark environment:

[0074]

[0075] 2) Centrifuge the eight-tube strip and then place it in the ABI QuantStudio 5 Real-time PCR instrument for qPCR reaction. The reaction conditions are as follows:

[0076]

[0077]

[0078] 9. Scratch test

[0079] 1) Prepare the chamber: Place the scratch chamber in a clean culture dish, sterilize with alcohol, irradiate under ultraviolet light for 30 minutes, and dry. Prepare a 24-well plate, draw a line on the bottom, stick the chamber on the drawn area, and continue to prepare under ultraviolet light.

[0080] 2) Cell plate: Take logarithmically growing MHCC-97H and MHCC-97H / REGO cells, add 1mL of trypsin, digest for 3min, after digestion is complete, add 1mL of complete medium to terminate digestion, collect cells and centrifuge at 200g for 5min, discard the supernatant, and resuspend the cells in medium. The cell suspension is configured to a concentration of 100,000 cells / mL. Add 350μL-400μL into the chamber and culture overnight.

[0081] 3) Scratch experiment record: On the second day, the chamber was removed, washed with PBS and then added with serum-free medium. At 0h, 24h, and 48h, the microscope was magnified 100 times and photographed. The black line drawn in advance was used as the positioning to ensure that each photograph was taken at the same position. This was to understand the changes in cell migration ability.

[0082] 10. Transwell migration assay

[0083] 1) Prepare the Transwell device: The Transwell device should be used immediately after opening. Place it directly into a 24-well plate and irradiate with ultraviolet light for 30 minutes.

[0084] 2) Cell plate: MHCC-97H and MHCC-97H / REGO cells in the logarithmic growth phase were selected. After the culture medium was removed, 1 mL of trypsin was added and digested for 3 min. Complete culture medium was added to terminate the digestion. The cells were collected and centrifuged at 200 g for 5 min. The supernatant was discarded and the cells were resuspended in serum-free culture medium. The cells were configured to 200,000 cells / mL and 400 μL of the cells were added to the upper chamber of the Transwell chamber. 1 mL of serum-containing culture medium was added to the lower chamber.

[0085] 3) Cell staining observation: After culturing in a 37°C 5% CO2 constant temperature incubator for 24 hours, the Transwell device was removed from the 24-well plate, the culture medium was aspirated, the cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min, washed with PBS, and stained in 0.5% crystal violet for 15 min. After staining, the cells were washed again with PBS, and the cells remaining in the upper chamber were slowly wiped with a hairless cotton swab until the cells in the upper chamber were completely wiped off. After treatment, the cells were photographed with a microscope at a magnification of 200 times, and 5 photos were taken at random positions. The obtained photos were counted to detect the cell migration ability.

[0086] 11. Statistical Analysis

[0087] Each set of experimental data was obtained through three independent repetitions, and the experimental results were expressed as mean ± standard deviation. The data were analyzed using IBM SPSS Statistics 20 software.

[0088] Example 1 Expression of TMEM14B gene in hepatocyte tissue and its relationship with the prognosis of hepatocellular carcinoma patients

[0089] According to the hepatocellular carcinoma samples and normal tissue sample data provided by the TCGA database (TCGA-LIHC) and the GTEx database, the gene expression level of TMEM14B in hepatocellular carcinoma tissues and normal tissues of hepatocellular carcinoma patients was analyzed. The results showed that the gene expression level of TMEM14B in hepatocellular carcinoma tissues was significantly upregulated compared with normal tissues. The correlation between the gene expression level of TMEM14B in hepatocellular carcinoma patients and their survival time was analyzed using TCGA-LIHC survival data. The results showed that compared with hepatocellular carcinoma patients with high expression of TMEM14B, hepatocellular carcinoma patients with low expression of TMEM14B in tumor tissues had significantly increased overall survival and disease-specific survival. (P=0.00045, Figure 2 A; P = 0.0005, Figure 2 B). The above results suggest that the expression level of TMEM14B may be associated with the occurrence, development and prognosis of hepatocellular carcinoma, and high expression of TMEM14B may promote the occurrence and development of hepatocellular carcinoma, and high expression of TMEM14B may be associated with poor prognosis of hepatocellular carcinoma patients.

[0090] Example 2 Expression of TMEM14B gene in hepatocyte epithelial cells

[0091] According to the single-cell sequencing data of hepatocellular carcinoma tissue and adjacent tissue provided by GEO database (GSE149614), 9 cell types were identified and annotated, including: NK cells, hepatocytes + hepatocellular carcinoma cells, monocytes, macrophages, stem cells, endothelial cells, stromal cells, T cells and B cells ( Figure 3A). We retained the hepatocytes ( Figure 3 B). The gene expression level of TMEM14B in hepatocellular carcinoma epithelial cells and normal liver tissue epithelial cells was analyzed. The results showed that the gene expression level of TMEM14B in hepatocellular carcinoma epithelial cells was significantly upregulated compared with that in normal liver tissue epithelial cells.

