| Objective Hepatocelluar carcinoma(HCC)is one of the most frequently diagnosed cancers worldwide and among the leading causes of cancer-related death.Although deregulation of microRNAs has been frequently described in HCC,imperfection is known about the precise molecular mechanisms by which microRNAs modulate the process of tumorogenesis and behavior of cancer cells.So we aimed to investigate the molecular mechanism of miR-758-3p targeting MDM2,mTOR and PI3K/AKT pathway and then demonstrate the the roles of miR-758-3p in the development of HCC.Methods Oncomine and cBioportal were used to delineate the m TOR and MDM2` impact on HCC,and CCK-8 assays,scratch assays,transwell migration and invasion assays were measured within HCC cells with elevated or decreased MDM2 or mTOR expression to detect the roles mTOR and MDM2 play in the proliferation,migration and invasion of HCC cells.Dual luciferase reporter assays,western blotting and qRT-PCR were used to validate the the targeting roles of miR-758-3p to MDM2 and mTOR.Then the expression of mi R-758-3p in HCC tissues and cells were both measured using qRT-PCR,and the correlation between miR-758-3p expression and clinical characteristics of HCC patients were analyzed.Finally,CCK-8 assays,scratch assays,transwell migration and invasion assays,colony formation assay and western blotting assays were measured within HCC cells whose miR-758-3p expression were elevated or decreased to investigate the roles miR-758-3p play in the proliferation,migration and invasion of HCC cells and the molecular biological mechanism.Results Firstly,MDM2 expression in HCC versus normal liver tissues was elevated and the alteration of MDM2 has a statistically-significant reduction in disease-free survival of HCC patients(P=0.00673)while there was just little elevation in mTOR expression but the alteration of mTOR has a statistically-significant reduction in survival of HCC patients(P=0.0221).And proliferation and migration of HCC cell line hepG2 were significantly decreased after knockdown of MDM2 and mTOR,and so as the invasion of another HCC cell line HCC-LM3.Taken together,these data suggest MDM2 and mTOR are closely associated with HCC development.Secondly,MiR-758-3p expression were significantly down-regulated in the whole examined HCC cell lines compared with the normal liver cell line LO2,and in consistence was reduced in HCC tissues,relative to the corresponding non-tumor samples.Additionally,miR-758-3p expression was associated with AFP levels in the serum(P =0.002)and TNM staging(P =0.027).Thirdly,mi R-758-3p could target and combine with the 3'untranslated regions of both MDM2 and m TOR,then suppress the activities of both MDM2 and mTOR at both mRNA and protein levels.Subsequently,over-expression of miR-758-3p considerably inhibited HCC cells proliferation,migration,invasion and development,while inhibition of miR-758-3p had the opposite effects.Finally,the results of western blotting testing the important genes of PI3K/AKT pathway showed over-expression of miR-758-3p successfully up-regulated the expressions of p53,AKT and PRAS40 while downregulated the expressions of p-p70S6 kinase(Ser371)and p-4E-BP1,indicating that MDM2-P53 and mTOR signaling way may be involved in the suppression of miR-758-3p on HCC.Conclusion MiR-758-3p was down-expressed in HCC tissues compared with the adjacent normal liver tissues,indicating it could be involved in the development of HCC.Additionally,inhibiting MDM2 and mTOR could suppress HCC cells` proliferation,migration and invasion.In whole,miR-758-3p suppresses HCC cells` proliferation,migration and invasion via targeting mdm2 and mTOR,and MDM2-p53 and mTOR pathway may be associated with this process. |
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