A Preliminary Study On The Up-regulation Of MiR-375 Expression Induced By Sorafenib And Inhibiting Angiogenesis In Hepatocellular Carcinoma

Objective:Micro RNA microarray and q RT-PCR were used to screen out differentially expressed miRNA expression profiles in HCC cells before and after treatment with sorafenib.To explore the regulatory function and potential molecular mechanism of miRNAs differentially expressed in angiogenesis of hepatocellular carcinoma.Methods:?.Sorafenib promotes miR-375 expression in hepatocellular carcinoma cells;1.Hep3 B and Hep G2 were treated with different concentrations of sorafenib for 48 h,and the IC50 of sorafenib to Hep3 B and Hep G2 was determined;2.RNA of hepatoma cells before and after treatment with sorafenib was extracted as the preparation of miRNA chip;Screened out the up-regulated miRNAs including miR-375;3.On the basis of miRNA chip,q RT-PCR was used to detect the expression of miR-375 in hepatocarcinoma cells before and after treatment with different concentrations of sorafenib;?.miR-375 inhibits angiogenesis in hepatocellular carcinoma and its potential molecular mechanisms1.q RT-PCR was used to detect the expression of miR-375 in hepatocellular carcinoma tissues and para-carcinoma tissues;Hep3B,Hep G2,Huh1,Huh7 and LO2;2.Using lentiviruses that overexpress miR-375 to infect hepatoma cells,q RT-PCR was used to detect the infection efficiency of miR-375;3.CCK-8 assay,plate clone assay,transwell assay,wound-healing assay,apoptosis assay in vitro were separately performed to investigate the influence of miR-375 on proliferation,clone formation,invasion,migration and apoptosis of HCC cells;4.The effect of miR-375 on angiogenesis was detected by the study of human umbilical vein endothelial cells(HUVECs)tube formation assays,Aortic Ring Sprouting Assay,and Chicken chorioallantoic membrane(CAM)assays;5.Bioinformatics software predicts the downstream target gene of miR-375;q RT-PCR,Western blotting and IHC(immunohistochemistry)were used to detect the expression of PDGFC in hepatocellular carcinoma tissues and para-carcinoma tissues;6.Using lentiviruses that overexpress miR-375 and miR-375 inhibitor to Promote and reduce the expression of miR-375;q RT-PCR,Western blotting and Elisa were used to detect the expression of PDGFC;7.Dual-luciferase reporter assay;8.The conditional medium of each group was used for Rescues assays and RNAi assays of human umbilical vein endothelial cells(HUVECs)tube formation assays,Aortic Ring Sprouting Assay,and Chicken chorioallantoic membrane(CAM)assay;9.Tumor xenograft model in vivo a were used to determine the effects of miR-375 on tumor growth,angiogenesis and survival time of nude mice;10.miR-375 inhibitors were transfected while adding sorafenib,q RT-PCR and Western blotting were used detect the expression of PDGFC;?.Preliminary study on miR-375 regulation of PDGFC downstream-8-signaling pathway in hepatocellular carcinoma cells1.The expression of P-AKT and P-ERK1 / 2 was detected by Western blotting after overexpression or down-regulation of miR-375;Result:?.Sorafenib promotes miR-375 expression in hepatocellular carcinoma cells;1.The IC50 of sorafenib to Hep3 B and Hep G2 were 2.77 u M,3.28 u M,respectively;2.The results of miRNA chip showed that sorafenib could promote miRNAs expression including miR-375;3.The results of q RT-PCR showed that the expression of miR-375 were increased in a concentration-dependent manner;?.miR-375 inhibits angiogenesis in hepatocellular carcinoma and its potential molecular mechanisms1.q RT-PCR results showed the expression of miR-375 in hepatocellular carcinoma tissues was significantly lower than that in para-carcinoma tissues;the expression of miR-375 in Hep3 B,Hep G2,Huh1 and Huh7 was significantly lower in LO2;2.We observed much enhanced Green Fluorescent Protein(e GFP)expression after After transfection;q RT-PCR results showed that miR-375 overexpression was successful;3.The results of CCK-8 assay and plate clone assay showed that miR-375 could significantly inhibit the proliferation and clonal formation of hepatoma cells.Transwell invasion assay showed that miR-375 significantly inhibited the invasion of hepatoma cells.Transwell migration assay and Wound-healing assay showed that miR-375 significantly inhibited the migration of hepatoma cells.Apoptotic assay showed that miR-375 significantly promoted the apoptosis of hepatocarcinoma cells.4.miR-375 significantly inhibited HUVE cells from forming tube,inhibited angiogenic sprouting of aortic Ring,inhibited the angiogenesis of chicken chorioallantoic membrane;5.Target genes of miR-375 were predicted by bioinformatic software targetscan 7.1 on line,which showed that PDGFC might be one of the potential downstream targets of miR-375;q RT-PCR,Western blotting and immunohistochemistry showed that the expression of PDGFC in hepatocellular carcinoma tissues was significantly higher than that in para-carcinoma tissues;6.q RT-PCR and Western blotting showed that the expression of PDGFC in Hep3 B,Hep G2,Huh1,and Huh7 was significantly higher than that in LO2;q RT-PCR,Western blotting and Elisa showed that miR-375 expression was negatively correlated with PDGFC expression;7.Dual-luciferase reporter assay showed that PDGFC was one of the downstream targets of miR-375;8.Rescue and RNAi assays of human umbilical vein endothelial cells(HUVECs)tube formation assays,Aortic Ring Sprouting Assay,and Chicken chorioallantoic membrane(CAM)assay showed that PDGFC promoted HUVE cells from forming tube,promoted angiogenic sprouting of aortic Ring;promoted the angiogenesis of chicken chorioallantoic membrane;9.miR-375 significantly inhibited tumor growth and reduced tumor angiogenesis,significantly prolonged the survival time of nude mice;The role of PDGFC is opposite to miR-375;10.q RT-PCR,Western blottng results showed that Sorafenib inhibits PDGFC expression by promoting miR-375 expression;?.Preliminary study on miR-375 regulation of PDGFC downstream signaling pathway in hepatocellular carcinoma cellsWestern blotting showed that overexpression of miR-375 significantly inhibited the expression of P-Akt and P-ERK1/2;anti-miR-375 significantly promoted the expression of P-Akt and P-ERK1/2;Conclusion:1.The expression of miR-375 was induced by sorafenib and the expression of miR-375 was increased in a concentration-dependent manner.2.miR-375 can inhibit the angiogenesis of hepatocellular carcinoma cells in vitro and in vivo by inhibiting the expression of PDGFC.3.Sorafenib inhibits PDGFC expression by inducing upregulation of miR-375 expression.4.miR-375 inhibits the activation of Akt and ERK1 / 2 signaling pathways.