MiR-126Regulates Angiogenesis By Target Vascular Endothelial Growth Factor-A In Gastric Adenocarcinoma

Background and objective:With the progress of human tumor research, tumor angiogenesis has been shownto play more and more important role in the tumor growth，appreciation，invasion andmetastasis. Vascular endothelial growth factor was the most important angiogenesisfactor and was at the heart of tumor angiogenesis. VEGF family has seven members,namely: VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, svVEGF and placentalgrowth factor. VEGFA play the most important role in angiogenesis. Anecdotalevidence suggested that over-expression of VEGF and VEGFR was closely relatedwith angiogenesis and malignant phenotype of gastric carcinoma and was anindependent predictor in prognosis of gastric cancer. The literature showed thatmiR-126was down-regulated in gastric adenocarcinoma tissue compared to nomalgastric tissue and it was involved in clinical features of tumors, including tumor size,lymph node metastasis, local invasion and TNM stages. But the role of miR-126played in the angiogenesis of gastric cancer was still unknown. In the current study,we planed to get gastric cancer cell lines of over-expression and low-expression ofmiR-126.and then determine the relationship between miR-126and VEGFA in gastriccancer and analyze its mechanism.Methods:1. The choice of cell lines:MKN SGC-7901-45,AGS, MKN-28cell lines weregot from digestive insitute of nanchang university.Realtime-PCR was used to detectthe expression of them.2.To detect the quality of GC cells after pretreatment: Gastric cancer cell linesSGC-7901，MKN-28and MKN-45were infected by recombinant lentivirus LV-miR-126and miRCURY LNA miR-126inhibitor. Realtime-PCR was used to confirmthe relative expression of miR-126.3. In vitro experiments:GC cells were divided to mock group,Lenti-miR-126group,Lenti-control group,LNA-miR-126group and LNA-control group. Cells wereharvested after72h pretreatment and then total protein were isolated. Western blot was used to detected the expression level of Akt, mTOR, Erk and VEGFA after theabove pretreatment.Results:1.The relative expression of miR-126in MKN-28,MKN-45, SGC-7901, GES-1,AGS cell lines.The range（from high to low）of miR-126expression in GC cells was AGS，MKN-45,SGC-7901,MKN-28（p＜0.05）.2.The expression of miR-126after pretreatment in GC cellsMKN-28,SGC-7901and MKN-45cells infected by LV-miR-126can over-express miR-126（p＜0.01）,and infected by miRCURY LNA miR-126inhibitorcan down-regulate the expression of miR-126(p<0.01).3. The effects of miR-126on the expression of Akt，mTOR，p-mTOR，Eek1/2and VEGFA in MKN-28,SGC-7901and MKN-45cells.The expression of mTOR, Akt, Erk1/2and VEGFA was inhibited as Over-expression of miR-126in gastric cancer lines MKN-28,SGC-7901and MKN-45(p<0.05or0.01), On the contrary,the expression of mTOR, Akt, Erk1/2and VEGFAwas promoted as low-expression of miR-126in MKN-28,SGC-7901and MKN-45cells(p<0.05or0.01).Conclusion：We hypothesize that miR-126may regulate angiogenesis by target VEGFAthough MAPK/Erk or(and) PI3K/Akt signal pathways in gastric adenocarcinomacell.