Effects Of Microspherule Protein 1 On Growth,Invasion And Migration Of Gastric Cancer Cells And Their Mechanisms

ObjectiveTo investigate the effect of microspherule protein 1(MCRS1)on proliferation,invasion and migration of gastric cancer cells,and to lay the experimental foundation for further exploring the regulation mechanism of MCRS1 on gastric cancer.Methods1.Cell culture and cell strain screening experiments: gastric cancer cells BGC-823,SGC-7901 and gastric mucosal epithelial cells GES-1 were cultured in vitro,and the expression of MCRS1 in three cells was detected by western blotting.Cells with low expression of MCRS1 were selected for subsequent overexpression experiments,and cells with high expression of MCRS1 were selected for subsequent down-regulation experiments.2.Construction and identification of pcDNA3.1-myc-MCRS1 recombinant plasmid.3.Gene transfection: pcDNA3.1-myc-MCRS1 plasmid and MCRS1 siRNA were separately transferred into corresponding gastric cancer cells by using the Lipofectamine2000 reagent.Overexpression experiments were divided into blank group,pcDNA3.1 empty vector transfection group and pcDNA3.1-myc-MCRS1 transfection group(overexpression group);siRNA interference experiments were divided into blank group,control siRNA transfection group and MCRS1 siRNA transfection group(down-regulation group).4.MTT assay was used to detect the effect of overexpression and down-regulation of MCRS1 on the proliferation of gastric cancer cells.5.Colony formation assay was used to detect the effect of overexpression and down-regulation of MCRS1 on the proliferation of gastric cancer cells.6.Cell scratch test was used to detect the effect of overexpression and down-regulation of MCRS1 on the migration ability of gastric cancer cells.7.Transwell assay was used to detect the effect of overexpression and down-regulation of MCRS1 on the invasion and migration of gastric cancer cells.8.Western blotting was used to detect the effect of overexpression and down-regulation of MCRS1 on the expression of epithelial-mesenchymal transition(EMT)-related protein(E-cadherin,N-cadherin,ZO-1,Snail1,Snail2 and Twist).Results1.Compared with the normal gastric epithelial GES-1 cells,the expression of MCRS1 protein in the gastric cancer BGC-823 cells was decreased(P < 0.01),but the expression of MCRS1 protein in the SGC-7901 cells was increased(P < 0.01).2.PCR identification and sequencing analysis showed that the MCRS1 recombinant plasmid was successfully constructed.3.The expression of MCRS1 protein in overexpression group was significantly increased compared with the blank group and the empty vector transfection group(P < 0.01),the expression of MCRS1 protein in down-regulation group was significantly decreased compared with the blank group and the control group(P < 0.01),indicating that the gene was transfected into gastric cancer cells successfully.4.The results of MTT assay confirmed that overexpression of MCRS1 inhibited the proliferation of gastric cancer BGC-823 cells(P < 0.01);while down-regulation of MCRS1 promoted the proliferation of gastric cancer SGC-7901 cells(P < 0.01).5.The results of colony formation assay confirmed that overexpression of MCRS1 decreased the number of gastric cancer BGC-823 cell clones(P < 0.01);while down-regulation of MCRS1 increased the number of gastric cancer SGC-7901 cell clones(P < 0.01).6.The results of cell scratch test confirmed that overexpression of MCRS1 increased the relative distance of cell scratches and decreased the migration rate of gastric cancer BGC-823 cells(P < 0.01);while down-regulation of MCRS1 decreased the relative distance of cell scratches and increased the migration rate of gastric cancer SGC-7901 cells(P < 0.01).7.The results of transwell assay confirmed that overexpression of MCRS1 decreased the number of invasion and migration of gastric cancer BGC-823 cells(P < 0.01);while down-regulation of MCRS1 increased the number of invasion and migration of gastric cancer SGC-7901 cells(P < 0.01).8.The results of western blotting method showed that the expression of E-cadherin and ZO-1 protein was increased and the expression of N-cadherin,Snail1,Snail2 and Twist protein was decreased when MCRS1 was overexpressed,compared with the blank group and empty vector transfection group(P < 0.01).The expression of E-cadherin and ZO-1 protein was decreased and the expression of N-cadherin,Snail1,Snail2 and Twist protein was increased when MCRS1 was down-regulationed,compared with the blank group and the control group(P < 0.01).ConclusionsIn this study,we confirmed that overexpression of MCRS1 significantly inhibited the proliferation,invasion and migration of gastric cancer cells,while down-regulation of MCRS1 promoted the proliferation,invasion and migration of gastric cancer cells which may be related to the inhibition of EMT in gastric cancer cells by MCRS1,suggesting that further exploration of the function of MCRS1 may be important for the early diagnosis and postoperative metastasis of gastric cancer.