NR_027324 regulates autophagy and apoptosis by combining with miR-103-3p as ceRNA to regulate ATG5 in cardiomyocytes during hypoglycemic and hypoxic injury

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 signicantly 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 rstly 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 conrmed. 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,


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Myocardial infarction refers to a fatal disease. The existing treatment of myocardial infarction is primarily to save the dying cardiomyocytes in the border zone of myocardial infarction [1]. In the process of pathophysiological variations in the border zone of myocardial infarction, autophagy and apoptosis of cardiomyocytes are critical to disease progression and prognosis. The autophagy and apoptosis of cardiomyocytes show a close relationship to the regulation of gene expression [2]. Long non-coding RNA (lncRNA) belongs to a type of non-coding RNA exhibiting a transcription length > 200 nt without coding protein [3]. It signi cantly impacts the pathophysiological process of myocardial infarction [4][5][6][7][8]. Their regulatory functions are primarily determined by various epigenetic regulatory mechanisms (e.g., transcriptional regulation, post-transcriptional gene regulation, competing endogenous RNAs(CeRNA), protein post-translational gene transcription regulation, as well as nuclear compartmentalization) [9]. According to the research ndings previously achieved by the authors [10], lncRNA has considerable differential expressions in the border zone of myocardial infarction in rats. Besides, by the bioinformatics analysis, lncRNA was found to play a potential role in various pathological mechanisms. To simulate myocardial ischemia in the border zone of myocardial infarction, H9C2 cells were administrated with hypoglycemia and hypoxia. Among the differentially expressed lncRNAs screened by lncRNA microarray, NR_027324 with a signi cant differential expression and an up-regulated expression in the border zone of rat myocardial infarction was taken as the research object. The aim was to explore the mechanism by which NR_027324 regulates the expression of miR-103-3p as CeRNA and then regulates the expression of downstream target ATG5 to control cardiomyocyte autophagy and apoptosis.

Transfection
H9C2 cells were plated in 6-well plates (1x105 per well) and subsequently incubated at 37 ℃ for 24 h under 5% CO2. By complying with the manufacturer's protocol, the cells underwent the transient transfection with a nal 20 nM dose of overexpression plasmid of LncRNA-NR_027324 (Lncm), empty plasmid/overexpression plasmid control (EPC), LncRNA small interfering RNA (si-Lnc), LncRNA small interfering RNA control (si-Lnc-NC), miR-103-3p mimics (miRm) or miR-103-3p mimics control (miR-NC) with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scienti c, Inc.). After the cells were incubated for 24 h, the different cell groups were subsequently analyzed. with the manufacturer's protocol, SYBRGREEN mastermix (Cat.No.SY1020; Solarbio technology co.LTD, Beijing, China) was adopted to amplify the cDNA samples. Besides, the uorescence quantitative analysis was conducted with the ExicyclerTM96 uorescence quantitative instrument produced by BIONEER company in Korea. In brief, the PCR reaction conditions of mRNA and LncRNA included 94 °C for 5 min, followed by 40 cycles of 94 °C for 10 sec and 60 °C for 20 sec. The PCR reaction conditions of miRNA included 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 sec and 60 °C for 15 sec. Moreover, ddH 2 O was used as a non-template control for each plate. 5S was used to normalize the expression levels of miR-103-3p. β-actin was employed for normalizing the expression levels of NR_027324 and Atg5. The relative expression was quanti ed by 2 -△△CT method, which was repeated three times. The median of the three results was obtained to calculate the relative expression level. The sequence list of PCR reaction primers is presented in Table 1.

Western blot analysis
The total protein was lysed with a whole protein extraction kit (Cat.No.WLA019; wanleibio Co., Ltd., Shenyang, China Co., Ltd., Shenyang, China) at 37°C for 45 min; afterwards, it was further washed 6 times with TBST. Besides, the ECL chemiluminescence kit (Cat.No. WLA003; wanleibio Co., Ltd., Shenyang, China) was employed to detect the blot, and the gel image processing system (Gel-Pro-Analyzer software) was adopted to analyze the optical density of the target band value.
2.6 Immuno uorescence 1-2 drops of cell suspension were dropped on the glass slides that were washed in advance, sterilized by pure alcohol and sterilized under high pressures, and the cell adhesion was observed the next day. After the cells were adhered to the wall and exhibited the required density, they were washed with 1x phosphate buffer (PBS) for 5min x 3 times and then xed in 4% paraformaldehyde for 15 min. Next, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min. The samples were blocked with goat serum at ambient temperature for 15 min and subsequently incubated with primary antibody (LC3-I/II) overnight at 4 ℃. On the following day, the samples were incubated with secondary antibodies for 1 h at ambient temperature in the dark. Lastly, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich; Merck KGaA) at ambient temperature for 5 min. After each step, the slides were washed with PBS for 5min x 3 times. The anti-uorescence quenching agent was dropped on the glass slide, and the slide upside down on the glass slide dripped with anti-uorescence quenching agent was blocked. Olympus BX53 uorescence microscope (Olympus corporation, Tokyo, Japan) was used to capture images.

