NORAD在高糖诱导小鼠肾小球系膜细胞增殖及凋亡中的作用及机制

The role and mechanism of NORAD in proliferation and apoptosis of mouse mesangial cells induced by high glucose

目的探讨DNA损伤激活的非编码RNA(NORAD)在高糖诱导小鼠肾小球系膜细胞增殖及凋亡中的作用及机制。方法根据培养基中葡萄糖浓度将小鼠肾小球系膜细胞SV40-MES-13分为高糖组及低糖组,处理24 h后转染NORAD小干扰RNA(si-NORAD)及Toll样受体4(TLR4)小干扰RNA(si-TLR4),实时荧光定量PCR(qRT-PCR)检测NORAD及TLR4的表达。在SV40-MES-13细胞中分别转染NORAD、TLR4过表达质粒及小干扰RNA。Cell Counting Kit-8(CCK-8)、5-乙炔基-2′脱氧尿嘧啶核苷(EdU)染色及流式细胞术检测细胞增殖和凋亡结果;共转染NORAD和miR-520h模拟剂,TLR4和miR-520h模拟剂后检测荧光素酶强度及三者表达情况;共转染si-NORAD和miR-520h抑制剂、TLR4过表达质粒和miR-520h模拟物以及共转染si-TLR4和miR-520h抑制剂后检测三者水平及细胞增殖水平来验证三者的调节关系。结果高糖处理小鼠系膜细胞SV40-MES-13后,NORAD(1.65±0.08比1.00±0.06,P=0.003)及TLR4(1.96±0.09比1.01±0.07,P=0.001)的表达均升高。NORAD及TLR4过表达后细胞增殖加快(1.34±0.04比0.85±0.04,P=0.001;1.33±0.02比0.82±0.03,P<0.001),细胞凋亡减少(0.45±0.03比0.94±0.06,P=0.001;0.51±0.05比0.99±0.03,P=0.001)。双荧光素酶报告基因结果显示,miR-520h能与NORAD及TLR4结合;NORAD与miR-520h表达相互影响,miR-520h模拟物可减少TLR4表达。与单独转染si-NORAD相比,共转染si-NORAD及miR-520h抑制剂时TLR4相对表达量升高(0.74±0.03比0.55±0.03,P=0.014);与单独转染miR-520h模拟物相比,共转染miR-520h模拟剂及TLR4过表达质粒后细胞增殖更快(0.73±0.01比0.61±0.02,P=0.007);与单独转染miR-520h抑制剂相比,共转染miR-520h抑制剂及si-TLR4后细胞增殖减少(1.31±0.04比1.55±0.04,P=0.013)。结论NORAD可结合miR-520h调节TLR4促进高糖诱导系膜细胞增殖及抑制凋亡。

Objective To uncover the role and mechanism of non-coding RNA-activated by DNA damage(NORAD)in proliferation and apoptosis of mouse mesangial cells induced by high glucose.Methods SV40-MES-13 cells were divided into high glucose group and low glucose group according to the concentration of glucose in the culture medium.After 24 hours of treatment,SV40-MES-13 cells were transfected with si-NORAD and si-Toll-like receptor(TLR4),and real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of NORAD and TLR4.NORAD,TLR4 overexpression plasmids and small interfering RNA were transfected into SV40-MES-13 cells respectively.Cell counting kit(CCK-8)assay,5-ethynyl-2′-deoxyuridine(EdU)assay and flow cytometry were performed to determine the results of cell proliferation and apoptosis.Luciferase activity and their mRNA levels were detected after cotransfection of pGL3-NORAD WT and miR-520h mimics or pGL3-TLR4 WT and miR-520h mimics.After cotransfection of si-NORAD and miR-520h inhibitors,TLR4 overexpression plasmids and miR-520h mimics,and cotransfection of si-TLR4 and miR-520h inhibitors,the levels of NORAD,miR-520h and TLR4,as well as cell proliferation were detected to verify the regulatory relationship among them.Results NORAD and TLR4 were upregulated in SV40-MES-13 cells in high glucose group(1.65±0.08 vs 1.00±0.06,P=0.003;1.96±0.09 vs 1.01±0.07,P=0.001).Overexpression of NORAD and TLR4 promoted the proliferative ability(1.34±0.04 vs 0.85±0.04,P=0.001;1.33±0.02 vs 0.82±0.03,P<0.001)and inhibited the apoptosis in SV40-MES-13 cells(0.45±0.03 vs 0.94±0.06,P=0.001;0.51±0.05 vs 0.99±0.03,P=0.001).The dual-luciferase reporter gene assay showed that miR-520h was confirmed to bind to NORAD and TLR4.The expression of NORAD and miR-520h affected each other,and miR-520h mimics reduced the expression of TLR4.The expression of TLR4 in cotransfected NORAD small interference RNA and miR-520h inhibitor was higher than that in NORAD small interference RNA alone(0.74±0.03 vs 0.55±0.03,P=0.014).Cotransfection of miR-520h mimics and TLR4 overexpression plasmids increased cell proliferation compared with miR-520h mimics alone(0.73±0.01 vs 0.61±0.02,P=0.007).Cotransfection of miR-520h inhibitors and TLR4 small interference RNA reduced cell proliferation compared with miR-520h inhibitors alone(1.31±0.04 vs 1.55±0.04,P=0.013).Conclusions The regulatory loop NORAD/miR-520h/TLR4 promotes the proliferative ability and inhibits apoptosis in glomerular mesangial cells induced by high glucose.