The Study Of Hypoxia In VM Formation And Its Mechanism Of Colorectal Cancer

Purpose The paper mainly researched the role of hypoxia in VM formation of colorectal cancer and relevant mechanisms. Firstly, we detected the expression of VM in the colorectal cancer and colorectal adenomas tissue in vivo and analyzed correlation of hypoxia inducible factor-lα(HIF-lα) with pathological data and its effect on the prognosis; Then by detecting the correlation among HIF-lα protein with EMT and VM-related proteins, we explored the role of HIF-lα in VM formation of colorectal cancer; Finally by mimcrying hypoxic microenvironment in vitro, we investigated the effect of HIF-lα on VM formation of colorectal cancer and its possible molecular mechanisms.Methods(1)For the collected 50 cases of colorectal cancer tissues and 50 cases of colorectal adenoma conducting HIF-lα, VEGF, FLK-1, VE-cad, MMP-2, E-cadherin, Vimentin and Snail Immunohistochemistry staining and CD31/PAS double staining, we detected the expression of these indicators.(2)Analysis the relationship of HIF-lα、MVD with clinical prognosis in colorectal cancer, and the correlation of HIF-lα expression with E-cadherin, Vimentin, VEGF, VE-cad, FLK-1, MMP-2, Snail.(3)Construction of hypoxic model in vitro, we detected the influence of hypoxia on HCT116 and HT-29 cells invasion, migration and tube change compared with normoxic conditions.(4)By WB and RT-PCR assays, we detected the expression of EMT marker(E-cadherin, Vimentin) protein and mRNA levels by Hypoxia-induced in HCT116 and HT-29 cells compared with normoxic conditions.(5)By WB assay, we detected the expression of VE-cad, MMP-2 and Snail by Hypoxia-induced in HCT116 and HT-29 cells compared with normoxic conditions.Results(1)HIF-1α expression in colorectal cancer was significantly higher than the adenomas. By CD31/PAS double staining, the results showed that VM was present in colorectal cancer with serious hypoxia, and adenoma had no VM.(2)Association between HIF-lα expression and pathological data Expression of HIF-lα at the age of ≦50 years or>50 years, male patients and female patients, tumor ≦5cm or>5cm patients, TNMⅠ/Ⅱor Ⅲ/Ⅳstage patients had no difference, and had no statistical difference significance. Expression of HIF-lα with poorly or moderate differentiated tumor was significantly more than well-differentiated tumor, and the difference was meaningful(P=0.04). Expression of HIF-lα with metastatic tissue was higher than no metastasis tissue, and the difference was meaningful(P=0.009). Survival analysis showed that the survival time of HIF-lα-positive patients was shorter than the survival time of HIF-lα-negative patients.(3)MVD in colorectal cancer was significantly higher than colorectal adenomas, and the difference was statistically significant. Then analyzed MVD≤20 and MVD> 20 patients survival time in colorectal cancer, we found MVD≤20 patients survival time was longer than MVD>20, MVD was negatively correlated with survival time.(4)Correlation among HIF-lα expression with VEGF, VE-cad, FLK-1 and MMP-2 in colorectal cancer. Expression of HIF-lα and VEGF was positively correlated(r=0.346, P=0.014); the expression of HIF-lα and FLK-1 was positively correlated(r =0.338, P=0.016); as well as expression of HIF-lα and VE-cad(r=0.406, P=0.003); the expression of HIF-lα and MMP-2 was also positively correlated(r =0.529, P=0.000).(5)The correlation of HIF-lα expression HIF-lα with E-cadherin、Vimentin、Snail. Expression of HIF-lα was positively related to Snail(r=0.371, P=0.004). HIF-lα-positive expression were 32 cases of the 50 cases colorectal cancer, 10 cases were Vimentin-positive expression(r=0.276, P=0.026) of 32 HIF-lα-positive cases and the 10 cases all were E-cadherin-negative, it showed that HIF-lα was positively related to EMT.(6)By constructing of hypoxic model in vitro, observing the effect of hypoxia on cell function,the result showed that the two cells invasion、migration and tube formation ability all were enhanced in hypoxic group compared with normoxic group.(7)WB results showed that E-cadherin expression was decreased, but HIF-1α, Vimentin, Snail, VE-cad and MMP-2 protein expression were increased in hypoxia group compared with normoxic group.(8)RT-PCR results showed that E-cadherin mRNA expression was decreased, but HIF-1α, Vimentin mRNA expression were increased in hypoxia group compared with normoxic group.Conclusions 1 HIF-1α was highly expressed was in colorectal cancer, and it was related to the prognosis of patients. 2 VM was present in colorectal cancer with serious hypoxia, and adenoma had no VM. 3 Hypoxia induced the invasion, migration and tube formation ability of colorectal cancer HCT116, HT-29 cells all enhanced. 4 Hypoxia induced the occurrence of EMT and promoted VM-related protein(VE-cad, MMP-2) highly expressed.