Effcts Of HIF-2α—SCD1 Signal Pathway On The Biological Behaviours Of Hepatoma Cells Induced By Hipoxia

Background and ObjectiveHepatocellular carcinoma(HCC) is one of the most common malignant digestive tumors. With a high incidence and mortality in China, HCC is a serious threat to people’s health. Therefore, it is necessary to explore the mechanism of biological behavior of liver cancer cells, so as to seek effective treatment for HCC.Many tumors contain relatively hypoxic microenvironment. Hypoxia inducible factor- 2 alpha(HIF-2α) which is an important transcriptional factor of cellular oxygen signaling pathway, is closely related to the outcome and prognosis of malignant tumors. Recent studies have reported that HIF-2α is over expressed in renal cell carcinoma, liver cancer and other tumors. It can promote the proliferation, metastasis and invasion of tumor cells by starting its downstream target genes for accelerating the deterioration of the disease. Previously, we have found that HIF-2α could promote hepatic steatosis by enhancing the expression of adipose differentiation-related protein in LO2 cells induced by hypoxia. However, there were few studies on whether HIF-2α can affect the biological behaviors through regulating lipid metabolism in HCC.It has been revealed that the hypoxia environment in cancer cells can aggravate intracellular lipid synthesis. Stearoyl- Co A desaturase1(SCD1) is the rate limiting enzyme for catalytic synthesis of Monounsaturated fatty acid(MUFA). We have found significantly elevated expression of SCD1 in HCC. And it could negatively regulate autophagy through inactivation of the AMP-activated protein kinase(AMPK) signaling pathway to suppress cell apoptosis and promote cell growth for the formation and development of HCC. However, the mechanism of SCD1 elevated expression in liver cancer cells is still not clear, besides, there were few studies on whether hypoxic environment mediated its expression by HIF-2α.In this study, we firstly observed the HIF-2α or SCD1 protein expression of Hep G2 and SMMC-7721 induced by 1% O2. Then, we employed the HIF-2α interference plasmid and CAY10566, a small molecule inhibitor of SCD1, to explore whether HIF-2α affect the behaviors of liver cancer cells induced by hypoxia through regulating SCD1 protein expression. This may provide new evidences of pathogenesis and potential therapeutic target for HCC.Method:一 、 The regulatory relationship between HIF-2α and SCD1 in hepatoma cells induced by hypoxia:1. Cell culture: Hep G2 and SMMC-7721 cell was respectively cultured in DMEM and RPMI 1640 containing 10% festal bovine serum, 0.0625g/L penicillin and 0.1g/L streptomycin.2. The time spectrum of HIF-2α and SCD1 protein expression in hepatocarcinoma cells induced by 1% O2: Hep G2 and SMMC-7721 cells were exposed to 1% O2 at times of 0 h, 3 h, 6 h, 12 h and 24 h. The total protein of each time point was extracted, and the protein expression level of HIF-2α and SCD1 in both Hep G2 and SMMC-7721 cell lines were determined by western blot(WB)analysis.3. Screening of the effective sh RNA-HIF-2α plasmid: There were blank, negative plasmid, sh RNA-HIF-2α A-D plasmid. In order to screen the most effective plasmid, fluorescence microscopy was used to observe the transfection efficiency and WB analysis was performed to detect the expression level of HIF-2α protein after 72 hours of transfection according to the instruction.4. The regulatory relationship between HIF-2α and SCD1 in hepatoma cells induced by hypoxia: According to the results of the last part and preliminary study [1], we selected 12 h as the hypoxia induced time and adopted 10 μM CAY10566 which is the inhibitor of SCD1 to stimulate the cells for 24 h. The research was divided into six group: blank group(without any treatment), hypoxic group(1% O2 for 12 h), negative control group(transfected with negative sh HIF-2α plasmid+1% O2 for 12 h), interference group(transfected with sh HIF-2α plasmid+1% O2 for 12 h), CAY group(CAY10566 10 μM+1% O2 for 12 h) and interference +CAY group(transfected with sh HIF-2α plasmid+CAY10566 10 μM+1% O2 for 12 h)。The protein expressions of HIF-2α and SCD1 in both Hep G2 and SMMC-7721 cell lines were determined by Western blot analysis.