The Mechanism Of LncRNA HOTTIP As A Cerna In The Development Of Clear Cell Renal Cell Carcinoma

Background: Renal cell carcinoma(RCC)is one of the most common urological malignant tumors and clear cell renal cell carcinoma(ccRCC)is the most common pathological type.Up till now,there is still no effective treatment for patients with metastatic and recurrent ccRCC.The ceRNA hypothesis reveals a new mechanism for RNA-RNA interactions.However,reseaches on lncRNA as a ceRNA in ccRCC are still rare.Therefore,investigating the aberrantly expressed lncRNAs with important biological functions and its mechanisms will help to clarify ccRCC pathogenesis,and provide new ideas and potential targets for the diagnosis and treatment of ccRCC.Objective: Screen abnormally expressed genes based on the bioinformatics analysis of ccRCC gene expression profile in TCGA database.Construct a ceRNA regulatory network of DElncRNA-DEmiRNA-DEmRNA in ccRCC and identify key genes in network closely associated with ccRCC prognosis.Then,one of the key lncRNA was selected as the target,and its role and mechanism as ceRNA inthe developmentc of ccRCC was validated by experiments.Methods:(1)The gene expression profiles and clinical information of ccRCC patients were downloaded from TCGA database.DElncRNAs,DEmRNAs and DEmiRNAs were identified using “edge” package in R software.GO and KEGG enrichment analyses were performed using STRING.A PPI network based on DEmRNAs was constructed using STRING,and the key DEmRNAs and their relative signal pathways were identified by “clusterProfiler” package in R version 3.6.0.Then,the independent prognostic RNAs related to overall survival(OS)in ccRCC were identified by univariate and multivariate COX regression analyses.One of the independent prognostic LncRNAs was identified as the target lncRNA and the correlation between its expression level and clinicopathological characteristics was also evaluated.(2)The expression levels of target lncRNA in ccRCC tissues and cell lines were detected by qRT-PCR,and the correlation between HOTTIP expression and clinical characteristics was also verified.(3)Establish cell lines with overexpressed or knockdown target lncRNA,and the biological functions of target lncRNA in ccRCC were assessed by MTT assay,colony formation assay,flow cytometry,Hoechst straining,Transwell assay,and xenograft mouse model.(4)Dual luciferase reporter gene assay,Fluorescence in situ hybridization(FISH),cell cytoplasm/nucleus separation experiment,AGO2-RNA binding protein immunoprecipitation(RIP),and otherexperiments were used to verify that target lncRNA was able to affect the development of ccRCC by sponging miRNA and targeting gene.Results:(1)A total of 1517 DElncRNAs,1331 DEmRNAs and 83 DEmiRNAs were confirmed by “edge” package in R version 3.6.0.A ceRNA network containing 100 DElncRNAs,14 DEmiRNAs and DEmRNAs was constructed.11 DElncRNAs,3 DEmiRNAs and 4DEmRNAs in ceRNA network were found to be closely related to ccRCC prognosis.LncRNA HOTTIP was identified as target gene,and its expression level was significantly associated with pathological grade,stage and recurrence/progression.And lncRNA HOTTIP/miR-506/VIM was selected to experiments validations.(2)The expression level of lncRNA HOTTIP in 28 pairs of ccRCC tissues and paracancerous tissues,as well as renal cell lines and normal renal tubular epithelial cell line was detected by qRT-PCR,which verified that the expression levels of lncRNA HOTTIP were significantly increased both in ccRCC tissues and cell lines,and was significantly correlated with Furhman pathological grade,clinical stage and T stage.In vitro experiments suggested that lncRNA HOTTIP could promote proliferation,migration,invasion,and inhibit apoptosis in ccRCC cells,while in vivo experiments confirmed its oncogenic roles in ccRCC.(3)The results of FISH and cell cytoplasm/nucleus separation experiment showed that lncRNA HOTTIP was mainly located in the cytoplasm in786-O cell.Dual luciferase reporter gene assay and RIP demonstrated thatlncRNA HOTTIP could directly bind to miR-506-3p,interacting with RNA-induced silencing complex.And VIM directly binding to miR-506-3p was also verified by dual luciferase reporter gene assay.Next,qRT-PCR and Western Blot were used to detect the expression changes of target gene VIM in each vector at the levels of RNA and protein respectively.The results showed that the expression of target gene VIM was positively correlated with lncRNA HOTTIP,while miR-506-3p could reduce the regulatory effect of lncRNA HOTTIP on the increase of VIM expression.Finally,the rescue experiments showed that ectopic expression of miR-506-3p reversed the effects of proliferation,invasion,migration,and apoptosis in Caki-1 cells.Conclusion: In our study,the ceRNA regulatory network was successfully constructed and lncRNA HOTTIP/miR-506/VIM was identified as the taget and validated by experiments.lncRNA HOTTIP could act as ceRNA,promoting ccRCC cell proliferation,invasion and migration by sponging miR-506-3p and targeting VIM.The present study provides a novel direction for the research of ccRCC molecular mechanism and provides a new potential target for ccRCC diagnosis and treatment.