MicroRNA-145Suppresses Hepatocellular Carcinoma By Targeting IRS1and Its Downstream Akt Signaling

BackgroundsHepatocellular carcinoma (HCC) is the fifth most frequent malignancy worldwide and the third most common cause for cancer related death. Although various genetic and epigenetic changes leading to HCC have been elucidated, the understanding on the molecular mechanisms underlying liver cancer has been growing. MicroRNAs are a class of highly conserved short noncoding RNAs, and recent research advances highlighted the role of dysregulated microRNAs (miRNAs) in HCC. MicroRNA-145(miR-145) has been highlighted because of its association with a variety of cancers.ObjectivesOur study focused on discussing the anti-tumor role and mechanism of miR-145in hepatocellular carcinoma (HCC), and the mechanism of miR-145by targeting IRS1and its downstream signaling pathways. Methods1) We test the expression level of miR-145and IRS1in48paired HCC tissues by quantitative PCR, and analyze their correlation statistically.2) Huh7and PLC HCC cell line were transfected with miR-145mimics for miR-145restoration, and study the role of miR-145in HCC cell line by CCK-8, flow cytomentry and colony formation.3) Bioinformatics softwares were applied to predict the possible targets of miR-145, and luciferase reporter experiments and western blot for gene validation.4) HCC cells were transfected with IRS1siRNA to interference the expression of IRS1, and CCK-8, flow cytomentry and colony formation were employed for testing the effects of IRS1on cell proliferation.5) Western blot was used to test the effect of miR-145restoration or IRS1silencing on IRS1and its downstream Akt/FOXO1pathway proteins.6) An additional rescue experiment was performed to validate the role of miR-145in IRS1/Akt/FOXO1pathway by IGF-1stimulation.Results1) Out of48paired HCC cancer tissues and adjacent non-tumor hepatic tissues,34(70.8%) cancer tissues presented with miR-145in down-regulated pattern compared with normal adjacent tissues. However, IRS1expression level was increased in66.7%(32/48) of HCC tissues, these results indicated that decreased miR-145expression is a frequent event in human HCC, and it was closely associated with IRS1over-expression.2) We restored miR-145expression by transfecting miR-145mimics to HCC cell lines, and the results show that miR-145increased the number of cells in G0/G1phase of cell cycle, the colonies formed by miR-145transfected HCC cells were markedly fewer and smaller, Cell growth assay showed that restored expression of miR-145could efficiently inhibit proliferation of Huh7and PLC cells.3) Bioinformatics software predicted IRSl was the possible target gene of miR-145, and we demonstrated it by luciferase reporter assay and western blot. IRS1silencing was performed to further confirmed that silencing of IRS1can inhibit the growth of HCC cell line and reduce the phosphorylation of Akt and FOXO1as well as the expression of cyclinD1.4) MiR-145restoration can reduce the phosphorylation of Akt and FOXO1as well as the expression of cyclin D1and it can be reversed by IGF-1stimulation.ConclusionsOur results suggested that miR-145could inhibit HCC through targeting IRS1and its downstream signaling, and the loss of miR-145regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.