| HHC(hepatocellular carcinoma)is one of the most common malignant tumor in the word, the deaths caused by liver cancer accounted for a third of the world cancer deaths, is the second major cause of cancer death in china. At present, the incidence of liver cancer appeared rising trend year by year, bring heavy social, medical and family burden. The main treatment of liver cancer is surgical resection, liver transplantation, interventional therapy, and the radiation and chemotherapy. Due to the molecular targeted drugs in recent years, the prognosis of patients with liver cancer has made a lot of improvement, but the effect is still poor. Because of the high degree of malignant, rapidly progress, late discovery, the tumor mortality rate is very high. Through investigating the mechanism of molecular regulatory in the process of the development of liver cancer, find the specificity sites of the treatment on liver cancer, and develop an liver cancer drugs, has the vital significance in the treatment of liver cancer research.c- FLIP(Fas-associated death domain- like interleukin 1 beta converting enzyme inhibitory protein) is a is an important member of the IAP, has reported in1997, it contains DED structure domain, widespread exists in many species,such as the virus, eukaryotes, mammals, closely relates with inflammation, tumor and autoimmune disease. In tumor, the c- FLIP involves in the apoptosis pathway, the NF-κB pathway, as well as autophagy and necrosis pathway There has evidence that the c-FLIP can effect many protein ubiquitin process. c-FLIP overexpress in liver cancer, can promote the proliferation of liver cancer cells, negative correlates with the prognosis of liver cancer. There has no related research about whether the c-FLIP can effect tumor glucose metabolism at present.Glucose as the main energy material of cells, play an important role in the process of tumor proliferation, it is different that the glucose metabolism of tumor from normal tissue. In tumor cells, glucose main by aerobic glycolysis, produce large amounts of lactic acid. To meet the demand of metabolism, tumor cell over expresses the glucose transporter to enhance the glucose uptake. Different tumor cells expresses different glucose transporter. The changes of glucose metabolism rely on the oncogenes and tumor suppressor genes working together. Glucose as energy material of tumor, We can inhibit tumor glucose metabolism, thereby inhibit the tumor proliferation,. through investigating the regulating mechanism of tumor glucose metabolism.c-FLIP as a molecule which can promote tumor proliferation, whether can effect the glucose metabolism in liver cancer. What is the influence mechanism?Objectives Clear whether the c- FLIPL can effect the glucose metabolism on liver cancer cell; Second, and investigate the mechanism of the c-FLIPL regulating the glucose metabolism in liver cancer cell; Study the significance of regulating mechanism in the development of liver cancer, and analyzer the correlation between the c-FLIPL clinical prognosis of liver cancer.Methods1. We interfered the c-FLIPL expression using the lentiviral vector.; and used the seahorse XF24 Analyzer to investigate the effect of c-FLIPL on glucose metabolism; We used glucose analogues 2-NBDG with fluorescent markers in the cell culture medium to analysis the glucose uptake in vitro, established a tumor-bearing mice mode, used the PET-CT to analysis the tumor glucose uptake in vivo; and investigated the effect of c-FLIPL on the proliferation of liver cancer using the tumor-bearing mice mode. 2. Used Western Blot and RT-PCT to detect glucose transporter expression, to analyze the effect of c-FLIPL on protein ubiquitination, we used the protein ubiquitin detection method; And used the immune coprecipitation and indirect immunofluorescence to detect the interactions of protein using. 3. We used the FACS to analyze the levels of liver cancer apoptosis under the condition of low glucose, and screened the liver cancer cell lines tolerate to low glucose, and analyzed the whether the cell line tolerate the low glucose through the c-FLIPL. 4. We detected the expression of c-FLIPL and relevant glucose transporter in liver cancer and adjacent tissues using the immunohistochemical technique, and analyzed the differences of expression between the tumor tissue and adjacent normal, statistic analyzed the correlation between the two protein expression, and detccted whether there was correlation between the c-FLIPL and the clinical staging of liver cancer.Results 1. We stably knocked down the c-FLIPL expression using the lentiviral vector, seahorse XF24 Analyzer found that c-FLIPL promote glycolysis; we further found that c-FLIPL promote glycolysis through promoting cell glucose uptake in vivo and vitro. we found c-FLIPL promote the liver cancer proliferation in tumor-bearing mice mode. 2. In liver cancer, c-FLIPL enhanced glucose uptake through interaction with SGLT1, thereby inhibited ubiquitination of SGLT1. 3. Different liver cancer cell line has different sensitivity to the low glucose, in hepatocellular carcinoma cell line Hep G2, MHC-97 h, Huh-7, SMMC- 7721, BEL- 7704 and Hep G2, the cell line MHC-97 h, Huh-7 were notsensitive to low glucose, and SMMC- 7721, BEL- 7704 were sensitive. In the sensitive cells line, the SGLT1 and c-FLIPL expression higher than the tolerance. And the further study indicated that SGLT1 play an important role in glucose sensitivity, c-FLIPL promote cell tolerance to low glucose through SGLT1. 4. Immunohistochemical detection found c-FLIPL and SGLT1 were overexpression in liver cancer, and SGLT1 expression correlated with the c-FLIPL, statistical analysis found that c-FLIPL and SGLT1 expression positive correlated with the clinical stage of liver cancer.Conclusions We found that c-FLIPL can promote hepatocellular carcinoma cell glucose uptake through inhibiting glucose transporter SGLT1 degradation, thereby promote the tolerance to the low glucose. In liver cancer, c-FLIPL and SGLT1 expression were higher than adjacent normal, and SGLT1 expression correlated with c-FLIPL, c-FLIPL and SGLT1 expression were positively related to the clinical stage of liver cancer. Through our research, we have indicated the mechanism of c-FLIPL regulation of glucose metabolism of liver cancer, and investigated the role of SGLT1 in the development of liver cancer. |
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