| Objective: Hepatocellular carcinoma(HCC)is one of the highest fatality rate sources of epithelial malignant tumors,the incidence of it in our country is still high recently.Lymphatic channel is a malignant tumor back way of transfer,which is one of the main reasons caused invasion,high recurrence and bad prognosis in liver cancer.Many research results show that the mechanism to the spread of the lymph node metastases regulated by many factors,such as many genes are activated or suppressed,they coordinate with each other or antagonistic to each other,including cell proliferation,adhesion,migration,extracellular matrix degradation and so on.Annexin A7 is one of the key genes identified to determine lymphatic channel in mice liver cancer by the potential group early,that impacts on differentiation,proliferation,secretion,apoptosis,invasion and migration ability for all kinds of tumor cell.Annexin A7 gene is a set ca2 + phosphatide protein,activated by ca2 +and relied on membrane fusion.Then it could promote the membrane aggregation,fusion,adhesion,transportation,etc.Otherwise it can activate the GTP enzyme made and mediate the ca2+/ GTP signal transduction pathways made.Adenylate cyclase-associated protein 1(CAP1)was found originally in yeast,in which it can adjust the Ras/cAMP signaling pathways.CAP1 in mammals have the same effect of regulating actin and restructuring,the impact on cell migration.In recent years there has many researches on relationship between CAP1 and tumor metastasis,however,it seems that the influence for cell biology function of different tumor cells is not the same.Focal adhesion kinase(FAK)is one of the types of the cytoplasm of body protein tyrosine kinase,expressing in a lot of epithelial origin of tumor cells,such as colon cancer,breast cancer,oral cancer,colon cancer,thyroid cancer,gastric cancer and ovarian cancer,etc.It may effect on tumor growth,proliferation,invasion,metastasis and angiogenesis.Src is one member of the SRC family of tyrosine kinase,involved in cell adhesion,proliferation,apoptosis,differentiation and cell movement process.Paxillin is the key bridging protein to spot signaling molecules stick together,Which is the focus of the downstream of FAK.Its abnormal expression is associated with tumor occurrence,proliferation,adhesion,invasion,skeleton restructuring and transfer ability of change.E-cadherin(CDH1)is closely related with tumor invasion and metastasis of a calcium mucin,which can participate in cell polarity,cell morphology and maintain the integrity of the cell structure.Methods: 1.Constructing PGPUP GFP-neo-shRNA-Annexin A7 and PGPUP-GFP-neo-shNC expression vector,and stable transfect the Hca-P cells in mice.Then we gained lower expression of Annexin A7 and irrelevant sequence Hca-P cell lines.Western Blot and qRT–PCR technique were used to validate the interference of Annexin A7 gene to CAP1,FAK,Src,E-cadherin gene and protein expression level.2.Building PGPUP GFP-neo-shRNA-CAP1 and PGPUP-GFP-neo-shNC expression vector,and stable transfect the Hca-P cells in mice.Finaly we obtained lower expression of CAP1 and irrelevant sequence Hca-P cell lines.Western Blot and qRT – PCR technique were applied to validate the interference of CAP1 gene to FAK,Src,Paxillin,E-cadherin gene and protein expression level.3.After obtained lower expression of CAP1 and irrelevant sequence Hca-P cell lines,CCK 8 cells detection and flow cell test were applied to validate the cell proliferation and apoptosis abilities.Mouse lymph node adhesion and TranswellChambers experimental methods was used to test the cell functions of adhesion and invasion.4.Western Blot and qRT-PCR were used to check the influence of cutting Annexin A7 to the gene and protein expression level of CAP1.Using cell immunofluorescence technique to detect Annexin A7 positioning with CAP1 expression.Results: 1.At the mRNA expression level,Annexin A7 in ShRNA group is relatively reduced 82.86% and 82.78% than A7 independent sequence cells group and Hca-P cells respectively(P < 0.05),but there is not obvious difference between A7 irrelevant sequence and Hca-P group(P > 0.05).FAK and Src gene expressed in ShRNA-A7 cell group is increased 1.88 times,1.67 times and 1.78 times,1.54 times than independent sequence A7 and Hca-P groups respectively,there is not obvious difference between unrelated sequence and Hca-P group(P > 0.05).E-cadherin in ShRNA-A7 cell group is reduced 49.88% and 43.01% than A7 irrelevant sequence cells and Hca-P cell group respectively(P < 0.05),no obvious difference between the A7 sequence and Hca-P group(P > 0.05).On the protein expression level,Annexin A7 in ShRNA-A7 group cell protein expression levels is reduced 62.75% and 60.34% than A7 irrelevant sequence and Hca-P cell group(P < 0.05),while it has no difference among the Hca-P and the A7 irrelevant sequence groups(P > 0.05).The protein expression of FAK and Src in ShRNA-A7 cell group is increased 1.80 times,1.52 times and 1.59,and 1.58 times than the sequence and Hca-P groups(P < 0.05),E-cadherin protein in ShRNA-A7 cell group is fell by 54.27% and 54.27% than A7 irrelevant sequence and Hca-P groups(P < 0.05),but these three proteins in Hca-P has no exist significant differences with the A7 irrelevant sequence group(P > 0.05).2.At the mRNA expression level,CAP1 in ShRNA-CAP1 cells group is relatively decreased by 60.73% and 65.25% than CAP1 sequence and Hca-P