ShRNA Inhibition Of 47kDa Annexin A7 Affects Apoptosis And Apoptosis Of Signal Pathway In Mouse Hepatocacinoma Hca-P Cells

Objective: belongs to malignant tumor which is generated from liver cell or liver Primary liver cancer bile duct's cell.According to the statistics,each year the mortality rate of primary liver cancer is the third in the digestive system,there are about 110000 people died of liver cancer in China,and about 250000 died of liver cancer each year in world.Although surgeonal resection is still the preferrest method of treatment,but the five year relapse rate and metastasic rate are still high.The mean survival time is between 1 to 4 months from diagnosis to death.Liver cancer belongs to the malignant tumors of epithelial origin which has high metastatic potential and metastasizes early to regional lymph nodes.The rate of lymph node metastasis is 7.45% in the primary liver cancerThe mouse ascitic hepatoma(H22)cells were implanted into inbred Chinese 615 mouse abdominal cavity.After many generations stably grew,the cells were injected into the foot-pad of inbred Chinese 615 mouse.So,the experimental lymphatic metastasis model was established,the cell line designated as H22-F25/L.5 cell clones(16A3,16C6,22C5,H7,and A2)with different metastatic ability have been separated from Hca-F25/L cell line.Hca-P cells showed a low metastatic potential(lymphatic metastasis rate<25%).In our previous study,we applied the suppressive subtracted hybridization technique,gene chip and quantitative proteomics technique to identify differentially expressed genes and proteins for lymphatic metastasis between the two cell lines.And there are 21 different genes and proteins were the highly expressed in Hca-F cells,whlie only 3 were the highly expressed in Hca-P cells.In our previous study,we have found these genes(Anxa7,Jnk1,Gsn and Rack1)may enhance the proliferative capability,apotosis and invasion potential of the Hca-F cells and Hca-P cells.In cancer,there is a loss balance between cell division and cell death.The problem can arise in any one step along the way of apoptosis.Lymphatic metastasis is a complex process involving multiple gene and their products.A detailed understanding of the mechanism of lymphatic metastasis is still lacking.Anxa7 is a ubiquitously expressed member of the multifunctional Ca/Phospholipid-binding annexin family.Both the isoforms 47 k Da and 51 k Da of Annexin A7 are expressed in liver cancer cells.This study aimed at elucidating which isoform of Annexin A7 affects the cell apoptosis and how to affect in the hepatocarcinoma cell line.Apoptosis associated pathways can be activated by: 1)the intrinsic mitochondrial pathway releasing pro-apoptotic molecules such as Cytochrome-C into the cytoplasm where it activates a cascade of caspases through Caspase-9.This pathway is regulated by the Bcl2 family.2)the intrinsic endoplasmic reticulum pathway that is believed to be Caspase-12 dependent and 3)the extrinsic death receptor pathway,such as Fas and its ligand Fas L,as well as cysteine proteases like caspase-8.To gain the insight into the role of 47 k Da Anxa7 in cancer,an RNA interference(RNAi)technique to downregulate 47 k Da Anxa7 expression was performed in Hca-P cell line.Our group focuses mainly on the anti-apoptotic mechanism of the 47 k Da isoform of Anxa7 in Hca-P cell line.Methods: 1.Short hairpin RNA sh RNA-Anxa7 expression plasmids and nonspecific-sequence control-sh RNA(sh RNA-NC)plasmids were constructed,and stable transfect the Hca-P(P)cells in mice.Then we gained lower expression of Anxa7(Pd)and irrelevant sequence Hca-P(Pdn)cell lines.2.Western Blot and q RT–PCR technique were used to validate the interference of Anxa7 gene and protein expression level.3.Western Blot were applied to validate 47 k Da and 51 k Da isoform Anxa7 protein expression level in the three cell groups.3.Extraction of cytoplasmic and mitochondrial proteins,compare 47 k Da and 51 k Da isoform Anxa7 location and protein expression level in the three cell groups.4.Flow cell test,Transwell and CCK 8 cells detection were applied to validate the cell apoptosis,invision and proliferation abilities.5.The expression of Bcl2,Bax,Cytochrome-C,Fas L/Fas,and Caspase-3,8,12 was studied by western blotting(WB)analysis in the three cell groups.6.Flow cell test were applied to validate the mitochondrial membrane potential in the three cell groups.7.Detecte the expression of Anxa7 and Bcl2,Bax coimmunoprecipitation,and the location and protein expression level of Bcl2 in the three cell groups..Results: 1 Automatic DNA sequencing confirmed the expression vector sequencing results of of p GPU6/GFP/Neo-sh RNA-Anxa7;and cell showed green fluorescent protein were regarded transfected efficiently.2.At the m RNA expression level,Pd group isobviously reduced than P and Pdn cells(P<0.05),but there is not obvious difference between Pdn and P cell groups(P>0.05).3.On the protein expression level,47 k Da Anxa7 in Pd is reduced than Pdn and P cell groups(P<0.05),while it has no difference between Pdn and P cell group groups(P>0.05).47 k Da Anxa7 is located in cytoplasm and mitochondria;and 51 k Da is mainly located mitochondria.4.The early apoptotic ability of Pd cell group is lower than P and Pdn groups(P<0.05).Down-regulated 47 k Da isoform,the transwell result showes that the number of cells of Pd group is obviously less than P and Pdn group(P< 0.05),but there is no significant difference between P and Pdn group(P>0.05).5.CCK8 proliferation test suggest that there is no significant difference amonge the three groups of Pd,P and Pdn within 24 hours(P > 0.05),and since the 48 hours they start to produce significant differences at each time point(P< 0.05),there is no significant difference between P and Pdn groups at each time period(P>0.05).6.WB shows down-regulated 47 k Da isoform,the expression of Bcl-2,Cyt-C in cytoplasm,Caspase-3 were higher in Pd than in P and Pdn(P<0.05);while the expression of Bax,Cyt-C in mitochondria?Caspase-12?Fas?Fas-L? Caspase-8 was almost the same among Pd,P and Pdn(P>0.05).7.Down-regulated 47 k Da isoform declined the mitochondrial membrane potential.Pd was lower than in P and Pdn(P<0.05).9.Anxa7 co-immunoprecipitate with Bcl2 in P,but not co-immunoprecipitate with Bax.By WB analysis,Anxa7 and Bcl2 co-expressed in both the mitochondria and the cytoplasm And down-regulated 47 k Da Anxa7 decreased the expression of Bcl2 both in the cytoplasm and the mitochondria.Conclusion: 1.The target gene was successfully built into p GPU6/GFP/Neo-sh RNA expression vector,stable transfection achieved.2.Both Real time q RT-PCR and Western blot confirmed successful down regulation of 47 k Da Anxa7.3.The expression of 47 k Da and 51 k Da Anxa7 are located in cytosolic fraction and mitochondria.4.47 k Da Anxa7 protein can decrease the apoptosis capability,enhance the proliferation and invasion potential of the Pd cells.5.These results indicate that 47 k Da Anxa7 could associated with the apoptosis of Hca-P cells through its interaction with Bcl2 by the mitochondrial pathway.6.These results indicate that 47 k Da Anxa7 donot induce the apoptosis of Hca-P cells through its interaction with Bax,caspase-8,caspase12,Fas,Fas-L by the intrinsic endoplasmic reticulum pathway and the extrinsic death receptor pathway.7.Anxa7 co-immunoprecipitated with Bcl2.8.We can infere that down regulated 47 k Da Anxa7 could potentially be used as the therapeutic target for tumor apoptosis treatment.