Mechanisms Of P62/SQSTM1and Autophagy In Colon Cancer Tumorigenesis

Objective:Colon cancer is one of the most common gastrointestinal malignancy, its pathogenesis has been widely studied, but the mechanism is still not very clear. Autophagy plays an important role in tumorigenesis. p62is involved in autophagy, a key protein in apoptosis and tumorigenesis. To investigate the relationship between p62protein, autophagy and colon cancer, have a positive meaning for elucidating pathogenesis of colon cancer and finding potential therapeutic strategies.Methods:The clinical data of119cases was collected in sporadic colon cancer patients, such as their gender, age, primary tumor site,tumor size, histological type and degree of differentiation of tumor, clinical stage and fresh surgical specimens. Through immunohistochemistry, real-Time PCR and western blot,p62protein expression was detected in all tumor samples and normal tissues. Autophagy marker protein LC3was detected by immunofluorescence and western blot. Association between levels of p62/LC3and clinical data of patients were statistical analyzed.Meanwhile, according to the mRNA sequence of human P62viral shRNA interference plasmid was constructed. Virus particles for knockdown p62were produced in293T cells.Then, HCT-116colon cancer cell lines were infected. Viral interference effect was detected through Real-time PCR and western blot. Cell proliferation, growth, cell migration were analyzed after knockdown p62.Moreover, Mouse xenograft carried out in nude mice. Effects on autophagy also were detected after p62knockdown. Proliferation of colon cancer cells was analyzed after p62knockdown with or without treatment of inhibitors of autophagy (3-MA).Results:Our results showed that P62mRNA and protein can be detected in colon cancer tissues and normal tiusses.119cases of colon cancer patients data show that P62mRNA and protein were increased compared with normal tissue. Meanwhile, puncta of LC3was significantly increased in colon cancer tiusses compared with normal tissues. Western blot results also show that the expression of LC3-II in colon cancer tissues was significantly higher than in normal tissues. These results show that autohphay is actived in colon cancer tissues. There are no significant correlation between expression level of p62, LC3and clinical data, suggesting that p62and autophagy may only play a role in the tumorigenesis of colon cancer.To further investigate the role of p62and autophagy in colon cancer, p62were knockdowned by shRNA plasmids virus after in vitro packaging and purification. We also found that growth,proliferation and clone formation of HCT-116cells was inhibited after knockdown p62in vitro and in vivo.Puncta of LC3was decreased. Meanwhile, the knockdown of p62, has a synergistic effect with autophagy inhibitor3-MA inhibiting effect on the growth of HCT-116cells.Conclusions:1.Abnormal aggregated p62actives autophagy which play a critical role in colon cancer tumorigenesis.2.Knockdown p62and autophagy inhibitors may have a therapeutic effect on colon cancer,and both have a synergistic effect.