靶向抑制人SREBP-1基因对高糖刺激下人肾小管上皮细胞脂滴形成的影响

Effects of SREBP-1 targeted RNAi on lipid droplet formation in HKC cells under stimulation of high glucose

目的构建人SREBP-1基因的shRNA载体并转染人肾小管上皮细胞(HKC)明确是否沉默SREBP-1基因的表达可减轻高糖环境引起的细胞内脂滴形成。方法设计两对针对人SREBP-1基因的干扰靶序列及短链寡核苷酸和线性化的pGenesil-1质粒进行连接,筛选鉴定并最终命名为pGenesil-1-SREBP1-1和pGenesil-1-SREBP1-2。体外培养人肾小管上皮细胞并随机分组,采用阳离子脂质体法转染细胞后给予高糖刺激48h,在倒置荧光显微镜下观察绿色荧光蛋白表达,半定量RT-PCR和Westernblot分析目标基因表达丰度的变化,并采用油红O染色检测细胞内脂滴。结果经酶切及测序鉴定证明构建的重组载体pGenesil-1-SREBP1-1、pGenesil-1-SREBP1-2序列正确;转染HKC细胞后均可表达绿色荧光;RT-PCR和Westernblot显示pGenesil-1-SREBP1-1、pGenesil-1-SREBP1-2能够有效抑制高糖环境下HKC细胞SREBP-1的表达,与阴性质粒对照组相比,差异有统计学意义。进一步研究发现在HKC细胞内,SREBP-1基因表达沉默明显抑制了脂肪酸合成酶(FAS)的转录,细胞内脂滴明显减少。结论有效沉默高糖刺激下HKC细胞SREBP-1的基因表达可通过抑制FAS转录而减少细胞内脂滴形成。

Aim To construct eukaryotic expression vector of shRNA （small hairpin RNA ） for human SREBP-1 （ sterol regulation element binding protein-1 ） gene and explore its effects on lipid droplet formation in human renal proximal tubular epithelial cell line （HKC） under the stimulation of high glucose. Methods Two eukaryotic expression vectors of shRNA were constructed for human SREBP-1 gene. The HKC cells were transfected with negative control plasmid （pGenesil-l-HK） and two recombinant vectors （pGenesil-1- SREBP1-1 and pGenesil-1-SREBP1-2） and then were cultured under the stimulation of high glucose for about 48 h. The expression of SREBP-1 mRNA and FAS mRNA was detected by RT-PCR and SREBP-1 protein expression was investigated by Western blot. Lipid droplets were detected by Oil Red O staining. Results DNA sequencing showed that the target segments were successfully cloned into pGenesil-1 vector respectively.RT-PCR indicated that two recombinant vectors could inhibit the expression of SREBP-1 mRNA and FAS mRNA in HKC cells under the stimulation of high glucose. Similarly, SREBP-1 protein was also inhibited by the transfection with recombinant vectors. Oil Red O staining found that silencing of SREBP-1 gene resulted in lipid droplets decrease. Conclusions The eukaryotic expression vector of shRNA for human SREBP-1 gene was successfully constructed, and the expression of SREBP-1 was inhibited effectively by the expressed siRNA in HKC ceils that resulted in lipid droplets decrease through FAS mRNA transcription inhibition.