Lactate dehydrogenase (LDH) assay
H9C2 cells were cultured with 10% fetal calf serum and were inoculated with 24-well plates (1x10 5 per well). After the cells were transfected, the LDH leakage assay was performed to determine cell injury with the LDH cytotoxicity assay kit (Cat.No. WLA072; wanleibio Co.,Ltd., Shenyang, China) by complying with the manufacturer's protocol. The absorbance was measured at 490nm enzyme-linked immunosorbent assay (ELX-800, BIOTEK, USA), which was repeated three times in each group.

MTT assay
The H9C2 cells were plated in 96-well plates (5×10 3 per well). In each group, 5 repeats were set, and a blank control was set with the culture medium only. After the cells were adhered to the wall, the cells received the transfection and culture at 37 ℃ for 24 h. The cells were administrated with hypoglycemia and hypoxia according to different groups. After reaching the time point, the culture medium was removed; 20 μl of MTT reagent was added to each well and then incubated at 37 ℃ and 5%CO 2 for 4 h; the supernatant was rigorously discarded; afterwards, 150 μl dimethyl sulfoxide (DMSO) was added and maintained in the dark for 10 min. The absorbance of each well was measured with a microplate reader at 570nm, and the average value of 5 wells was determined.

Statistical analysis
Data were analyzed with SPSS version 16.0 (IBM corp., Armonk., NY, USA). All data are expressed as the mean ± standard deviation. Signi cant differences were identi ed by Student's t-test or one-way analysis of variance followed by Tukey's test. P<0.05 was considered to exhibit a statistically signi cant difference.

Results
Variations of NR_027324, miR-103-3p and ATG5 in hypoglycemic and hypoxic H9C2 cells To simulate the state of ischemia and hypoxia in the border zone of myocardial infarction, H9C2 cells were cultured under high glucose (4.5g/L) and normoxic conditions for 24 h; then, they were cultured in low glucose (1g/L glucose) DMEM and in an hypoxic incubator at 37℃, 1% O 2 , 94% N 2 , and 5% CO 2 for 6 h. High glucose (4.5g/L) and normoxic conditions were classi ed as controls. As revealed from the results, the cell viability was reduced, and LDH increased in hypoglycemia and hypoxia group. The expression of NR_027324 was signi cantly up-regulated, complying with the results of microarray. The expression of miR-103-3p decreased, while the expression of ATG5 was up-regulated, complying with the results of Zhang et al [11]. Fig. 1.
Effect of overexpression of NR_027324 on viability of hypoglycemic and hypoxic H9C2 cells Fig. 3 shows that H9C2 cells were transfected with NR_027324 overexpression plasmid for 24 h and then cultured in hypoglycemia and hypoxia for 6 h. Compared with the control group, the expression of NR_027324 in the hypoglycemia and hypoxia group was up-regulated; compared with the hypoglycemia and hypoxia + transfection empty plasmid group, the NR_027324 in the hypoglycemia and hypoxia + NR_027324 overexpression group was further up-regulated, while the expression of miR-103-3p decreased. Compared with the control group, the relative viability of cells in the hypoglycemia and hypoxia group was reduced signi cantly. Compared with the hypoglycemia and hypoxia + transfection empty plasmid group, the cell viability in the hypoglycemia and hypoxia + NR_027324 overexpression group increased compared with the control group, while the LDH exhibited the corresponding variations.
(P < 0.001) Effect of interfering with NR_027324 expression on the viability of hypoglycemic and hypoxic H9C2 cells As suggested in the mentioned experiment, compared with the control group, the viability of H9C2 cells was reduced, and LDH increased after cultured for 6 h in hypoglycemia and hypoxia group. 24 h after transfection of si-NR_027324, the cells were cultured for 6 h in hypoglycemia and hypoxia group.
Compared with the hypoglycemia and hypoxia + si-NR_027324-NC group, the expression of NR_027324 was down-regulated, while that of miR-103-3p increased and that of ATG5 decreased in the hypoglycemia and hypoxia + si-NR_027324 group. After interfering with the expression of NR_027324, the relative viability of (MTT) was further reduced, and LDH showed a further increase in the hypoglycemia and hypoxia group. (P < 0.001) Fig. 4.
Interference with NR_027324 expression regulates autophagy and apoptosis in hypoglycemic and hypoxic H9C2 cells As presented in Fig. 5, compared with the control group, Bax, Cleaved-caspase3, Cleaved-caspase9 (i.e., apoptosis-promoted proteins) increased in hypoglycemia and hypoxia group, while the expression of apoptosis inhibitor protein Bcl2 was down-regulated, suggesting that the apoptosis was activated. After hypoglycemia and hypoxia, compared with si-NR_027324-NC, the expression of ATG5 was downregulated after interfering with the expression of NR_027324, while those of Bax, Cleaved-caspase3 and Cleaved-caspase9 showed further up-regulations (P < 0.001), and that of Bcl2 further decreased (P < 0.001). It was therefore suggested that interfering with the expression of NR_027324 can down-regulate the expression of Atg5, inhibit cell protective autophagy and promote apoptosis. No signi cant change was identi ed in the mentioned apoptosis-promoted genes between Gdh group and si-Lnc-NC+Gdh group (P > 0.05). As revealed from the results of LC3-I/II immuno uorescence, compared with the control group, LC3-I/II protein in the hypoglycemia and hypoxia group (Gdh group) exhibited an increased expression, while in the Gdh+ si-NR_027324 group, the expression of LC3-I/II was down-regulated. (Fig. 6) NR_027324 regulates ATG5 expression by binding mir-103-3p as CeRNA As shown in Fig. 7, compared with the control group, the expression of miR-103-3p decreased, and that of ATG5 was up-regulated after transfection with overexpression of NR_027324 (P < 0.001). After NR_027324 showed an up-regulated expression, compared with miR-103-3p-mimic-NC, transfection with miR-103-3p-mimic led to the increased expression of miR-103-3p (P < 0.001), while the expression level of ATG5 decreased (P < 0.001). No signi cant variation was identi ed in the above expression between Con group and Epc group, and between Lncm group and Lncm+miR-NC group. P>0.05

