KLF9 Mediates MiR-140-5p To Be Involved In The Progression Of Renal Cell Carcinoma

Objectives: Renal cell carcinoma(RCC)accounts for approximately 4% of all cancers and has the highest mortality rate among all urological cancers.However,the pathogenesis of RCC is still unclear.micro RNAs(mi RNAs)belong to nc RNAs,which play important roles in the pathogenesis of RCC,and were reported to be involved in tumorigenesis by modulating the expression of downstream target genes.In the previous study,we found that the expression of mi R-140-5p in RCC tissues was significantly higher than that in adjacent normal tissues,and mi R-140-5p promoted the growth and metastasis of RCC in vitro.Additionally,Krüppel-like factor 9(KLF9)is one of target genes of mi R-140-5p.Therefore,whether KLF9 mediates mi R-140-5p to participate in the progression of RCC needs to be further confirmed,and the transcriptional role of KLF9 in RCC worth further exploring.Methods: All clinical samples(216 cases)of renal cell carcinoma underwent radical nephrectomy in Peking University Shenzhen Hospital from 2014 to 2018 were collected,and the paracancerous tissues obtained were more than 5cm away from the edge of the cancer tissue.This study was approved by the Ethics Committee of Peking University Shenzhen Hospital.All patients agreed that their samples could be used for experimental research and paper presentation,and signed informed consent before sampling.Carcinoma tissues and adjacent paracancerous tissues of 74 patients with RCC were randomly selected from 216 cases.The overall survival of the patients within3～5 years after surgery was followed up by telephone and outpatient service.None of the patients did receive radiotherapy,chemotherapy,immunotherapy or targeted therapy before or after surgery.We detected the expression of the target gene in RCC tissues and cells by quantitative real-time PCR(RT-q PCR).The proliferation of RCC cells was detected by cell counting kit-8(CCK-8)assay and colony formation assay.The migration and invasion ability of RCC cells were assessed by wound-healing assay and transwell assay.Western blotting and immunohistochemistry(IHC)were used to detect the expression level or enrichment of proteins.Chromatin immunoprecipitation assay(Ch IP)and dual-luciferase assay were used to investigate the molecular mechanism of mi R-140-5p in RCC.Tumor formation assay and tumor metastasis model in vivo were constructed in nude mice.The data were analyzed using SPSS 22.0.Student’s t test,ANOVA or chi-square test was used to analyze group differences.Overall survival and disease-free survival curves were analyzed by the Kaplan-Meier method and log-rank test.Results: RT-q PCR showed that the expression of mi R-140-5p was significantly increased in RCC tissues and cell lines.And the tumor diameter and postoperative metastasis rate of RCC patients in the group with high mi R-140-5p expression were significantly higher than those in the group with low mi R-140-5p expression.Survival analysis showed that overall survival time was significantly reduced in the group with high mi R-140-5p expression.Knockdown of mi R-140-5p significantly reduced the proliferation,migration and invasion ability of RCC cell lines.Accordingly,overexpression of mi R-140-5p significantly enhanced the proliferation,migration and invasion of RCC cells.We predicted candidate target genes for mi R-140-5p by bioinformatics analysis,in which KLF9 expression was negatively correlated with mi R-140-5p.RT-q PCR and Western blot assay showed that the expression levels of mi R-140-5p and KLF9 were inversely correlated in RCC in vitro.Moreover,dual-luciferase assay confirmed two effective binding sites between mi R-140-5p and KLF9 in the 3’UTR of KLF9.The inhibitory effect of knockdown of mi R-140-5p on the malignant phenotype(proliferation,migration and invasion)of RCC cells could be reversed by the knockdown of KLF9.In combination with bioinformatics,RT-q PCR and western blotting assay,we found that KLF9 was positively correlated with post-transcriptional expression of KCNQ1(also known as KV7.1 and KVLQT1)in RCC tissues and cells,and may be a transcriptional activator of KCNQ1.Further CHIP assay and dual-luciferase assay revealed significant KLF9 enrichment at-841/-827 in the KCNQ1 promoter region.In addition,the overexpression of KCNQ1 expression significantly inhibited the proliferation,migration and invasion of RCC cells.In xenograft tumor formation experiment and lung metastasis experiment in nude mice,knockdown of mi R-140-5p significantly inhibited the proliferation and metastasis of RCC in vivo,and the expressions of KLF9 and KCNQ1 were correspondingly increased in xenograft tumor tissues.Conclusions: In the present study,we found that mi R-140-5p promoted the proliferation and metastasis of RCC cells in vivo and in vitro.Further studies showed that mi R-140-5p inhibited the expression of KLF9 by specifically binding to the 3’UTR of KLF9,thereby regulating the growth and metastasis of RCC cells.KLF9 promotes the transcription of KCNQ1,through binding to the promoter region(-841/-827)of KCNQ1.In summary,KLF9 mediates mi R-140-5p to be involve in the progression of RCC,and promotes the transcription of downstream target gene KCNQ1 in RCC.Our study provides a potential target for the diagnosis and therapy of RCC.