Study Of The Effect And Molecular Regulation Mechanism Of ANXA5 Expression On The Invasion,Metastasis And Cisplatin Resistance Of HepG2 Cells

Background: Annexin A5(ANXA5),was widely expressed in a variety of tissues.The dyeregulation of ANXA5 was related with malignant phenotype of neoplasms.The expression level of ANXA5 in PVTT samples was much higher than their matched primary tumor samples.In addition,ANXA5 was also high expressed in cisplatin-NPC cell line.More than that,our pilot study indicated that ANXA5 promoted abilities of proliferation,migration,invasion and other biological behaviors of murine hepatocarcinoma Hca-F/Hca-P cells.This article discussed the effect and the underlying mechanism of ANXA5 on human Hep G2 cells.Objective: 1.Construct p GPU6/GFP/Neo-sh RNA-ANXA5/NC expression vectors,then stably transfected into Hep G2 cells and obtained monoclonal Hep G2 cells with stable downregulation of ANXA5 and control cells.2.Studied the influence of ANXA5 on the in vitro proliferation,migration,invasion,adhesion and skeleton formatting abilities,as well as their potential molecular mechanisms of ANXA5 regulating the malignant behaviors of Hep G2 cells.3.Investigated the effect of ANXA5 knockdown on resistance to cisplatin.Methods: 1.Designed specific ANXA5 si RNAs and a negative control(NC)sequence according to m RNA sequence of human ANXA5 which was checked in Gen Bank and inserted into p GPU6/GFP/Neo plasmids to construct p GPU6/GFP/Neo-sh RNAANXA5/NC expression vectors.2.Transfected reconstructed plasmids into Hep G2 cells and obtained monoclonal Hep G2 cells with stable downregulation of ANXA5 and negative control cells confirmed by Western Blotting.3.MTT and colony formatting assays were performed to detect the impaction of ANXA5 knockdown on the proliferation capacity of Hep G2 cells.4.Transwell and wound healing assays were measured to test the influence of ANXA5 depletion on the migration and invasion abilities.5.Learned the effect of ANXA5 downregulation on abilities of Hep G2 cells adhering on Fn gel,HUVECs and murine lymph nodes by adhesion tests.6.Actin cytoskeleton staining was applied to observe the transform of actin skeleton in Hep G2 cells after ANXA5 silencing.7.MTT assay combined with Hoechst33342 staining were performed to investigate the drug resistance and anti-apoptosis induced by cisplatin for Hep G2 cells.8.Western Blotting were used to examine protein expression level changes of Raf,p-Raf(Tyr341),p-MEK1/2(Ser217/221),ERK2,p-ERK2(Thr185/Tyr187)and c-Myc in Hep G2-sh ANXA5.9.Quantitative real-time reversed transcription-PCR(q RT-PCR)was used to examine m RNA expression level change of E-Cadherin.Results: 1.The pGPU6/GFP/Neo-shRNA-ANXA5/NC vectors were successfully constructed.Monoclonal Hep G2 cells with stable ANXA5 downregulation(Hep G2-sh ANXA5)and negative control cells(Hep G2-sh NC)were obtained.The expression level of ANXA5 was significantly downregulated for about 100.0%,compared with Hep G2-sh NC cells.2.ANXA5 downregulation inhibited the capacity of proliferation on Hep G2 cells.Besides,the colony formatting rate was decreased by24.7% in Hep G2-sh ANXA5 cells,compared with Hep G2-sh NC cells.3.The migration ability of Hep G2-sh ANXA5 cells was reduced by almost 70.0%.4.ANXA5 silencing suppressed invasion potential of Hep G2 cells by 85.0%.5.The adhesion abilities on Fn gel,HUVEC cells and murine lymph nodes of Hep G2-sh ANXA5 cells were inhibited by 10.2%,30.2% and 48.0%,respectively.6.Following ANXA5 silencing,the actin microfilaments was induced to disorganize.7.Hep G2-sh ANXA5 cells showed poor sensitivity towards cisplatin treatment.8.Western Blot and q PCR showed that the expression level of p-Raf,p-MEK1/2(Ser217/221),p-ERK2(Thr185/Tyr187),c-Myc were suppressed and E-Cadherin was evidently upregulated followed by ANXA5 knockdown.Conclusion: 1.Monoclonal Hep G2 cell line with stable knockdown of ANXA5 and negative control cell line were obtained.2.Knockdown of ANXA5 impaired in vitro proliferation ability of Hep G2 cells.3.ANXA5 depletion suppressed in vitro migration,invasion and adhesion abilities of Hep G2 cells and inhibited rearreagment of actin skeleton in Hep G2 cells.4.ANXA5 might regulate proliferation,migration,invasion,adhesion and actin skeleton rearreagment abilities via ERK signaling pathway.5 ?Knockdown of ANXA5 strengthened cisplatin chemoresistance for Hep G2 cells.