MiRNA-3679 Promotes Hepatocellular Carcinoma Proliferation By Inhibiting Downstream ZADH2-target Genes

Objective:1.Verify the relationship between miRNA-3679 and hepatocellular carcinoma;2.Verify the role of miRNA-3679 in the occurrence and development of hepatocellular carcinoma;3.Find the downstream target genes of miRNA-3679 and verify the relationship,and verify miRNA-3679 exerts its effect through downstream target genes;4.The influence of miRNA-3679 on tumor occurrence and development after inhibition of miRNA-3679,in order to find potential therapeutic targets and prognostic indicators of HCC.Materials and Methods:Collect 50 cases of hepatocellular carcinoma tissues and adjacent tissues from West China Hospital of Sichuan University,and use q PCR to detect the expression of miRNA-3679 in hepatocellular carcinoma tissues and adjacent tissues.Further detect the expression in common hepatocellular carcinoma cell lines,and to identify two cell lines with significantly elevated expression levels as the follow-up mechanism study cells.The survival data of liver cancer patients in the TCGA database were extracted through the ENCORI platform,and the survival curves of patients with different miR-3679 expression levels were obtained through the visual analysis of the ENCORI platform（URL:http://starbase.sysu.edu.cn/pan Mir Survival Exp.php）.Hep3B,SMMC-7721 cell lines were transfected with miRNA-3679 inhibitor(P3000^TMreagent),and the transfection efficiency was measured by q PCR;CCK-8experiment and Edu staining were used to detect the effect of miRNA-3679 inhibitor on cell proliferation after transfection;The cloning experiment detects the effect of transfection of miRNA-3679 inhibitor on cell cloning ability;TUNEL staining and flow cytometry detect the effect of transfection of miRNA-3679 inhibitor on cell apoptosis;flow cytometry detects the effect of transfected miRNA-The effect of3679 inhibitor on the cell cycle.Three databases of ENCORI,miRDB and Target Scan were used to predict the downstream target genes of miR-3679,and the influence of target genes was screened in the downstream of Hep3B cells after transfection of miRNA-3679inhibitor by q PCR.Use a mutation kit to perform site mutations on potential binding sites,transfect the constructed recombinant plasmid into Hep3B cells,and divide them into ZADH2 mutant group（ZADH2-MUT）and ZADH2 wild type group（ZADH2-WT）,and determine the luciferase Whether the activity can be affected by the miR-3679 inhibitor simultaneously transfected.Western-blot detects the expression of ZADH2 protein after transfection of miRNA-3679 inhibitor.CCK-8experiment and Edu staining were used to detect the effects of transfection of miRNA-3679 inhibitor and simultaneous transfection of miRNA-3679 and ZADH2inhibitor on cell proliferation.The clone formation experiment detects the effect of transfection of miRNA-3679 inhibitor and simultaneous transfection of miRNA-3679 and ZADH2 inhibitor on the ability of cell clone formation.TUNEL staining and flow cytometry were used to detect the effects of transfection of miRNA-3679 inhibitor and simultaneous transfection of miRNA-3679 and ZADH2inhibitor on cell apoptosis.The tumor formation experiment in nude mice detected the effects of inhibiting miRNA-3679 and inhibiting miRNA-3679 and ZADH2 on tumor mass and volume.q PCR was used to detect the expression levels of miRNA-3697 and downstream ZADH2-target genes in tumor tissues of each group.Western-blot detection of inhibition of miRNA-3679 and inhibition of miRNA-3679,ZADH2 on the downstream target protein expression in mouse tumor tissue.Western-blot detected the expression of tumor tissue-related apoptosis proteins（pro-Caspase-9,cleaved-Caspase-9,pro-Caspase-3,cleaved-Caspase-3,Bax,Bcl-2）in each group.Immunohistochemical staining was used to determine the expression of Caspase-3,a key protein in apoptosis.Ki-67 immunohistochemical staining was used to analyze the cell proliferation in the tumor tissue sections of each group.Results:The expression level of MiRNA-3679 in cancer tissues of patients with hepatocellular carcinoma was significantly higher than that in adjacent tissues（P<0.0001）,and in liver cancer cell lines（SNU-475,SNU-398,Hep3B,Smmc-7721,Hep G2）The expression level of miR-3679 was higher than that of normal human liver cell lines（P<0.05）,and the high expression in Hep3B and Smmc-7721 was particularly significant（P<0.01）;the high expression of miRNA-3679 in the HCC group was at different stages.The survival rate was lower than that of the low expression group,but the difference was not statistically significant（P<0.05）.The expression level of miRNA-3679 in Hep3B and Smmc-7721 in the