Investigation On The Related Molecular Regulation Mechanisms Of ANXA3 33-kDa On The Malignant Properties Of Human Hepatocarcinoma HepG2 Cells

Background:Annexin A3 33-kDa（ANXA3 33-kDa）,one isoform of annexin A3 which plays important roles in tumor development and progression,drug resistance,and lymphatic metastasis.Previous studies always concentrate on the function of ANXA3 whole level or 36-kDa,while studies of ANXA3 33-kDa in tumor are few.Recent researches have indicated that ANXA3 33-kDa levels significantly ascend in HCC tissues and primary RCC cell cultures,which come from human RCC cell cancer,suggesting that ANXA3 33-kDa may be tightly associated with tumor progression.Objective:1.To detect ANXA3 33-kDa levels in HepG2 cells.2.To construct p GPU6/GFP/Neo-shANXA3 expression plasmids,and transfected into HepG2 to obtain monoclonal HepG2 cells with stably ANXA3 33-kDa knockdown by screening.3.To investigate the effect of ANXA3 33-kDa knockdown on cell proliferation,colony formatting,migration,invasion,cytoskeleton,angiogenesis,apoptosis,and drug resistance capacities of HepG2.4.To explore the mechanism of ANXA3 33-kDa regulating the malignant properties and chemoresistance of HepG2.5.To explore the influence of ANXA3 33-kDa knockdown on the growth of nude mice xenograft of HepG2 cell and its possible mechanism.Methods:1.Western Blot was performed to detected ANXA3 33-kDa level in HepG2 cell.2.Based on ANXA3 mRNA sequence（NM₀05139）,3 shRNA targeting ANXA3 and one scrambled control（shControl）sequence were designed by Sidirect and Whitehead Target Designer software,then were inserted into p GPU6/GFP/Neo vectors for establishing eukaryotic expression vectors of p GPU6/GFP/Neo-shANXA3 and p GPU6/GFP/Neo-shControl.3.Using Lipofectamine? 2000 Reagent,the vectors were transfected into HepG2 cells.Western Blot was performed to detect ANXA333-kDa levels in monoclonal HepG2 cells and to obtain the stably monoclonal HepG2 cells with ANXA3 33-kDa knockdown by screening.4.MTT and colony formatting assays were applied to investigate the effect of ANXA3 33-kDa knockdown on proliferation and colony formatting abilities of HepG2 cells.5.Wound scratch,FITC-phalloidin staining and Transwell chamber assays were performed to determine the influence of ANXA3 33-kDa downregulation on the migration and invasion of HepG2 cells in vitro.6.Tube formation in vitro assay was conducted to measure the effect of ANXA3 33-kDa downregulation on angiogenesis in vitro of HepG2 cells.7.Annexin V/PI apootosis staining assay was performed to determine the effect of ANXA3 33-kDa downregulation on HepG2 cell apoptosis.8.MTT and trypan blue coloring methods were conducted to investigate the effect of ANXA3 33-kDa downregulation on the drug resistance for HepG2 cells.9.Hoechst 33258 staining and Annexin V/PI apootosis staining assays were used to measure the anti-apoptosis induced by platinum and 5-fluorouracil for HepG2 cells.10.Western Blot was performed to determine the molecular mechanism of ANXA3 33-kDa regulating the malignant properties and chemoresistance of HepG2 cells.11.Tumor xenograft experiment was used to investigate the effect of ANXA3 33-kDa downregulation on the growth of nude mice xenograft of HepG2 cells.12.Immunohistochemical staining was performed to detect the levels of ANXA3、Ki-67、Bcl-2 and Bax in nude mice xenograft tissues to explore its possible mechanism.Results: 1.ANXA3 33-kDa was high abundance expression in HepG2 cells.2.p GPU6/GFP/Neo-shAnxa3/Control vectors were established.2 monoclonal HepG2cells（shANXA3-1 and shANXA3-2）were obtained by screening,which ANXA333-kDa levels were respectively decreased by ¹00% and ⁹8.11±0.03% without significant change of 36-kDa levels as compared with shControl group.3.As compared with shControl group,the in vitro proliferation,colony formatting,migration and invasion capacities of shANXA3-1 group were decreased by ²0.8% （P<0.0001）,⁴4.54%（P=0.0066）,⁵8.5%（P=0.0005）,⁴4.99%（P=0.0195）,respectively.the in vitro proliferation,colony formatting,migration and invasion abilities of shANXA3-2 group were decreased by ¹5.7%（P=0.0016）,⁴2.13%（P=0.0148）,⁵5.13%（P=0.0041）,⁴1.45%（P=0.0004）.4.The migration distances of shANXA3-1 and shANXA3-2 groups were significantly shortened and the wound-healing potentials were noticeably weakened as compared with shControl group（P=0.005,P=0.0082）.The actin cytoskeleton of shANXA3-1 and shANXA3-2groups became more disorganized and vaguer,even part of microfilament disappeared compared with shControl group.5.As compared with shControl group,the distribution of HUVECs for shANXA3-2 group was scattered,which the angiogenesis capacity of it became inferior.6.The total and early/metaphase apoptosis rates for shANXA3-2 group were significantly higher than shControl group（P=0.0269,P=0.0243）,whereas the late apoptosis rates were no statistic difference（P=0.1885）.7.Following platinum and 5-fluorouracil treating cells,the IC50 values of shANXA3-1and shANXA3-2 groups were noticeably bigger than shControl group（P=0.0056,P=0.0114;P=0.0456,P=0.0023）.8.Compared with shControl group,that the apoptosis induced by cisplatin and 5-fluorouracil for shANXA3-2 group was not obvious,which the tolerance for cisplatin and 5-fluorouracil was stronger.Following platinum stimulation,the total and early/metaphase apoptosis rates for shANXA3-2group were smaller than shControl group（P=0.0060,P=0.0111）,without statistical significance between the late apoptosis rates（P=0.317）.9.ANXA3 33-kDa isoform down-regulation could regulate the levels of molecular proteins in ERK,PI3K/Akt and endogenous apoptosis pathways.10.As compared with shControl group,the growth of nude mice xenograft for shANXA3-2 group became slower.12.ANXA3,Ki-67 and Bcl-2 were positively expressed in shControl group while they were negative expression in shANXA3-2 group.Bax levels in two groups were negatively expressed.Conclusion: 1.pGPU6/GFP/Neo-shANXA3 and pGPU6/GFP/Neo-shControl vectors were successfully established.Meanwhile,2 monoclonal HepG2 cells（shANXA3-1and shANXA3-2）were obtained by screening.2.ANXA3 33-kDa downregulation inhibited HepG2 cell proliferation,colony formatting,tube formation,migration and invasion,promoted apoptosis,and enhanced resistance to cisplatin and 5-fluorouracil.3.ANXA3 33-kDa regulated HepG2 cells malignant properties via ERK and PI3K/Akt pathways,and modulated the drug resistance for HepG2 cells through endogenous apoptosis pathway.4.ANXA3 33-kDa knockdown suppressed the growth of nude mice xenograft of HepG2 cells,and achieved the inhibiting effect through regulating cell proliferation and apoptosis of HCC.