Role Of CCN3(NOV) In Clear Cell Renal Cell Carcinoma

Renal cell carcinoma (RCC, also known as renal adenocarcinoma) is a kidney cancer that originates in the lining of the proximal convoluted tubule.85%of RCCs are clear cell RCC (ccRCC), others are granular cell carcinoma or mix. RCC is the most common malignancy in adult kidney, represent3%of all human malignancy, and accounts for over85%of renal cell carcinomas. Furthermore, approximately25%of patients will present with metastatic disease. The metastatic may happened in the very early stage of RCC, even when the size of tumor is very small. Thus an accurate prognosis may be difficult to establish only by TNM or histological grade grading of tumor. Currently, there are several molecular markers can be used as predictors of survival for patients with RCC, including Ki67, VEGF, P27. However, these markers often provide low accuracy in predicting prognoses of RCC, and an independent, reliable prognostic marker which can reflect the tumor metastasis ability is needed by clinical doctors. We used to choose the surgery procedure (nephrectomy or nephron sparing surgery) only by the size of the tumor (whether the tumor is above4cm or not). However, the limit of this method has been recently proposed by some researchers. A novel marker which can reflect the tumor metastasis ability and prognoses is urged by doctor for choosing the surgery procedure.The ccn3gene (NOV nephroblastoma overexpressed gene) is one of the members of the CCN family. The CCN family comprises cysteine-rich61(CYR61/CCN1), connective tissue growth factor (CTGF/CCN2), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1(WISP-1/CCN4),-2(WISP-2/CCN5) and-3(WISP-3/CCN6). The proteins that constitute the CCN family are secreted regulatory proteins. These proteins play roles in a lot of important physiological activities including stimulate the development of cardiovascular system of embryon, increase the formation of bones and cartilage, and stimulate the vessel formation, tissue repair, inflammation in adult. In recent research, The CCN3expression plays a role in carcinoma on proliferation, adhesion, migration and invasion in many carcinoma cell types. It has a great value to the diagnosis and prognosis of carcinoma. According to our earlier study, CCN3has an abnormal expression in ccRCCs, suggesting that CCN3may play a role in process of ccRCC development, such as initial, progress, metastasis, of cancer. Prognostic value of CCN3in osteosaroma and breast cancer has been reported these days. The level of CCN3expression has some relationship with ccRCC's staging and grading, suggesting that CCN3may be a potential prognosis factor of ccRCC and has a great clinical value.In this study, we made CCN3overexpressed in clear cell RCC cell line786-0by using lipofectin transfection method, then studied its effect on cell proliferation, adhesion, migration and invasion. The molecular biological mechanism has also been studied. The expression of matrix metalloproteinases1,2,3,7,9,13(MMP-1,2,3,7,9,13), Ki67, VEGF, COX-2, P27in these3groups has been tested by using Real-time PCR. On the other hand, a custom tissue array was constructed from40ccRCC patients who underwent nephrectomy. Clinical and pathological variables of all patients were retrospectively reviewed. Immunohistochemistry was performed to study the prognosis value of CCN3, the correlation of CCN3and other bio-markers such as Ki67, VEGF, COX-2, P27also has been studied.Part1. The role of nephroblastoma overexpressed gene on clear cell renal cell carcinoma in proliferation, adhesion, migration and invasion.Objective To investigate the effects of nephroblastoma overexpressed (NOV) on proliferation, adhesion, migration and invasion of ccRCC cells.Methods We constructed a NOV expression plasmid and transfected the plasmid into ccRCC cell line786-0, then used the fluorescence microscope to monitor the transfection rate, and used G418to screen cells. CCN3overexpression has been verified by RT-PCR (gene level) and Western-Blot (Protein level). Then we analyzed the effects of NOV expression on proliferation, adhesion, migration and invasion of RCC cells by drawing growth curve, WST-1assay, cell adhesion assay, matrigel invasion