二、The effect of HIF-2α—SCD1 pathway on the biological behaviors of Hep G2 and SMMC-7721 cells under hypoxia:The cell proliferation rates were tested by CCK8. The cell apoptosis were detected by flow cytometry with Annexin V/PE double-staining, and the invasiveness were detected with transwell assay.Results:一 、 The regulatory relationship between HIF-2α and SCD1 in hepatoma cells induced by hypoxia:1. The time spectrum of HIF-2α and SCD1 protein expression in hepatocarcinoma cells induced by 1% O2: After being exposed to hypoxia for 0 h, 3 h, 6 h, 12 h and 24 h, the levels of HIF-2α and SCD1 protein in both hepatocarcinoma cell lines were increased in a time dependent manner(P<0.05). While in SMMC-7721 cells, the level of HIF-2α protein reached a platform at 12 h. Therefore, the optimal hypoxic induction time of SMMC-7721 and Hep G2 cells is 12 h.2. Screening of HIF-2α effective interference plasmid : The transfection efficiency of every plasmid reached about 90% after 72 h of transfection. WB showed that sh RNA-HIF-2α C plasmid had the best inhibitory effect on HIF-2α protein expression as compared to the blank(P<0.05). Therefore, HIF-2α C plasmid was selected for the study.3. The regulatory relationship between HIF-2α and SCD1 in hepatoma cells induced by hypoxia: WB showed that the expression levels of SCD1 protein was significantly decreased after HIF-2α being interfered in Hep G2 and SMMC-7721 cells induced by hypoxia(P<0.05). However, there was no significant effect of SCD1 inhibition on HIF-2α protein expression in both hepatocellular carcinoma cells(P>0.05). When inhibited HIF- 2α and SCD1 expression at the same time, the reduction level of SCD1 protein in both hepatocellular carcinoma cell lines were more obvious than that of inhibiting SCD1 or HIF-2α alone(P<0.05). It suggested that HIF-2α can regulate SCD1 expression of hepatocellular carcinoma cells induced by hypoxia.二、The effect of HIF-2α—SCD1 pathway on the biological behaviors of Hep G2 and SMMC-7721 cells under hypoxia:1. CCK8 detection of the hepatocellular carcinoma cell proliferation: Compared with the blank group, the cell proliferation rates increased in SMMC-7721 and Hep G2 cells of the hypoxic group and negative control group(P<0.05). Furthermore, the proliferation rates of SMMC-7721 and Hep G2 cells which HIF-2α or SCD1 expression were restrained were lower than those in the hypoxic group and negative control group. And the proliferation rates of both hepatocellular carcinoma cell lines in the interference +CAY group decreased more significantly than those in hypoxic group, interference group and CAY group(P<0.05).There was no significant difference between hypoxic group and negative control group(P>0.05).2. The detection of apoptosis ratio of every group with flow cytometry: Compared with the blank group, the apoptosis ratios decreased in the hypoxic group and negative control group of both Hep G2 and SMMC-7721 cells(P<0.05). Moreover, the apoptosis ratios in both Hep G2 and SMMC-7721 cells which HIF-2α or SCD1 expression were inhibited were higher than those of the hypoxic group and negative control group. What’s more, the apoptosis ratios of both hepatocellular carcinoma cell lines in the interference +CAY group increased more significantly than those in the hypoxic group, interference group and CAY group(P<0.05). There was no significant difference between hypoxic group and negative control group(P>0.05).3. Transwell analysis of liver cancer cell invasion: The invasion index in the hypoxic group and negative control group were enhanced as compared with the blank group of both Hep G2 and SMMC-7721 cells. In contrast with the hypoxic group and negative group, the invasiveness were weakened after HIF-2α or SCD1 being inhibited in both Hep G2 and SMMC-7721 cells. While the invasiveness of both hepatocellular carcinoma cell lines weakened more significantly in the interference + CAY group than those in the hypoxic group, interference group and CAY group of both hepatoma cell lines(P<0.05).There was no significant difference between hypoxic group and negative control group(P>0.05).Conclusion:1. Hypoxia can up-regulate the expression level of HIF-2α and SCD1 protein of hepatocellular carcinoma cells.2. Hypoxia may regulate the energy metabolism of hepatocellular carcinoma cells though HIF-2α—SCD1 pathway, and then affect its biological behaviors.