Construction Of LncRNA-Based Prognosis Prediction Model And Studies Of Functional Role And Regulatory Mechanism Of LncRNA RCAT1 In Renal Cell Carcinoma

BackgroundRenal cell carcinoma(RCC),arising from the tubular epithelium of kidney,is one of the most common malignancies of the urinary system and accounts for 80%to 90%of renal malignancies.Based on the latest statistics of the American Cancer Society,renal cell carcinoma ranks sixth among male malignant tumors and ranks ninth among female malignant tumors,posing a serious threat to human life quality and physical condition.In China,there were 66,800 new cases of renal cell carcinoma and 23,400 death cases every year,and the incidence and mortality of renal cell carcinoma increased year by year,which became a major threat and heavy social burden to national health.Renal cell carcinoma is highly resistant to conventional chemoradiotherapy.Surgical resection is the major treatment for patients with renal cell carcinoma.Kidney is located in the retroperitoneal cavity and its growth space is large,so the early symptoms are more insidious.When patients present with symptoms,the tumor is already large or has distant metastases.It is estimated that about 30%of patients have metastases at the time of initial diagnosis,and 20%to 30%of patients have local recurrence or distant metastases during postoperative follow-up.The prognosis of patients with metastatic renal cell carcinoma is really poor,and the average 3-year survival rate is less than 5%.Recently,although some progress has been made in the term of immunotherapy and targeted therapy for renal cell carcinoma,the prognosis of patients with renal cell carcinoma is still unsatisfactory due to recurrence and metastasis.Therefore,it is of great significance to explore the biological behavior of renal cell carcinoma.Clarifying the molecular mechanism of cancer recurrence and metastasis and searching for efficient biomarkers and specific therapeutic targets is important for improving the prognosis of patients.Previous studies have shown that a large number of genes have no protein coding ability,which is called non-coding RNA(ncRNA),showing important roles in the occurrence and development of tumors.Among them,long noncoding RNA(lncRNA)is a class of RNA transcript with a length of more than 200 nucleotides and no protein-coding ability.Many studies reported that lncRNA could regulate the expression and activity of protein-coding genes through a variety of ways,such as transcriptional regulation of gene expression,adsorption of miRNA,binding protein and other ways,thus playing significant roles in a variety of biological processes.As a class of novel RNA molecules with spaceand time-specificity and high stability,lncRNA is aberrantly expressed in various tumors and could regulate tumor growth,metastasis,drug resistance and other aspects.It is expected to find novel molecular markers and therapeutic targets for renal cell carcinoma from the perspective of lncRNA,thus providing breakthrough for the diagnosis,treatment and prognosis of renal cell carcinoma in the future.The genome analysis technologies-based bioinformatics mining is a kind of effective and high throughput technology to obtain biological information,which can comprehensively detect and analyze the differentially expressed genes and the minor variations of oncogenes and tumor suppressors,leading to deeper understanding of the mechanism of cancer occurrence and development and providing novel diagnosis and treatment methods.The Cancer Genome Atlas(TCGA)and The Human Tumor-related Gene Expression Omnibus(GEO)databases are two largest genomics databases in the world.Bioinformatics mining on the genomic databases could help to free researchers from the laborious task of tracking genes one by one,promoting quickly,accurately and specifically find potential tumor molecular markers or therapeutic targets.Therefore,it is expected to make great breakthrough in the study of the relationship between lncRNA and renal cell carcinoma by using biological information mining as the entry point.In this study,TCGA and GEO database(GSE96574)were used to analyze the differential expression of 21,590 lncRNAs between renal cell carcinoma tissues and normal tissues,to screen out candidate lncRNAs whose expression was significantly up-regulated or down-regulated in renal cell carcinoma tissues.Afterwards,the prognostic model based on the expression of lncRNAs was preliminarily established by using the prognostic information from TCGA database.Furthermore,the prognosis-related lncRNA RCAT1 was further screened out based on the prognostic model and database prediction.After literature search,no relevant literature report of lncRNA RCAT1 was found.Furthermore,in vivo and in vitro experiments were performed to clarified the function of lncRNA RCAT1 in the progression and metastasis of renal cell carcinoma.We also clarified the specific molecular regulatory mechanism of lncRNA RCAT1,which could provide important molecular markers for the prognosis assessment of