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.
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。.