Expression Of Rictor And Its Role In Invasion And Metastasis Of Renal Cell Carcinoma

ObjectivesMetastasis is a major cause of death among renal cell carcinoma (RCC) patients, but the mechanism is still unclear. In the present study, we detected the expression pattern of Rictor in human renal normal and tumor tissues, and evaluated its correlation with survival potential of RCC patients. Then we used the siRNA technology to knockdown the expression of Rictor to investigate the influence of Rictor down-regulation on chemotaxis and the related molecular mechanism.Methods1. The expression of Rictor was assessed by immunohistochemistry in a tissue microarray constructed from216RCC samples and46normal renal tissue as control to analyze the relationship between Rictor expression and clinicopathologic parameters, and evaluate the relationship between Rictor and prognosis by Kaplanr-Meier survival curve.2. Western Blotting analysis showed expression of Rictor in all the five RCC cell lines (786-O,769-P, ACHN, SN12C, HTB47).3. We designed a Stealth RNA duplex targeted against human Rictor to attenuate Rictor expression in ACHN cells, which was identified by Western Blotting analysis.4. Using MTT to detect the influence of Rictor down-regulation on ACHN cells proliferation.5. Chemotaxis assay and wound healing assay were performed to detect the role of Rictor in chemotaxis ability and cell movement.6. Adhesion assay was used to examine the effect of Rictor expression on cell adhesion of RCC cells to the extracellular matrix.7. Matrigel invasion assay was used to examine the effect of Rictor down regulation on ACHN cells invasion ability.8. Western Blotting analysis was used to detect the activation of Akt, Integrin β1and cofilin in control cells and siRictor cells.Results 1. There was a significant correlation between the strong Rictor expression and high tumor grades, metastasis (P=0.006). Kaplan-Meier survival analysis showed lower probability of survival in Rictor positive group compared with the negative group.2. Rictor protein was detected in five RCC cell lines by Western Blotting analysis. Three high metastatic human RCC cell lines, ACHN, SN12C and HTB47, apparently expressed higher level of Rictor than two primary RCC cell lines,786-0and769-P.3. Western blotting assay demonstrated reduction of Rictor protein levels in siRNA transfected ACHN cells in comparison with SCR cells and untransfected cells.4. Down regulation of Rictor impair the ACHN cells proliferation(P<0.001).5. Rictor-reduced ACHN cells showed a significant decreased chemotaxis and directional migration ability compared with controls(P<0.001).6. The number of adherent Rictor-reduced ACHN cells was significantly decreased compared with controls(P<0.001).7. Rictor-reduced ACHN cells showed a significant decreased invasion ability compared with controls(P<0.001)8. Western Blotting analysis revealed that down-regulation of Rictor abolished Akt-Ser473, cofilin and integrin β1phosphorylation in ACHN cells.Conclusions1. High expression of Rictor is associated with metastasis and poor prognosis of RCC. Rictor expression level is an independent prognostic factor in RCC.2. Down-regulation of Rictor impaired RCC cells chemotaxis, proliferation, migration, invasion ability, demonstrate that Rictor is involved in the regulation of RCC cells chemotaxis and matastasis.3. Rictor is an upstream signaling molecule of Akt. Rictor was involved in EGF-induced chemotaxis through the regulation of cofilin and integrin β1phosphorylation.4. Rictor serves as a novel and promising metastasis therapeutic target for RCC.