Discussion
In the previous research of the authors [10], the lncRNA expression pro le was analyzed in the border zone of myocardial infarction. It was reported that lncRNAs were differentially expressed in the border zone of myocardial infarction. Unlike the results of other existing studies on myocardial infarction area [5,7], the results achieved by the authors revealed that the total number of differentially expressed lncRNAs in the border zone was more than that of the myocardial infarction area, demonstrating more occurrences of active and more complex biological processes. For instance, LncRNA: AY212271, showing a coexpression relationship to Alox5ap, might be involved in various in ammatory reactions [12][13][14][15][16]. EF424788 and MRAK088538 were co-expressed with Itgb2, and Itgb2 was reported to be a risk factor for myocardial infarction and atherosclerotic thrombotic cerebral infarction [17][18][19]. Moreover, Itgb2 was involved in the reduction of the risks of myocardial infarction by statins [20]. BC166504 was co-expressed with 4 mRNAs to participate in myocardial infarction, which included B4galt1, Eln, Il1b and Nfkbiz. B4galt1 and Il1b signi cantly impacted in ammation [21]. Eln (elastin), expressed as tropoelastin in smooth muscle, was associated with the remodeling of the extracellular matrix (ECM) of the vessel wall, which is considered a vital step in atherosclerosis and likely to predict potential cardiovascular events [22]. Nfkbiz refers to a nuclear inhibitor of NF-κB protein(IκB), which may reduce the susceptibility of myocardial infarction by reducing NFκB, i.e., a critical factor of potentially activated in ammation [23]. Both GO analysis and Pathway analysis suggested that LncRNA is involved in the apoptosis signaling pathway.
LncRNA could signi cantly regulate the expression of miRNAs by acting as competing endogenous RNAs, and then it could regulate the expression of target genes at the level of post-transcriptional translation [5,24,25]. MiRNAs refers to a type of small RNA that has been newly discovered in recent years, and some miRNAs are involved in the pathophysiological process of cardiovascular disease [26][27][28]; MiRNAs may also be a novel target for treatment. MiRNAs exhibits the function of regulating gene expression at the post transcriptional translation level. Mature miRNAs are not fully complementary to the target mRNA, thereby inhibiting the expression at the protein translation level, which may affect the stability of mRNA and cause the degradation of target mRNA. Existing studies reported that various miRNAs regulate apoptosis in cardiomyocytes. For instance, miR-25 prevented oxidative damage of cardiomyocytes by inhibiting mitochondrial apoptosis [29]. MiR-21 was signi cantly up-regulated during heart failure and inhibited cardiomyocyte apoptosis by suppressing PDCD4 [30,31]. MiR-1 could promote cardiomyocyte apoptosis after myocardial infarction, while miR-133 could select caspase-9 as the target gene to inhibit cardiomyocyte apoptosis after myocardial infarction [32]. MiRNA acts as the target of intervention therapy, and there may be too many pathways regulated by the identical miRNA simultaneously, causing too much in uence. However, the conservatism of LncRNA is poor, and the impact is signi cantly smaller. Thus, as a target of gene therapy, LncRNA may be more suitable. Apoptosis and autophagy participate in the overall pathophysiology in the border zone of myocardial infarction. Abnormal expression of many apoptosis-related genes could promote or inhibit cardiomyocyte apoptosis and autophagy (e.g., ATG5, Bax, Bcl-2, Caspase-3 and Caspase-9) [33].
