Research On The Expression And Function Of ITGA3 In Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma(ICC)is one malignant type of liver cancer,deriving from the intrahepatic biliary epithelial cells,whose morbidity and mortality rates ranking only second to hepatocellular carcinoma(HCC).China has a huge population of liver cancer.The incidence of intrahepatic cholangiocarcinoma and its mortality rate are in the front ranks of the world.With the deeper recognization of tumors,advance of medical technology and the cooperation of multiple disciplines in recent years,comprehensive therapic strategies based on the operations have acquired better effect for cancer treatment.Nevertheless,the prognosis of ICC is still not satisfying.Patients' five-year survival rate is only 20-40%after complete resection.Those people who have lost the operation opportunity usually survive no longer than six months.There is still a lack of effective treatments for ICC.At present,we are still in the period of exploration in the pathogenic mechanisms of intrahepatic cholangiocarcinoma.Integrins are one kind of membrane proteins.Recently,increasing evidences reveal that integrins play important roles in a range of malignant tumors'progression.ITGA3(Integrin subunit alpha 3),as one kind of the integrins,has been found to express aberrantly in many malignant human tumors and influence tumor cells' behavior to function in the origination and development,including colorectal cancer,melanoma,and prostate cancer.However,the pathogenic mechanism of ITGA3 in human ICC hasn't been reported so far.In our research,we explored the clinical correlation and pathogenic mechanism of ITGA3 in human intrahepatic cholangiocarcinoma for the first time.Part ? The expression of ITGA3 and its associations with clinical and pathological features in intrahepatic cholangiocarcinomaAims:Expression of ITGA3 in intrahepatic cholangiocarcinoma tissues and relevance between ITGA3 levels and clinicopathological features are confirmed in this paper.Methods:We detected the expression level of ITGA3 in 46 pairs of ICC tumor tissues and paired peritumor tissues by Immunohistochemical technique.In addition,we carried out the survival analysis and explored potential relevance between its expression level and clinicopathological features.Results:The immumohistochemical staining results indicated the expression level of ITGA3 was higher than in paired peritumor tissues in 32 ICC patients' tumor tissues(70%),while the expression level of ITGA3 in tumor tissues was lower compared with paired peritumor tissues in 14 ICC patients(30%).Besides,the analysis of the relevance between the ITGA3 expression and clinicopathological features of ICC in 46 patients showed that high level of ITGA3 was significantly correlated with larger tumor size(p=0.034),more existence of lymph node metastasis(p=0.034),and advanced TNM stages(p=0.027).In addition,the survival analysis showed that patients with the higher expression level of ITGA3(n=32)underwent worse prognosis than those with lower expression level of ITGA3(n=14;p=0.015).Conclusion:ITGA3 is overexpressed in ICC tumor tissues and significantly associates with the larger tumor size,more existence of lymph node metastasis,advanced TNM stages and worse prognosis of the patients.Part II The expression of ITGA3 and its associations with tumor cells' biological behavior in intrahepatic cholangiocarcinomaAims:Expression of ITGA3 in intrahepatic cholangiocarcinoma(ICC)cell lines and relevance between ITGA3 levels and ICC cells' biological behavior are confirmed in this paper.Methods:Western-Blotting assay was used to detect the ITGA3 expression level in the ICC cell lines RBE,ccLp,Hccc-9810,HuccT-1.Knockdown of ITGA3 cell models were established by using small interfering RNA(si-RNA).The effect of ITGA3 on cell viability was assessed by EdU assay and colony formation assay before and after treatment.We detected the cell cycle distribution by flow cytometry.Western-Blotting assay was used to detect the expression of cell cycle related proteins,and the potential mechanism was discussed.The effect of ITGA3 on migration and invasion ability was assessed transwell assays before and after treatment.Results:The expression level of ITGA3 was higher in ICC cell lines Hccc-9810 and HuccT-1 than cell lines RBE and ccLp in protein level.The efficiency of knockdown of ITGA3 in ICC cell lines Hccc-9810 and HuccT-1 was detected by Western blotting assay.EdU assay and colony formation assay results show that cell proliferation activity was restricted significantly in small interfering RNA treatment group compared with NC group both in HuccT-1(p<0.05)and Hccc-9810(p<0.01)cell lines.Cell cycle analysis results indicated that knockdown of ITGA3 had a significant influence on the cell cycle distribution in HuccT-1 and Hccc-9810 cells.The results showed that more cells were trapped in G1 phase and fewer entered into S phase,compared with negative control(NC)group.Western-Blotting results indicated that by knockdown of ITGA3,the expression levels of cyclin-dependent kinase(CDK)2,CDK4,and CDK6 downregulated significantly,as well as cyclin D1 and El in HuccT-1 and Hccc-9810 cells.Transwell results showed that the number of cells passed through the membrane showed no significant difference between the siRNA and the NC group in both HuccT-1(p>0.05)and Hccc-9810(p>0.05)cell lines,which indicated that the overexpression of ITGA3 in ICC cells had no influence on the abilities of the invasion or migration through the epithelial-mesenchymal transition process.Conclusion:ITGA3 is overexpressed in HuccT-1 and Hccc-9810 cell lines.High expression level of ITGA3 promotes proliferation and cell cycle progression in ICC.Furthermore,high expression level of ITGA3 didn't involve in the migration and invasion of ICC cells.