Effects Of MiRNA-124-3p On Proliferation,Invasion,Migration And Lymphatic Metastasis Of Hepatocellular Carcinoma Cells Through Targeting ANXA7

Background:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in clinic.It has the characteristics of high invasion,early metastasis,high recurrence and mortality.At present,radical surgery is still the first choice for the treatment of hepatocellular carcinoma,but the 5-year survival rate of HCC is still very low.Tumor metastasis is an important cause of death in HCC patients.Lymphatic metastasis is an important way of hepatocellular carcinoma metastasis.The degree and extent of lymphatic metastasis is an important factor determining the prognosis of patients,thus studying on the mechanism of lymphatic metastasis of hepatocellular carcinoma is of great value for understanding the biological characteristics of hepatocellular carcinoma and guiding clinical treatmentAnnexin A7(ANXA7)is an important member of the annexin family in vertebrate cells,which plays an important role in cytoskeleton activity,membrane phospholipidization,membrane receptor regulation and mitosis.Recent studies have found that Annnexin A7 plays an important role in tumorigenesis,development,invasion and metastasis.In the past,our group screened genes and proteins that expressed differentially in Hca-F cells(lymph node metastasis rate>70%)and Hca-P cells(lymph node metastasis rate<30%)by gene chip,protein spectrum and suppression subtraction.ANXA7 gene(ANXA7)is one of the important differentially expressed genes affecting lymphatic metastasis potential of tumors.Up-regulation of ANXA7 gene expression enhanced the proliferation and migration ability of Hca-F cells and Hca-P cells,while down-regulation of ANXA7 gene expression reduced the proliferation and migration ability of Hca-F cells and Hca-P cells.It is suggested that ANXA7 may play a role in promoting lymphatic metastasis of hepatocellular carcinomaMicroRNA(miRNA)is an endogenous non-coding small RNA molecule with 18-24 nucleotide lengths.Relevant studies have shown that microRNAs can bind to target genes completely or incompletely and play a role in regulating gene expression Currently,studies have shown that microRNAs have significant effects on signal transduction pathways,cell apoptosis and metabolism,myogenesis,cancer,viral infection,and body development.miRNAs may interact with ANXA7 and play an important role in tumorigenesis,development,metastasis and invasion.However,the interaction between ANXA7 gene and microRNAs has not yet been involved.In this study,we analyzed the interaction of ANXA7 with microRNAs,and further observed the effect and mechanism of interaction of ANXA7 with microRNAs on proliferation,invasion,migration and lymphatic metastasis of hepatocellular carcinoma cells.It is of great significance to elucidate the biological characteristics of hepatocellular carcinoma and the molecular biological mechanism of lymphatic metastasisObjective:1.ANXA7-interacting microRNAs were predicted and screened by bioinformatics methods,and the targeting relationship was validated2.To investigate the effects of miR-124-3p on proliferation,apoptosis,invasion and migration of hepatocellular carcinoma cells in vitro and its mechanism3.To investigate the effect of miR-124-3p on the expression of transplanted tumor,lymph node metastasis and tumor-related molecules in Hca-F cells in vivo through targeting ANXA7.Methods:1.ANXA7-interacting miRNAs were predicted and screened by bioinformatics.2.The expression of miRNA-124-3p and ANXA7 in high lymphatic metastasis potential cells(Hca-F),low lymphatic metastasis potential cells(Hca-P)and normal hepatocytes of mice(N cell)were detected by qRT-PCR,and the expression of ANXA7 protein in Hca-F,Hca-P and N cells were detected by Western Blot.3.ANXA7 3'-UTR wild-type(WT)plasmid and mutant(MUT)plasmid were harvested and sequenced