The Effect Of Klotho And IGF1R Signaling In The Proliferation Of The Normal Liver Cells And Hepatocellular Carcinoma Cells

Background:Hepatocellular carcinoma (HCC) is one of the most common cancer, and the third most frequent cause of cancer-related death worldwide. Deaths cause by HCC in China account for more than half of the world every year. Despite overall improvements in early diagnosis, cancer therapy and liver transplantation, there are still many problems such as high recurrence rate, and short 5-year survival rate. So further study of the pathogenesis of HCC, and find effective treatment approach is still the top priority.Klotho gene discovered by Kuro-o et al. in 1997 is closely related to human aging phenotypes. The gene mutated in a mouse strain exhibited short life span and complex phenotypes resembling human premature aging syndrome. In recent years, klotho has been identified as a tumor suppressor in several human malignances including HCC. It has been reported that exogenous klotho gene expression significantly inhibited cancer cells proliferation, decreased migration and induced apoptosis and autophagy, through the regulation of IGF1R phosphorylation and subsequent activation of downstream ERK and AKT signaling. However, there is no study shows the role of klotho in normal liver cells. In this paper, we knockdown klotho in cells by small interfering RNA (siRNA), and try to investigate the role of klotho in normal liver cells or HCC cells proliferation and the expression of HCC-related oncogene as well as its associated signal transduction pathways.Objective:1. Construct klotho specific small interfering RNA (siRNA) and the control siRNA.2. Transfect human normal liver cell line LO2 cells and human HCC cell line HepG2 cells with klotho specific siRNA. Detect the cell proliferation, oncogene expression and the expression and phosphorylation of IGF1R signaling pathways related proteins. Investigated the role of klotho in cell proliferation and oncogenesis, as well as associated signal transduction pathways.Methods:In this study, klotho specific siRNA, constructed to knockdown klotho in vitro, were used to transfect normal liver cell line LO2 cells and liver cancer cell line HepG2 cells. A cell counting (CCK-8) assay was used to measure the cell viability. Expression and phosphorylation of klotho and IGF1R signaling pathways related proteins were assessed using Western blot assays. The expression levels of oncogene transcripts were evaluated by quantitative reverse transcription polymerase chain reaction (qPCR).Results:The results show that the klotho gene expression in liver cancer cells is obviously lower than that in normal liver cells. However, the phosphorylation levels of IGF-1 receptor is the opposite. Klotho silencing decreased LO2 cells proliferation, whereas promoted the proliferation of HepG2 cells. The results of qPCR show that after interfering klotho expression, the expression of oncogenes in normal cells has a clear upward trend. The results of Western blot show that phosphorylation level of IGF1R signal pathway related molecules such as IGF1R, AKT, ERK is correlated reciprocally with klotho expression in liver cancer cells, however, that is positively correlated in normal liver cells.Conclusion:Klotho can promote the proliferation of normal liver cells, while decreased cancer cells proliferation. Defect of klotho gene may lead to more oncogene expression in the normal liver cells. Klotho may work through regulation of IGF1R signaling pathway and subsequent activation of downstream AKT and ERK signaling.