Study On The Corresponding Molecular Regulation Mechanisms Of ANXA3 On The Malignant Behaviors Of Murine Hepatocarcinoma Cell Lines

Background: Annexin A3(ANXA3) is a member of the annexins that are a structurally homologous super-family of calcium and membrane-binding proteins. There are two isoforms of 36 and 33 k Da annexin proteins due to the alternative splicing of exon III in Anxa3 gene. The deregulation of ANXA3 are closely associated with the tumor development and progression, tumor metastasis and drug resistances. Hca-F and Hca-P are a pair of syngeneic murine hepatocarcinoma cell lines with differed lymphatic metastasis potentials. ANXA3 is abundantly expressed in both Hca-F and Hca-P cells,the remarkable different ANXA3 expression levels in them making the cell line pair the ideal cell models for studying the development and progression of lymphatic metastasis for hepatocarcinoma.Objective: 1. To construct p GPU6/GFP/Neo-sh RNA-Anxa3 expression plasmids, to stably transfect them into Hca-F and Hca-P cells and to obtain monoclonal Hca-F and Hca-P cell lines with stable knockdown of ANXA3. 2. To construct p EGFP-N1-Anxa3 and pc DNA3.1/V5-His B-Anxa3 eukaryotic expression vectors, to transiently transfect them into Hca-P cells and to overexpress ANXA3 in Hca-P cells. 3. To investigate the influence of ANXA3 knockdown on the in vitro proliferation, migration and invasion abilities of Hca-F cells. 4. To investigate the influence of ANXA3 knockdown on the in vitro proliferation, clone formation, migration, invasion and chemoresistance properties of Hca-P cells. 5. To confirm the subcellular localization of ANXA3 expression. 6. To investigate the impact of ANXA3 over- expression on the in vitro proliferation, cloneformation, migration and invasion of Hca-P cells. 7. To explore systemically the molecular action mechanisms of ANXA3 regulating the malignant behaviors of Hca-F and Hca-P cells.Methods: 1. The targeting sh RNAs of Anxa3 and a negative control(NC) sequence were designed and inserted into p GPU6/GFP/Neo vectors to construct the expression vectors of p GPU6/GFP/Neo-sh RNA-Anxa3 and p GPU6/GFP/Neo-sh RNA-NC. 2. The p GPU6/GFP/Neo- sh RNA-Anxa3/NC vectors were stably transfected into Hca-F and Hca-P cells. The monoclonal Hca-F and Hca-P cell lines with stable ANXA3 knockdown rates were obtained by G418 screening selection combined with limited dilution method. 3. The primers were designed and the Anxa3 CDS was constructed into p EGFP-N1 and pc DNA3.1/V5-His B eukaryotic expression vectors using genetic engineering construction method and subsequently transiently transfected into Hca-P cells. 4. CCK-8 assay was performed to investigate the influences of ANXA3 knockdown on the proliferations of Hca-F and Hca-P cells, and ANXA3over-expression on the proliferation of Hca-P cells. Soft agar colony formation assay was performed to investigate the effects of ANXA3 knockdown and overexpression on the colony formatting capacity of Hca-P cells. 5. Transwell chamber assay was measured to determine the influences of ANXA3 on the in vitro migration and invasion abilities of Hca-F and Hca-P cells. 6. CCK-8 assay combined with Hoechst 33258 staining method were performed to investigate the influences of ANXA3 knockdown on the drug-resistance and anti-apoptosis induced by cisplatin for Hca-P cells. 7. The subcellular localization of ANXA3 was determined by using laser scanning confocal microscope(LSCM). 8. Western Blot assay was performed to detect the protein expression level changes of the related molecules involved in the signaling pathways for regulating the malignant behaviors of Hca-F and Hca-P cells.Results: 1. The p GPU6/GFP/Neo-sh RNA-Anxa3/NC vectors were successfully constructed. Monoclonal Hca-F and Hca-P cell lines with stable ANXA3 knockdown were obtained and named as F-ANXA3-sh RNA, P-ANXA3-sh RNA1 and P-ANXA3-sh RNA2. 2. Enzymatic digestion and sequencing detection indicated that thep EGFP-N1-Anxa3 and pc DNA3.1/V5-His B-Anxa3 eukaryotic expression vectors were successfully constructed. 3. The proliferation, migration and invasion abilities of FANXA3-sh RNA cells were 1.62-, 1.59- and 2.98- folds of those from F-Control-sh RNA cells, respectively. ANXA3 knockdown promoted the malignant behaviors of Hca-F cells through activating the Erk and PI3K/Akt pathways. 4. The proliferation, colony formation, migration and invasion abilities of P-ANXA3-sh RNA1(2) cells were 1.13-(1.08-), 4.06-(4.86-), 4.98-(4.75-) and 2.00-(2.77-) folds of those from P-Control-sh RNA cells, respectively. ANXA3 knockdown enhanced the malignant behaviors of Hca-P cells through activating the Erk and JNK pathways. 5. Following ANXA3 over-expression, the proliferation, colony formation, migration and invasion abilities of pc DNA-Mock cells were 1.19-, 1.32-, 2.92- and 3.4- folds of those from pc DNA-ANXA3 cells. ANXA3 over-expression reduced the malignant behaviors of Hca-P cells through inhibiting the Erk and JNK pathways. 6. Confocal laser scanning microscope result indicated ANXA3 was mainly expressed in cytoplasm. 7. ANXA3 knockdown enhanced drug resistance against cisplatin treatment in Hca-P cells, while showed no effect on 5-FU resistance. ANXA3 might regulate cisplatin resistance of Hca-P cells via the intrinsic apoptotic pathways.Conclusion: 1. The p GPU6/GFP/Neo-sh RNA-Anxa3/NC, p EGFP-N1-Anxa3 and pc DNA3.1/V5-His B-Anxa3 eukaryotic expression vectors were successfully constructed.2. Monoclonal cell lines with stable ANXA3 knockdown were obtained. 3. ANXA3 knockdown promoted the in vitro proliferation, migration and invasion malignant behaviors of Hca-F cells through activating the Erk and PI3K/Akt pathways. 4. ANXA3 knockdown increased the in vitro proliferation, colony formation, and migration and invasion abilities of Hca-P cells. Correspondingly, ANXA3 over-expression inhibited the in vitro proliferation, colony formation, and migration and invasion abilities of Hca-P cells mainly via Erk and JNK pathways. 5. ANXA3 was mainly expressed in cytoplasm. 6. ANXA3 knockdown enhanced the resistance against cisplatin of Hca-P cells via the intrinsic apoptotic pathways.