Long Noncoding RNA SOX2-OT Exacerbates Hypoxia-Induced Cardiomyocytes Injury by Regulating miR-27a-3p/TGFβR1 Axis

Background Myocardial infarction (MI) was a severe cardiovascular disease resulted from acute, persistent hypoxia, or ischemia condition. Additionally, MI generally led to heart failure, even sudden death. A multitude of research studies proposed that long noncoding RNAs (lncRNAs) frequently participated in the regulation of heart diseases. The specific function and molecular mechanism of SOX2-OT in MI remained unclear. Aim of the Study. The current research was aimed to explore the role of SOX2-OT in MI. Methods Bioinformatics analysis (DIANA tools and Targetscan) and a wide range of experiments (CCK-8, flow cytometry, RT-qPCR, luciferase reporter, RIP, caspase-3 activity, trans-well, and western blot assays) were adopted to investigate the function and mechanism of SOX2-OT. Results We discovered that hypoxia treatment decreased cell viability but increased cell apoptosis. Besides, lncRNA SOX2-OT expression was upregulated in hypoxic HCMs. Hereafter, we confirmed that SOX2-OT could negatively regulate miR-27a-3p levels by directly binding with miR-27a-3p, and miR-27a-3p also could negatively regulate SOX2-OT levels. Furthermore, knockdown of SOX2-OT promoted cell proliferation, migration, and invasion, but limited cell apoptosis. However, these effects were reversed by anti-miR-27a-5p. Besides, we verified that miR-27a-3p binding with the 3′UTR of TGFBR1 and SOX2-OT regulated TGFβR1 level by collaborating with miR-27a-3p in HCMs. Eventually, rescue assays validated that the influence of SOX2-OT silence or miR-27a-3p overexpression on cellular processes in cardiomyocytes injury was counteracted by TGFBR1 overexpression. Conclusions Long noncoding RNA SOX2-OT exacerbated hypoxia-induced cardiomyocytes injury by regulating miR-27a-3p/TGFβR1 axis, which may provide a novel insight for heart failure treatment.


Introduction
Myocardial infarction (MI) referred to the necrosis of myocardium caused by severe persistent hypoxia or ischemia due to coronary occlusion and interruption of blood flow [1,2]. MI brought numerous damages to the heart muscle and has become the leading cause of morbidity and mortality among diseases worldwide [3,4]. Several risk factors such as excessive labor, irregular diet, smoking, and drinking were reported to be closely associated with the initiation or progression of MI [5,6]. Although the overall mortality of MI patients was obviously reduced by coronary reperfusion, the additional reperfusion injuries such as cardiomyocyte dysfunction and death were frequently reported [7,8]. erefore, better understanding and further exploration of molecular mechanisms in MI are urgently needed. e long noncoding RNAs (lncRNAs) belonged to a category transcript with no less than 200 nucleotides in length and without protein-coding capacity [9,10]. LncRNAs were proved to take part in the regulation of several biological processes such as cell proliferation, apoptosis, migration, and invasion in a variety of diseases or tumors [11,12]. Long noncoding RNA HOTAIR was reported to accelerate the proliferation, migration, and invasion of head and neck squamous cell carcinoma (HNSCC) cells by sponging microRNA-206 and targeting STC2 [13]. Additionally, long noncoding RNA ZFAS1 facilitated chondrocytes proliferation, migration, and apoptosis of in osteoarthritis [14]. In MI, silence of lncRNA XIST inhibited myocardial cell apoptosis through regulating miR-449 in the MI rat model [15]. Recently, lncRNA SOX2 overlapping transcript (SOX2-OT) was proposed to promote a series of tumors progression such as esophageal squamous cell carcinoma, lung squamous cell carcinoma, and cholangiocarcinoma [16][17][18]. Although SOX2-OT was identified to be upregulated in ischemic heart failure [19], the specific function and regulator mechanism of SOX2-OT remains elusive.
MicroRNAs (miRNAs) were another subgroup of noncoding RNAs with about 22 nucleotides in length [20]. MiRNAs were shown to bind to the 3′ untranslated region (3′-UTR) of target gene to modulate its level and thereby regulate cell proliferation, differentiation, and apoptosis [21]. Numerous miRNAs were proposed to be implicated in the development of MI. As examples, miR-208, miR-494, miR-499, and miR-1303 played key roles in early diagnosis of MI [22]. miR-130 exacerbated myocardial injury caused by acute myocardial infarction via targeting PPAR-c [23]. Recently, the downregulation of miR-27a-3p suppressed inflammatory response and apoptosis of hippocampal neuronal cell in epilepsy via increasing mitogen-activated protein kinase 4 (MAP2K4) [24]. In addition, miR-27a-3p interacted with hsa_circ_0026480 and hsa_circ_0046159 to modulate chronic thromboembolic pulmonary hypertension [25]. Nevertheless, the biological role of miR-27a-3p was unclear in MI.
In conclusion, the present project was aimed to explore the function and mechanism of SOX2-OT in MI. We found out that SOX2-OT exacerbated hypoxia-induced cardiomyocytes injury by promoting proliferation, migration, and invasion but inhibiting apoptosis via miR-27a-3p/TGFβR1 axis, which may offer a novel and possible tactic for MI treatment.

