The Regulatory Mechanism Of Stem Cell Factor In Hepatocellular Carcinoma Under Hypoxia

ObjectiveHepatocellular carcinoma is common in gastrointestinal cancer. Despite improvement of therapeutic methods including Surgery, radiotherapy and chemotherapy, the malignant tumor remains a poor prognosis. Hypoxia is a common microenvironment of Hepatocellular carcinoma. Hypoxia plays a central role in the development and behavior of hepatocellular carcinoma, but the adaptive mechanism of hepatocellular carcinoma in this environment remains to be defined.Previous data indicate that hypoxia induces the expression of SCF, an important cytokine for tumor growth and angiogenesis. We have found that hypoxia increased the expression of HIF and SCF mRNA in SMMC-7721 cancer cells, which contribute to upregulate the transcription of SCF. Whatever, a hypoxia-response element locat in the promoter region of SCF has been found. We speculate SCF maybe involved in the adaptive mechanism of hepatocellular carcinoma in hypoxia environment.The aim of this study is to uncover the pathophysiologial function and the regulatory mechanism of HIF-1/HIF-2 and SCF in Hepatocellular carcinoma. Chromatin inununoprecipitationassay(ChIP) and luciferase assay were performed to detect the binding of HIF-1/HIF-2 to SCF promoter in live cells. Furthermore, we confirm the relationsl.ip between HIF-1/HIF-2 and SCF in the tissue samples. Functionaly analysis the SCF promoted the growth of SMMC-7721 cells by autocrine methods and the migration and tumor formation of human umbilical venous endothelial cells by paracrine methods. We look forword to provide a new target for the treatment of HCC.Methods1. To explore the expression of HIF-1/HIF-2 and SCF in Hepatocellular carcinoma under hypoxia.1) We choose human hepatocellular carcinoma cell lines:HepG2, SMMC-7721, Bel 7402 as object. They were maintained in RPMI 1640 with 10% heat-inactivated fetal bovine serum. All cells were cultured at 37℃in a humidified incubator containing 5% CO2. Mixed gas (1%O2,5%CO2, 94%N2) was used for the hypoxia incubator;2) Knockdown of HIF-1α/HIF-2αby RNA interference;3) SCF, HIF-lα, HIF-2α, VEGF andβ-actin mRNA and Protein levels were evaluated by realtime PCR and Western blot; 4) The level of culture supernatants SCF protein were evaluated by ELISA.2. Investigate the regulatory mechanism of HIF-1/HIF-2 and SCF in Hepatocellular carcinoma under hypoxia.1) Chromatin inununoprecipitationassay (ChIP) was performed to detect the binding of HIF-1/HIF-2 to SCF promoter in SMMC-7721 cell;2) HIF-1/HIF-2 expression plasmids and firefly 1 uciferase reporters carring SCF promoters were co-transfected into SMMC-7721 cell and regulation of SCF gene was examined by luciferase assay.3. Uncover the pathophysiologial function of SCF in HCC.1) The expression of SCF, HIF-1α, HIF-2α, CD34 were examined by immunohidtochemistry in tumor tissues of 104 HCC patients. The results analyzed by relating them to the clinical stage and pathological grade;2) Analysis the SCF promoted the growth of SMMC-7721 cells by autocrine methods and the migration and tumor formation of human umbilical venous endothelial cells by paracrine methods.ResultsHypoxia up regulated the expression of SCF. Knockdown of Hypoxia-inducible factor-2α(HIF-2α) but not HIF-lαby RNA interference decreased the expression of SCF. Furthermore, HIF-2αcould directly bind to the hypoxia-response element located in the promoter region of SCF and upregulate the transcription of this promoter. Immunohistochemical analysis showed that HIF-2a was overexpressed in Hepatocellular carcinoma and correlated to the expression of SCF. Meanwhile, HIF-2αand SCF were associated with the Grade, TNM stage, venous invasion and the state of liver cirrhosis. The Spearman analysis showed that the level of SCF was significantly associated with HIF-2αexpression. Functionaly analysis showed that SCF promoted the growth of SMMC-7721 cells by autocrine methods and the migration and tumor formation of human umbilical venous endothelial cells by paracrine methods.ConclusionIn conclusion, SCF is an important factor for the growth and angiogenesis of Hepatocellular carcinoma; HIF-2 is a key transcription factor for SCF expression; HIF-2αand SCF might be a potential target for the treatment of Hepatocellular carcinoma.