Reduced Expression Of ARID1A Promotes Gastric Cancer Cells Proliferation And Invasion By Epithelial-mesenchymal Transition And Involved Primary Mechanisms

Objective: The gastric cancer cell lines AGS and SGC7901 were transfected with lentiviral vector(containing shRNA specifically targeting the human ARID1 A gene)to construct stable cell lines with ARID1 A silencing.To explore the effect of silencing ARID1 A gene on the process of epithelial mesenchymal transition(EMT)and to promote the proliferation and invasion of gastric cancer cells,and to explore the possible molecular mechanism of the effects of silencing ARID1 A on EMT through protein level.Methods: 1.The expression of ARID1 A mRNA and protein in gastric cancer cells was detected by RT-qPCR and Western blot.2.Construction of lentivirus(shRNA containing specific targeting human ARID1 A gene)vector,transfected gastric cancer cell lines AGS and SGC7901,and puromycin screening.The ARID1 A gene knockdown effect was verified by RT-qPCR and Western blot from mRNA and protein levels,and a stable gastric cancer cell line that silenced ARID1 A gene was obtained.3.The effect of ARID1 A expression on the process of epithelial-mesenchymal transition(EMT)was observed by inverted microscope.Western blot was used to detect epithelial marker E-cadherin,a marker of interstitial N-cadherin and Vimentin.4.The CCK8 and Transwell assays were used to detect the biological behaviors of gastric cancer cells such as proliferation activity and cell invasion after ARID1 A gene silencing.5.Silencing ARID1 A caused the changes of PI3 K / Akt protein in EMT-related signaling pathway: PI3 K,Akt and p-Akt(S473)were detected by Western blot.Results: 1.ARID1 A gene was expressed in gastric cancer cell lines,select the relatively high expression of ARID1 A gastric cancer cells AGS and SGC7901 for follow-up experiments.2.The recombinant ARID1A-shRNA recombinant lentiviral vector was transfected into gastric cancer cell lines,and a gastric cancer cell line that stably silenced ARID1 A was selected.RT-qPCR results showed that: AGS and SGC7901 cells transfected with lentivirus,compared with negative empty vector control group,gene silencing group ARID1 A mRNA expression was significantly down-regulated(P<0.01).Western blot results showed that ARID1 A protein expression was down-regulated in gene silencing group compared with negative control group(P<0.01,P<0.001).3.After the silence of ARID1 A gene,the morphology of cells changed from tight epithelial morphology to loose interstitial morphology.The protein expression of epithelial marker E-cadherin was observed by Western blot in the process of epithelial-mesenchymal transition(EMT)Decreased,interstitial markers N-cadherin,Vimentin increased,suggesting that knockdown ARID1 A gene induced epithelial-mesenchymal transition.4.The cell proliferation activity assay by CCK8 method showed that: AGS,SGC7901 cell seed plate early(within 12h),ARID1 A gene silencing group compared with the negative empty vector group,the cell proliferation activity had no statistically significant difference(P>0.05).At 24 h,48h,and 72 h,compared with the negative empty vector group,the ARID1 A gene silencing group had an increased proliferative activity.Transwell invasion assay results showed that compared with the negative empty vector group,more gastric cancer cells in the gene silencing group passed through the Matrigel,and the cell invasion ability was significantly increased.Our results suggest that silencing ARID1 A gene can promote the proliferation and invasion of gastric cancer cells.5.Western blot results showed that compared with the negative empty vector group,ARID1 A expression was significantly reduced in the SGC7901 cells stably silenced ARID1 A.At the same time,PI3 K expression was up-regulated,and total Akt protein expression was not different,but the phosphorylation level of Akt(pAKT-Ser473)was significantly increased.Suggesting that silencing ARID1 A may activate the PI3 K / Akt pathway by up-regulating PI3 K protein expression and thereby affecting downstream Akt phosphorylation.Conclusions: 1.Knockdown of ARID1 A may promote gastric cancer cell proliferation and invasion.2.Silencing ARID1 A may induce epithelial-mesenchymal transition of gastric cancer cells by up-regulating the expression of PI3 K,affecting downstream Akt phosphorylation,activating PI3K/Akt pathway,and further promoting cell proliferation and invasion.