| Background: Renal cell carcinoma(RCC)is one of the most common tumor of genitourinary cancers and the 7th most commonly confirmed male cancers in America.Renal cell carcinoma has the highest incidence and mortality in malignant tumor of genitourinary tract.Cancer statistical data have indicated an estimated 58,000 new cases and 13,000 deaths from renal cell carcinoma occurred in 2011 worldwide.Furthermore,Renal cell carcinoma is more prevalent than other urinary tract carcinomas,make up 3.0 percent of all cancers in China and the occurrence has gradually increased.Clear cell renal cell carcinoma(ccRCC)is the primary histopathological subtypes of renal cell carcinoma.Many risk factors play important roles in the formation and promotion of clear cell renal cell carcinoma,which include exposure to diabetes,adiposis,hypertension,cancer-causing chemicals and smoking.However,the explicit mechanisms of the formation of clear cell renal cell carcinoma are not fully understood.Therefore,it is imperative to identify the mechanisms of clear cell renal cell carcinoma and to detect the possible signaling molecular connections with development of cancer.Janus kinase2/signal transducer and activator of transcription3(JAK2/STAT3)signaling pathway have been found to play an important role in the occurrence and development of a variety of tumors.Recent study have revealed that the proliferation,migration and invasion of renal carcinoma cells were reduced via the inhibition of JAK2/STAT3 signaling pathway.Therefore,the inhibition of JAK2/STAT3 is an effective therapeutic target on account of the signaling pathways are often overexpression in numerous cancers.Klotho,a transmembrane protein,was initially verified in 1997.Klotho is an newly anti-aging gene,which expressed in various organs and tissues including the distal renal tubules,thyroid gland,brain,lung and endocrine organ.An extracellular domain and an intracellular domain constitute the basic structure of Klotho,and the former is composed of two homologous domains(KL1 and KL2),however there is no clear functions exist in the intracellular domain.Klotho have been proved to involve in various diseases which containing acute kidney injury,chronic kidney diseases,cardiovascular disease,adiposis,cerebral arterial thrombosis and diabetes.Furthermore,Klotho has been shown to be down-regulated in various malignant tumors with poor prognosis,including cervical cancer lung cancer,breast cancers,liver cancer,colorectal carcinoma and pancreatic carcinoma.Overexpression of Klotho can inhibit the proliferation of lung cancer cells and caused cell growth arrest or apoptosis,which could serve as a potential target for cancer therapy.However,the biological effect of Klotho in clear cell renal cell carcinoma and the correlation between Klotho and JAK2/STAT3 signaling pathway have not been elucidated.The purpose of our study was to investigate the effects of Klotho of anti-tumour and explore its cellular mechanism in human renal carcinoma cells.In the present study,we found that the expression of Klotho significantly decreased in human renal carcinoma tissues and JAK2/STAT3 were identified as a direct downstream target of Klotho.Overall,the results suggest that Klotho is a vital element regulating the tumor proliferation,metastasis and invasion of clear cell renal cell carcinoma via JAK2/STAT3 signaling pathway,representing that Klotho could be supposed to be a potential therapeutic agent in the treatment of cell renal cell carcinoma.Objective: In our study,we investigate the effect of Klotho overexpression on cell viability,apoptosis,migration and invasion of 786-O and A498 cells.Further investigation of the underlying mechanisms indicates JAK2/SAT3 pathways play key roles in these actions.Methods: Real-time quantitative PCR and immumohistochemical staining was used to detect the expression of Klotho in para-carcinoma tissue and tumor tissue,and all the tissues were derived from the pathological specimens of 58 cases of ccRCC patients.Using two different renal cancer cell lines,A498 and 786-O transfected with Klotho,the anti-tumor effect of Klotho overexpression was determined by CCK-8assay and the apoptosis induced by Klotho overexpression was detected by cytometric apoptosis assays in vitro.Then,transwell invasion and migration assay were used to detect anti-metastasis effect of Klotho overexpression.the protein level of JAK2/TAT3 and epithelial-mesenchymal transition(EMT)-associated proteins were examined by Western blot analysis.A xenografl model was used to determine whether Klotho overexpression inhibits tumor growth in vivo.Results: The result of Real-time quantitative PCR and immumohistochemical staining indicated that the expression level of Klotho in RCC tissue is significantly lower than that in para-carcinoma tissue.With cell viability and flow cytometry assays,we found that Klotho overexpression potently suppressed cell growth of A498 and 786-O cells.The result of transwell assay and western blot assay suggested that the migration,invasiveness and epithelial-mesenchymal transition of tumor cells were.inhibited by Klotho overexpression.Consistently,the xenograft model performed in nude mice exhibited reduced tumor growth with Klotho overexpression treatment.Furthermore,We examined the change of JAK2/STAT3 phosphorylation caused by the treatment with Klotho overexpression.We found that the levels of phosphorylated JAK2 and STAT3 were remarkably decreased both in vitro and in vivo.To determine whether Klotho overexpression played its anti-tumor effects via JAK2/STAT3 pathway,we applied IL-6 to activate JAK2/STAT3 signaling pathway in786-O cells.IL-6 could significantly improve phosphorylation of STAT3 in 786-O cells.And Klotho inhibited proliferation and metastasis in 786-O cells could be reversed by IL-6,confirming that STAT3 plays a crucial role in the viability,proliferation and metastasis in 786-O cells.Conclusions: Firstly,our study have verified that Klotho expression in RCC tissues and cells was down-regulated compared with that in normal kidney tissue and normal renal tubular epithelial cells.Subsequent our study demonstrates that Klotho overexpression can markedly exhibit pro-apoptotic and metastasis inhibitory effect on ccRCC cells.In addition,we found JAK2/STAT3 pathways account for the anti-tumor effects of Klotho overexpression.Our study provides evidence for the therapeutic potential of Klotho as renal cancer treatment. |
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