NR_027324 regulates autophagy and apoptosis by combining with miR-103-3p as ceRNA to regulate ATG5 in cardiomyocytes during hypoglycemic and hypoxic injury
Authors: Meng Qingkun , Tan Hao , Gu Hui , Luo Jiaying , Wang Jingjing , Wang Chuanhe , Han Su , Sun Zhijun 
Published: November 2020

Abstract
Background: Myocardial infarction (MI) refers to a fatal disease, and the border zone (BZ) of myocardial infarction is of high importance to the prognosis of myocardial infarction patients. Autophagy and apoptosis can significantly impact cardiovascular diseases. In the previous studies conducted by the authors, plenty of differential expressions of long non-coding RNA (lncRNA) were reported in the border zone of myocardial infarction. As revealed from the results of bioinformatics analysis, LncRNA may take up a vital part in the pathological process of cardiovascular disease by regulating apoptosis and autophagy. This study aimed to firstly conduct bioinformatics analyses to predict that NR_027324, as a competitive endogenous RNA (ceRNA), binds to miR-103-3p and subsequently regulates the expression of downstream target gene ATG5, to secondly verify the mentioned regulatory relationship by molecular biological experiments, and thirdly to investigate that the above pathways transmit apoptosis and autophagy signals in cardiomyocytes during hypoglycemic and anoxic injuries. Methods: The binding of NR_027324 to miR-103-3p was predicted by the bioinformatics analysis, and then the expression of downstream ATG5 was regulated. By the dual luciferase assay, the binding of NR_027324 to miR-103-3p was confirmed. H9c2 cells underwent the culture under low glucose and hypoxia to simulate cardiomyocytes under ischemia. NR_027324 siRNA and overexpression plasmid vector and miR-103-3p (mimics and inhibitors) were adopted to transfect cells to examine its function in cardiomyocytes. By the reverse transcription-quantitative polymerase chain reaction (RT-PCR), the expression levels of NR_027324, miR-103a-3p and ATG5 were assessed. Cell viability was measured by the MTT assay, and cell injury was analyzed by the lactate dehydrogenase assay (LDH). The western blotting assay was performed to detect the expressions of Bcl2, Bax, Atg5, cleaved-caspase3 and cleaved-caspase9. Moreover, the immunofluorescence assay was used to detect the expression of LC3-I/II as a marker of autophagy.Results: In hypoglycemic and anoxic H9C2 cells, the expressions of NR_027324 and ATG5 were up-regulated, while miR-103-3p showed a down-regulated expression. By the dual luciferase assay, the binding of NR_027324 to miR-103-3p was confirmed. The overexpressed NR_027324 could enhance the reduced cell viability of hypoglycemic and anoxic H9C2 cells and reduce cell damage. Interfering with the expression of NR_027324 could further reduce the viability of hypoglycemic anoxic H9C2 cells, increase injury, inhibit protective autophagy and accelerate apoptosis of hypoglycemic anoxic H9C2 cells. The mentioned pathophysiological processes were achieved by NR_027324 binding to miR-103-3p and then regulating the expression of target gene ATG5.Conclusion: In hypoglycemic and anoxic H9C2 cells, the expressions of NR_027324 and ATG5 are up-regulated, while the expression of miR-103-3p decreases. NR_027324 acts as ceRNA combined with miR-103-3p to regulate ATG5 and control autophagy and apoptosis in cardiomyocytes during hypoglycemic and anoxic injuries.