Study On The Effect Of Timeless On Proliferation And Apoptosis In Hepatocellular Carcinoma Cells HepG₂

Aims:Hepatocellular carcinoma is one of the highest mortality cancers, mostly found in China, the Sahara Desert of Africa and Southeast Asia, barely found in Oceania, Europe and America. There are over600,000new cases of hepatocellular carcinoma every year in the world, with mortality over300,000, half of the number occur in China. But so far, the mechanism of tumorigenesis and progression of hepatocellular carcinoma hasn’t been fully elucidated, and it greatly delays the development of prevention and treatment level for hepatocellular carcinoma. Circadian rhythm is a common phenomenon existing in many species, it regulates lots of living activities. The disfunction of the regulation of circadian rhythm is related to tumorigenesis and progression, but the studies on relationship between circadian genes and hepatocellular carcinoma hasn’t been studied widely. Timeless, an important circadian gene, directly couples the cell cycle and the circadian cycle, it can also influence expression level of other circadian genes (PER family and CRY family). Meanwhile, Timeless can promote repair of damaged DNA through ATR-Chkl and ATM-Chk2pathways. Because of Timeless’s biological functions in circadian rhythm, cell cycle and embryonic development, as well as diverse expression pattern in different cancerous tissues, we want to explore the biological function of Timeless on cell proliferation and apoptosis in hepatocellular carcinoma cells.Methods:We checked the expression level of TIMELESS in Hepatocellular carcinoma cell lines HepG2and SMMC-7721by western-blot. The expression level of TIMELESS was regulated by transfection of HepG2cells with Timeless siRNA or recombinant plasmid pcDNA4-Flag-Timeless. Then we analyzed the effect of down-regulated expression level of TIMELESS on proliferation and apoptosis in HepG2cells by MTT, clone formation, AnnexinV-FITC staining and flow cytometry.Rusults:(1) The endogenous TIMELESS expression was detected in hepatoma carcinoma cell lines HepG2and SMMC-7721;(2) We successfully down-regulated the expression level of TIMELESS in HepG2cells by transfection of siRNA ts2(200and400pmol) and ts3(50-300pmol) and up-regulated the expression level of TIMELESS in HepG2cells by transfection of recombinant plasmid pcDNA4-Flag-Timeless;(3) The results of MTT suggested that the proliferation of HepG2cells was significantly enhanced by down-regulated expression level of TIMELESS. Compared with negative control siRNA (NC,200pmol), the growth rates of HepG2cells transfected with ts2(200pmol)(P<0.01) in the3rd,4th and5th day were enhanced by15.82%,20.26%and11.17%respectively, its proliferation in vitro increased significantly. The growth rates of HepG2cells transfected with ts2(400pmol)(P<0.05) were enhanced by10.20%,6.32%and6.31%, so it has the same effect. Besides, the down-regulation of expression level of TIMELESS could significantly enhance the capacity of HepG2cells’ clone formation. Compared with negative control siRNA (NC,200pmol), the rates of clone formation of HepG2cells transfected with ts2(200pmol and400pmol) were enhanced by97%(P<0.05) and125%(P<0.01), respectively;(4) Analysis of AnnexinV-FITC staining and flow cytometry demonstrated that down-regulated expression level of TIMELESS could inhibit the apoptosis of HepG2cells. The apoptosis rates of HepG2cells were6.79%and5.0%respectively after transfection with ts2(200pmol and400pmol) for5days, and the apoptosis rate of HepG2cells transfected with the NC (200pmol) was12.01%.Conclusions:Down-regulated expression level of TIMELESS in HepG2cells could enhance proliferation and clone formation and inhibit apoptosis of the cells. These results could provide the experimental basis to identify the effect of circadian gene Timeless in treatment of hepatocellular carcinoma.