Correlation Of HIF-1α And MiRNA-200b Expression In Human Colorectal Cancer Tissues And Hypoxia-induced Colon Cancer Cell Lines

The common high-risk gastrointestinal cancer in our country is colorectal cancer(CRC),which decides the main traits of it,such as a result of illness hidden,and the early symptoms are not obvious.The five year survival rates are more than 90% for localized CRC(stage I)and who had reached middle or late period are only about 65% for CRC.After surgical resection or radio-chemotherapy,the prognosis is not sure.It would be the main reason to cause death in patients with colorectal cancer,which the disease recurrence and tumor metastasis.Therefore,it is particularly important to explore the early diagnosis and treatment of colorectal cancer high risk individuals,recent research suggests that: Hypoxic microenvironment frequently exists in many solid tumours and it may be one of the initiating factors of malignant tumor progression.What’s more,it’s a very important factor leading to tumor aggressiveness,metastasis promotion.Howeve,the EMT(epithelial mesenchymal transition)also plays a key role in the invasion and metastasis of tumor.In this process,the cancer cells in the central plains are transformed from quiescent epithelial cells into mesenchymal cells that have the ability to invade.Recent studies have indicated that EMT occurrence is closely related to the microenvironment of the tumor.Hypoxia is one of the most important microenvironment in colorectal cancer,and HIF-1α is the most critical hypoxia-responsive transcription factor.Related studies show that HIF-1α will be expressed abnormally at the early stage of tumors,and the abnormal expression of HIF-1 is related to the postoperative survival rate of the patients.So it can be used as an effective prognostic marker,and also play a key role in the occurrence and development of tumor.The mi RNAs are also involved in the process of EMT,and plays a key role in EMT.Especially micro RNA-200 family,they down-regulated expressed in invasive tumors,they are reacted as inhibitors of EMT,tumor cell adhesion,migration,invasion and metastasis.So the microRNA-200 family members are the typical tumor suppressor gene.Recent reports that miR-200 b can target vascular endothelial growth factor(EGFV),vascular endothelial growth factor receptors(Flt 1 and DRK)to inhibit their expression,resulted as negative regulation of tumor angiogenesis,and have effect on invasion and metastasis of tumor.What’s more,miR-200 b expression is significantly down-regulated under hypoxia,which induces angiogenesis.We have confirmed that the expression of mi RNA-200 in cancer tissues was significantly lower than that in normal tissues.However HIF-1α expression in cancer tissues was significantly higher than that in normal tissues.Several potential binding sites of HIF-1α are found in the mi R-200 b promoter using bio-informatics techniques.The target of mi RNA-200 family members are recognized widely,but the mechanism of how miR-200 b is regulated is not completely clear.Therefore,we examined HIF-1α and mi R-200 b expression in colorectal cancer and their adjacent tissues,and then to detect HIF-1α and mi R-200 b expression in colorectal cancer cell lines under different hypoxia conditions and then to explore the relationship between HIF-1α and mi R-200 b in colon cancer cells.This data will provide evidence of relationship between HIF-1α and mi R-200 b in solid tumors.MethodsThe real-time quantitative PCR was used to detect the expression of HIF-1α and mi RNA-200 b in colorectal cancer tissues and adjacent tissues from 50 patients.The hypoxia model of cultured cells was set up by the usage of cobalt chloride(Co Cl2)in the culture medium.Quantitative real-time PCR and Western blot were used to detect HIF-1α protein and mi RNA-200 b levels in CoCl2-induced Caco-2 cells.ResultHIF-1α m RNA level in colorectal cancer samples was significantly higher than peri-cancerous mucosa(P<0.01),whereas,miRNA-200 b level in colorectal cancer samples was significantly lower than peri-cancerous mucosa(P<0.01).HIF-1α mRNA level in colorectal cancer samples was inversely correlated with miRNA-200 b level(r=-0.324,P<0.05).HIF-1α protein level in CoCl2-induced Caco-2 cells had increased after 2 h of exposure to CoCl2,miRNA-200 b levels had decreased during the first 2 hours hypoxia.Within 4-12 h further hypoxia HIF-1α protein levels had decreased slowly,whereas mi RNA-200 b levels had increased,finally,HIF-1α rapidly increased to second peak after 16 hours hypoxia.On the contrary,mi RNA-200 b level decreased rapidly,and reached lowest level at 16 hour due to hypoxia.HIF-1α protein level in CoCl2-induced Caco-2 cells was inversely correlated with mi RNA-200 b level(r=-0.7422,P<0.05).ConclusionHIF-1α and miRNA-200 b were inversely expressed in colon cancer tissues and CoCl2-induced Caco-2 cells,the data indicated that HIF-1α might regulate mi RNA-200 b expression in direct or indirect mechanism.The exact mechanism needs to be further explored.