The Research Of ANKRD6 Regulates The Mechanism In The Proliferation Of Colorectal Cancer Cells

ObjectiveColorectal cancer is one of the common malignant tumors of the digestive tractin the world today,which seriously endangers human health.Colorectal cancer has become the leading cause of death among cancer patients worldwide.In Western countries,colorectal cancer is the second leading cause of cancer death.The latest report shows that the incidence of colorectal cancer is on the rise in Asia.Colorectal cancer is the fifth leading cause of cancer death in China.In economically developed cities,this proportion is even higher.At present,the treatment of colorectal cancer includes surgical treatment,radiotherapy and chemotherapy,of which surgery is the main treatment,but there are still 20-30%of patients with recurrence.Therefore,the study of tumor-specific markers of colorectal cancer provides new targets and new ideas for clinical diagnosis and treatment,and has important theoretical and practical significance.Diversin is also known as ankyrin repeat domain protein 6(ANKRD6).Previous studies have shown that Diversin is significantly higher in non-small cell lung cancer,breast cancer,and bladder cancer than in adjacent tissues.Our previous study found that Diversin is overexpressed in non-small cell lung cancer and plays a role in the development of tumorigenesis.At the same time,the gene chip results show that the target gene ANKRD6 regulates the cell cycle on the cell:G1-S phase checkpoint.At present,the expression and significance of Diversin in human colorectal cancer are still unclear.Whether it has the role of regulating tumor cell proliferation and its molecular mechanism remains to be further explored.This study aimed to investigate the role of Diversin in the proliferation of colorectal cancer cells and to explore its molecular mechanisms.MethodsThe specimens from the Affiliated Central Hospital of Shenyang Medical College were surgically removed from 2010 to 2012,and the pathologically confirmed wax specimens of colorectal cancer were used as research objects.The study was reviewed and approved by the local institutional ethics committee of Shenyang Medical College.We collected cancer and paracancerous tissue samples from the same patient and detected the expression level of Diversin in colorectal cancer tissues and adjacent tissues by immunohistochemistry.To further investigate the effect of Diversin on cell proliferation,we used human colorectal cancer cell HCT116 to infect Diversin overexpression lentivirus(LV-ANKRD6)and negative control lentivirus(LV-NC)and human colorectal cancer cell LOVO cells to infect Diversin knock Low lentivirus(LV-ANKRD6-RNAi)and negative control lentivirus(LV-siNC).MTS,flow cytometry and Western blotting were used to compare the effects of up-regulation and down-regulation of Diversin on proliferation,cell cycle and related protein expression of colorectal cancer cells.ResultsImmunohistochemistry showed that Diversin was highly expressed in human colorectal cancer tissues and was significantly associated with malignant tumor differentiation,clinical stage and lymph node metastasis(P<0.05).However,there was no statistical difference between Diversin overexpression and age,gender,and tumor location.MTS results showed that overexpression of Diversin promoted the proliferation of colorectal cancer cells(P<0.05),while down-regulation of Diversin inhibited the proliferation of colorectal cancer cells.The distribution of cell cycle stages measured by flow cytometry showed that the percentage of G1 phase cells in HCT116 cells with Diversin overexpression was decreased compared with control cells,while the percentage of cells in S phase was significantly increased(G1:64.26%vs 47.42%,S:14.53%vs 36.89%,P<0.05),while in Diversin knockdown LOVO cells,the percentage of cells in G1 phase increased,and the percentage of cells in S phase decreased(G1:30.15%vs 81.8%,S:52.80%vs 9.14)%,P<0.05).The results of western blot showed that the expression level of P21 and P27 protein in the colorectal cancer cell HCT116 overexpressing Diversin was decreased,and the expression activities of cyclinD,CDK4 and cyclinE,CDK2 were increased.At the same time,the expression level of ?-catenin protein did not change significantly,while the ratio of p-JNK/JNK increased.In the down-regulation of Diversin's colorectal cancer cells LOVO,P21,P27 protein expression levels increased significantly,cyclinD,CDK4 and cyclinE,CDK2 expression levels decreased.At the same time,there was no significant change in the expression level of ?-catenin protein,while the ratio of p-JNK/JNK decreased.ConclusionDiversin is highly expressed in human colorectal cancer tissues and is positively correlated with malignant tumor differentiation,clinical stage and lymph node metastasis.Diversin regulates CRC cell proliferation through P21,P27,cyclinE,CDK2,cyclinD,CDK4,and p-JNK.We speculate that Diversin may function through G1-S checkpoints and non-canonical JNK pathways.