转化生长因子β诱导肾小球系膜细胞增生中的长链非编码RNA差异表达研究

Analysis of the differential expression of long noncoding RNAs in experimental mesangial cells proliferation induced by TGF- β

目的检测长链非编码RNA（lncRNA）在转化生长因子β（TGF—β）诱导的肾小球系膜细胞增生中的改变，探讨lncRNA在系膜增生性肾小球肾炎发病中的作用。方法用TGF—β刺激建立系膜细胞增生模型。采用细胞增殖实验筛选TGF-β诱导肾小球系膜细胞增生的最佳浓度；用芯片技术筛选TGF-β刺激组与对照组有差异表达的lncRNAs；采用生物信息学分析法分析lncRNAs芯片结果，用RT-PCR法验证差异表达的lncRNAs，再根据生物信息学分析结果推测靶基因，用RT-PCR法验证。结果细胞增殖实验结果显示，10ng／mlTGF-β为刺激细胞增殖最佳浓度。芯片筛选结果显示，TGF—β刺激组和对照组约30000多个lncRNAs表达，TGF—β刺激组有5550个lncRNAs差异表达于对照组，其中119个和147个lncRNAs上调或下调超过2倍。根据lncRNA筛选原则选取部分lncRNAs用RT—PCR法验证。结果提示：与对照组相比，TGF—β刺激组细胞uc．60、MRAK079149、MRAK029456、XR_005507、XR_007641、UC_14和uc．412的表达明显上调；BC088254、DQ402472、BC098733、BC158832、BC098746表达明显下调，与芯片检测结果一致。生物信息学分析结果显示uc．412和MRAK079149的下游靶基因分别为抗凋亡转录因子（AATF）和NimA相关激酶8（NeK8），RT—PCR验证结果提示：与对照组比较，TGF—β刺激组AATF和NEK的表达显著上调（P〈0．05）。结论体外TGF—β诱导的系膜细胞增生模型中存在lncRNAs的差异表达。

Objective Long noncoding RNA （lncRNA） has been identified to regulate DNA methylation, histone acetylation, and gene post-transcriptional regulation in kinds of diseases, including tumorigenesis, obesity and so on. Therefore, lncRNAs might be the potential targets of mesangial cells proliferation. Methods Mesangial cells were exposed to suitable concentration of TGF-β through cell proliferation assay; then the lncRNAs expression levels were detected by microarray in experimental group and control group separately; finally the differentially expressed lncRNAs were identified by RT-PCR; meanwhile, and the expression levels of target genes were also detected by RT-PCR. Results Cell viability assay confirmed that 10 ng/ml TGF- β could promote mesangial cell proliferation significantly. Totally, over 30 000 lncRNAs were detected in TGF- 15 treated MCs and control group cells separately. Compared to the control group, 5550 lncRNAs differentially expressed in TGF- β treated MCs, including 119 up- regulated and 147 down-regulated over 2 fold. RT-PCR results appeared that uc.60, MRAK079149, MRAK029456, XR 005507, XR_007641, uc.14, and uc.412 were significantly up-regulated in TGF-β treated MCs, and BC088254, DQ402472, BC098733, BC158832, BC098746 were stably down-regulated. Compared to the control group, the mRNA expression levels of AATF and NEK were increased in the TGF-β treated mesangial cells （P 〈 0.05）. AATF and NEK were downstream target genes of uc.412 and MRAK079149 respectively. Conclusion The differential expression of long noncoding RNAs presents in the experimental mesangial cells proliferation induced by TGF- β.