Long Non-coding RNA ZFAS1 Promotes Proliferation And Metastasis Of Clear Cell Renal Cell Carcinoma Via Targeting MiR-10a/SKA1 Pathway

Objective: Renal cell carcinoma(RCC)is one of the most common malignant tumors of the genitourinary system,of which 60-85% is clear cell renal cell carcinoma(ccRCC).Nearly one-third of ccRCC patients have metastases at diagnosis and have a poor prognosis.Early diagnosis and effective targeted therapy are important means to improve its survival.NFX1-type zinc finger-containing protein 1 antisense RNA 1(ZFAS1)is highly expressed in many malignant tumor tissues and is closely related to poor prognosis,but its role in ccRCC is still unclear.The aim of this study was to investigate the role of ZFAS1 in ccRCC and to investigate the regulatory network of lncRNA-miRNA-mRNA present in ccRCC by studying the relationship between ZFAS1 and miR-10a/SKA1 pathway,to reveal the molecular mechanisms of ccRCC and seek for new molecular therapeutic targets.Methods: In this study,60 human ccRCC tissues,20 adjacent cancer tissues,human ccRCC cell lines 786-O,Caki-1,ACHN,A498 and human renal tubular cell line HK-2,and ccRCC xenogeneous subcutaneous transplantation and metastatic tumor mouse model were studied.Real-time PCR was used to detect the expression of ZFAS1,miR-10 a and SKA1 mRNA in human ccRCC tissues and adjacent tissues,ccRCC cell lines 786-O,Caki-1,ACHN,A498 and human renal tubular cell line HK-2.Lentiviral transfection was used to construct stable ccRCC cell lines with ZFAS1 or SKA1 knockdown.miR-10 a inhibitor was transiently transfected to knocked down miR-10 a expression in ccRCC cells.The CCK-8 assay was used to detect the proliferation of ccRCC cells.Transwell assay was used to analyze the migration and invasion abilities of ccRCC cells.Western blot was used to detect the expression of SKA1 protein in cell lines.The luciferase reporter gene assay was used to analyze the targeted binding of ZFAS1 to miR-10 a and miR-10 a to SKA1.RNA immunoprecipitation(RIP)assay was used to analyze the direct binding of ZFAS1 to miR-10 a.ccRCC subcutaneous xenograft and lung metastasis of BALB/c nude mice models were used to observe the tumor growth and metastasis.Statistical analysis was performed using SPSS 20.0 statistical software,and the values were expressed as mean±standard deviation(mean±SD).Comparation among multiple groups was performed using one-way analysis of variance(ANOVA).Comparison between two samples was performed using t test.Survival rates were analyzed by Kaplan-Meier method and survival rates were compared by log-rank test.Correlation analysis was performed using the Pearson test.P<0.05 was considered statistically significant.Results: 1.The expression of ZFAS1 in ccRCC was significantly higher than that in adjacent tissues.The high expression of ZFAS1 was closely related to tumor size,lymph node metastasis,smoking and poor prognosis.2.The expression of ZFAS1 in 786-O,Caki-1,ACHN and A498 cell lines was higher than that in HK-2 cell line.ccRCC cell lines with ZFAS1 stably knocked down(Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh)were successfully constructed by lentiviral transfection of shRNA targeting ZFAS1,and ZFAS1 expression was significantly decreased in Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh than that in control cells with shNC transfected.The proliferative viability of Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh were significantly lower than that in control cells with shNC transfected.The migration and invasion abilities of Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh were significantly lower than that in control cells with shNC transfected.ccRCC subcutaneous xenograft and lung metastasis of BALB/c nude mice models were successfully established with Caki-1-ZFAS1-sh,and ZFAS1 expression was significantly decreased in tumor tissues of Caki-1-ZFAS1-sh group than that in shNC group.The size and weight of tumors in Caki-1-ZFAS1-sh group were lower than that in shNC group.The incidence of lung metastasis in Caki-1-ZFAS1-sh group was significantly lower than that in shNC group.3.There are a theoretical binding site between ZFAS1 and seed region of miR-10 a.Luciferase reporter gene analysis found that miR-10 a could target to ZFAS1.RIP experiments confirmed that ZFAS1 can directly bind to miR-10 a,The expression of miR-10 a in Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh were higher than that in control cells with shNC transfected,miR-10 a expression in ccRCC tissues was lower than that in adjacent tissues and negatively correlated with ZFAS1 expression.The miR-10 a expression of Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh with miR-10 a inhibitor transfected were significantly lower than that in control cells with inhibitor control transfected.The proliferation,migration and invasion ability of Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh with miR-10 a inhibitor transfected was significantly higher than that in control cells withinhibitor control transfected.4.There is a theoretical binding site between SKA1 and seed region of miR-10 a.Luciferase reporter gene analysis revealed that miR-10 a binds to SKA1.The protein and mRNA expression of SKA1 in Caki-1 and ACHN with miR-10 a mimics transfected were significantly lower than that in control cells with mimics control transfected.mRNA expression of SKA1 in ccRCC tissues is lower than that in adjacent tissues and negatively correlated with miR-10 a expression.The expression of SKA1 in Caki-1-ZFAS1-sh and ACHN-ZFAS1-sh with miR-10 a inhibitor transfected were significantly higher than that in control cells with inhibitor control transfected.The expression of SKA1 in ccRCC tissues was positively correlated with the expression of ZFAS1,and both were negatively correlated with the expression of miR-10 a.5.The ccRCC cell line with SKA1 stablely knockdown(ACHN-SKA1-sh)was successfully constructed by lentiviral transfection of shRNA targeting SKA1.The expression of SKA1 in ACHN-SKA1-sh was significantly decreased than that in control cells with shNC transfected.The proliferation,migration and invasion ability of ACHN-SKA1-sh was lower than that in the control cells with shNC transfected.Conclusion: 1.ZFAS1 was overexpressed in ccRCC and correlated with tumor size,lymph node metastasis,smoking and poor prognosis,indicating that ZFAS1 can be used as a marker to judge the prognosis of ccRCC patients.2.When the expression of ZFAS1 in ccRCC cells was down-regulated,the proliferation,migration and invasion ability of ccRCC cells were significantly decreased in vitro,and the growth and metastasis of ccRCC cells were significantly attenuated in vivo,indicating that ZFAS1 can induce cell growth and metastasis of ccRCC,and play a role in promoting development and progression of ccRCC.3.After down-regulation of ZFAS1 expression in ccRCC cells,the expression of miR-10 a increased and the expression of SKA1 decreased,the inhibition of miR-10 a expression partially restored the pro-carcinogenesis of ZFAS1,the inhibition of SKA1 expression reduced the proliferation,migration and invasion ability of ccRCC cells,indicating that the miR-10a-SKA1 pathway may mediate the pro-carcinogenesis of ZFAS1 in ccRCC,suggesting that ZFAS1-miR-10a-SKA1 pathway can be used as a new target for the prevention and treatment of ccRCC.