MiR-221-3p Promotes The Proliferation,Metastasis And Invasion Of Human Hepatoma Cells By Inhibiting The Target Gene MGMT

Background and objective:hepatocellular carcinoma is one of the high incidence rate and mortality of malignant tumors in 2017 in China,The National Cancer Center released report shows that the incidence of HCC was third in male and female was sixth,and the mortality of HCC is highest in third.There are no obvious symptoms in the early stage of HCC.Many patients have been found in the late stage,only 30%to 40% of the patients can receive radical treatment.For radical treatment of HCC,no matter whether liver transplantation or surgical resection and radiofrequency ablation can be avoided,the recurrence rate is relatively high.The recurrence rate in 5 years is up to 70%.Although the concepts and methods of precise hepatectomy and anatomical hepatectomy have been widely applied and promoted,none of them have significantly improved the recurrence rate and prognosis of HCC.With the breakthrough of targeted therapy and immunotherapy in the treatment of cancer,the search for the target of cancer has become a new research focus.However,there is still no clear target for the study of HCC,and the lack of effective systemic drugs has become the bottleneck ofthe comprehensive treatment of HCC.Therefore,exploration is of great importance to the new targeted therapy in non-traditional sense,and to strengthen the research on the molecular biological mechanism of the occurrence and development of HCC.McroRNAs(miRNAs)is composed of a group of 22-28 nucleotides,a small molecule,which is a non coding RNA(non-codingRNA,ncRNA).It can specifically inhibit the expression of target genes through the principle of complementary bases and the binding of target genes to mRNA.More and more studies have shown that the abnormal expression of miRNA is closely related to the occurrence and development of tumor.The relationship between miRNA and HCC and its regulatory mechanism have become one of the research hotspots in recent years.In recent years,a large number of abnormal expression of miRNA in HCC has been found,and it has been proved to be involved in the regulation of HCC.The sequence of miR-221-3p gene of AGCTACATTGTCTGCTGG GTTTC,recently found its expression is upregulated in prostate cancer,bladder cancer,breast cancer and other tumors,and promote tumor development is closely related to the function of miRNA mainly depends on its target gene regulation of target genes,so study regulation is very important,therefore,it is necessary to explore the prediction the influence of miR-221-3p on the biological behavior of HCC and its target gene.In this study,we proposed by real-time quantitative qRT-PCR detection of miR-221-3p in HCC tissues and cell lines expression;method using lentiviral transfection enhanced endogenous miRNA function by MTT,cell cycle,cell migration and invasion experiment method to study the effect of miR-221-3p on proliferation and metastasis of HCC;according to the bioinformatics prediction results by quantitative real-timetechnology,protein electrophoresis,process of luciferase report experiments to identify the target gene of miR-221-3p.The solution of these questions would indicate the function of miR-221-3p in HCC growth and metastasis.The results may provide reference for new targets and mocular markers of HCC in clinical diagnosis and treatments.Methods:1.miR-221-3p expression in tissues and cellsFresh specimens were collected: 20 the expression of miR-221-3p was detected by q RT-PCR in hepatocellular carcinoma and its corresponding adjacent tissues and 10 normal liver tissues.Then qRT-PCR was used to detect its expression in normal liver cells and hepatoma cell lines.2.Effect of miR-221-3p on proliferation and metastasis of hepatocellular carcinoma cellsUse the miR-221-3p lentivirus control group and blank lentivirus transfected into miR-CON hepatoma cell lines SMMC-7721 and BEL-7404 respectively,two cell lines,then we used MTT cell proliferation assay,cell cycle experiment,cell apoptosis assay,wound healing assay,Transwell invasion assay and cloning assay for the role of miR-221-3p in hepatocellular carcinoma cell proliferation,migration and invasion effect.3.Prediction and identification of target gene of miR-221-3p(1)The downstream target genes of miR-221-3p were screened through prediction software RNA hybrid and related prediction sites microRNA,Pictar and TargetScan.