in human embryonic kidney cells(293T).The targeting relationship between miR-124-3p and ANXA7 was verified by double luciferase reporter assay.4.Hca-F and Hca-P were transfected with miRNA-independent sequence,miR-124-3p mimics and miR-124-3p inhibitor by Lipofectamine 2000,respectively,to establish two cell lines of NC group,miR-124 mimics group and miR-124 inhibitor group.qRT-PCR and Western Blot were used to detect the expression of ANXA7 and ANXA7 in each group.5.CCK-8 cell proliferation test was used to detect the cell proliferation ability,cell apoptosis was detected by flow cytometry;and cell migration and invasion ability was detected by Transwell test.The expression levels of Bax,Bcl-2,MMP-9 and CXCL12 were detected by Western blot of each group in two cell lines.6.Hca-F and Hca-P were transfected with shRNA sequences of ANXA7-independent sequence and ANXA7 gene interference target by Lipofectamine 2000 respectively.The control group(Hca-F cells/Hca-P cells),N-Control group(transfection-independent sequence),shRNA-Anxa7 group(transfection of shRNA sequence of ANXA7 gene interference target)were established.The expression of ANXA7,Bax,Bcl-2,MMP-9 and CXCL12 in each group was detected by qRT-PCR and Western blot.7.Hca-F were taken to establish lymphatic metastasis models in 615 mice,including NC group,miR-124 mimics group and miR-124 inhibitor group.The growth and lymph node metastasis of transplanted tumors were observed.The expression levels of ANXA7,MMP-9 and CXCL12 in transplanted tumors were detected by Western blot.The expressions of ANXA7,MMP-9 and CXCL12 in lymph node metastasis foci were observed by immunohistochemistry.Results:??Bioinformatics Prediction and Targeted Regulation Validation of ANXA7-interacting miRNAs1.ANXA7 was used as target gene,five miRNAs were screened by bioinformatics may have potential regulatory effects on ANXA7 gene,which were mmu-mir-19-3p,mmu-mir-124-3p,mmu-mir-135-5p,mmu-mir-190-5p and mmu-mir-323-3p.miR-124-3p was selected for validation experiment.2.The expression levels of ANXA7 mRNA and ANXA7 protein in Hca-F and Hca-P were significantly higher than those in N cell,and in Hca-F were significantly higher than those in Hca-P(P<0.05).The expression levels of miR-124-3p in Hca-F and Hca-P were significantly lower than those in N,and in Hca-F were significantly lower than those in Hca-P(P<0.05).3.Luciferase reports showed that the relative luminescence values of co-transfected plasmids of miR-124 mimics and WT-ANXA7 3'-UTR were significantly lower than those of co-transfected NC mimics and WT-ANXA7 3'-UTR plasmids(P<0.05),while the relative luminescence values of co-transfected plasmids of miR-124 mimics and MUT-ANXA7 3'-UTR were unconventional compared with those of NC mimics and MUT-ANXA7'-UTR plasmids(P>0.05).4.The levels of ANXA7 mRNA and protein in miR-124 mimics group were significantly lower than those in NC group(P<0.05),and the levels of ANXA7 mRNA and protein in miR-124 inhibitor group were significantly higher than those in miR-124 mimics group and NC group(P<0.05).??Effect of miR-124-3p targeted regulation of ANXA7 on the biological behavior of Hca-F/P in Vitro.1.CCK-8 experiment showed that the proliferation ability of miR-124 mimics group in Hca-F and Hca-P was lower than that of NC group and miR-124 inhibitor group,and that of miR-124 inhibitor group was higher than that of NC group(P<0.05).2.Cell flow cytometry showed that the apoptotic rate of the miR-124 mimics group was significantly higher than that of the NC group and the miR-124 inhibitor group in Hca-F and Hca-P,and the apoptotic rate of the NC group was significantly higher than that of the miR-124 inhibitor group(P<0.05).3.Western blot showed that the expression level of Bax protein in miR-124 mimics was significantly higher than that in NC group and miR-124 inhibitor group,and the expression level of Bcl-2 protein was