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Abstract
This study included 196 patients presenting to Heidelberg UniversityHospital, Germany to be treated with either hepatic resection or hepatic transplantation or targeted therapy. Pathological biopsy taken from the patients was analyzed for mutation in the circadian clock genes and tissue microarray was done to identify the expression of the circadian proteins.CircadianClock genes mRNA expression analysis were carried out on 7 normal liver samples and 37 HCCs.Different HCC cell lines were used to identify to protumerogenic function and effect of circadian genes in HCC using MTT assay, flow cytometry, western blot and immunoprecipitation. Immunofluorescent and migration assays were used to test the effect of gene knockdown on migration. RT-PCR, nuclear cytoplasmic fractionation and EEF1A2 DNA transfection were used to examine the mechanism of the protumerogenic function of TIMELESS. Results: The analysis of the mutations in studied 68 liver tumors tissue (HCC) showed that: 35.3% of the samples showed mutation in Per1 gene, 8.8% of the samples showed mutation in Per2 gene, 23.5% of the samples showed mutation in Clock gene, 11.8% of the samples showed mutation in Timeless gene and none was in form of deletion mutation, 13.2% of the samples showed mutation in RORA gene, 20.6% of the samples showed mutation in CSNK1E gene.No significant associations were found with gender, etiology, vascular invasion, and tumor size (p>0.05).On analysis the of the function of the oncogenic circadian genes in HCC cell linesthe mammalian timeless (TIM) protein interacts with proteins of the endogenous clock and essentially contributes to the circadian rhythm. TIM was found to be overexpressed in a subset of human HCCs both at the mRNA and the protein level. Also it was found that siRNA-mediated knockdown of TIM reduced cell viability due to the induction of apoptosis and G2 arrest which was mediated via CHEK2 phosphorylation. Moreover, the cells with silenced Timeless showed a significantly reduced migratory capacity and reduced expression levels of various proteins