(2)The expression of MGMT protein in 20 cases of HCC and its corresponding para cancerous tissue and 10 cases of normal liver tissues were detected by immunohistochemical method.(3)Methods using lentiviral transfection,miR-221-3p was transfected into SMMC-7721 and BEL-7404 cells,the expression of candidate target genes MGMTmRNA and MGMT protein qRT-PCR and Western Blot miR-221-3p up and miR-221-3p down detection group and its corresponding NC cells,preliminary identification of the target gene miR-221-3p.(4)Using the double fluoro enzyme reporter gene system to identify whether there is a complementation between miR-221-3p and MGMT,which proves that MGMT is a target gene for miR-221-3p in hepatoma cells.Results:1.Expression of miR-221-3p in hepatocellular carcinoma and hepatoma cell lines.(1)Expression of miR-221-3p in tissues was detected by using qRT-PCRThe expression of miR-221-3p in 20 cases of liver cancer,corresponding paracancerous tissue and 10 normal liver tissues was detected by qRT-PCR.The results showed that the expression level of miR-221-3p was significantly increased in HCC tissues(P<0.01).The expression of miR-221-3p in adjacent tissues was not significantly different from that in normal liver tissues(P>0.05).(2)Expression of miR-221-3p in normal liver cells and hepatoma cell linesThe expression of miR-221-3p in four hepatoma cell lines was higher than that in normal cell line HL-7702(P<0.01),and was highly expressed in SMMC-7721 and BEL-7404 hepatoma cells by q RT-PCR detection...2.The effect of miR-221-3p on the proliferation and metastasis of hepatoma cells.(1)MiR-221-3p was transfected into SMMC-7721 and BEL-7404 cells by lentivirus vector,and the transfection efficiency was detected by qRT-PCR.The results showed that: after transfection of miR-221-3p,the expression level ofmiR-221-3p in SMMC-7721 and BEL-7404 cells was upregulated significantly,indicating that the interference effect was significant(P<0.05).(2)MTT assay was used to detect the SMMC-7721 and BEL-7404 cells transfected with miR-221-3p,and the proliferative ability was detected.The change of absorptivity of the absorbance varied with time on the wavelength of490 nm.The results showed that the proliferation of BEL-7402 and SMMC-7721 cells in miR-221-3p down group was significantly inhibited after 48 hours of proliferation test,and the proliferation ability of miR-221-3p up group increased significantly(P<0.05)compared with group NC.It is suggested that miR-221-3p has the function of promoting the proliferation of hepatoma cells.(3)FACS cell apoptosis test,lentivirus infection SMMC-7721 and BEL-7404 cells,5 days after culture,miR-221-3p down group and NC group apoptosis rate comparison.Compared with group NC,the number of apoptotic cells in miR-221 down group increased(SMMC-7721,5.48% ± 0.07% vs4.02% ± 0.265%,P<0.05,BEL-7404,9.17% ± 0.189% vs,3.92% ± 00955,P<0.05),while in miR-221-3p up group,there was no significant difference between apoptotic cells and those in the P<0.05 group.(4)In the cell cycle test,Lentivirus infect SMMC-7721 cells.After 5 days of culture,miR-221 up,miR-221 down group and NC group are in G1,S and G2/M phase.Compared with the NC group,the cells in the miR-221-3p up and miR-221-3p down group had no significant changes in the S phase,and the cells in the G1 phase decreased(P > 0.05)and were at the G2/M stage(P<0.05).In the BEL-7404 cells of the lentivirus infection,compared with the NC group,the cells in the miR-221-3p up group were in the S phase(P<0.05),the cells in the G1 phase decreased(P<0.05),and the cells at the G2/M stage had no significant changes.The cells in the miR-221-3p down group were not significantlychanged in the S phase.More(P<0.05).This suggests that it may not affect hepatocellular carcinoma cells by affecting cell cycle..