[0092] Example 3 Relationship between KEGG pathways and Hallmarkers included in differentially expressed genes and KEGG pathways and Hallmarkers regulated by TMEM14B

[0093] The relevant pathways contained in DEGs were analyzed by KEGG and Hallmarker enrichment. Hallmarker analysis showed that DNA repair, peroxisome, MYC-target-V1, and oxidative phosphorylation were activated in hepatocellular carcinoma ( Figure 4 A), while KEGG results showed that the oxidative phosphorylation pathway was activated in hepatocellular carcinoma ( Figure 4 B). TMEM14B-related genes were analyzed by Hallmark. The experimental results showed that DNA repair genes, MYC-tagget-V1 genes and oxidative phosphorylation genes were enriched in TMEM14B-related genes ( Figure 5 A). At the same time, we constructed the regulatory network between TMEM14B and DNA repair genes, MYC-tagget-V1 genes and oxidative phosphorylation genes ( Figure 5 B-5D). The relationships between TMEM14B-DNA repair genes, TMEM14B-MYC-target-V1 genes and TMEM14B-oxidative phosphorylation genes were further verified by TCGA-CIHC. The experimental results showed that 8 of the 11 TMEM14B-DNA repair gene interactions were significantly correlated by TCGA-LIHC data; 34 of the 46 TMEM14B-MYC-target-V1 gene interactions were significantly correlated by TCGA-LIHC data; and 11 of the 15 TMEM14B-oxidative phosphorylation gene interactions were significantly correlated by TCGA-LIHC data. The experimental results showed that TMEM14B in tumor tissues was positively correlated with oxidative phosphorylation, MYCtarget V1 and DNA repair-related genes.

[0094] Example 4 Relationship between TMEM14B and the proliferation ability of LM3 and HepG2 cells

[0095] To investigate the effect of TMEM14B on the proliferation of hepatocellular carcinoma cells, this study used hepatocellular carcinoma cells with TMEM14B knockdown to perform CCK8 experiments. TMEM14B in HepG2 and LM3 cells was knocked down using siRNA. Cell proliferation was assessed using CCK-8 assay at 24, 48, and 72 hours. The experimental results showed that TMEM14B knockout inhibited the proliferation of HepG2 and LM3 cells ( Figure 6 A- Figure 6 B).

[0096] Example 5 Relationship between TMEM14B and LM3 and HepG2 cell migration ability

[0097] To explore the effect of TMEM14B on the migration ability of hepatocellular carcinoma cells, this study used hepatocellular carcinoma cells with TMEM14B knockdown to perform scratch assays and Transwell migration assays. TMEM14B in HepG2 and LM3 cells was knocked down using siRNA. The cell migration ability was evaluated using a scratch healing assay at 24 and 48 hours. The experimental results showed that TMEM14B knockout inhibited the migration of HepG2 and LM3 cells in a two-dimensional plane ( Figure 7 A- Figure 7 B). The cell migration ability was evaluated by Transwell migration assay at 24 hours. The experimental results showed that TMEM14B knockout inhibited the migration of HepG2 and LM3 cells in three-dimensional space ( Figure 8 A- Figure 8 B)

[0098] Summary of the invention

[0099] For the first time, small interfering RNA was used to reduce the expression of TMEM14B in LM3 and HepG2 cells. It was found that knocking down TMEM14B can significantly inhibit the proliferation of hepatocellular carcinoma cells and inhibit the migration ability of hepatocellular carcinoma cells in two-dimensional planes and three-dimensional spaces. It was confirmed that the small interfering RNA that knocked down TMEM14B has a clear effect of inhibiting the proliferation of hepatocellular carcinoma cells; the small interfering RNA that knocked down TMEM14B has a clear effect of inhibiting the migration and invasion of hepatocellular carcinoma cells. Therefore, the small interfering RNA that knocked down TMEM14B can be used to prepare drugs for the treatment of hepatocellular carcinoma; the small interfering RNA that knocked down TMEM14B can be used to prepare drugs for the treatment of metastasis and recurrence of hepatocellular carcinoma.

Claims

1. Application of small interfering RNA for knocking down TMEM14B in the preparation of drugs for the treatment of hepatocellular carcinoma.

2. Use of the small interfering RNA for knocking down TMEM14B as claimed in claim 1 in the preparation of a drug for treating hepatocellular carcinoma, characterized in that the sequence of the small interfering RNA for knocking down TMEM14B is shown in the sequence listing SEQ ID NO:

1.

3. Use of the small interfering RNA for knocking down TMEM14B as claimed in claim 1 in the preparation of a drug for treating hepatocellular carcinoma, characterized in that the sequence of the small interfering RNA for knocking down TMEM14B is shown in the sequence listing SEQ ID NO:

2.

4. Application of small interfering RNA for knocking down TMEM14B in the preparation of preparations for inhibiting the proliferation, migration and EMT of hepatocellular carcinoma cells.

5. Use of the small interfering RNA for knocking down TMEM14B as claimed in claim 4 in the preparation of a preparation for inhibiting proliferation, migration and EMT of hepatocellular carcinoma cells, characterized in that the sequence of the small interfering RNA for knocking down TMEM14B is shown in the sequence table SEQ ID NO:

1.

6. Use of the small interfering RNA for knocking down TMEM14B as claimed in claim 4 in the preparation of a preparation for inhibiting proliferation, migration and EMT of hepatocellular carcinoma cells, characterized in that the sequence of the small interfering RNA for knocking down TMEM14B is shown in the sequence listing SEQ ID NO:

2.

7. Application of small interfering RNA for knocking down TMEM14B in the preparation of drugs for treating metastasis and recurrence of hepatocellular carcinoma.

8. Use of the small interfering RNA for knocking down TMEM14B as claimed in claim 7 in the preparation of a drug for treating metastasis and recurrence of hepatocellular carcinoma, wherein the sequence of the small interfering RNA for knocking down TMEM14B is as shown in the sequence listing SEQ ID NO:

1.

9. Use of the small interfering RNA for knocking down TMEM14B as claimed in claim 7 in the preparation of a drug for treating metastasis and recurrence of hepatocellular carcinoma, wherein the sequence of the small interfering RNA for knocking down TMEM14B is as shown in SEQ ID NO: 2 in the sequence listing.

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