The Effect Of Downregulation Of MTDH On Proliferation And Apoptosis Of Breast Cancer MCF-7Cell Via ShRNA Under Hypoxic Conditions

Background and Significance: Breast cancer is the world’s highest incidence ofmalignant tumors of women. Numerous studies indicate that tumor microenvironmentcan cause genomic instability of tumor cells, the hypoxic tumor microenvironmentimportant feature. Under hypoxic conditions, part of the tumor cell genome changesand showing a stronger survival and invasion, including hypoxia inducible factor1-α(HIF-1α) plays a major regulatory role. Under hypoxic conditions, HIF-1α play animportant role of promoting tumor cell metabolism, angiogenesis and proliferation,invasion, metastasis and inducing drug resistance to tumor cells，and the expressionlevel was positively correlated with poor prognosis tumors.Metadherin(MTDH)was low or no expression in normal breast tissue and breasthyperplasia, but overexpression in breast cancer tissue, which was positively correlated with the malignant behavior of breast cancer. Studies have demonstratedMTDH overexpression can promote the expression of HIF-1α; in additional studiesalso showed that after down-regulate HIF-1α by using siRNA, expression of MTDHdecreases. The researchers speculate that MTDH and HIF-1α forms a positivefeedback loop of mutual regulation, suggesting MTDH gene under hypoxic conditionsto promote the malignant behavior of tumor cells may play an important role.Therefore, if we know MTDH gene’function in breast cancer cells under hypoxicenvironment, we can understand more about the impact of hypoxic microenvironmenton breast cancer cells.In this study, cobalt chloride simulated hypoxic microenvironment, down regulateMTDH gene expression by shRNA; changes of proliferation and apoptosis biologicalbehavior and related changes in the expression levels of molecules were observed inhuman breast cancer cell line MCF-7in order to initially understanding the effect ofregulation of MTDH gene under hypoxic environment on breast cancer cells.Objective: To investigate the effect of down-regulation of MTDH on proliferationand apoptosis of human breast cancer MCF-7cell in hypoxia environment.Method:1. Cobalt chloride was used to simulated hypoxic environment, RT-PCRand Western blot to detect changes in expression levels of HIF-1α and MTDH gene inthe hypoxic environment;2. MTDH-shRNA plasmid and control-shRNA weretransfected into MCF-7cells, transfection efficiency was observed after24h;Expression changes of MTDH gene in mRNA and protein levels were analysis byRT-PCR and Western blot to verify silencing effect;3.MTT experiment was used todetect the impact of proliferation after the downward MTDH gene expression ofMCF-7cell under hypoxia detection, Hoechst33258staining and flow cytometry wereused to detect apoptosis circumstances;4. Changes of HIF-1α mRNA and proteinlevels was observed after down-regulatetion of MTDH gene’s expression by RT-PCRand Western blot.Results:1. In the anoxic environments which was imitated by cobalt chloride, theexpression of HIF-1α at the mRNA levels did not change in MCF-7cells, theexpression of HIF-1α at the protein levels was increased with prolonged hypoxia, the expression levels of MTDH at the mRNA and protein were gradually increased;2.Transfection efficiency of70%, MTDH gene expression at the mRNA and proteinlevels were lower in MTDH-shRNA group;3. MCF-7cell’s proliferation capacitydiminished and the number of apoptotic cells increase after down regulate theexpression of MTDH gene;4. Down regulate MTDH gene expression had no effecton HIF-1α mRNA, but can reduce HIF-1α protein expression.Conclusion: Under hypoxic conditions, the level of HIF-1α protein expression ofMCF-7could be regulated by MTDH gene, while the capacity of cell proliferationwas restricted and apoptosis rate increase.