(5)Scratch assay,miR-221-3p lentivirus infected SMMC-7721 cells after scratch,photographed at 0h and 24 h,the migration ability of miR-221-3p group down(mobility ratio calculation for different camera observing time migration distance with "0 hour" scratch width value)showed: miR-221-3p,SMMC-7721 and BEL-7404 in cell migration obviously enhanced compared with the NC group(SMMC-7721:0.98 ± 0.007 vs 0.9 ± 0.009,P<0.05 ± 0.02 vs ± 0.43;BEL-7404:0.65 0.02;P<0.05;miR-221-3p),down after the migration ability of NC group of two HCC cell lines compared are obviously weaker(SMMC-7721:0.58 ± 0.007 vs 0.9 ± 0.009;0.43 ± 0.02 BEL-7404:0.24 ± 0.02 vs P<0.05;).It is suggested that miR-221-3p promotes cell migration in hepatocellular carcinoma cells.(6)Transwell invasion assay showed that in SMMC-7721 and BEL-7404 cells,the number of transmembrane cells miR-221-3p up transfection significantly more than group NC(SMMC-7721:71 ± 1.68 vs 51 ± 0.9;BEL-7404:184 ± 9.63VS125 ± 1.64,P < 0.05),also found that miR-221-3p cells in down group was significantly lower than the invasive ability of cells in NC group(SMMC-7721:28.2.1vs51 ± BEL-7404:65 ± 11.84vs125 ± 0.9;1.64;P<0.05).The results suggest that miR-221-3p can enhance the invasiveness of SMM-C7721 and BEL-7404 cells in vitro,and down regulation of miR-221-3p can inhibit the invasiveness of hepatoma cells.(7)The clone formation was tested in the SMMC-7721 and BEL-7404 cell groups of the lentivirus infection: 3 days after infection,the seed was planted for16 days.Compared with group NC,the number of clones in miR-221-3p up group increased(SMMC-7721:116 ± 3 vs 89 ± 4;BEL-7404:160 ± 5vs123 ± 6;P<0.05),and the number of clones in miR-221-3p down group decreased(SMMC-7721:49±6 vs 89±4;BEL-7404:85±4vs123±6;P<0.05).It is suggested that miR-221-3p promotes the cloning of hepatoma cells,and the cloning ability of the hepatoma cells is obviously inhibited after the downregulation.3.Prediction of target gene of miR-221-3p(1)Using bioinformatics prediction software microRNA,TargetScan,Pictar and RNA hybrid to predict the target genes regulated by miR-221-3p.According to the role of miR-221-3p in promoting cancer,the possibility of predicting the tumor suppressor gene MGMT from the primal gene is the most likely.(2)Immunohistochemical staining suggested that the positive expression of MGMT protein was brown yellow granules,mainly located in the cytoplasm.The immunohistochemical results showed positive expression in normal liver tissue,positive expression in liver cancer group,and there was significant difference between the two groups(P < 0.05).(3)This study used q-PCR on human hepatocellular carcinoma cell line SMMC-7721 after transfection and BEL-7404 MGMTmRNA expression levels were detected,found that the expression level of miR-221-3p in down group was significantly higher than that of NC group(P < 0.05);and the miR-221-3p up group was significantly lower than that in group NC showed significant difference(P < 0.05)(4)The application of Western were detected by Blot and MGMT expression level of different forms of miR-221-3p hepatocellular carcinoma cell line SMM(5)The results of dual luciferase system showed that there was a significant difference between 3'UTR-NC+miRNA group and 3' UTR+miRNA(P<0.05),indicating the binding of miR-221-3p to 3'UTR of target gene MGMT,and inhibiting its expression.significantly lower than that in the other two groups(P <0.05),indicating that the effect of siRNA was significant.Conclusions1.The expression of 1.miR-221-3p was elevated in the liver cancer tissues,and the expression of Hep G2,BEL-7404,Huh-7 and SMMC-7721 in hepatoma cells was significantly higher than that in normal liver cells..2.miR-221-3p is a tumor promoting factor of liver cancer.Down regulation can inhibit the proliferation,invasion and metastasis of hepatoma cells..3.MGMT is one of the target genes of miR-221-3p.By inhibiting the target gene MGMT,miR-221-3p plays a role in promoting the proliferation,invasion and metastasis of cancer cells in the liver cancer.