As indicated from the reannotation analysis, NR_027324 was a segment of H9 gene, and mir-103-3p was evaluated as its binding target. MiR-103-3p could down regulate the protein expression of Beclin1, ATG5, and inhibit autophagy [11]. The authors inferred that NR_027324, a section of the H9 gene, may target miR-103-3p via CeRNA binding, adjust its expression, and subsequently regulate the downstream target gene ATG5 of miR-103-3p. Based on the dual luciferase reporter system, the inference of targeted binding of NR_027324 to miR-103-3p was directly con rmed. Through the overexpression of NR_027324 and by interfering with the expression of NR_027324 in hypoglycemic and hypoxic H9C2 cells, it was veri ed that NR_027324 could regulate the viability, apoptosis and autophagy.
Existing studies suggested that after the primary rat cardiomyocytes was administrated with OGD (oxygen-glucose deprivation) for 4 h, the cell survival rate decreased, LDH release was improved, and autophagy was activated [34]. The same method was adopted in this study to simulate ischemia and hypoxia in the border zone of myocardial infarction in rats, and the role of NR_027324 in the zone was studied. According to some existing studies, autophagy of H9C2 cells was activated at 4 h and inhibited from 8 h to 12 h after being administrated with OGD. Administration of rapamycin, an autophagy activator, could inhibit the activation of NFκB [35]. Some studies reported that NR_027324 can protect the hypoxic injury of cardiomyocytes [36,37]. Through the results of this study, it was inferred that NR_027324 promotes autophagy. Autophagy increases after the overexpression and decreases after interfering with the expression. After the intervention of hypoxia and hypoglycemia for 6 h, the expression of Atg5 was up-regulated, and the stress level of autophagy rose, which might be the self-protective mechanism of cardiomyocytes. After the overexpression of NR_027324, autophagy was further activated, which could protect the cells. The cell survival rate increased, and the release of LDH decreased. After transfection with interfering NR_027324, the expression of Atg5 was down-regulated, protective autophagy was inhibited, apoptosis was promoted, and the expressions of Bax and cleaved-caspase3 were up-regulated; thus, the cell survival rate decreased, and the release of LDH increased.
In brief, this study reported that NR_027324 binds to miR-1033p by acting as CeRNA, and then regulates the downstream target gene ATG5 to control cardiomyocyte apoptosis and autophagy in rats H9C2 cells with hypoglycemia and hypoxia. In hypoglycemic and hypoxic rat cardiomyocytes, LncRNA is likely to act as a possible therapeutic target to promote cardiomyocyte autophagy, reduce cardiomyocyte apoptosis and save ischemic and hypoxic cardiomyocyte. It cannot be ignored that with the time evolution of myocardial infarction, autophagy and apoptosis also have dynamic changes, and the role of LncRNA may also show dynamic variations, which should be con rmed by further research. The microarray analysis suggested that more LncRNAs may play a part by regulating in ammatory response, abnormal contraction and reducing scarring and other different pathways. More research and evidence are required to delve into the functions of other lncRNAs.

Conclusions
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. Availability of data and material: We declare that materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant con dentiality.  Expression of apoptosis-related proteins and autophagy-related proteins in H9C2 cells under hypoglycemia and hypoxia, and transfected with si-NR_027324 or si-NR_027324-control. A-E, Quantitative evaluation of protein expression levels of ATG5, Bax, Bcl2, Cleaved-caspase3 and Cleaved-caspase9 proteins was quantitatively studied. F, Protein expression levels of ATG5, Bax, Bcl2, Cleaved-caspase3 and Cleaved-caspase9 were determined by the western blotting assay. Con, normal control group, high glucose normoxic group; Gdh, hypoglycemic hypoxia group; si-LncNC,LncRNA interference control group;