长链非编码RNA XIST靶向miR-150-5p调控狼疮性肾炎系膜细胞增殖凋亡的研究

Long-chain noncoding RNA XIST regulates cell proliferation and apoptosis of mesangial cells in lupus nephritis by targeting miR-150-5p

目的 观察长链非编码RNA X-非活性特异性转录物(lncRNA XIST,XIST)、miR-150-5p对狼疮性肾炎(lupus nephritis,LN)系膜细胞增殖凋亡的影响并探究其机制。方法 分离培养LN小鼠和正常小鼠的系膜细胞。pcDNA组(转染空载体)、pcDNA-XIST组(转染pcDNA-XIST)、antagomiRNA组(转染antagomiRNA)、antagomiR-150-5p组(转染antagomiR-150-5p)、pcDNA-XIST+agomiRNA组(共转染pcDNA-XIST和agomiRNA)、pcDNA-XIST+agomiR-150-5p组(共转染pcDNA-XIST和agomiR-150-5p)用脂质体法转染至LN系膜细胞;实时荧光定量反转录聚合酶链式反应(real time fluorescence quantitativereverse transcription polymerase chain reaction,RT-qPCR)检测XIST、miR-150-5p的表达;双荧光素酶报告试验检测细胞的荧光活性;细胞计数试剂盒(cell counting kit,CCK8)、膜联蛋白V-异硫氰酸荧光素-碘化丙锭(annexin V-fluorescein isothiocyanate propidium iodide,annexin V-FITC/PI)检测细胞增殖、凋亡;蛋白免疫印迹(Western bloting,WB)实验检测细胞周期依赖性蛋白激酶抑制因子1A(cyclin-dependent kinase inhibitor 1A,P21)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、BCL2关联X蛋白(BCL2-associated X protein,Bax)的蛋白表达。结果 与正常小鼠肾组织或系膜细胞相比,LN小鼠肾组织或系膜细胞XIST表达显著升高,miR-150-5p表达显著降低(P<0.001);过表达XIST或抑制miR-150-5p均明显促进LN系膜细胞增殖、抑制凋亡,促进PCNA、Bcl-2蛋白表达,抑制P21、Bax蛋白表达(P<0.001)。miR-150-5p明显抑制野生型XIST细胞的荧光活性,并负向调节XIST(P<0.001)。过表达miR-150-5p明显抑制过表达XIST对LN系膜细胞增殖凋亡的影响(P<0.001)。结论 LncRNA XIST促进LN系膜细胞增殖,抑制凋亡,其机制与直接靶向miR-150-5p有关。

Objective To observe the effects of long-chain noncoding RNA X-inactive specific transcripts(LncRNA XIST,XIST)and miR-150-5p on the proliferation and apoptosis of mesangial cells in lupus nephritis(LN)and to explore its mechanism of action.Methods Mesangial cells from LN mice and normal mice were isolated and cultured.pcDNA group(transfected empty vector),pcDNA-XIST group(transfected pcDNA-XIST),antagomiRNA group(transfected antagomiRNA),antagomiR-150-5p group(transfected antagomiR-150-5p),pcDNA-XIST+agomiRNA group(co-transfected pcDNA-XIST and agomiRNA),pcDNA-XIST+agomiR-150-5p group(co-transfected pcDNA-XIST and agomiR-150-5p)were transfected into mesangial cells of LN by liposome method.Real time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the expression of XIST and miR-150-5p.Dual luciferase assay was used to detect the fluorescence activity of cells,and cell counting kit(CCK8)and annexin V-fluorescein isothiocyanate propidium iodide(annexin V-FITC/PI)were used to detect cell proliferation and apoptosis.Protein expressions of cyclin-dependent kinase inhibitor 1A(p21),proliferating cell nuclear antigen(PCNA),B-cell lymphoma-2(Bcl-2)and BCL2-associated X protein(Bax)were detected by Western blotting(WB).Results Compared with normal mouse renal tissue or mesangial cells,the expression of XIST significantly increased and the expression of miR-150-5p significantly decreased in renal tissue or mesangial cells of LN mice(P<0.001).Overexpression of XIST or inhibition of miR-150-5p significantly facilitated the proliferation and inhibited apoptosis,up-regulated the expression of PCNA and Bcl-2 protein,and down-regulated the expression of p21 and Bax protein of mesangial cells of LN(P<0.001).MiR-150-5p significantly inhibited the fluorescence activity of wild-type XIST cells and negatively regulated XIST(P<0.001).Overexpression of miR-150-5p significantly inhibited the effect of overexpression of XIST on the proliferation and apoptosis of mesangial cells of LN(P<0.001).Conclusion LncRNA XIST can promote the proliferation and inhibit apoptosis of mesangial cells of LN,and its mechanism may be related to directly targeting miR-150-5p.