长链非编码RNA6030408B16RIK结合微小RNA-326-3p参与腹膜透析超滤衰竭发生的机制

Mechanism of long noncoding RNA 6030408B16RIK binding to miRNA-326-3p involved in the development of ultrafiltration failure by peritoneal dialysis

目的研究长链非编码RNA6030408B16RIK(LncRNA6030408B16RIK)结合微小RNA(miRNA-326-3p)致大鼠尿毒症模型腹膜纤维化的机制,为预防超滤衰竭提供新的依据。方法将36只造模成功的尿毒症大鼠分为六组,每组6只大鼠,分别为:尿毒症组(不行腹膜透析治疗),空白组(腹膜透析4周),NC组(腹膜透析4周+转染空质粒),inhibitor NC组(腹膜透析4周+转染空质粒抑制剂),miR-326-3p mimic组(腹膜透析4周+miR-326-3p模拟物),miR-326-3p inhibitor组(腹膜透析4周+miR-326-3p抑制剂)。构建LncRNA6030408B16RIK原始序列载体及LncRNA6030408B16RIK突变体载体,将miR-326-3p模拟物及模拟物阴性对照(mimic NC)分别与荧光素酶报告载体共同转入HEK-293T细胞中;以双荧光素酶报告实验、RNA pull down实验验证LncRNA6030408B16RIK与miR-326-3p的靶向关系,用实时荧光定量逆转录聚合酶链式反应(qRT-PCR)和蛋白质印迹法(Western blotting)分析包括α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异性蛋白-1(FSP1)、波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)等在内的多种腹膜纤维化相关因子的表达情况。结果双荧光素酶实验结果:pWt-LncRNA6030408B16RIK(野生型LncRNA6030408B16RIK)的荧光素酶活性[(0.49±0.05)比(1.01±0.11)]明显降低(P<0.05),而pMut-LncRNA6030408B16RIK(突变型LncRNA6030408B16RIK)的荧光素酶活性[(1.00±0.10)比(1.03±0.12)]没有明显变化(P>0.05)。与尿毒症组相比,miR-326-3p mimic组中α-SMA、FSP1、波形蛋白表达量[(0.51±0.06)、(0.22±0.03)、(0.61±0.06)比(0.81±0.09)、(0.62±0.08)、(0.96±0.10)]明显下降,E-钙黏蛋白[(1.99±0.21)比(1.14±0.14)]明显上升(P<0.05),而与空白组相比,miR-326-3p inhibitor组中α-SMA、FSP1、波形蛋白表达[(1.57±0.16)、(1.16±0.18)、(1.72±0.17)比(0.92±0.09)、(0.64±0.07)、(0.92±0.10)]明显上升,E-钙黏蛋白表达[(0.62±0.06)比(1.14±0.15)]明显下降(P<0.05)。结论LncRNA6030408B16RIK可直接结合miR-326-3p,而过表达miR-326-3p可抑制尿毒症大鼠腹膜透析超滤衰竭的发生。

Objective To investigate the mechanism of long noncoding RNA 6030408B16RIK(lncRNA6030408B16RIK)binding to microRNA(miRNA-326-3p)causing peritoneal fibrosis in a rat uremic model to provide a new basis for the prevention of ultrafiltration failure.Methods A total of 36 successfully modeled uremic rats were divided into 6 groups with 6 rats in each group:the uremic group(without peritoneal dialysis treatment),blank group(peritoneal dialysis for 4 weeks),NC group(peritoneal dialysis for 4 weeks+transfected empty plasmid),inhibitor NC group(peritoneal dialysis for 4 weeks+transfected empty plasmid inhibitor),miR-326-3p mimic group(peritoneal dialysis for 4 weeks+miR-326-3p mimic),and miR-326-3p inhibitor group(peritoneal dialysis for 4 weeks+miR-326-3p inhibitor).The lncRNA6030408B16RIK original sequence vector and lncRNA6030408B16RIK mutant vector were constructed,and the miR-326-3p mimic and mimic negative control(mimic NC)were transferred into HEK-293T cells together with the luciferase reporter vector respectively.Dual-luciferase reporter assay and RNA pull down assay were used to verify the targeting relationship between lncRNA6030408B16RIK and miR-326-3p,and the expression of various peritoneal fibrosis-related factors including α-smooth muscle actin(α-SMA),fibroblast-specific protein-1(FSP1),vimentin(Vimentin),and E-calmodulin(E-cadherin)α-SMA,FSP1,vimentin and E-cadherin were analyzed by RT-qPCR and Western blotting.Results The dual-luciferase assay showed that the relative luciferase activity of pWt-lncRNA6030408B16RIK(wild-type lncRNA6030408B16RIK)was significantly lower[(0.49±0.05)vs.(1.01±0.11)](P<0.05),while pMut-lncRNA6030408B16RIK(mutant lncRNA6030408B16RIK)had no significant change in luciferase activity[(1.00±0.10)compared to(1.03±0.12)](P>0.05).Compared with the uremic group,the expression of α-SMA,FSP1 and vimentin[(0.51±0.06),(0.22±0.03),(0.61±0.06)vs.(0.81±0.09),(0.62±0.08),(0.96±0.10)]was significantly decreased in the miR-326-3p mimic group,and E-cadherin[(1.99±0.21)vs.(1.14±0.14)]was significantly increased(P<0.05).Compared with the blank group,in the miR-326-3p inhibitor group,the expression of α-SMA,FSP1 and vimentin[(1.57±0.16),(1.16±0.18),(1.72±0.17)vs.(0.92±0.09),(0.64±0.07),(0.92±0.10)]significantly increased in the miR-326-3p inhibitor group,and E-cadherin[(0.62±0.06)vs.(1.14±0.15)]was significantly decreased(P<0.05).Conclusion LncRNA6030408B16RIK directly binds miR-326-3p,and overexpression of miR-326-3p inhibits the development of peritoneal dialysis ultrafiltration failure in uremic rats.