The Functional And Mechanism Investigation Of Rictor In Renal Cell Cancer Metastasis

Background and Objective: Renal cell carcinoma(RCC) is the most common tumor in kidney tissue, and cancer metastasis accounts for more than 50% of the death among renal cell carcinoma patients. Recently, more and more research indicated cytokines, especially TNFa could increase renal cell carcinoma metastasis, but the underlying molecular mechanism is largely unknown. Rictor is the key molucular of m TORC2,and our previous study showed Rictor played an important role in breast cancer metastasis, however, the function and potential mechanism of Rictor in RCC metastasis remains to be determined.In this study, we first investigate the functional role of Rictor durning the renal cell carcinoma metastasis in vivo and in vitro. In the second part of this study, we focus on the alteration of RCC cells in metastasis ability after TNFa treatment using cell biology and molecular biology experments. Further more, we investigate the role of Rictor durning this process for the purpose of understanding the molecular mechanism of Rictor in the phenomenon that TNFa change tumor cell metastasis ability. We hope this research can deepen our understanding in the molecular mechanisms of tumor metastasis, and strive to provide new ideas for the early detection and treatment for clinicians in tumor metastasis.Methods: 1) Western Blotting and RT-PCR were used to detect Rictor down-regulation efficiency in ACHN cells after lenti-virus infection.2) Scratch assay, chemotaxis assay and invasion assay were uesd to detect changes in metastasis ability of Rictor stable knock-down cells in vitro.3) Xenograft tumor transplant mouse model in SCID mice was uesd to detect changes in metastasis ability of Rictor stable knock-down cells in vivo.4) Scratch assay, chemotaxis assay and invasion assay were applied to investigate the effects of TNFa treatment on ACHN cell motility, and the function of Rictor in this process.5) Western Blotting and RT-PCR were used to detect the expression of Rictor in m RNA and protein level after TNFa treatment.6) Western Blotting and immunofluorescence techniques were used to investigate the expression and the phosphorylation of IkBa, and the translocation of p65 in ACHN cells after TNFa treatment.7) Dual luciferase repoter assay and ChIP assay were used to investigate the transcriptional regulation of RICTOR.Results: 1) After lenti-virus infection, the expression of Rictor was knock-down in ACHN cells.2) Down-regulation of Rictor impaired tumor cell migration, chemotaxis and invasion ability in vitro.3) Down-regulation of Rictor impaired tumor cell metastasis in vitro.4) TNFa treatment could increase ACHN cell migration, chemotaxis and invasion ability, whereas down-regulation of Rictor could impaired the enhancement of these ability.5) The expression of Rictor is increased in mRNA and protein level after TNFa treatment.6) After TNFa treatment, IkBa could be degraded, and the phosphorylation of IkBa could enhanced in ACHN cells, besides, p65 could translocate from the cytoplasm to the nucleus.7) Dual luciferase reporter assay showed-51 bp to-301 bp region in the upstream of RICTOR played a vital role in transcriptional activity. And p65 could enrich at the predictive binding site in this region.Conclusions: 1) Rictor played a key role in ACHN cell metastasis in vivo and in vitro.2) TNF? could promote ACHN cells metastasis in vitro, probably by means of up-regulation Rictor expression through NF-?B pathway.