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目的 探讨长链非编码RNA RMRP(LncRNA RMRP)通过调控miR-766-5p对氧糖剥夺/再灌注(OGD/R)诱导的小鼠HL-1心肌细胞铁死亡的影响及机制。方法 体外培养HL-1细胞，并构建OGD/R模型，qRT-PCR检测不同再灌注时间点HL-1细胞中LncRNA RMRP表达水平。将LncRNA RMRP小RNA干扰片段(si-RMRP)及其阴性对照(si-NC)、miR-766-5p抑制剂(miR-766-5p inhibitor)及其阴性对照(inhibitor-NC)转染至HL-1细胞中，再进行OGD/R处理。CCK-8检测细胞存活率；试剂盒检测细胞上清液中乳酸脱氢酶(LDH)水平及细胞中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)和亚铁离子(Fe2+)水平；qRT-PCR检测细胞中LncRNA RMRP和miR-766-5p表达水平；Western blot检测细胞中铁死亡相关蛋白谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)、铁蛋白重链1(FTH1)表达水平；双荧光素酶报告基因实验检测LncRNA RMRP与miR-766-5p之间的海绵吸附关系。结果 HL-1细胞中LncRNA RMRP表达水平随着再灌注时间的延长逐渐升高(P<0.01)。OGD/R处理可降低HL-1细胞存活率及细胞中miR-766-5p表达水平(P<0.01);提高上清液中LDH及细胞中MDA和Fe2+含量，抑制细胞中SOD和GSH活性(P<0.01);同时下调细胞中GPX4、SLC7A11和FTH1蛋白表达水平(P<0.01)。沉默LncRNA RMRP可提高OGD/R诱导后HL-1细胞的存活率及细胞中miR-766-5p表达水平(P<0.01);降低上清液中LDH和细胞中MDA和Fe2+含量，增加细胞中SOD和GSH活性(P<0.01);同时上调细胞中GPX4、SLC7A11和FTH1蛋白表达水平(P<0.01)。双荧光素酶报告基因实验证实，LncRNA RMRP可海绵吸附调控miR-766-5p表达，且抑制miR-766-5p表达可部分抵消LncRNA RMRP沉默对OGD/R诱导的HL-1细胞铁死亡的改善作用。结论 沉默LncRNA RMRP可抑制OGD/R诱导的HL-1细胞铁死亡，其作用机制与海绵吸附调控miR-766-5p表达有关。
Abstract：Objective To investigate the effect and mechanism of long non-coding RNA RMRP(LncRNA RMRP) on oxygen-glucose deprivation/reperfusion(OGD/R)-induced ferroptosis in mouse HL-1 cardiomyocytes by regulating miR-766-5p. Methods HL-1 cells were cultured in vitro, and OGD/R models were established. The expression levels of LncRNA RMRP in HL-1 cells at various reperfusion time points were subsequently quantified using qRT-PCR. The LncRNA RMRP small RNA interference fragment(si-RMRP) and its corresponding negative control(si-NC), as well as the miR-766-5p inhibitor and its respective negative control(inhibitor-NC), were transfected into HL-1 cells. Subsequently, the cells were subjected to OGD/R treatment. CCK-8 assay was employed to evaluate cell viability. Assay kits were employed to measure the levels of lactate dehydrogenase(LDH) in the cell supernatant, as well as the intracellular levels of malondialdehyde(MDA), superoxide dismutase(SOD), glutathione(GSH), and ferrous ion(Fe2+). qRT-PCR analysis was conducted to assess the expression levels of LncRNA RMRP and miR-766-5p. Western blot analysis was conducted to assess the expression levels of proteins associated with ferroptosis including GPX4, SLC7A11, and FTH1. Dual-luciferase reporter assays were performed to investigate the sponge adsorption relationship between LncRNA RMRP and miR-766-5p. Results As reperfusion time extended, the expression level of LncRNA RMRP in cells progressively increased(P<0.01). Treatment with OGD/R significantly inhibited the viability of HL-1 cells, reduced the expression of miR-766-5p(P<0.01), elevated the levels of LDH in the supernatant, as well as MDA and Fe2+ levels within the cells, and decreased the activities of SOD and GSH in cells(P<0.01). Additionally, OGD/R treatment downregulated the protein expression levels of GPX4, SLC7A11, and FTH1(P<0.01). Silencing LncRNA RMRP reversed these effects by enhancing the viability of HL-1 cells, increasing miR-766-5p expression(P<0.01), reducing LDH in the supernatant, as well as MDA and Fe2+ levels within the cells, and promoting SOD and GSH activities in cells(P<0.01). Furthermore, silencing LncRNA RMRP upregulated the protein expression levels of GPX4, SLC7A11, and FTH1(P<0.01). The dual-luciferase reporter assay confirmed that LncRNA RMRP could regulate the expression of miR-766-5p through a sponge adsorption mechanism. Partial inhibition of miR-766-5p inhibitor expression could mitigate the improvement effect caused by LncRNA RMRP silencing on OGD/R-induced ferroptosis in HL-1 cells. Conclusion Silencing LncRNA RMRP inhibits OGD/R-induced ferroptosis in HL-1 cells, potentially through the sponge-mediated regulation of miR-766-5p expression.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.12.003
中图分类号:R54
引用信息:
[1]何蕾,孙兴兰,吴应兴,等.LncRNA RMRP通过调控miR-766-5p对氧糖剥夺/再灌注诱导的小鼠HL-1心肌细胞铁死亡的影响[J].安徽医科大学学报,2025,60(12):2207-2214.DOI:10.19405/j.cnki.issn1000-1492.2025.12.003.
基金信息:
江西省自然科学基金青年基金项目(编号：20232BAB216 008); 中国国家留学基金项目(编号：202506820050)~~