The Expressions Of Calcium-related Proteins And The Relationships With ANXA7

Backgroud:Primary hepatocellular carcinoma is a malignant tumor of the origin of the epithelium,it is one of the most serious malignant tumors in the world,and also is the most common tumor in our country.As an important determinant of the prognosis of hepatocellular carcinoma,lymphatic metastasis is an important factor in the prognosis of HCC.Therefore,the study of the molecular mechanism of lymphatic metastasis of liver cancer has become the focus of the study.Mouse ascites hepatoma cells Hca-F cell line and Hca-P cell line have the same or similar backgroud,previous our team used a variety of molecular biology techniques to find out the difference between Hca-F cell line and Hca-P cell line.Among them,we found the significant differences in the levels of gene and protein in ANXA7.At the same time,through up regulated of ANXA7 expression in Hca-F cells and down regulated expression of ANXA7 in Hca-P cells to study its effect on the behavior of cell metastasis,the results indicated that ANXA7 plays an important role in the lymphatic metastasis of mouse ascites hepatoma.This study through observing the expression of the calcium-ralated protein STIM1?CAPN2 and PYK2 in high and low lymphatic metastasis potential of mouse ascites hepatoma Hca-F and Hca-P cells,and the relationship between ANXA7 to further elucidate the molecular mechanism of lymphatic metastasis of hepatocellular carcinoma(HCC).Objective:The expression of calcium related proteins STIM1 ?CAPN2 and PYK2 in Hca-F cells and Hca-P cells was determined by real-time quantitative PCR and Western Blot.Using ANXA7-upregulated Hca-F cells and ANXA7-downregulated Hca-P cells to observe the relationship of ANXA7 and calcium related proteins,then analyzes the effects of changes in the expression of ANXA7 on calcium related proteins expression,and identify the calcium related proteins which are closely related to ANXA7.Inhibiting the activity of calcium related proteins,through the cell function tests to verify whether the capacity of cell invasion and proliferation is consistent with the results of down-regulated ANXA7.Method:Using real-time fluorescence quantitative PCR and protein immune blotting respectively to detect the different expressions of calcium related proteins STIM1 ?CAPN2 and PYK2 in Hca-F cells?Hca-P cells ?ANXA7-upregulated Hca-F cells and ANXA7-downregulated Hca-P cells.After determing the calcium related proteins which are closely related to ANXA7,using the protein inhibitor to culture the cells,CCK8 experiment is used to detect cell optical density changes for different concentration gradient in Hca-P cells for12 h,24h,36 h,48h,Calculating the IC50 to find the best inhibition concentration,testing the effects of protease inhibitors on the proliferation of tumor cells.Transwell assay are performed to observe the impact of protein inhibitor on cells migration and invasion.Result:Detection the expression of the genes in Hca-F and Hca-P cells at the m RNA level,the expression of STIM1 in Hca-F cells is 1.45 times of in Hca-P clells and the expression of CAPN2 in Hca-F cells is 1.67 times of in Hca-P cells,PYK2 in Hca-F cells is 1.98 times of in Hca-P cells;The results of Western Blot showed that the expression of ANXA7 in Hca-F cells was 1.9 times higher than that of in Hca-P cells,STIM1 in Hca-F cells was 1.38 times of in Hca-P cells,CAPN2 in Hca-F cells was 3.2 times Hca-P cells,PYK2 in Hca-F cells expression was 2.5 times of in Hca-P cells;The results of immunohistochemistry showed that the expression of calcium related proteins STIM1?CAPN2 and PYK2 in solid tumor Hca-F cells were higher than that of in Hca-P cells,the results were statistically significant.There was no difference of the expression of STIM1 in ANXA7 up and down regulated cells.The expression of PYK2 was decreased in ANXA7-upregulated Hca-F cells and increased in ANXA7-down regulated Hca-P cells.But the expression of CAPN2-calcium neutral protease showed a consistent results with the change of ANXA7,which means CAPN2 is related to ANXA7.The results of CCK8 indicated that the calcium neutral protease inhibitor ALLN reduced the proliferation of tumor cells.Transwell experiments showed that the CAPN2 protease inhibitor significantly inhibited tumor metastasis and invasion.Conclusion:The expression of calcium related proteins STIM1?CAPN2 and PYK2 in mouse ascites hepatoma Hca-F cells was higher than that in Hca-P cells,which was consistent with the expression of ANXA7 in Hca-F cells and Hca-P cells.The relative high expression of calcium related proteins STIM1?CAPN2 and PYK2 in high lymphatic metastasis cells suggests that calcium related proteins STIM1?CAPN2 and PYK2 may promote the lymphatic metastasis of tumor cells.The expression of CAPN2 was closely related to the expression of ANXA7,which suggesting that CAPN2 may be located at the downstream functional pathway of ANXA7 or ANXA7 may through some mechanism regulate the expression of CAPN2.The results of CCK8 and Transwell tests showed that the proliferation ability,migration and invasion ability of mouse ascites hepatoma cells decreased significantly under the action of calcium neutral protease inhibitors ALLN,which were consistent with the function of ANXA7 down regulated hepatocellular carcinoma cells.These results suggest that CAPN2 may be involved in the metastasis of hepatocellular carcinoma with ANXA7.