HIF-2α Predominates In The Regulation Of Hypoxic Microenvironment Through C-Myc In Colon Cancer Cells

Global incidence of colorectal cancer was increased year by year. It ranks third in all malignant tumors, and the mortality rate ranks fourth. As there are lack of specific symptoms in the early stage of colorectal cancer, majority of them has already progressed to advanced stage at diagnosis. Surgery was the first-line treatment option for colorectal cancer, cooperated with chemotherapy, radiotherapy and targeted therapy in the comprehensive treatment. Searching for biological target for cancer monitoring and intervention become an important study field for the prevention and treatment of colorectal cancer.The development and progression of colorectal cancer were closely related to the mutation of oncogene and inactivation of tumor suppressor gene. C-Myc is a recognized proto-oncogene, which was overexpressed in many malignant tumors. It has a dual effect on tumor cells, either stimulating cell proliferation or promoting cell apoptosis. Evaluating the role of c-Myc in tumor and relative regulatory mechanisms would be valuable in the diagnostic and therapeutic work-ups of colorectal cancer. As the enlargment of colon cancer, it will develop with the necrosis and hypoxic condition in tumor area. Hypoxic microenvironment is involved in the regulation of proliferation, apoptosis, migration, invasion and angiogenesis of tumor cells. Hypoxic inducible factors (Hifs) were widely expressed in various tissues of mammals, and responsible for the hypoxic adaptation. They are key regulators of coordination changes at the transcription level, and of great significance for subject survival and tumor growth under hypoxia condition. In this study, we would assess HIF-la/HIF-2a and c-Myc under hypoxic stress to elucidate the expression changes of HIF-1α/HIF-2α and c-Myc. Then we interfere the expression of HIF-1α/HIF-2α by siRNA to determine the regulation of HIF-1α/HIF-2a on c-Myc. A proteasome inhibitor, MG132, was also use to clarify the role of ubiquitination in the degradation of c-Myc in hypoxic environment.Material and Methods:1. Extracted total protein from HCT116and SW480colorectal cancer cells at different time points of hypoxic treatment. The expression changes of HIF-la/HIF-2a, c-Myc and p-c-Myc were assessed by Western blot.2. Extracted total RNA from colorectal cancer cells HCT116and SW480at different time points of hypoxic treatment.the mRNA change of c-Myc were measured by qPCR. The promoter transcription activity was evaluated by dual luciferase reporter assays.3. The protein levels of c-Myc was detected after Interfered the expression of HIF-la/HIF-2a by siRNA with HCT116and SW480incubated in24h hypoxic condition.4. MG132was administrated into the culture medium of HCT116and SW480cells, which incubated under nomoxia or hypoxia for24h. the protein levels of c-Myc were detected.Results:1. Expression changes of HIF-la/HIF-2a and c-Myc in colon cancer cells under hypoxic microenvironment. In the time course of hypoxia, HIF-la had its peak level at4hours and then decreased gradually as the hypoxia prolonging. While HIF-2a was consistantly increasedwithin24hours hypoxia. c-Myc and p-c-Myc was downregulated gradually with the prolonging of hypoxia.2. Expression changes of c-Myc mRNA in colon cancer cells under hypoxic condition. In the time course of hypoxia, the mRNA level of c-Myc was decreased.The luciferase activities of c-Myc promoter in hypoxic cells were strikingly lower compare to those cells in normoxic condition after24hours.3. Regulation of c-Myc by hypoxic inducible factors. Knockdown of either HIF-la or HIF-2a in HCT116and SW480cells would upregulate the protein expression of c-Myc. The change was more dramatic when HIF-2a was interfered.4. The effect of MG132on the expression of c-Myc. Pretreatment with MG132wound increase the protein expression of c-Myc in a dose-dependent way.Conclusion:1. Under chronic hypoxia, HIF-2α is the predominant hypoxic inducible factor in colon cancer cells, accompanied by impaired transcription activity, mRNA and protein of c-Myc.2. The expression of c-Myc in colorectal cancer cells was affected by hypoxia inducible factors and ubiquitination, and the effect of HIF-2a was more obvious than HIF-1α.