SLC9A3-AS1调控miR-148a-3p/ROCK1信号轴影响肾癌细胞生物学功能

SLC9A3-AS1 Regulates miR-148a-3p/ROCK1 Axis to Affect Biological Function of Renal Carcinoma Cells

目的检测lncRNA SLC9A3-AS1在肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)组织与肾癌细胞中的表达水平,探讨其促进肾癌细胞恶性生物学行为的机制。方法应用GEPIA2在线软件(http://gepia2.cancer-pku.cn/)分析TCGA数据库中SLC9A3-AS1在ccRCC组织中的表达水平;采用实时荧光定量聚合酶链反应(qRT-PCR)检测SLC9A3-AS1在不同肾癌细胞系、24例ccRCC组织与癌旁正常肾脏组织中的表达水平;应用细胞增殖/毒性检测试剂盒(cell counting kit-8,CCK-8)和Transwell小室迁移实验检测敲低SLC9A3-AS1表达对肾癌细胞增殖与迁移的影响;应用蛋白质免疫印迹法检测增殖与迁移相关信号通路蛋白的表达水平;采用双荧光素酶报告基因实验验证SLC9A3-AS1与miR-148a-3p/ROCK1轴的靶向调控关系。结果GEPIA2软件分析结果显示,相较正常肾脏组织,SLC9A3-AS1在肾透明细胞癌组织中表达显著上调(P<0.01)。qRT-PCR结果显示,相较癌旁正常肾脏组织,SLC9A3-AS1在24例ccRCC组织中表达显著上调(P<0.01);相较永生化肾小管上皮细胞,SLC9A3-AS1在4种肾癌细胞系中表达均显著上调,以786-O细胞最为显著(P<0.01)。干扰SLC9A3-AS1表达,可显著抑制786-O细胞的增殖与迁移能力,上调E-cadherin的蛋白表达水平,而下调N-cadherin、MMP2的蛋白表达水平(均P<0.05);过表达miR-148a-3p可显著抑制786-O细胞的增殖与迁移能力(P<0.01)。双荧光素酶报告基因检测结果表明,SLC9A3-AS1可特异性结合miR-148a-3p,后者进一步靶向结合ROCK1 mRNA的3′UTR区域;过表达miR-148a-3p可明显下调ROCK1 mRNA的表达水平,敲低miR-148a-3p表达则产生相反的效应;敲低SLC9A3-AS1表达可显著下调786-O细胞中ROCK1 mRNA与蛋白的表达水平(P<0.01);下调miR-148a-3p表达可部分逆转SLC9A3-AS1沉默对786-O细胞增殖与迁移的抑制作用(P<0.05)。结论SLC9A3-AS1在ccRCC中通过调控miR-148a-3p/ROCK1轴发挥促癌作用,有望成为ccRCC的一个新的分子标志物。

Objective To examine lncRNA SLC9A3-AS1 expression levels within clear cell renal cell carcinoma(ccRCC)tissues and cells,aiming to elucidate its role in advancing the malignant progression of renal cancer cells.Methods GEPIA2 online software(http://gepia2.cancer-pku.cn/)was used to analyze the expression level of SLC9A3-AS1 in ccRCC tissues from the TCGA database.Else,real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was utilized to measure the expression levels of SLC9A3-AS1 in various renal cancer cell lines as well as ccRCC and adjacent normal kidney tissues from 24 cases.To evaluate the impact of SLC9A3-AS1 on renal cancer cell proliferation and migration,CCK-8 cell proliferation assay and Transwell chamber migration assay were conducted.Western blot was employed to assess the expression levels of proteins related to proliferation and migration signaling pathways.Additionally,dual-luciferase reporter gene assay was conducted to validate the targeted regulatory relationship between SLC9A3-AS1 and the miR-148a-3p/ROCK1 axis.Results The GEPIA2 software analysis revealed a significant upregulation of SLC9A3-AS1 expression in ccRCC tissues compared to normal kidney tissues(P<0.01).The qRT-PCR analysis showed that the expression of SLC9A3-AS1 was significantly upregulated in ccRCC tissues compared to adjacent normal kidney tissues in the 24 cases(P<0.01).When compared to immortalized renal tubular epithelial cells,higher expressions of SLC9A3-AS1 were detected in four different types of renal cancer cell lines,with the most pronounced increase observed in 786-O cells(P<0.01).Interfering with SLC9A3-AS1 expression had a notable inhibitory effect on the proliferation and migration of 786-O cells.This intervention led to an increase in E-cadherin protein expression and a decrease in N-cadherin and MMP2 protein expression levels(both P<0.05).Likewise,the overexpression of miR-148a-3p exerted a similar inhibitory effect on the proliferation and migration of 786-O cells(P<0.01).The results of dual-luciferase reporter gene test suggested that SLC9A3-AS1 could specifically bind to miR-148a-3p,which further targeted the 3′UTR region of ROCK1 mRNA.Overexpression of miR-148a-3p significantly downregulated the expression level of ROCK1 mRNA,while knockdown of miR-148a-3p had the opposite effect.The silence of SLC9A3-AS1 significantly suppressed the expression level of ROCK1 mRNA and protein in 786-O cells(P<0.01).Moreover,the data revealed that downregulation of miR-148a-3p could partially counteract the inhibitory effects of SLC9A3-AS1 silencing on the proliferation and migration of 786-O cells(P<0.05).ConclusionSLC9A3-AS1 has a promoting effect on progression of ccRCC by modulating the miR-148a-3p/ROCK1 axis.It has the potential to serve as a novel molecular marker for ccRCC.