renal cell carcinoma patients and provide novel molecular targets for the development of targeted drugs for renal cell carcinoma.Part ⅠConstruction of a lncRNA-based prognostic model for renal cell carcinoma by bioinformatics analysisObjective1.Analyze the differential expression of lncRNAs between renal cell carcinoma tissues and normal renal tissues.2.Construct the lncRNA-based prognostic model for renal cell carcinoma.3.Analyze the differential expression of miRNAs and mRNAs between renal cell carcinoma tissues and normal renal tissues,and construct the ceRNA regulatory network.MethodsBased on TCGA database and GEO database,we analyzed the expression of lncRNAs in renal cell carcinoma tissues and normal tissues using limma package of R soft.A total of 190 differentially expressed lncRNAs were obtained by taking intersection of the differentially expressed genes in two databases.The COX univariate regression analysis,Lasso analysis,and COX multivariate regression analysis were used to screen the lncRNAs closely associated with the prognosis of renal cell carcinoma patients and establish a prognostic prediction model.The optimal cut-off value was used to divide the patients with renal cell carcinoma into high-risk group and low-risk group.The predictive value of this prediction model was evaluated by ROC analysis and Kaplan-Meier analysis.Based on TCGA database,the miRNA and mRNA differentially expressed miRNAs and mRNAs were screened using limma package of R soft.According to the prediction results of DIANA-LncBase v2,miRDB,miRTarBase,TargetScan,and StarBase databases and the results of differential analysis,the lncRNA regulatory network in renal cell carcinoma was constructed and visualized by Cytoscape.The potential functions of genes in this regulatory network were evaluated by GO and KEGG analyses.ResultsThe differential analysis on TCGA database revealed 2,809 differentially expressed lncRNAs.The differential analysis on GEO database revealed 413 differentially expressed lncRNAs.A total of 100 commonly up-regulated lncRNAs and 90 commonly downregulated lncRNAs were screened through the integration analysis on the two databases.COX univariate regression analysis,Lasso analysis and COX multivariate regression analysis were performed,and 5 lncRNAs which could independently affect the prognosis of patients with renal cell carcinoma were screened out and a prognostic prediction model was subsequently established.The ROC analysis showed that the prognostic model could well predict the prognosis of patients.The Kaplan-Meier analysis showed that the prognostic model was able to divide patients into two groups,and the high-risk group had significantly lower prognostic outcomes compared to the low-risk group.The differential analysis on TCGA database revealed 109 differentially expressed miRNAs and 5,100 differentially expressed mRNAs.Based on the predicted results of various databases,the lncRNA regulatory network was constructed,including 4 lncRNAs,15 miRNAs and 153 mRNAs.GO and KEGG analyses revealed that genes in this regulatory network were involved in multiple tumor-related signaling pathways,such as DNA-binding transcriptional activator activity,proximal promoter sequence-specific DNA binding,PI3K-Akt signaling pathway,and microRNA.Conclusions1.There were different expression patterns of lncRNA,miRNA,and mRNA between renal cell carcinoma tissues and normal renal tissues.2.The lncRNA-based prognosis prediction model for patients with renal cell carcinoma was constructed according to the results of prognosis analysis.3.The lncRNA regulatory network in renal cell carcinoma was constructed based on the differential analysis and prediction results.Part ⅡScreen the key lncRNA in renal cell carcinoma progression and explore the molecular mechanismObjective1.Screen the key lncRNA in renal cell carcinoma.2.Detect the expression of key lncRNA RCAT1 in renal cell carcinoma cells and renal cell carcinoma tissues.3.Evaluate the correlation between the expression of key lncRNA RCAT1 and the prognosis of patients with renal cell carcinoma.4.Elucidate the specific molecular regulatory mechanism of lncRNA RCAT1 in the progression and metastasis of renal cell carcinoma.MethodsThe expression of key lncRNA RCAT1 in renal cell carcinoma cells and renal cell carcinoma tissues was detected by real-time quantitative PCR.Based on the expression of lncRNA RCAT1 in renal cell carcinoma tissues,patients in TCGA cohort were divided into two groups:lncRNA RCAT1 high expression group and lncRNA RCAT1 low expression group.Kaplan-Meier analysis was used to evaluate the prognosis of patients with renal cell carcinoma in the two groups.The correlation between lncRNA RCAT1 expression and clinicopathologic features of different renal cell carcinoma patients were also analyzed.Based on the constructed lncRNA regulatory network,the potential regulatory pathways of lncRNA RACT1 in renal cell carcinoma were screened.Luciferase assay,RNA binding protein immunoprecipitation assay,qRT-PCR,and Western blot were used to verify the