| ObjectiveTo study the effect of CRYAB gene silencing on proliferation and migration of gastric cancer cell line SGC-7901 in vitro by lentivirus mediated RNA interference technique.Methods1.A lentiviral mediated CRYAB-shRNA was constructed and successfully transfected into SGC-7901 cell,and fluorescence microscopy was used to detect the transfection efficiency.The empty vector was used as the negative control group(sh-ctrl group),The experimental group was CRYAB virus transfection group(sh-CRYAB group).2.The expression of CRYAB on mRNA and protein level were detected by Real-time PCR and western blot analysis,respectively.3.Cell proliferation was detected by CCK8 assay and Cell cloning experiment.4.The cell migration was analyzed by transwell migration assay and wound healing assay.Results1.The transfection rate of SGC-7901 cells with infection number(MOI)of 20 was greater than 80%.2.After lentivirus was transfected into SGC-7901 cells,the expression of CRYAB was decreased on mRNA and protein level significantly(P<0.01,P<0.001).A stable transfected CRYAB-sh RNA gastric cancer cell line was successfully constructed.3.The colony formation experiment showed that the number of sh-CRYAB group was obviously reduced(P < 0.01)compared with sh-control.4.The cell proliferation was determined by CCK-8.The results show that the cell proliferation rate of sh-CRYAB group was significantly lower in fourth day,fifth day compared with sh-control.(P <0.05,P <0.01).5.The results of the cell migration show that the cell migration rate of sh-CRYAB group was significantly lower than sh-ctrl group(P<0.01).the number of migration cells of sh-CRYAB was(49.5±2.0),and the number of migration cells of sh-control was(370.0±2.9)(P<0.01).6.The results of the wound healing assay showed that the closure of sh-ctrl group wassignificantly faster than sh-CRYAB group at 24,48 and 72 h(P<0.01).The wound of sh-ctrl group was basically covered by cells,and the wound of sh-CRYAB group was still clear,the relative blank areas of 0,24,48 and 72 h were measured.ConclusionSilencing of CRYAB expression might effectively inhibit cell proliferation and migration in SGC-7901 cell.CRYAB gene might be a promising novel target in gene therapy of gastric cancer. |
