The Study On Phosphorylation And Distribution Of Rb In The Tumorigenesis And Development Of Colorectal Carcinoma

Recently, the incidence of colorectal carcinoma(CRC) is increasing steadily in China, and the mortality of CRC is ranking the top of malignant tumor. With the development of comprehensive treatment, the 5-year survival rate of CRC is about 30%, and the recurrence and metastasis are the most common reasons of death which affect long-term survival rate. Therefore, further research of the tumorigenesis and development can pave a way for the new therapy and improve the prognosis of CRC.Retinoblastoma gene (RB) is a tumor suppressor gene which was first cloned in the world, and it plays an important role in the regulation of cell cycle. Rb regulates each part of the cell cycle, and makes the cell cycle steady. Cdk4 which regulate the G1 phase is one of the members of Cyclin-dependent kinase. Augmentation, mutation and high expression of Cdks were found in some cancer cell lines. Cdk4 is relative to the phosphorylation and nuclear export of Rb, and the phosphorylation of Rb induced by Cdk4 leads to the inactivation of Rb. In the S phase, Rb is in the state of under-phosphorylation and exclusively distributes in the nucleus, but it becomes over-phosphorylated in the S phase and inactivated. There are some researches showing that Rb distributes strictly in the cell nucleus in each phase of the fibroblasts, and if induced with cancer gene, the distribution of Rb changes obviously.This research studies the mechanism and the effect of phosphorylation of Rb induced by cdk4 on the process of tumorigenesis and difference of the expression of Rb among normal mucosa, adenoma and cancer. Meanwhile, we explore whether the phosphorylation and change of distribution of Rb induced by Cdk4 may affect the malignancy of CRC. As a result, we want to learn about the tumorigenesis mechanism of CRC and make some theories for the targeted therapy. Objective:To learn if the expression of Rb and Cdk4 is different in the tissue of normal mucosa, adenoma and CRC.The tissues harvested in surgical operation by section of general surgery and endoscopy center of Zhongshan Hospital were collected. The expression of Rb and phosphorylated Rb in different tissue were evaluated by Western blot. The expression of Cdk4 is evaluated by Western blot and realtime RT-PCR.1. The expression of Rb and phosphorylated Rb in tissue of malignant tumor is higher than that in adenoma and normal mucosa, and there is no significant difference of expression between adenoma and normal mucosa.2. The expression of Cdk4 successively gets higher in normal mucosa, adenoma and malignant tumor.1. The expression of Rb and phosphorylated Rb is high in malignant tumor of CRC, indicating that as a tumor suppressor gene, Rb expresses high in the late stage of adenoma or the early stage of malignant tumor, and in that stage Rb gets phosphorylated and inactive, so that the tumorgenesis happends,but this phenomenon doesn't seen in the change from nomal mucosa to adenoma.2. The expression of Cdk4 successively gets higher in normal mucosa, adenoma and malignant tumor, and this indicates that Cdk4 has positive correlation with the process of tumogenesis of CRC. To observe the change of cell cycle and invasive activity of Love cell through regulation of Rb nucleus export and over-expression.Lovo cells are treated with Leptomycin B (LMB) to restrict RB in the cell nucleus.A plasmid with Cdk4R24C(an arginine-to-cysteine exchange at residue 24) was constructed, and transfect the Lovo cell with this plasmid. At last, we evaluate the change of cell cycle, and the invasive ability of the cell in both situations were evaluated through Transwell.1. Rb can be well restricted in the nucleus of Lovo cells treated with 30nM LMB.2. Lovo cells which Rb is strictly restricted in the nucleus show no significant change in the cell cycle and invasive ability when campared with the untreated cells.3. Lovo cells transfected with the Cdk4R24C plasmid show a significant change in the cell cycle and higher invasive ability when campared with untransfected cells.1. Alteration of distribution of Rb does not change the cell cycle and invasive activity of Lovo cells.2. Over-phosphorylated Rb in Lovo cells can change the cell cycle, and make a transition from G1 to S phase. Over-phosphorylated Rb is positively relative with invasive ability of Lovo cell.