Silencing ENO1 By ShRNA Inhibits The Proliferation,Invasion,Migration Of Gastric Cancer Cells

Background and purpose:Gastric cancer is one of the most common malignant tumor in the worldwild,the mortality is the second of malignant tumor in the world.There are wide distribution characteristics with the incidence of gastric cancer,about two-thirds of the gastric cancer patients in developing countries,including China's stomach cancer patients(42%).There are some high-risk areas in worldwide,including Eastern Asia,Eastern Europe and some areas of Central and Southern of the United States.The incidence of gastric cancer ranks in the first of all kinds tumors in China.It is about 170,000 people died of stomach cancer each year,nearly a quarter of deaths of all malignant tumors.ENO1 is high expression in the stomach cancer,and is associated with the differentiation of gastric cancer cells.ENO1 is closely related to invasion and metastasis in non-small cell lung cancer.By this study,we will build a lentivirus vector of enzyme-alpha(enolase-a,ENO1),and transfect MKN45 cell lines of gastric cancer and explore the influence about proliferation,migration,invasion of human gastric cancer cells in vitro.Methods:The protein level of ENO1 in MKN28,MKN45,SGC7901,BGC823 was detected by Western Blot.The constructed PLKO.1-ENO1shRNA vector was transfected into 293T cells and used to infect gastric cancer cells MKN45 by using lentiviruses.The positive clones were selected with puromycin to establish stable transfection cell lines.Negative controls were generated by infection with viruses containing empty vector PLKO.1-scramble shRNA by the same protocol,and using wide type MKN45 cells as blank control.The silencing effect was confirmed by reverse transcription-PCR and Western blotting at mRNA and protein levels,respectively.Cell proliferation ability and chemosensitivity were tested by MTT assay.Cell migration and invasion ability were detected by using scratch experiment and transwell experiment.Results:ENO1 was expressed lower in high differentiated gastric cancer cell line MKN28,on the contrary,ENO1 was expressed relatively higher in poor differentiated gastric cancer cell lines MKN45,SGC7901 and BGC823.ENO1 ShRNA lentivirals vector was successfully transfected into MKN45 cells,the protein level and mRNA level of ENO1 after silencing ENO1 was assayed by western blot and RT-PCR.The results showed that ENO1 expression was significantly inhibited.MTT methods displayed that the growth of ENO1 was significantly inhibited after silencing ENO1 in MKN45.MTT was used to detect the drug sensitivity to 5-Fu and DDP,the results showed that compared with Scr and MKN45 group,ENO1 ShRNA group for 5-Fu and DDP showed significantly inhibited,there was statistical significance(P<0.05)and the inhibition of MKN45-ENO1-shRNA significantly decreased.The cloning assays results showed that the tumor formation ability of ENO1 ShRNA was obviously lower than Scr and MKN45 group,there was statistical significance(P<0.05).The results of invasion and migration showed that ENO1 ShRNA was significantly decreased cell invasion and migration ability(P<0.05).Silencing ENO1 was significantly inhibited gastric cancer cell proliferation,increaseed the sensitivity to chemotherapy drugs and reduced the migration and invasion ability of the gastric cancer cells in vitro.Conclusions:ENO1 is associated with of invasion and migration of gastric cancer.Our results suggested that the decrease of ENO1 was effectively inhibited gastric cancer cell proliferation,invasion and migration,and was sensitivity to chemotherapy drugs.