The Molecular Mechanism Of MiR-185 Regulating The Invasion And Migration Of Hepatoma Carcinoma Cell

Objectives In this study, analysing the different expression of miR-185 between hu man hepatocellular carcinoma cell line and normal liver cell line and the regulatin g effect of miR-185 on the proliferation, invasion and migration of hepatocellular ca rcinoma cell line Hep G2 and SMMC-7721, combined with the identification of the direct target gene of miR-185 will providing a theoretical basis for specific molecul ar mechanisms of miR-185 on the occurrence and development, invasion and migrati on of HCC, and to provide theoretical foundation for understanding the early diagnosi s and metastatic and therapeutic target of liver cancer.Methods 1 The over-expression plasmid of miR-185(pc DNA3/pri-miR-185) was constructed and the antisense oligonucleotide of miR-185(ASO-185) was synthesized, then the human hepatoma cell line Hep G2 and SMMC-7721 were transfected with pc DNA3/pri-miR-185 plasmid(miR-185 group), or pc DNA3 empty vector(pc DNA3 group), or miR-185 antisense oligonucleotides(ASO-185 group), or out-of-order oligonucleotides(ASO-NC) respectively. 2 Real-time PCR was used to detect the expression levels of miR-185 m RNA after over-expression or suppression of miR-185 in hepatocellular carcinoma cells. 3 The effect of miR-185 on the proliferation of Hep G2 and SMMC-7721 cells were detected with CCK-8 assay and colony formation assay. 4 The transwell assay was adopted to analysis the function of miR-185 on the invasion and migration of Hep G2 and SMMC-7721 cells. 5 The candidate target gene of miR-185 was predicted with bioinformation and combined with c DNA microarray analysis, then the direct target gene NR1D1 was verified by fluorescent reporter assay. 6 The m RNA and protein levels of target gene NR1D1 after over-expression or suppression of miR-185 in hepatocellular carcinoma cells were detected with real-time PCR and Western blot.Results 1 Compared with pcDNA3 group, miR-185 was significantly up-regulated in miR-185 group in Hep G2(P < 0.01). miR-185 was significantly down-regulated in miR-185 group in Hep G2 compared with ASO-NC group(P < 0.01). 2 CCK-8 assay showed that the cell growth activity of Hep G2 and SMMC-7721 after over-expresssion of miR-185 were significantly inhibited compared with pc DNA3 group(P < 0.01). The growth activity of Hep G2 and SMMC-7721 were promoted compared with ASO-NC group after suppression of miR-185(P < 0.01). 3 The colony formation assay showed that the cell colony formation activity of Hep G2 and SMMC-7721 were significantly inhibited compared with pc DNA3 group after over-expresssion of miR-185(P < 0.01). The colony formation activity of Hep G2 and SMMC-7721 were promoted compared with ASO-NC group after suppression of miR-185(P < 0.01). 4 The Transwell assay showed that the invasion and migration activity of Hep G2 and SMMC-7721 were significantly inhibited compared with pc DNA3 group after over-expresssion of miR-185(P < 0.01). The invasion and migration activity of Hep G2 and SMMC-7721 were promoted compared with ASONC group after suppression of miR-185(P < 0.01). 5 NR1D1 was identified as the direct target gene of miR-185 with c DNA microarray analysis, combined with bioinformation and fluorescent reporter assay. 6 The m RNA and protein levels of NR1D1 were both inhibited in Hep G2 and SMMC-7721 tranfected with pc DNA3/pri-miR-185, and the m RNA and protein levels of NR1D1 were both upgraded in Hep G2 and SMMC-7721 tranfected with miR-185 ASO.Conclusions 1 miR-185 inhibits the proliferation and colony formation activities of Hep G2 and SMMC-7721. 2 miR-185 inhibits the invasion and migration of Hep G2 and SMMC-7721. 3 miR-185 negatively regulates NR1D1 m RNA and protein levels, indicating that NR1D1 is the direct target gene of miR-185 in SMMC-7721 and Hep G2 and in HCC cells miR-185 might function as tumor suppressor genes by targeting NR1D1.