| Background and Objective:Renal cell cancer accounts for about 3% of all cancer, its mortality rate is as high as 40%, in recent years the incidence of kidney cancer steadily increase 2 ~ 4% a year. To the current method for the treatment of kidney cancer, the medical community now agree that radical nephrectomy is possibly a method of curing kidney cancer [7-11], but a variety of surgical procedures has a relatively good results only for those in a early clinical stage. While in clinical practice, a considerable part of patients emerged distant metastasis, venous thrombosis or manifestations of later period when treatment, surgery can not resect tumors in vivo completely, surgical results are poor, 5-year survival of patients is not high. Therefore, application of molecular targeted therapy becomes more and more widespread and the study of the mechanism in renal cell carcinoma proliferation and invasion is particularly necessary for the next targeted therapy.Rho associated coiled-coil forming protein kinase 2 plays a key role in the Rho / Rock signaling pathway and the development of cancer. what’s more, Class A scavenger receptor member 5, SCARA5, related with varieties of tumor’s formation,invasion and metastasis and so on, known as "understated tumor suppressor gene", plays an inhibition action in the proliferation of tumor cells. These two important genes play a very extensive and important effect and are closely related to the occurrence and development of tumors in our body. RCC has a strong ability of proliferation, we infer that Rock2 probably regulate the proliferation of kidney cancer by adjusting the expression of SCARA5.Reducing Rock2 expression in renal cancer cells can lead to increased expression of SCARA5 through gene chip filter in our pre-period. This study detected ROCK2 expression in Clinical renal cancer tissue firstly, and retrospectively analyzed the clinical characteristics and prognosis of these patients, and thereby analyzd the relationship between ROCK2 and survival in renal cell cancer patients; Secondly, we made the effect on renal cell cancer proliferation of ROCK2’s regulating the expression in SCARA5 clear through experiments in vitro or vivo; Finally, we discussed the molecular mechanisms about how Rock2 regulate SCARA5. Method:1.In our study, fluorescence quantitative PCR, Western blot and immunohistochemical method were performed to detect the expression levels of Rock2 mRNA and protein in 118 cases of renal carcinoma tissues through surgery resection,adjacent tissues and renal carcinoma 786-O, 769-1 and Caki cells from patients who were treated at the Department of Urology, the Second Affiliated Hospital of Nanchang University betwen January 2002 to January 2010, then, analyzing the results statistically, follow-up patients with phone, and retrospectively analyzing the clinical and pathological features in all patients, summarizing the relationship between ROCK2 expression and clinicopathological features and survival rate in patients of renal cell carcinoma;2. Lower expression of Rock2 in renal carcinoma, use the EDU and the plate colony experiment to observed renal carcinoma cell proliferation’s change; Build subcutaneous tumor model of mice with stably transfected shRock2 renal carcinoma cell line and non-transfected renal cell carcinoma strain, observe the growth change of mice subcutaneous tumors;3.After silencing and overexpressing Rock2 in renal cancer cells, fluorescence quantitative PCR and Western blot were utilized to detect the change of SCARA5 mRNA and protein’s expression in renal cell carcinoma; When silencing the expression of Rock2, we suppressed the expression of SCARA5, and while overexpressing Rock2, we increased the expression of SCARA5. We observed ROCK2 and SCARA5 expression’s changes and renal cancer cell’s proliferation.4.Using co-immunoprecipitation method to confirm whether ROCK2 had a direct effect on SCARA5, western blot and luciferase gene reRock2 port or not, we proved that the regulation of SCARA5 by Rock2 was achieved through β-catenin / TCF4 pathway.Result:1. ROCK2 was high expression in 71 cases of renal cell carcinoma(positive rate 60.2%), and only 25 cases of the adjacent cancer tissue were found expression(positive rate 21.2%); The differences of positive expression in ROCK2 has nothing to do with patients’ age, sex, symptoms, tumor size and location, whether or not having invasion of adrenal or vascular histologically or tumor’s necrosis, but closely related to the clinical stage, tumor grade, tumor recurrence and the risk factor; what’s more, positive expression of the gene are closely related to the low survival rate of the group of kidney cancer patients.2. Transfecting shRock2 into 786-O cells and 769-1 cells or adding Rock2 specific inhibitor(Y27632) to the two cell renal carcinoma cell, proliferation decreased significantly(p <0.05); Also animal experiments found that silenceing Rock2 expression group established nude mice subcutaneous tumor decreased significantly(p <0.05) in terms of both volume and weight compared to the control group, which the tumor was formed by simple kidney cancer cells.3. We also discovered that after reducing the expression of ROCK2 in kidney cancer 769-1 and 786-O cells, SCARA5 mRNA and protein expression were increased significantly(p <0.05);but after stabilizing overexpression of ROCK2, SCARA5 mRNA and protein expression significantly reduced. Instead, reducing the expression of SCARA5 can reverse the lowering of proliferation of cancer cells caused by silencing Rock2 expression, and increasing expression SCARA5 can reverse the enhancement of renal cell carcinoma proliferation caused by Rock2’s overexpression.4. Co-immunoprecipitation, luciferase gene reporter and Western blot confirmed that Rock2 does not act upon SCARA5 directly in the proliferation of renal cell carcinoma, its regulating the expression of SCARA5 is achieved throughβ-catenin / TCF4 signal path. Conclusion:Rock2 can regulate the proliferation of kidney cancer, that the mechanism is achieved throughβ-catenin / TCF4 pathway’s acting upon SCARA5. This mechanism is expected to provide new ideas for the studies of renal cancer cell proliferation reasearch, also being potential to open a new chapter for kidney cancer molecular targeted therapy. |
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