miRNA-3679 inhibitor group transfected with miRNA-3679 inhibitor was significantly lower than that in the blank control group and the transfection negative control（Inhibitor NC）group（P<0.01）;CCK-8 Experimental detection of cell proliferation showed that the absorbance at the wavelength of 450nm in the miRNA-3679 inhibitor group was lower than that of the NC group and the Inhibitor NC group,the difference was statistically significant（P<0.05）;Edu staining detected cell proliferation,suggesting The number of positive cells in the miRNA-3679inhibitor group was lower than that in the NC group and the Inhibitor NC group（P<0.01）;the clone formation experiment detected the effect of miRNA-3679inhibition on the cloning ability of Hep3B and Smmc-7721 cells,and the cells in the miRNA-3679 inhibitor group The number of clones was significantly lower than that of the NC group and Inhibitor NC group（P<0.01）;TUNEL staining was used to detect cell apoptosis,and the TUNEL staining positive cell count（%）of the miRNA-3679 inhibitor group was significantly higher than that of the NC group and Inhibitor NC group（P<0.01）;Apoptosis was detected by flow cytometry,the number of apoptosis in miRNA-3679 inhibitor group was significantly higher than that in NC group and Inhibitor NC group（P<0.01）;Flow cytometry detects the cell cycle.After miRNA-3679 is inhibited,there is no difference in the cell counts of Hep3B and Smmc-7721 cells in the interphase（P>0.05）;but the cell count in the G0/G1 phase is significantly reduced（P<0.05）,And the cell count in the cell division phase（M phase）increased significantly,the difference was statistically significant（P<0.05）;Western-blot detected apoptosis-related proteins（Caspase-9,cleaved-Caspase-9,pro-Caspase-3,cleaved-Caspase-3,Bax,Bcl-2）expression levels,showed similar trends in Hep3B and Smmc cell lines,Bax,cleaved-Caspase-9,and cleaved-Caspase-3 in the miRNA-3679 inhibitor group The level was significantly higher than that of NC group and Inhibitor NC group（P<0.05）,but the Bcl-2 level was lower than that of NC group and Inhibitor NC group（P<0.05）.Bioinformatics analysis of the downstream target genes of miRNA-3679,screening 6 down-regulated genes in liver cancer:GLUD1,B3GAT1,SLC46A3,MAP2K3,ATF5,ZADH2;q PCR detection of downstream target genes of Hep3B cells transfected with miRNA-3679 inhibitor（GLUD1,B3GAT1,SLC46A3,MAP2K3,ATF5,ZADH2）expression levels,the results showed that the expression of downstream target genes SLC46A3,ATF5 was not different after miRNA-3679was inhibited（P>0.05）,but GLUD1,B3GAT1,The expression of MAP2K3 and ZADH2 increased significantly（P<0.05）,and the difference between B3GAT1,MAP2K3,and ZADH2 was particularly significant（P<0.01）,and the expression of ZADH2 was higher than that of B3GAT1,MAP2K3;Luciferase activity was used to detect the expression of ZADH-2 in Hep3B cell lines ZADH2-WT and ZADH2-MUT.After transfection with miR-3679 inhibitor,luciferase activity increased significantly（P<0.01）,while ZADH2 gene and miR-3679 After the binding site mutation of miR-3679 inhibitor,transfection of miR-3679 inhibitor no longer affects the luciferase activity（P>0.05）;Western-blot detects the expression of ZADH2 protein in the NC group and miR-3679 inhibitor group,the results show:The expression of ZADH2 protein in the miR-3679 inhibitor group was significantly higher than that in the NC group（P<0.01）;CCK-8 experiment detected the miR-3679 inhibitor group,inhibited miR-3679 and ZADH2（miR-3679inhibitor+si-ZADH2）group and blank The proliferation of the control group（NC group）,the results showed that the absorbance of the miR-3679 inhibitor group was significantly lower than that of the NC group（P<0.01）at the wavelength of 450nm when the cells were cultured for 72 hours,but miR-3679 inhibitor+si-The absorbance of the ZADH2 group was higher than that of the miR-3679 inhibitor group（P<0.05）;Edu analysis detected the proliferation of miR-3679 inhibitor group,miR-3679 inhibitor+si-ZADH2 group and NC group.The results showed that the number of positive cells in miRNA-3679 inhibitor group was lower than that of NC group and Inhibitor NC group（P<0.05）.The number of positive cells in the inhibitor+si-ZADH2 group was higher than that in the miRNA-3679 inhibitor group,and the difference was statistically significant（P<0.05）;the clone formation experiment detected miR-3679 inhibitor group,miR-3679 inhibitor+si-ZADH2group and NC group The effect on the clone formation ability of Hep3B and Smmc-7721 cells,the results showed that the clone count in the miR-3679 inhibitor group was significantly less than that in the NC group（P<0.01）,but the clone count in the miR-3679 inhibitor+si-ZADH2 group was significantly