Effects Of Hypoxia On DEC1Expression And Cell Proliferation And Apoptosis In Human Gastric Cancer Cells

Gastric cancer is a common form of malignant tumor all over the world, the development of molecular mechanism has always been the focus of scientific research. Abnormal transcription factors are closely related to the occurrence of tumor development.Furthermore, studying the specific expression mode of those transcription factors in tumors, may provide a new forecast index for tumor treatment and intervention targets.Differentiated embry-ochondrocyte expressed gene1(DECl), also known as SHARP-2or Stral3.DEC1is a kind of bHLH transcription factor which is isolated from human cartilage cells by Shen. It is widely expressed in most normal tissues, and regulating the body’s normal circadian rhythms, autoimmune and the stability of immune cells internal environment. Recent studies have found that DEC1are high expressions in many tumors, and is closely related to the development of tumors. Hypoxia inducible factor-la (HIF-la) can induce gene expression in the case of hypoxia, and it is closely related to tumor growth and metastasis, proliferation and apoptosis. Various extracellular stimuli can induce DEC1expression, and one of the most important research is the correlation between DEC1and tumor tissue hypoxia. Many studies have showed that DEC1can be induced in many cell lines,such as lung adenocarcinoma, renal cell carcinoma, and bladder cancer. All of these suggest that DEC1and HIF-1α common lead to the occurrence and development of the tumor. Zheng Y, et al have showed that there are high expression of DEC1in gastric cancer cell lines, but the expression of DEC1and its effect on cell proliferation and apoptosis under the condition of hypoxia in gastic cancer lines is unclear. This is the key problem of the research of this article.Objective1. To detect the expressions of DEC1in the conditions of normoxic and hypoxia in gastric cancer cell lines BGC823and HGC27.To study the correlation between DEC1and HIF-l1α， and further explore the effect of hypoxia on DEC1and its possible mechanism.2. To construct a lentiviral DEC1expression vector and then establish a gastric cancer cell line with stable expression of DEC1. This will provide a good cell model for further study of DEC1.3. To explore the influence of DEC1on cell proliferation and apoptosis caused by hypoxia in gastric cancer,and provide experimental evidence for whether DEC1can be used as a novel target for cancer therapy.Methods1. The human gastric cancer cell line BGC823and HGC27were cultured and passed.The experimental groups were cultured in the hypoxia incubator,and the control groups were exposed to the normal oxygen condition.2. The levels of mRNA and protein of HIF-1α and DEC1were determined by RT-PCR, Western blotting and immunofluorescence technique,and their correlations were analyzed.3. Construct a lentiviral DEC1expression vector (pGV-DEC1) and negative control vector (pGV).293T cells were transfected using Lipofectamine2000and packaged for the recombinant lentivirus particles. The lentivirus were used to infect the HGC27cells and establish a gastric cancer cell line with stable expression of DEC1. Western Blotting was used to confirm the validation of lentivirus.4. The infection cell lines were divided into overexpress group (pGV-DEC1) and light group (pGV). And the two groups were further devided into the normoxic groups (pGV-N, pGV-DEC1-N) and hypoxia groups(pGV-H, pGV-DEC1-H), respectively. The proliferation rates of cells were analyzed by CCK-8.The apoptosis rates of cells were analyzed by flow cytometry using Annexin V-PE/7-AAD staining.Results1. The results of Western Blotting and RT-PCR show that the expressions of both mRNA and protein of DEC1and HIF-1α were upregulated significantly compared with the control groups under the hypoxia condition(p<0.05),and their expressions have significant correlation(p<0.05).2. The results of Western Blotting show that the over-expression group(pGV-DEC1) appears a stripe with DEC1and GFP fusion protein,and the light group(pGV) only appears GFP stripe.3. The results of CCK-8show that the proliferation rate of hypoxia group(pGV-H) is higher than normoxic group(pGV-N) among light groups within24h.But the proliferation rate of hypoxia group(pGV-H) is lower than normoxic group(pGV-N) among light groups between24h and48h(p<0.05). The proliferation rate of over-expression group(pGV-DEC1-N) is higher than light group(pGV-N) among normoxic groups,and the increase is more significant with the extention of time(p<0.05). The proliferation rate of over-expression group(pGV-DEC1-H) is higher than light group(pGV-H) among hypoxia groups(p<0.05).4. Annexin V-PE/7-AAD staining show that the apoptosis rate of over-expression group(pGV-DEC1-N) is lower than light group(pGV-N) among normoxic groups(p<0.05). And the apoptosis rate of hypoxia group(pGV-H) is higher than normoxic group(pGV-N) among light groups(p<0.05).And the apoptosis rate of over-expression group(pGV-DEC1-H) is lower than light group(pGV-H) among hypoxia groups(p<0.05).5. Conclusions1. Hypoxia can induce the expressions of HIF-1α and DEC1, which have significant correlation.2. The human DEC1lentiviral expression plasmid was successfully constructed, HGC27cells were successfully infected and stable expression of DEC1was confirmed by Western blotting.3. DEC1can promote cell proliferation and against the apoptosis which is induced by serum starvation in gastric cancer.