RASAL2 Regulates The Proliferation Of Cervical Cancer Hela Cells And Its Mechanism

ObjectiveTo investigate the effect of RAS activator 2(RASAL2)on the proliferation,invasion and metastasis of cervical cancer HeLa cells and its mechanism of action,to lay a foundation for exploring the regulatory mechanism of RASAL2 on cervical cancer HeLa cells.Methods1.Culture cervical HeLa cells in vitro.2.The overexpression plasmid GV40-myc-RASAL2 was constructed.Full-length human RASAL2 amplification,construction and identification of GV40-myc-RASAL2 eukaryotic expression vector.3.Gene transfection: The RASAL2 gene was transfected into HeLa cells using transfection reagent LIP3000.The experiments were divided into untransfected group,empty vector transfection group and RASAL2 gene transfection group.4,MTT assay overexpression of RASAL2 on the proliferation of HeLa cells.5.Scratch test to detect the effect of overexpression of RASAL2 on the migration of HeLa cells.6.Protein expression of RASAL2,E-cadherin,N-cadherin,SNAIL,ZO-1,WEE1 and CCNB1 were detected by Western blotting.7.Co-localization of immunofluorescence confirmed the presence of RASAL2 and WEE1 colocalization in cervical HeLa cells.Results1.Identification of GV40-myc-RASAL2 recombinant plasmid: PCR and sequencing analysis showed that the GV40-myc-RASAL2 recombinant plasmid was successfully constructed.2.Overexpression of RASAL2 by gene transfection technology: There was no significant difference in RASAL2 protein expression between the empty vector transfection group and the untransfected group(p>0.05),and the expression of RASAL2 protein was significantly increased in the GV40-myc-RASAL2 transfection group.p < 0.01).This indicates successful transfection of RASAL2 gene.3.MTT results: The proliferation rate of HeLa cells transfected with GV40-myc-RASAL2 group was significantly lower than that of GV40 empty vector control group and blank control group at 24 h,48 h and 72 h(p < 0.01).4.Scratch test results: Compared with the blank control group,the RASAL2 gene transfection group significantly inhibited the migration of HeLa cells(p<0.01).5.Western blotting results: Compared with GV40 empty vector transfection group and blank control group,E-cadherin protein expression was increased in RASAL2 gene transfection group,and N-cadherin,SNAIL and ZO-1 protein expression was decreased.At the same time,overexpression of RASAL2,WEE1 and CCNB1 protein expression was increased(p < 0.01).6.Immunofluorescence co-localization results: RASAL2 and WEE1 are mainly co-localized in the nucleus in cervical cancer HeLa cells.ConclusionsThe results of this study indicate that RASAL2 inhibits the proliferation and migration of HeLa cells,suggesting that RASAL2 may be involved in the developpment of cervical cancer;its mechanism may be related to the interaction of RASAL2 and WEE1.