LUCAT1 Promotes Proliferation And Invasion Of Clear Cell Renal Cell Carcinoma Cells

ObjectivesRenal cell carcinoma(RCC)is the third most common genitourinary cancer and about 70%of RCC are clear cell RCC(ccRCC).Approximately one-third of ccRCC patients present with metastasis at the time of diagnosis,which is associated with a poor prognosis.Long noncoding RNA LUCAT1 have been found to play critical roles in non-small cell lung cancer and ovarian cancer.However,the role of LUCAT1 in ccRCC remains unclear.Our present study aimed to explore the roles of LUCAT1 in ccRCC.Moreover,we aimed to investigate the potential regulatory network of LUCAT1-miRNA-mRNA in ccRCC by involved miR-495-3p and SATB1 to reveal the underlying mechanisms.Methods1)LUCAT1 levels in clinical sample tissues including 64 ccRCC tissues and 20 adjacent normal renal tissues from ccRCC patients were determined by the qRT-PCR assay.Data of LUCAT1 levels in 448 ccRCC tissues and 67 adjacent normal renal tissues were download from TCGA.Then the relationship between LUCAT1 expression and clinical features of ccRCC patients was analyzed by chi-square test.The prognostic value of LUCAT1 for ccRCC was analyzed by Kaplan-Meier and Cox regression analysis.The potential diagnostic value of LUC ATI was estimated for ccRCC by receiver operating characteristic(ROC)curve.Moreover,LUCAT1 levels in different human ccRCC cell lines(786-O,Caki-1 and ACHN)and human renal tubular epithelial cells(HK-2)were detected by qRT-PCR.2)LUCAT1 of 786-0 and Caki-1 was inhibited by LUCAT1 siRNA,the siRNA knockdown efficiency was confirmed by qRT-PCR.Then,CCK8 and EdU assays were performed to evaluate the effect of LUCAT1 inhibition on cell proliferation.In addition,matrigel-coated transwell assay was carried out to study the impacts of LUCAT1 inhibition on cell invasion.Furthermore,xenografts in nude mice was used to evaluate the effect of LUCAT1 inhibition on tumor growth of ccRCC in vivo.3)Targetscan bioinformatics software was used to identify the target binding sequences between miR-495-3p and SATB1.DIANA bioinformatics software was used to identify the target binding sequences between miR-495-3p and LUCAT1.Then,dual-luciferase reporter assay was used to analyze the binding effect and binding site between miR-495-3p and LUC AT1,and the role of LUC AT1 in the regulation of SATB1 expression through miR-495-3p.4)To determine whether LUC AT1 exerts biological functions through miR-495-3p,we performed rescue experiments by inhibiting miR-495-3p expression in LUCAT1-knockdown cells.miR-495-3p was inhibited in LUCAT1-knockdown cells by tranfection of cells with miR-495-3p inhibitor,miR-495-3p inhibitive efficiency was confirmed by qRT-PCR.Then,CCK8 assay was performed to examine cell proliferation,matrigel-coated transwell assay was carried out to detect cell invasion.Moreover,SATB1 expression were detected by qRT-PCR and western blot,the correlation between SATB1 and miR-495-3p,SATB1 and LUC AT1 were analyzed by pearson correlation analysis.Results5)LUC AT1 was significantly up-regulated in ccRCC tissue compared with adjacent normal tissue both in validated cohort and TCGA cohort.LUCAT1 showed potential diagnostic value for cRCC by ROC curve.Increased expression of LUCAT1 was correlated with larger tumor size,more distant metastasis and advanced clinical stage in TCGA cohort,and correlated with larger tumor size,more lymph node metastasis advanced clinical stage and more smoke in validated cohort.Kaplan Meier analysis indicated that patients with higher LUCAT1 expression had a worse overall survival both in validated cohort and TCGA cohort.In addition,multivariate analysis revealed that increased expression of LUCAT1 was an independent predictor of overall survival in validated cohort.Moreover,the levels of LUCAT1 in the ccRCC cell lines including 786-0,Caki-1 and ACHN were signifcantly up-regulated when compared to that in normal human proximal tubule epithelial cell lines HK-2.6)CCK8 assay showed the average OD values at 450 nm were significantly lower in LUCAT1 siRNA-transfected cells than that of the control group in both types of ccRCC cells.EdU assays showed reduced positive cells numbers after depletion of LUCAT1.Knockdown of LUCAT1 significantly decreased the invasion abilities of ccRCC cells.We further found the tumor growth and tumor weight were significantly reduced after LUCAT1 was knocked down.7)Prediction by DIANA tool found miR-495-3p was he potential target of LUCAT1.Next,we found expression of miR-495-3p was significantly downregulated in ccRCC samples.Pearson's correlation analysis revealed an inverse correlation between miR-495-3p and LUCAT1 in these 64 clinical ccRCC tissues.We further analyzed miR-495-3p expression in LUCAT1-knockdown ccRCC cells and found that miR-495-3p was boosted after LUCAT 1 downregulation,while ectopic overexpression of miR-495-3p did not affect LUCAT1 expression.Next,we found the miR-495-3p mimic reduced the luciferase activity of pmirGLO-LUCAT1-wt but not of pmirGLO-LUCAT1-mut.8)To determine whether LUCAT 1 exerts biological functions through miR-495-3p,we performed rescue experiments by inhibiting miR-495-3p expression in LUCAT 1-knockdown cells.We found proliferation was decreased in LUCAT 1-knockdown Caki-1 and 786-0 cells,whereas miR-495-3p inhibition partially reversed the reduction of proliferation.Furthermore,transwell invasion assays revealed that LUCAT 1 knockdown significantly inhibited ccRCC cell invasion,while miR-495-3p inhibition partially rescued the reduction of invading cells.9)A bioinformatics analysis using TargetScan and DIANA indicated that SATB1 and LUCAT 1 owned same binding site of miR-495-3p.We then found that expression of SATB1 mRNA was significantly upregulated in ccRCC tissues.We further found reverse correlation between SATB1 and miR-495-3p and positive correlation between SATB1 and LUCAT 1 were found in clinical ccRCC samples.The luciferase reporter assay showed that the miR-495-3p mimic reduced the luciferase activity of pmirGLO-SATB1-wt,but not of pmirGLO-SATB1-mut,and the down-regulation of LUCAT 1 had the same effect.We next found knockdown of LUCAT 1 significantly repressed SATB1 expression,whereas SATB1 expression was partially rescued after inhibition of miR-495-3p,demonstrating that the LUCAT 1 has a pivotal role in ccRCC progress by regulating SATB1 mediated by miR-495-3p.Conclusions1)LUCAT 1 is one of the up-regulated IncRNAs in ccRCC.Its abnormal expression can predict the progression of ccRCC.2)LUCAT1 acts as an oncogenic IncRNA that promotes the tumorigenesis and progression of ccRCC.3)LUCAT1 functions as as an oncogenic IncRNAin ccRCC through miR-495-3p-SATB1 axis.4)LUCAT1 could be a useful marker and potential therapeutic target for ccRCC.