cell groups(P <0.05),but there is not obvious difference between the CAP1 irrelevant sequence and Hca-P groups(P > 0.05).FAK,Src and Paxillin gene expressed in ShRNA-CAP1 cellgroup is increased 2.47 times,2.18 times and 2.18 times,1.76 times and 1.67 times,1.76 times than independent CAP1 irrelevant sequence and Hca-P groups(P <0.05),but these three genes in the CAP1 irrelevant sequence group have no difference with Hca–P group(P > 0.05).E cadherin in ShRNA-CAP1 cell group is decreased by40.11% and 43.01% respectively than CAP1 irrelevant sequence and Hca-P cell groups(P < 0.05),but there is not obvious difference between the CAP1 irrelevant sequence and Hca-P groups(P > 0.05).On the protein expression level,CAP1 protein expression level in ShRNA-CAP1 cell group is reduced by 50.83% and 50.29% than CAP1 sequence and Hca-P groups(P < 0.05),while there is no obvious difference between the CAP1 irrelevant sequence groups and Hca-P cell groups(P > 0.05).The protein expression of FAK,Paxillin and Src in ShRNA-CAP1 cell group is increased1.64 times and 1.60 times,1.67 times and 1.67 times,1.43 times and 1.43 times(P <0.05),E-cadherin protein expression levels in the group of ShRNA-CAP1 is decreased 39.6% and 48.22%(P < 0.05),but these four proteins in Hca-P has no exist significant differences with the A7 irrelevant sequence group(P > 0.05).3.CCK8 proliferation test was detected in the experimental groups of the ShRNACAP1,CAP1 irrelevant sequence and Hca-P cell setting in 0 h,24 h,48 h,72 h,96 h period of absorbance value.ShRNA-CAP1 cell group absorbance values point mean standard deviation at each time are 0.106±0.015 ? 0.152±0.010 ? 0.522±0.049 ?0.651±0.040 ? 0.651±0.040 ? 0.708±0.037;CAP1 irrelevant sequence cell group absorbance values point mean standard deviation at each time are 0.095±0.017 ?0.144±0.024?0.374±0.027?0.485±0.018?0.535±0.039;Hca-P cell group absorbance values mean standard deviation at each time point are 0.101±0.014?0.153±0.006?0.410±0.021?0.553±0.022?0.601±0.030.The results suggest that there is no significant difference between these three kinds of cells of Hca-P,ShRNA-CAP1,unrelated CAP1 sequence within 24 hours(P > 0.05),and since the 48 hours they start to produce significant differences at each time point(P < 0.05),chief of all,the proliferation ability of ShRNA-CAP1 cells is always higher than CAP1 irrelevant sequence and Hca-P cellgroups,there is no significant difference between independent sequence CAP1 and Hca-P groups at each time period(P > 0.05).4.Flow cytometry instrument was used to detect the groups of Hca-P cell,the ShRNA-CAP1 cell and the CAP1 irrelevant sequence cell on the early apoptosis function.The results show that the early apoptosis cells of three groups are6.33%±0.17%,3.41%±0.22% and 6.43%±0.25%.After lowering the gene expression of CAP1,the early apoptotic ability of ShRNA-CAP1 cell group is lower than CAP1 irrelevant sequence and Hca-P cell groups(P < 0.05).5.Lymph node adhesion experiment test was applied to inspect the cell adhesion ability of the Hca-P cell group,the ShRNA-CAP1 group and the CAP1 irrelevant sequence group.The lymph node set of three kinds of cells adhesion number of cells in the lymph nodes are 255.67±28.43 ? 734.33±67.50?228.33±20.03.It shows that the adhesion abilityfof ShRNA-CAP1 cell group is stronger than the CAP1 irrelevant sequence and Hca-P cell groups(P < 0.05).6.Transwell Chambers cell invasion experiment shows that the membrane cell number through the small room of the ShRNA-CAP1 cell,Hca-P cell and the CAP1 irrelevant sequence group are 61.00±8.51?173.00±18.03?58.00±6.40,Which means the number of cells through the Chambers of ShRNA-CAP1 group is obviously more than CAP1 irrelevant sequence group and Hca-P group(P < 0.05),but there is no significant difference between the other two groups(P > 0.05).7.On the analysis of the relationship between Annexin A7 and CAP1 shows that the after cutting the expression of Annexin A7 gene,the level of gene and protein expression of in ShRNA-A7 cell are lower than the A7 irrelevant sequence group with falling 17.21%and 46.86% respectively(P < 0.05).This means that low expression of Annexin A7 gene causing CAP1 gene and protein level dropped.Moreover,by immunofluorescence method expresses the Annexin A7 and CAP1 mainly locate in the cytoplasm of the Hca-P cell and a small amount of expression pitches in the nucleus.The image results mean that the two proteins may position in cytoplasm of the approximation region.Conclusion: Annexin A7 and CAP1 gene can regulate the expression of FAK,Src,Paxillin and E-cadherin in mRNA and protein level.FAK,Src,and Paxillin may get a high expression with cutting the Annexin A7 and CAP1 gene expression.However,E –cadherin obtains a lower expression in both after reducing the Annexin A7 and CAP1 gene expression.The proliferation,lymph node adhesion and invasion ability of Hca-P cell is increased,but the early apoptosis ability is inhibited within cutting Annexin A7 and CAP1 genes expression in the mRNA and protein level.Otherwise,the two genes are expressed in a common position location of Hca-P cell.So we speculate there may has some relevance between Annexin A7 gene and CAP1 gene by the reason of they have produced a consistent effect of the molecular mechanism of cell adhesion molecules,and which is closely related to the biological behavior of liver cancer Hca-P cell. |
PDF Full Text Request