The Study On Function And Molecular Mechanisms Of MiR-192 And MiR-99a In Hepatocellular Carcinoma Invasion And Metastasis

Primary liver caner,especially hepatocellular carcinoma(HCC),is one of the lethal cancers worldwide.The incidence of HCC is higher in Asia and Africa,andit is the second highest tumor mortality in China.Metastasis is a multiple and complicated process,and an important causion of cancer related death.In this study,three HCC cell lines(MHCC-97 L,MHCC-97 H and HCC-LM3)with gradually increased metastatic potentials were used to identify differentially expressed micro RNAs(mi RNAs)associated with HCC metastasis.Microarray data showed that 27 mi RNAs were differentially expressed with 19 mi RNAs gradually increased and with 8 mi RNAs gradually decreased in these three HCC cell lines.Firstly,we analyzed the expression levels of eight gradually decreased mi RNAs in TCGA liver cancer database.Six mi RNAs,mi R-489,mi R-30e-3p,mi R-194-3p,mi R-200a-3p,mi R-192 and mi R-574-3pwere decreased in HCC.After preliminary screening,we focused on mi R-192 in this study.Further analysis of TCGA data showed that mi R-192 was negatively correlated with the vascular cell invasion of HCC.Transwell migration and invasion assaysdemonstrated that mi R-192 could significantly inhibit HCC cell migration and invasion.Orthotopic transplantion assays showedthat mi R-192 also inhibited metastasis in vivo.Molecular mechanistic investigation showed that SLC39A6(solute carrier family 39,member 6)was a direct target of mi R-192.Knocking down SLC39A6 expression notably decreased HCC cell migration and invasion,whereas ectopic expression of SLC39A6 was able to enhance HCC cell migration and invasion.Moreover,restoration of SLC39A6 in mi R-192 overexpressing cells could partially antagonize mi R-192 migratory and invasive inhibition.In addition,mi R-192 could upregulate Snail expression and downregulate E-cadherin expression.GSK-3 inhibitors treatment onmi R-192 overexpression cells rescued cell migration and invasion.We found that mi R-192 could reduce thephosphorylation level of GSK-3?-Ser9,and SLC39A6 re-introduction partially recovered GSK-3?-Ser9 phosphorylation.Importantly,two independent HCC corhorts demonstrated that the expression level of mi R-192 was positively correlated with the overall survival timeof HCC patients.Next,we further analyzed mi R-99 a which owned higher basal expression and did not reported to regulate liver cancer metastasis.In vitro and in vivo experiments revealed that mi R-99 a significantly promoted HCC cell invasion and metastasis.After integration of Target Scan prediction,microarray data,transwell cell migration and dual-luciferase reporterassays,we identified CTDSPL(carboxy-terminal domainsmall phosphatase-like)asa mi R-99 a direct target in HCC.CTDSPL inhibited HCC cell migration and re-expression of it partially reversed mi R-99a-induced migratoty promotion.CTDSPL is a phosphatase which decrease the phosphorylation levels of Smad1-Ser463/465 and Smad1-Ser206.Interestingly,mi R-99 a overexpression increased Smad1-Ser463/465 and Smad1-Ser206 phosphorylation.Furthermore,Smad1 downstream factors BMP2 and ID3 expression were also upregulated due to mi R-99 a overexpression.Taken together,in the present study,we identified that mi R-192 was a potential metastasis suppressor and mi R-99 a was a candidate metastasis promoter of HCC.Our findings provided new clues for clarifying molecular mechanisms of HCC metastasis.