Molecular Mechanism For TIMELESS-mediated Glucose Metabolic Reprogramming In Hepatocellular Carcinoma Cells

? Background ? With the growth of population and aging in China,the morbidity and mortality of cancer are increasing year by year,and it has become an important cause of serious harm to the health of our country people.According to incomplete statistics,China's new cancer cases about 3 million 70 thousand in 2012,of which about 2 million 200 thousand patients died of cancer,is expected to 2030,Chinese new cancer patient will reach to 5million people,which will bring huge pressure to the national health and economy in China.At present,the incidence of liver cancer in China is about 25.7/10 million,becoming the third most serious malignant tumor after gastric cancer and lung cancer.China is the world's highest incidence of liver cancer and the largest number of deaths.Therefore,it is of far-reaching significance for us to investigate the regulatory mechanism of liver cancer,find the molecular markers and the new targets for biological targeted therapy of HCC(hepatocellular carcinoma).Circadian rhythms can ensure the coordination of the organism's life activities with the changes in the environment,and play an important role in maintaining the normal physiological activities of organisms.Until now,ten core circadian genes have been found:NPAS2,CLOCK,BMAL1,PER1,PER2,PER3,CRY1,CRY2,TIMELESS and CKI.Cell life activities of these core circadian genes directly involved in the regulation of apoptosis,including cell growth,metabolism,and immune cell transfer,and once the circadian gene disturbance in the body,it will lead to and promote the occurrence and progress of tumor.TIMELESS is an important member of core circadian rhythm gene.It is involved in many physiological activities such as genomic stability,DNA replication,DNA damage repair,cell differentiation and so on.The abnormality of TIMELESS including embryonic development,lung and kidney epithelial cell morphogenesis,depression and insomnia,breast cancer,cervical cancer,prostate cancer,nasopharyngeal cancer,leukemia and cancer and other diseases are closely related.Our preliminary results show that the expression of TIMELESS is up-regulated in HCC and promotes glucose uptake in HCC cells.At present,there are few studies on TIMELESS in HCC,and the regulation of TIMELESS on HCC may play an important role.Therefore,it is important to investigate the effect of TIMELESS on glucose metabolism in HCC cells.?Aims?1.To analyze the expression of TIMELESS in HCC patients and cells;To analyze the relationship between the expression of TIMELESS and the prognosis of HCC patients.2.To analyze the effect of TIMELESS on glucose metabolism in HCC cells.3.To elucidate the molecular mechanism of TIMELESS regulating glucose metabolism in HCC cells.?Methods? 1.The collected samples of HCC tissue and public datasets were used as the research object.QRT-PCR,Western Blot and immunohistochemical staining were used to detect the expression of TIMELESS in HCC.2.The effect of TIMELESS on glucose metabolism in HCC cells was analyzed by glucose uptake,lactic acid production,p H value of extracellular fluid and oxygen consumption.3.The molecular mechanism of TIMELESS on glucose metabolism in HCC cells was analyzed by q RT-PCR,Western,Blot,MTS and other methods to analyze?Results? 1.The expression of TIMELESS in HCC tissues was significantly higher thanthat in adjacent tissues,and the overall survival and recurrence free survival of HCC patients with high expression of TIMELESS were poor than those in patients with TIMELESS.2.TIMELESS promotes HCC cell glycolysis,and inhibitis oxidative phosphorylation.3.TIMELESS inhibits the expression of p53,and then promotes the aerobic glycolysis and the growth of HCC cells through the p53 dependent glycolysis pathway.?Conclusion?Through the detection of clinical tissue samples,bioinformatics analysis and in vitro cell experiments,we found that the expression of TIMELESS was elevated in HCC tissues,and the expression level of HCC was negatively correlated with the prognosis of HCC patients.TIMELESS through the inhibition of p53 to promote HCC cell glycolysis,and inhibit oxidative phosphorylation,promote cell growth and tumor progression.Therefore,TIMELESS may be a molecular marker of HCC prognosis and a new target for HCC biological targeted therapy.