| 李欣绪,周忠启,王志奎,等.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[J].安徽医药,2023,27(2):265-270. |
| 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 |
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| DOI：10.3969/j.issn.1009-6469.2023.02.012 |
| 中文关键词: 腹膜纤维化 尿毒症 超滤 腹膜透析 microRNA-326-3p 双荧光素酶报告实验 |
| 英文关键词: Peritoneal fibrosis Uremia Ultrafiltration Peritoneal dialysis MicroRNA-326-3p Dual-luciferase reporter as-say |
| 基金项目:山东省自然科学基金面上项目( ZR2019MH126) |
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| 中文摘要: |
| 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-326-3pmi，mic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L，ncRNA6030408B16RIK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(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法-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-Ln-cRNA6030408B16RIK(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。 |
| 英文摘要: |
| 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 ultrafiltra-tion failure.Methods A total of 36 successfully modeled uremic rats were divided into 6 groups with 6 rats in each group: the uremicgroup (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 con-structed, and the miR-326-3p mimic and mimic negative control (mimic NC) were transferred into HEK-293T cells together with the lu-ciferase reporter vector respectively. Dual-luciferase reporter assay and RNA pull down assay were used to verify the targeting relation-ship 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 lucifer-ase 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 vi-mentin [(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. |
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