higher than miR-3679inhibitor group（P<0.01）;TUNEL staining analysis detects the effects of miR-3679inhibitor group,miR-3679 inhibitor+si-ZADH2 group and NC group on Hep3B and Smmc-7721 cell apoptosis.The results show:miR-The number of apoptosis in the3679 inhibitor group and miR-3679 inhibitor+si-ZADH2 group was greater than that in the NC group（P<0.01）,and the miR-3679 inhibitor+si-ZADH2 group was significantly lower than that in the miR-3679 inhibitor group（P<0.01）;Flow cytometry（FITC-Annexin V/PI）detected the apoptosis of the three groups.The results showed that the apoptosis rate of miR-3679 inhibitor group was higher than that of NC group（P<0.01）,miR-3679 inhibitor+si-ZADH2 group Significantly lower than the miR-3679 inhibitor group（P<0.01）.Inhibition of miRNA-3679 and miRNA-3679 and ZADH2 have an effect on tumor formation in nude mice.The volume and mass of tumors in mice were measured at 7,14,24,and 28 days.The results showed that miR-3679 was at 28 days.The tumor volume（mm³）of inhibitor group and miR-3679 inhibitor+si-ZADH2group was smaller than that of NC group,but the tumor volume of miR-3679inhibitor group and NC group（P<0.01）,miR-3679 inhibitor+si-ZADH2 group was obvious Greater than the miR-3679 inhibitor group（P<0.01）.In terms of tumor mass（mg）,the tumor mass of the miR-3679 inhibitor group and miR-3679 inhibitor+si-ZADH2 group was less than that of the NC group,but the miR-3679 inhibitor group and NC The group difference was statistically significant（P<0.01）.The tumor mass in the miR-3679 inhibitor+si-ZADH2 group was significantly greater than that in the miR-3679 inhibitor group（P<0.01）;q PCR detected the expression levels of miRNA-3679 and downstream ZADH2-target genes in the tumor tissues of each group.The expression of miRNA-3679 in the miR-3679 inhibitor group and miR-3679 inhibitor+si-ZADH2 group was significantly lower than that in the NC group（P<0.01）,the expression of ZADH2 in the miR-3679 inhibitor group was significantly higher than that in the NC group（P<0.001）,and the expression of ZADH2 in the miR-3679 inhibitor+si-ZADH2 group was lower than that in the miR-3679 inhibitor group（P<0.001）;Western-blot The effect of inhibiting miRNA-3679 and inhibiting miRNA-3679 and ZADH2 on the expression of downstream target proteins in mouse tumor tissues was detected.The results showed that the expression of ZADH2 protein in the miR-3679 inhibitor group was significantly increased（P<0.001）,while miRNA-3679.After the expression of ZADH2,the expression of ZADH2 protein was lower than that of the miR-3679inhibitor group（P<0.001）;Western-blot detected tumor tissue-related apoptosis proteins（pro-Caspase-9,cleaved-Caspase-9,pro-Caspase-3,cleaved-Caspase-3,Bax,Bcl-2）expression levels,the results showed:Bax,Caspase-3,Caspase-9 protein expression in the miR-3679 inhibitor group was significantly higher than that in the NC group（P<0.05）,Bax,Caspase-3,Caspase-9 protein expression in miR-3679inhibitor+si-ZADH2 group was significantly lower than miR-3679 inhibitor group（P<0.05）,but Bcl-2 protein expression was significantly lower in miR-3679 inhibitor group In the NC group（P<0.05）,the miR-3679 inhibitor+si-ZADH2 group was significantly higher than the miR-3679 inhibitor group（P<0.05）;Immunohisto-chemical staining was used to determine the expression of the key protein Caspase-3in apoptosis.The results showed that the expression of Caspase-3 protein in the miR-3679 inhibitor group was significantly higher than that in the NC group（P<0.05）;miR-3679 inhibitor+si-ZADH2 The expression of Caspase-3 protein in the miR-3679 inhibitor group was significantly lower than that in the miR-3679 inhibitor group（P<0.001）;Ki-67 immunohistochemical staining was used to analyze the cell proliferation in tumor tissue sections of each group,and the results indicated that tumor cells were inhibited after miRNA-3679 The number was significantly less than the NC group（P<0.01）;while the miR-3679 inhibitor+si-ZADH2 group had more tumor cells than the miR-3679 inhibitor group（P<0.01）;the miR-3679inhibitor+si-ZADH2 group had more tumor cells Lower than the NC group,but the difference was not statistically significant.Conclusion:1.MiRNA-3679 is highly expressed in cancer tissues of HCC patients,which is related to the occurrence and development of HCC;2.Inhibiting the expression of miRNA-3679 can inhibit the proliferation and survival of liver cancer cell lines;3.Inhibiting the expression of miRNA-3679,Can promote the apoptosis of cancer cells and affect the cell cycle;4.miRNA-3679 can promote the occurrence and progression of HCC by inhibiting the downstream target gene ZADH2;5.miRNA-3679 can be used as a potential HCC therapeutic target.