KPNA2 Expression Pattern And Its Role In The Carcinogensis Of Colon Cancer

Colon cancer is one of the most common malignancy worldwide. In China, the incidence of colon cancer is increasing rapidly. Colon cancer is the most common malignancy in China. By now, the mechanism of colon cancer is still not elucidated. In cancer, dysfunction of the cellular transport machinery is commonly observed. The consequences for the localization and function of tumor-suppressors and oncogenes have lead to discussions of the therapeutic potential of targeting this machinery.In this study, we focused on karyopherin family, which comprises both import factors(importins) and export factors(exportins) and more than 20 members of the family have been described. We used Real-time PCR to detect the expression patterns of KPNA2, KPNA4, KPNA5, KPNA7 and KPNB1 in 40 paired colon cancer tissues and its adjacent normal tissues. Then Western blot analysis was performed to furthure validate the KPNA2 protein level in 40 paird specimens. We used immunohistochemical staining to detect KPNA2 gene in 203 tissue microarrays, and therefore find the correlations of KPNA2 expression with colon caner patients’ clinical pathology parameters by survival analysis and Cox hazards regression analysis. Subsequently, we conducted lentivirus vectors inhibiting KPNA2 to infect colon carcinoma cells. And then, we assessed the role of KPNA2 interfering colon cancer cell in proliferation, colony formation assay, migration, as well as the impact on tumorigenesis abilities in vivo animal models.The results showed that: 62% of colon cancer patients with elevated mRNA levels of KPNA2 expression, 45% of patients showed a more than five times elevated KPNA2 mRNA level. Western blot verification showed that most colon cancer tissues significantly have higher KPNA2 protein expression than normal tissues. Immunohistochemistry in TMA also verified that KPNA2 expression was significantly higher in colon cancer tissues than in normal tissue; and KPNA2 expression can be used as an independent predictor of colon cancer patients. In vitro experiments showed that knockdown of KPNA2 expression inhibited colon cancer cells proliferation, colony formation and migration ability; in vivo experiments shows that knockdown of KPNA2 expression inhibited colon cancer cell tumorigenicity in nude mices.This study is focused on the expression pattern of KPNA2 in colon cancer tissues and its role in carcinogensis of colon cancer, therefore layed a theoretical foundation for KPNA2 in clinical prognosis and therapy.Part Ⅰ. Expression patterns of five genes in colon cancerAim: To examine the expression pattern of KPNA2, KPNA4, KPNA5, KPNA7 and KPNB1, and find out the important karyopherin in colon cancer.Methods: Real-time PCR to detect the expression patterns of KPNA2, KPNA4, KPNA5, KPNA7 and KPNB1 in 40 paired colon cancer tissues and its adjacent normal tissues. Then Western blot analysis was performed to furthure validate the KPNA2 protein level in 40 paird specimens.Results: 1. 62% of colon cancer patients with elevated m RNA levels of KPNA2 expression, 45% of patients showed a more than five times elevated KPNA2 m RNA level.2. Western blot verification showed that most colon cancer tissues significantly have higher KPNA2 protein expression than normal tissues.Conclusions: KPNA2 is the most important karyopherin in colon cancer, and it showed that KPNA2 m RNA and protein levels was significantly higher in colon cancer tissues than in normal tissues.Part Ⅱ. KPNA2 expression in TMA and its relationship with clinicopathological featuresAim: To examine the expression of KPNA2 in TMA and the relationship between KPNA2 expression and clinicopathological factors.Methods: We used immunohistochemical staining to detect KPNA2 gene in 203 tissue microarrays, and therefore find the correlations of KPNA2 expression with colon caner patients’ clinical pathology parameters by survival analysis and Cox hazards regression analysis.Results: 1. Colon cancer tissues significantly have higher KPNA2 protein expression than normal tissues.2. KPNA2 elevated expression was associated with AJCC stage, T stage, N stage, M stage, histological differentiation and Ki-67 expression.3. Patients with positive KPNA2 expression showed an obviously poorer 5 year overall survival and disease-free survival; KPNA2 expression can be used as an independent predictor of colon cancer patients.Conclusions: KPNA2 could serve as an independent marker for diagnosis and prognosis for colon cancer, and KPNA2 might be a new gene related to colon cancer.Part Ⅲ. The role of KPNA2 in the carcinogensis of colon cancerAim: To further explore the role of KPNA2 in colon cancer, we constructed KPNA2 interfered colon cancer cell lines.Methods: We constructed two KPNA2 knock-down cell lines by conducting lentivirus vectors inhibiting KPNA2 to infect colon cancer cells. We explored the role of KPNA2 knockdown in colon cancer cells by cell biological experiments, such as CCK-8 assays, colony formation assays, Transwell migration assays and tumorigenesis abilities in vivo animal models.Results: 1. We constructed 4 candidate target for RNAi lentiviral vector of KPNA2, and then KPNA2-RNAi(23866-1) was found as the most effective target for knock-down KPNA2 expression by Real-time PCR and Western Blot.2. The efficiency of KPNA2 expression knock-down was more than 70%.3. In vitro experiments showed that knockdown of KPNA2 expression inhibited colon cancer cells proliferation, colony formation and migration ability.4. In vivo experiments shows that knockdown of KPNA2 expression inhibited colon cancer cell tumorigenicity in nude mices.Conclusion: KPNA2 knock-down significantly weakened tumor characteristics of colon cancer cell, and cell malignancy was also reduced.