assay and transwell migration assay.Results CCN3expression in p-EGFP-Cl-NOV transfected786-0cells (786-O-CCN3) were higher than the empty plasmid transfected786-0(786-O-empty vector) and control (786-0) on gene level and protein level (P<0.05), while there was no difference between empty plasmid transfected786-O and control.The proliferation activities of786-O-CCN3 cells were inhibited by29.14%and32.46%at48h and72h, respectively, compared to786-0cells (P<0.05); while the The proliferation activities of786-O cells transfected with empty vector(786-O-empty vector) were9.25%and-8.16%,and have no difference with786-O cells (P>0.05). Adhesion assay indicated significantly increased adhesion of786-O-CCN3cells to fibronectin (0.26±0.03) and laminin (0.28±0.04, compared to786-O cells(0.15±0.01,0.12±0.10) and786-O-empty vector cells(0.14±0.02,0.13±0.08). Invasion assay displayed that the numbers of cells penetrated through matrigel membrane were significantly higher in786-O-CCN3cells (240.25±23.12) compared to786-0cells(56.16±6.25) and786-O-empty vector cells(50.28±7.13). Migration assay displayed that the numbers of cells passed through polycarbonate filters were significantly higher in786-O-CCN3cells (267.25±20.94) compared to786-O cells(66.10±5.68) and786-O-empty vector cells (56.28±4.11)Conclusions Our data indicated that CCN3exhibits anti-proliferative effects on RCC cells, however, it promotes adhesion, migration and invasion of RCC cells Part2. Correlations of CCN3expression and other bio-markers such as Ki67, VEGF, P27,COX-2and MMPs expression in ccRCC.Objective To study the correlations of CCN3expression and other bio-markers such as Ki67, VEGF and P27expression in ccRCC.Methods Real-time PCR has been performed to stuay mRNA level of MMPs, Ki-67, P27, COX-2and VEGF in786-O,786-O-empty vector and786-O-CCN3cells. A custom tissue array (TMA) was constructed from40ccRCC patients who underwent nephrectomy.2benign renal tissue also has been subjected as control. Clinical and pathological variables of all patients were retrospectively reviewed and a data base has been established. Immunohistochemistry was performed for CCN3expression. Staining intensity are given as positive rate. CCN3expression levels were measured by using image pro-plus software. Other bio-markers such as Ki67, VEGF, P27and COX-2also have been stained by using Immunohistochemistry. The correlation of CCN3expression and these bio-markers has been studied. Results The expression of MMP-1, MMP-3, MMP-9, P27and COX-2in786-O-CCN3cells were significantly higher than786-O-empty vector and786-O cells (P<0.05). The expression of Ki-67, VEGF in786-O-CCN3cells were significantly lower than786-O-empty vector and786-O cells (P＜0.05). The Ki67, VEGF, P27and COX-2expression in ccRCC tissues has significant correlations with CCN3expression according to Pearson correlation test (P<0.05).Conclusions The CCN3expression level has correlation with Ki67, VEGF, P27, COX-2and MMPs indicating they may might be the molecular biology basis of the function of CCN3on ccRCC. Part3. The prognosis value of CCN3in clear cell renal cell carcinomaObjective To study the prognosis value of CCN3in clear cell renal cell carcinoma by using custom tissue array and clinical information.Methods A custom tissue array (TMA) was constructed from482RCC patients who underwent nephrectomy.10benign renal tissues also have been subjected as control. Clinical and pathological variables of all patients were retrospectively reviewed and a data base has been established. Immunohistochemistry was performed for CCN3expression. Staining intensity are given as expression scores.CCN3expression was categorized as high if ccRCC tissue stained stronger than the respective level of the corresponding benign tissue and categorized as low if ccRCC tissue stained less than or equal to the corresponding benign tissue. Differences in caner-specific survival (CSS) were analyzed by log-rank analysis.Results The CSS of high CCN3expression groups were significantly lower than low CCN3expression groups.Conclusions The expression of CCN3has a close relation with the prognosis of ccRCC, high CCN3expression level indicate poor prognosis of ccRCC, it can be used as a novel prognosis factor.