Meng Qingkun, Wang Chengfu, Gao Yang, Zhu Yu, Meng Jin, Luan Bo, Sun Zhijun
Department of Cardiology, The People's Hospital of China Medical University.
Department of Cardiology, Shengjing Hospital of China Medical University.
Int Heart J. 2025;66(4):660-669. doi: 10.1536/ihj.24-374.
This study explored the relationship among lncRNA NR_027324, miR-103-3p, and autophagy-related protein 5 (ATG5) in cardiomyocytes under hypoglycemic/hypoxic conditions.Bioinformatics and luciferase assays were used to investigate the interaction between NR_027324 and miR-103-3p. H9C2 cells cultured under hypoglycemic/hypoxic conditions were transfected with NR_027324 siRNA, overexpression vectors, and miR-103-3p mimics/inhibitors. Cell viability and damage were measured using MTT and LDH assays, respectively. qRT-PCR was performed to assess the mRNA levels of NR_027324, miR-103a-3p, and ATG5. Protein expressions were analyzed by Western blotting, and immunofluorescence was used to observe LC3-I/II expression.In hypoglycemic/hypoxic conditions, H9C2 cell viability decreased significantly, and LDH levels increased, indicating cell damage. Simultaneously, NR_027324 and ATG5 expressions were upregulated, while miR-103-3p was downregulated. Overexpression of NR_027324 enhanced cell viability and reduced LDH release. Conversely, knockdown of NR_027324 resulted in decreased cell viability and increased LDH release. In addition, knockdown of NR_027324 led to upregulation of miR-103-3p and downregulation of ATG5. Furthermore, the luciferase activity was significantly lower when miR-103-3p interacted with wildtype NR_027324, validating the binding between NR_027324 to miR-103-3p. The overexpression of NR_027324 led to downregulation of miR-103-3p, while its knockdown resulted in the upregulation of miR-103-3p. Lastly, NR_027324 overexpression alone significantly upregulated ATG5 expression, which was counteracted when NR_027324 and miR-103-3p were co-overexpressed.The findings of this study highlight the significance of NR_027324, miR-103-3p, and ATG5 in mediating the autophagy and apoptosis response of H9C2 cells under hypoglycemic/hypoxic stress, providing valuable insights into potential targets for therapeutic interventions in related cardiovascular conditions.
本研究探讨了低血糖/低氧条件下心肌细胞中长链非编码RNA NR_027324、miR-103-3p和自噬相关蛋白5（ATG5）之间的关系。采用生物信息学和荧光素酶测定法研究NR_027324与miR-103-3p之间的相互作用。将在低血糖/低氧条件下培养的H9C2细胞用NR_027324 siRNA、过表达载体以及miR-103-3p模拟物/抑制剂进行转染。分别使用MTT和LDH测定法测量细胞活力和细胞损伤情况。进行qRT-PCR以评估NR_027324、miR-103a-3p和ATG5的mRNA水平。通过蛋白质印迹分析蛋白质表达情况，并使用免疫荧光法观察LC3-I/II的表达。
在低血糖/低氧条件下，H9C2细胞活力显著降低，LDH水平升高，表明细胞受到损伤。同时，NR_027324和ATG5的表达上调，而miR-103-3p的表达下调。NR_027324的过表达增强了细胞活力并减少了LDH释放。相反，敲低NR_027324导致细胞活力降低和LDH释放增加。此外，敲低NR_027324导致miR-103-3p上调和ATG5下调。此外，当miR-103-3p与野生型NR_027324相互作用时，荧光素酶活性显著降低，证实了NR_027324与miR-103-3p之间的结合。NR_027324的过表达导致miR-103-3p下调，而其敲低则导致miR-103-3p上调。最后，单独过表达NR_027324显著上调了ATG5的表达，而当NR_027324和miR-103-3p共过表达时这种上调作用被抵消。
本研究结果突出了NR_027324、miR-103-3p和ATG5在介导低血糖/低氧应激下H9C2细胞自噬和凋亡反应中的重要性，为相关心血管疾病治疗干预的潜在靶点提供了有价值的见解。