role of lncRNA RC AT1/miR-214-5p/E2F2 regulatory pathway in renal cell carcinoma.ResultsBased on the above results and literature search,lncRNA RCAT1 was finally selected for further analysis.The qRT-PCR results showed that the expression of lncRNA RCAT1 in renal cell carcinoma cells was significantly higher than that in normal renal cells,and the expression of lncRNA RCAT1 in renal cell carcinoma tissues was significantly higher than that in normal renal tissues.According to the expression level of lncRNA RCAT1,patients were divided into two groups.The prognosis of patients with high expression of lncRNA RCAT1 was significantly lower than that of patients with low expression of lncRNA RCAT1.In addition,the expression level of lncRNA RCAT1 was closely correlated with tumor grade,tumor size,distant metastasis and clinical stage of renal cell carcinoma patients,suggesting that lncRNA RCAT1 may play an oncogenic role in renal cell carcinoma.Based on the constructed lncRNA regulatory network in renal cell carcinoma,we preliminarily selected the lncRNA RCAT1/miR-214-5p/E2F2 pathway for further verification.Luciferase reporter assay results showed that lncRNA RCAT1 and E2F2 shared the common miR-214-5p binding site,and miR-214-5p overexpression could reduce the luciferase of wild type reporters.The results of RNA binding protein immunoprecipitation assay showed that lncRNA RCAT1 and miR-214-5p could form RISC complex and participate in regulating the expression of E2F2.Conclusions1.LncRNA RCAT1 was preliminarily screened as the key lncRNA associated with renal cell carcinoma.2.The expression of lncRNA RCAT1 in renal cell carcinoma cells was higher than that in normal renal cells.3.The expression of lncRNA RCAT1 was negatively correlated with the prognosis of patients,and is closely related to the progression and metastasis of renal cell carcinoma,which may act as an oncogene in renal cell carcinoma.4.LncRNA RCAT1 could regulate miR-214-5P/E2F2 pathway in renal cell carcinoma.Part ⅡFunctional study on lncRNA RCAT1/miR-214-5p/E2F2 pathway in the progression and metastasis of renal cell carcinomaObjective1.Analyze the effects of lncRNA RCAT1 on the proliferation and metastasis of renal cell carcinoma cells in vitro.2.Elucidate the specific molecular regulatory mechanism of lncRNA RCAT1 in the progression and metastasis of renal cell carcinoma.3.Explore the role of lncRNA RCAT1 in the progression and metastasis of renal cell carcinoma in vivo.MethodsLncRNA RCAT1 was knocked down or overexpressed in renal cell carcinoma cells(786-O,769-P),and the transfection efficiency was detected by qRT-PCR.The effects of lncRNA RCAT1 on the proliferation of renal cell carcinoma cells were detected by MTT,colony formation,and EdU experiments.Flow cytometry was used to detect the effect of lncRNA RCAT1 on cell apoptosis.The effects of lncRNA RCAT1 on the migration and invasion of renal cell carcinoma cells were detected by scratch assay and transwell assay.The effect of lncRNA RCAT1 on the proliferation of renal cell carcinoma in vivo was detected by subcutaneous tumor formation in nude mice.The effect of lncRNA RCAT1 on the metastasis of renal cell carcinoma in vivo was detected using caudal vein pulmonary metastasis model in nude mice.ResultsThe qRT-PCR assay was performed to confirm the transfection efficiency of knockdown or overexpression of lncRNA RCAT1 in renal cell carcinoma cells(786-O and 769-P).The results of MTT,colony formation assay,and EdU experiments showed that knockdown of lncRNA RCAT1 could significantly reduce the proliferation ability of renal cell carcinoma cells.The Flow cytometry results showed that knockdown of lncRNA RCAT1 obviously increased the apoptotic level of renal cell carcinoma cells.On the contrary,overexpression of lncRNA RCAT1 significantly upregulated the proliferation ability of renal cell carcinoma cells and reduced the apoptotic level of renal cell carcinoma cells.The results of scratch assay and transwell migration and invasion assay showed that knockdown of lncRNA RCAT1 could inhibit the migration and invasion abilities of renal cell carcinoma cells,and overexpression of lncRNA RCAT1 could enhance the migration and invasion ability of renal cell carcinoma cells.Overexpression of miR-214-5p could partially reverse the promoting effect of lncRNA RCAT1 on the proliferation and metastasis abilities of renal cell carcinoma cells.E2F2 also acts as an oncogenic gene in renal cell carcinoma.In addition,in vivo experiments have further confirmed that lncRNA RCAT1 could promote the proliferation and metastasis of renal cell carcinoma.Conclusions1.The in vitro experiments indicated that lncRNA RCAT1 could significantly promote the proliferation and metastasis abilities of various renal cell carcinoma cells,and inhibit the cell apoptosis.2.LncRNA RCAT1 may play a tumor-promoter role in renal cell carcinoma by influencing the miR-214-5p/E2F2 pathway.3.The in vivo experiments showed that lncRNA RCAT1 could significantly promote the proliferation and metastasis of renal cell carcinoma.