LncRNA PVT1靶向调控Sgl基因在肾细胞癌侵袭和凋亡的机制研究

The mechanism of targeting Sgl gene by LncRNA PVT1 in the invasion and apoptosis of renal cell carcinoma

目的探究LncRNAPVT1靶向调控Sgl基因在肾细胞癌侵袭和凋亡的机制,以期为临床上肾癌的治疗提供新的靶点。方法选择2015年12月至2018年12月本院收治的58例肾细胞癌患者为研究对象,术中收集患者的肿瘤组织和癌旁组织。利用GEO数据库中肾癌组织中LncRNAs数据,通过高通量数据分析肾癌组织中LncRNAs的差异性表达,利用实时荧光定量PCR(RT-qPCR)检测肾癌组织中LncRNA PVT1的相对表达量。通过siRNA干扰技术以及基因过表达技术构建LncRNA PVT1的沉默载体和过表达载体,通过慢病毒转染人肾癌细胞系ACHN细胞,根据是否转染慢病毒以及慢病毒载体的类型,将ACHN细胞分成空白对照组、siRNA沉默组和siRNA过表达组。利用RT-qPCR和Western blot法检测Bcl-2、Bad、Bax和Caspase-3的表达水平。利用Transwell试剂盒检测细胞的侵袭,利用AV-PI试剂盒检测细胞的凋亡。以裸鼠为研究对象,进行种植瘤研究。结果肿瘤组织中LncRNA PVT1的表达量明显升高(P<0.05)。RT-qPCR结果显示,siRNA沉默组的LncRNA PVT1.Sgl和促凋亡基因Bad、Bax和Caspase-3的mRNA表达量要明显低于空白对照组和过表达组(P<0.05);siRNA沉默组的抑凋亡基因Bcl-2的mRNA表达量要明显高于空白对照组和过表达组(P<0.05)。Western blot结果显示,siRNA沉默组的LncRNA PVT1、Sgl和促凋亡基因Bad、Bax和Caspase-3的蛋白表达量要明显低于空白对照组和过表达组(P<0.05);siRNA沉默组的抑凋亡基因Bcl-2的蛋白表达量要明显高于空白对照组和过表达组(P<0.05)。Transwell结果显示,siRNA沉默组细胞的侵袭能力要明显高于空白对照组和过表达组(P<0.05);过表达组细胞的侵袭能力要明显低于空白对照组(P<0.05);AV-PI试剂盒检测结果显示,siRNA沉默组细胞的凋亡率要明显低于空白对照组和过表达组(P<0.05);过表达组细胞的凋亡率要明显高于空白对照组(P<0.05)。siRNA沉默组裸鼠移植瘤的直径明显大于空白对照组和过表达组(P<0.05);过表达组裸鼠移.植瘤的直径要小于空白对照组和siRNA沉默组(P<0.05)。结论LncRNA PVT1通过靶向调控Sgl基因调控肾细胞癌的侵袭和凋亡。

Objective To explore the mechanism of targeting Sgl gene by LneRNA PVT1 in the invasion and apoptosis of renal cell carcinoma,in order to provide a new target for clinical treat-ment of renal carcinoma.Methods From December 2015 to December 2018,58 patients with renal cell carcinoma admitted to our hospital were selected as the research objects,and tumor tssues and pa-ratumoral tssues of the patients were collected intraoperatively.The data of LncRNA in renal cell carcinoma were collected from GEO database.The differential expression of LncRNA in renal cell carcino-ma was analyzed by high throughput data.The relative expression of LncRNA PVT1 in renal cell carci-noma was detected by RT-qPCR.The silencing vector and over expression vector of LncRNA PVT1 were constructed by siRNA interference technology and gene over expression technology,and the hu-man renal cancer cell line ACHN cells were transfected by lentivirus.The expression levels of Becl-2,bad,Bax and Caspase-3 were detected by RT-qPCR and Westerm blot.The invasion of cells was detec-ted by Transwell kit and apoptosis was detected by AV-PI kit.And take nude mice as the research ob-ject,in order to carry on the research of planting tumor.Results The expression of LncRNA PVT1 was significantly increased in tumor tissues.The results of the RT-qPCR showed that the mRNA ex-pression of LncRNA PVT1,Sgl and pro apoptotic genes Bad,Bax and Caspase-3 in siRNA silenced group was significantly lower than that in blank control group and the over-expression group(P<0.05).The mRNA expression of anti apoptotic gene Bcl-2 in siRNA silenced group was significantly higher than that in the blank control group and the over expression group(P<0.05).The results of the Westerm blot showed that the protein expression of LncRNA PVT1,Sgl and pro apoptotic genes Bad,Bax and Caspase-3 in siRNA silenced group was significantly lower than that in the blank control group and the over-expression group(P<0.05).The protein expression of anti apoptotic gene Bcl-2 in siRNA silenced group was significantly higher than that in the blank control group and the over ex-pression group(P<0.05).The results of Transwell showed that the invasiveness of cells in siRNA si-lencing group was significantly higher than that in the blank control group and the over expression group(P<0.05).The invasiveness of cells in over expression group was significantly lower than that in blank control group(P<0.05).The apoptosis rate of cells in siRNA silencing group was significantly lower than that in blank control group and over expression group(P<0.05).The apoptosis rate of o-ver expression group was significantly higher than that of blank control group(P<0.05).The diame-ter of transplanted tumor in siRNA silenced group was significantly larger than that in blank control group and over expression group(P<0.05),and the diameter of transplanted tumor in the overex-pressed group was larger than that in the blank control group and the siRNA silenced group(P<0.05).Conclusions The LncRNA PVT1 regulates the invasion and apoptosis of renal cell carcinoma by targeting Sgl gene.