PLEKHO1 Rugulates RCC Cell Viability In Vitro And In Vivo,Potentially By The Hippo And MAPK/JNK Pathways

Objective: Kidney cancer involves nearly 337900 people worldwide annually and leads to almost 143400 deaths each year according to the latest data released by IARC in 2012.The disease is commonly palliative and uneventful course without distinct clinical symptoms and signs,which leads to the outcome that patients failed to be diagnosed in the initiated stage.For the inchoate small solid tumors,surgical excision by partial or radical nephrectomy is now recognized as a prefered choice.However,for the patients with metastatic neoplasm or unresectable tumor initially,the systemic treatment is needed.But,in the renal cell carcinoma(RCC),the most commonly histological type is clear cell renal cell carcinoma(ccRCC)that accounts for up to 90% of all renal malignancies.The ccRCC is originated from proximal convoluted tubule that highly express the multidrug resistant protein(MDR-1)which in turn lead to a high resistance of RCC to chemotherapy.On the other hand,although the development of drugs targeting VEGF and mammalian target of mTOR as well as the recent immunotherapy showing the promise,the complete responders are still rare.Although advances in signals and mechanisms of biological molecules have been brought into clinical practice,partial therapeutic effect to RCC patients who finally develop into drug resistance and relapse is still fairly far from desirable.As a consequence,it is essential to get insight into the mechanisms or the key regulator of RCC tumorigenesis and development,and to seek for novel potential curative target or to advance the present therapies to improve the clinical prognosis of RCC.Methods: 1.We downloaded RNA-seq raw data of KIRC from TCGA,and performed differencial expression analysis in R-project environment.Subsequently,we selected a number of differencially expressed genes into high-throughput content screening(HCS)for the genes obviously affecting RCC cell growth viability.2.We selected the genes functioning in RCC cell growth as the target genes.Combining the clinical information of RCC patients in TCGA,Kaplan-Meier and Log-Rank analysis was performed to demonstrate the relationship between the target gene and the clinical prognosis.Simultaneously,RT-PCR was performed to validated the expression profile of the target gene.3.The role in vitro of the target gene in the proliferation,apoptosis,metastasis was assessed by CCK-8,Celigo,flow cytometry and transwell assay.Meanwhile,we established xenograft nude mice model to investigate the proliferation function of the target gene in vivo.4.To explore the potential mechanism underlying the target gene promoting ccRCC cell growth,we performed mRNA microarray and co-expression analysis on the basis of TCGA data.Results: 1.Analyzing TCGA data,we detected that PLEKHO1 was distinctly upregulated in the tumor tissues.Additionally,we also found that PLEKHO1 downregulation in 786-O cell apparently abolished cell growth viability in HCS analysis.2.Kaplan-Meier and Log-Rank analysis showed: patients with highl PLEKHO1 expression posessed shorter median survival time than those with low PLEKHO1 expression.RT-PCR analysis showed the same PLEKHO1 expression profile as the result of TCGA analysis,3.With the help of RNAi technology,we artificially downregulated endogenous expression level of PLEKHO1 and found that Caki-1 and 786-O cells treated with RNAi showed growth activity dinstinctly blocked.4.Flow Cytometry assay showed that PLEKHO1 knockdown promoted Caki-1 and 786-O cell apoptosis.5.Xenograft tumor model showed that stable downregulation of PLEKHO1 in 786-O cells blocked the xenografts formation in which the 786-O cells treated with RNAi developed xenografts with lighter weight and smaller volume than those treated with non-RNAi.6.Through mRNA microarray and co-expression analysis,we predicted that PLEKHO1 potentially functioned through Hippo and MAPK/JNK signaling pathway.Conclusions: Collectively all the results revealed that PLEKHO1,as a stimulative factor implicated in renal cell carcinoma cell growth and apoptosis through regulating Hippo and MAPK/JNK signaling pathway,which was independent of the conventional viewpoint that PLEKHO1 usually acts as a negative regulator in tumors.The present experimental data provide an alien mechanistic insight into the RCC advance and disclosed a potential novel therapeutic guidance to the kidney cancer.