Effects Of ALDH2 On The Migration And Invasion Of Hca-P Cells And The Interaction With AnnexinA7

Objective:Liver cancer is one of the most common malignant tumors in China.According to the cancer data released by the National Cancer Center,the new cases of liver cancer ranked the fourth place in malignant tumors,while the death cases ranked the second place in malignant tumors.Invasion and metastasis are the two basic characteristics to distinguish benign and malignant tumors.In addition to intra-hepatic metastasis direct spread and implanted metastasis through the branches of the portal vein,the approaches of extra-hepatic metastasis through the lymphatic channels mainly include the hilar lymph nodes,the upper abdominal lymph nodes and the retroperitoneal lymph nodes.The range of lymphatic metastasis mostly determines the prognosis.Mitochondrial ALDH2 is widely expressed in various tissues,especially in the liver.It has been documented that the differential expression of ALDH2 can cause a series of diseases,such as cardiovascular disease,diabetes and tumor.It is mainly responsible for alcohol degradation in the liver,and a positive regulator of adipocyte differentiation as well.As a member of ALDH family,ALDH2 has been widely studied in many kinds of tumors in recent years.It has been found that the expression of ALDH2 in liver cancer,renal clear cell carcinoma,lung cancer and other tumor tissues is lower than that in the corresponding adjacent tissues,while the expression in meningioma is up-regulated;and the polymorphism of ALDH2 Gene is related to the occurrence of gastric cancer,esophageal cancer,pancreatic cancer,nasopharyngeal cancer and other tumors.The differential expression of ALDH2 in Hca-P and Hca-F was screened by quantitative proteomics and gene chip in the early stage of the group.It was found that the expression of ALDH2 in Hca-P cells was 2.12 times higher than that in Hca-F cells in 163 differential protein spots.Daidzin is a kind of isoflavone extractedfrom the roots and flowers of Pueraria lobata.Daidzin can bind to the catalytic site of ALDH2.It is an efficient reversible competitive inhibitor of ALDH2,which can effectively inhibit the biological activity of ALDH2.Annexin A7/ANXA7,is a kind of Ca2 + phospholipid aggregation protein,which has Ca2 + dependent membrane fusion activity,can promote membrane aggregation,fusion,adhesion,transportation,etc.,also can activate GTP enzyme and mediate Ca2 + /GTP signal transduction pathway.Previous studies in the group showed that the expression of annexin A7 in Hca-F was higher than that in Hca-P(about 3.0 times).It may be one of the key potential genes to determine the lymphatic metastasis of mouse liver cancer.Objective: This study was conducted by Western blot Methods,q PCR,ELISA,Transwell,immunocoprecipitation and immunofluorescence co location,were aimed to observe and elucidate the expression regulation of ALDH2 in hepatoma cells and its influence on tumor migration and invasion,to explore the interaction between ANXA7 and ALDH2,and then to study the influence of ANXA7 and ALDH2 on hepatoma,so as to provide a new target for further study of hepatoma.Method:1.Cells Hca-P and Hca-F were resuscitated and cultured;2.Western blot and q PCR were used to detect the expression of ALDH2 in Hca-P and hca-F;3.The best working concentration of daidzin was selected by ELISA,and the expression of ALDH2 was detected by Western blot and q PCR after adding different concentrations of dadzin;4.Construct the ALDH2 silence transfection Hca-P cell line,and detect the expression of ALDH2 and ANXA7 by q PCR and Western blot;5.Construct the ANXA7 overexpression gene transfection Hca-P cell line,and detect the expression of ANXA7 and ALDH2 by q PCR and Western blot;6.Transwell was used to detect the effect of ALDH2 on the migration and invasion of Hca-P;7.The interaction of ALDH2 and A7 in Hca-P and Hca-F was confirmed by immunoprecipitation and immunofluorescence.Results:1.The results of differential expression of ALDH2 in Hca-P and Hca-F: Western blot and q PCR showed that the expression of ALDH2 in Hca-P was higher than that in Hca-F(about 2-fold difference)2.The optimal working concentration of aidzin was 60 umol / L,and the Western blot and q PCR results showed that there was no difference in the expression of ALDH2 at protein and m RNA levels after adding different concentrations of inhibitors(0,20,40,60,80,100 umol / L);3.Results of interaction between ANXA7 and ALDH2: the results of q PCR and Western blot showed that the expression of ANXA7 in ALDH2 silence group was significantly higher than that in Hca-P and PNC group,while the expression of ALDH2 in ANXA7 overexpression group was significantly lower than that in Hca-P and PNC group.4.The results of Transwell showed that the invasion and migration of Hca-P cells were enhanced after adding inhibitors;5.The results of immunocoprecipitation showed that ANXA7 and ALDH2 co precipitated in Hca-P cells,but not in the control group(Ig G group);the results of immunofluorescence co localization showed that ANXA7 and ALDH2 Co located on the cell membrane of Hca-P / F cells.Conclusion:The expression of ALDH2 in Hca-P cells with high lymphatic metastasis potential is higher than that in Hca-F cells with low lymphatic metastasis potential;the inhibitor daidzin can inhibit ALDH2 not by changing its expression,but by inhibiting its enzyme activity;ALDH2 has the potential to inhibit the invasion and migration of Hca-P / F;Interaction between ANXA7 and ALDH2 may play a role in the occurrence and development of liver cancer.