significantly lower than that in NC group and miR-124 inhibitor group in Hca-F and Hca-P(P<0.05);the expression level of Bax protein in NC group was significantly higher than that in miR-124 inhibitor group,and the expression level of Bcl-2 protein was significantly lower than that in miR-124 inhibitor group(P<0.05).4.Transwell migration and invasion experiments showed that,miR-124 mimics could significantly reduce the number of penetration,and miR-124 inhibitor could significantly increase the number of penetration in Hca-F and Hca-P(P<0.05).5.Western blot showed that the expression levels of MMP-9 and CXCL12 in miR-124 mimics group were significantly lower than those in NC group and miR-124 inhibitor group(P<0.05),while the expression levels of MMP-9 and CXCL12 in NC group were significantly lower than those in miR-124 inhibitor group(P<0.05).6.ANXA7 was down-regulated in Hca-F and Hca-P,qPCR and Western blot showed that there were no significant changes in Bax,Bcl-2,MMP-9,CXCL12 gene and protein levels in Hca-F/Hca-P after transfection of unrelated sequence(P>0.05),while Bcl-2,MMP-9,CXCL12 gene and protein levels in Hca-F/Hca-P decreased significantly after transfection of ANXA7(P<0.05),there was no significant change of Bax gene and protein levels in Hca-F/Hca-P after transfection of ANXA7(P>0.05).??Effect of miR-124-3p targeted regulation of ANXA7 on the expression of tumor-related molecules and Lymph Node Metastasis in Hca-F Cell.1.The transplanted tumors were visible on the 6th to 12th day after inoculation.The volume of transplanted tumors increased with time.The tumors in miR-124 mimics group were smaller than those in the NC group.The tumors in the miR-124 inhibitor group were larger than those in the NC group(P<0.05).On the 28th day,mice were executed.The volume and average weight of transplanted tumors in the miR-124 mimics group were smaller than those in the NC group,and the transplanted tumors in the miR-124 inhibitor group were larger than those in NC group(P<0.05)2.On the 28th day of inoculation,15 mice all did not die.Tumor formation rate was 100%in NC group and miR-124 inhibitor group,and 80%in miR-124 mimics group The lymph node metastasis rate was lower in miR-124 mimics group than that in NC group,higher in miR-124 inhibitor group than that in NC group;the average weight of lymph nodes in miR-124 mimics group was smaller than that in NC group,and the average weight of lymph nodes in miR-124 inhibitor group was larger than that in NC group(P<0.05)3.Western Blot showed that the expression levels of ANXA7,MMP-9 and CXCL12 protein in transplanted tumors of miR-124 mimics group were significantly lower than those of NC group,while ANXA7,MMP-9 and CXCL12 protein in transplanted tumors of miR-124 inhibitor group were significantly higher than those of NC group(P<0.05)4.Immunohistochemical staining showed that IOD values of ANXA7,MMP-9 and CXCL12 protein in lymph node metastases in miR-124 group were significantly lower than those in NC group,while IOD values of ANXA7,MMP-9 and CXCL12 protein in lymph node metastases in miR-124 inhibitor group were significantly higher than those in NC group(P<0.05)Conclusions:In vivo and in vitro experiments have consistently confirmed that miRNA-124-3p can inhibit the proliferation,migration,invasion and lymphatic metastasis of hepatocellular carcinoma,and promote the apoptosis of hepatocellular carcinoma cells Inhibiting the expression of miRNA-124-3p can promote the proliferation,migration,invasion and lymphatic metastasis of hepatocellular carcinoma,and inhibit the apoptosis of hepatocellular carcinoma cellsmiRNA-124-3p can target and regulate ANXA7,reduce the expression of ANXA7 and Annexin A7 protein,and then reduce the expression levels of Bcl-2,MMP-9 and CXCL12,which promote the apoptosis of Hca-F/Hca-P,and inhibit the proliferation,migration,invasion and lymphatic metastasis of Hca-F.