| Background: Hca-P cells and Hca-F cell is our Task Force by the same mouse ascites hepatoma cells continue footpad of mice inoculated with 615,while the screening and isolating high and low lymphatic metastatic potential of two different cell strains subclones,among them,Hca-P cells of lymph node metastasis rate <30% is the low lymphatic metastasis cells,Hca-F cells in lymph node metastasis rate of> 70% is high lymphatic metastatic cells.In the previous study,we found that expression of Hca-F cells in the Annexin A7 mRNA is Hca-P cells in it 3.48 times,the protein expression of Hca-F cells in the Annexin A7 is Hca-P cells in it 3.0 times,and Annexin A7 After downregulated,the ability of mouse hepatoma cell proliferation,migration and invasion were decreased,while promoting apoptosis.Prompting ANXA7 may be one key genes of lymphatic metastatic potential of HCC cells.Core fucose glycosylation is a post-translational modification of proteins,abnormal expression is closely related to its level of malignancies,Fut8(a-1,6 fucose glycosyltransferase shift)is only glycosyltransferase of the catalytic core fucose synthesis,GDP-fucose is one of the substrate in the core fucose glycosylation process.In the Golgi Fut8 catalytic from fucose residues transferring to the GDP-fucose to-acetylglucosamine(GIc NAc)or more complex N-glycans constituting core fucose.The study found,Annexin A7 contains insulin,Ca2 + and phospholipid binding sites and it involves in mediating Ca2 + / GTP signaling pathway,which is equivalent to activate the enzyme GTP.Therefore,the study of interrelated Annexin A7 and core fucose glycosylated protein between the tumor to investigate the mechanism plays an important role,and it can provide new treatment ideas for clinical application.Objective: In this study,cell transfection,Western Blot,qRT-PCR,immunofluorescence techniques,contrast observation in mice overexpressing hepatoma cells and down-regulated gene regulation ANXA7 Hca-P for RBM4,HnRNP A2B1,PDLIM1 gene and protein expression levels influence,and then analyze the role and relationship ANXA7 core fucose glycosylated proteins.Method: 1,Build PG-CMV-EGFP-Kan / neo-ANXA7 expression vector,stably transfected into Hca-P cells to obtain ANXA7 overexpressed Hca-P cell lines;Construction pGPU6-GFP-neo-sh RNA-ANXA7 expression vector,stably transfected into Hca-P cells to obtain ANXA7 downregulation of Hca-P cell lines;using Western Blot,qRT-PCR technology to verify the impact of gene regulation ANXA7 HnRNP A2 / B1,RBM4,PDLIM1 protein and mRNA expression levels.2,Construction pGPU6-GFP-neo-sh RNA-HnRNP A2 / B1 expression vector,stably transfected into Hca-P cells to obtain HnRNP A2 / B1 downregulation of Hca-P cell lines;using CCK-8 kit and Transwell chamber Compare HnRNP A2 / B1 proliferation,invasion and migration of down and control groups,analysis HnRNP A2/ B1 on biological behavior of Hca-P cells.Results: 1,ANXA7 successful low expression at the mRNA level,ANXA7 down regulation group compared with the control group PNC1 cell group,gene expression levels of ANXA7 decreased to 10% of the original level,gene expression levels of HnRNP A2 / B1 decreased to 29% of the original level,RBM4 gene expression was reduced to 22% of the original level,decrease the level of gene expression PDLIM1 to 57% of the original level;at the protein level,compared ANXA7 down cell group with the control group PNC1 cells,ANXA7,HnRNP A2 / B1 protein expression decreased to 35% of the original levels,decreased protein expression levels RBM4 to the original level of 40%,a decrease of protein expression levels PDLIM1 to the original level of 56%.These results suggest that after the inhibition of expression,ANXA7 expression HnRNP A2 / B1,RBM4,PDLIM1 reduced.2,ANXA7 successfully over expressed at the mRNA level,comparing PANXA7 up regulation group with the control group PANXA7 unrelated sequence cells,increased gene expression levels ANXA7 to the original level of 2.69 times,HnRNP A2/ B1 gene expression level rise up to 1.81 times the original level of gene expression,the gene expression of RBM4 increased to 1.41 times the original level and the gene expression level of PDLIM1 increased to the original level of 1.5 times;at the protein level,comparing ANXA7 up regulation group with the control group PANXA7 unrelated sequence cell group,elevated levels of protein expression ANXA7 to 1.8 times the original level,elevated protein level HnRNP A2 / B1 to 2times the original level,increased protein expression levels RBM4 to the original level of 1.35 times,increased protein expression levels PDLIM1 to 1.2 times the original level.These results indicate that,after the ANXA7 overexpression,the expressing of HnRNP A2 / B1,RBM4,PDLIM1 increases.3,After the transfection of HnRNP A2 / B1,comparing HnRNP A2 / B1 down regulation group with the control group PNC2 cell group,at the mRNA level,HnRNP A2/ B1 gene expression level decreased to 20% of the original level at the protein level on,HnRNP A2/ B1 protein expression decreased to 35% of the original level,the results show that cell lines HnRNP A2 / B1 expression in the low PHnRNP A2 /B1 down regulation successfully constructed.4,CCK-8 cell experiments were measured the absorbance of P group,HnRNP A2/ B1 down cell group,PNC2 group of cells at 0 hours,24 hours,48 hours,72 hours,96 hours,120 hours,P cells group were 0.113 ± 0.010,0.156 ± 0.023,0.467 ± 0.013,0.572± 0.012,0.668 ± 0.009,0.712 ± 0.014;HnRNP A2 / B1 down cell group were 0.116 ±0.020,0.148 ± 0.013,0.241 ± 0.013,0.390 ± 0.012,0.460 ± 0.009,0.469 ± 0.014;PNC2cell group were 0.126 ± 0.013,0.166 ± 0.021,0.478 ± 0.013,0.570 ± 0.013,0.650 ±0.010,0.690 ± 0.013,results suggest that the growth rate of HnRNP A2 / B1 group was slower than P cell cell group and PNC2 cell group.5,In the transwell cell migration experiments,average number of the cell P group to penetrate membrane of the cell was 90.25 ± 4.56,average number of the HnRNP A2 / B1 down cell group to penetrate membrane of the cell was 28.96 ± 3.24,average number of the PNC2 cell group to penetrate membrane of the cell was 102.7 ± 2.82;In the transwell cell invasion experiment,average number of the cell P group to penetrate membrane of the cell was 80.15 ± 3.56,average number of the HnRNP A2 /B1 down cell group to penetrate membrane of the cell was 21.54 ± 4.24,average number of the PNC2 cell group to penetrate membrane of the cell was 85.07 ± 2.74,these results indicate that,after HnRNP A2 / B1 low expression,comparing HnRNP A2 / B1 down cell group with P group and the group of PNC2 cell,migration,invasive ability all decreased.Conclusion: ANXA7 gene regulated the expression of RBM4,HnRNP A2B1,PDLIM1 and with the lower ANXA7 reduced,mRNA and protein levels was high expressd when ANXA7 was over expressed;after HnRNP A2 / B1 low expression,Hca-P cell proliferation,migration and invasion were significantly decreased;ANXA7,RBM4,HnRNP A2B1,PDLIM1 may be involved in the biological activity of tumor cells and ANXA7 may be involved in core fucose glycosylated protein modification process,and tumor closely related to the occurrence and development. |
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