Cell and Cell Treatment.
Human cardiomyocyte primary cells (HCM) were obtained from Cellprogen (Torrance, USA) and cultivated in human cardiomyocytes primary cell culture media (Cellprogen) in a humid incubator at 37°C with 5% CO 2 . HCMs were incubated in an incubator with 1% O 2 , 5% CO 2 , and 94% N 2 for different times (12,24, and 48 h) to induce hypoxia.

Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-qPCR).
e extraction of total RNA from HCMs was performed using Trizol reagent ( ermo Fisher, USA) under the manufacturer's instructions. en, SuperScript ® III First-Strand Synthesis System ( ermo Fisher) RNA was used for reverse-transcription. Real-time PCR was operated by QuantiTect SYBR Green PCR Kit (Qiagen, Germantown, USA) on a QuantStudio 3 Real-Time PCR System ( ermo Fisher). e primers would be provided under requirement. GAPDH served as an internal control for lncRNA and mRNA. U6 served as internal control for miRNA. e relative RNA levels were analyzed using the 2 −ΔΔCt method.

RNA Immunoprecipitation (RIP) Assay.
Magna RNA immunoprecipitation (RIP) kit (Millipore, Billerica, USA) was adopted in RIP assay. Magnetic beads containing Ago2 or IgG (negative control) antibodies were added into HCM cells lysate which was preserved in RIP buffer before. e relative level of SOX2-OT and miR-27a-3p were detected by RT-qPCR assay. Input served as control.

Apoptosis Assay by Flow Cytometry.
Apoptosis was analyzed through dual staining with the Annexin V-FITC/ staining kit ( ermoFisher). According to the manufacturer's instructions, Annexin V-FITC and PI were added to the cellular suspension for staining, FACSCalibur flow cytometer in CellQuest 3.0.1 software (BD Biosciences, USA) was used to analyze HCMs. Percentages of apoptosis HCMs were detected by dual-color analysis.

Caspase-3 Activity Assay.
e caspase-3 assay kit (colorimetric) (Abcam, Cambridge, USA) was applied to measure caspase-3 activity in line with manufacturer's protocol. In brief, transfected HCMs were lysed in lysis buffer and then added to 96-well plate after cultivation for 48 hours. Subsequently, caspase-3 catalytic substrate DEVD-pNA, 2× reaction buffer, and DTT were cocultivated with HCMs in each well at 37°C for 2 h. Finally, the measurement of OD value at 405 nm was performed in a microplate reader.

Western Blot.
Total protein was extracted from heart tissues and cells by using RIPA lysis buffer (Santa Cruz Biotechnology, USA). e proteins were isolated by SDS-PAGE and then moved onto PVDF membranes (Millipore, MA). Subsequently, Tris-buffered saline 0.01% Tween 20 (Santa Cruz Biotechnology) with 5% defatted milk was used to block the membranes for one hour at room temperature. Next, primary antibodies including anti-TGFBR1 (ab31013, Abcam) and anti-GAPDH (ab181602, Abcam) were cultured with membranes at 4°C overnight. After cultivation with the secondary antibody for 1 h, the blots were visualized with a Clarity ™ Western ECL Blotting Substrates (Bio-Rad) under manufacturer's instruction, and protein levels were quantified by using ImageJ software. GAPDH functioned as an internal control.

Trans-Well Assay.
e trans-well assays were operated in 24-well trans-well chambers (8 μm; Corning, Shanghai, China). e upper chambers with (or without) 1 mg/mL Matrigel were used to detect cell invasion (or migration). 10, 000 HCMs in medium were seeded into the upper chambers, whilst medium supplemented with 10% FBS was added to the bottom chambers. 24 hours later, the migrated or invaded HCMs were immobilized in 75% methanol and stained with 0.1% crystal violet. e number of migrated and invaded cells in each field was counted under a light microscope.
2.11. Statistical Analysis. All data were displayed as means ± SD. Statistical analysis was progressed using the SPSS (Chicago, IL, USA) and GraphPad Prism 5 software (San Diego, CA). Experiments were operated for three times. Significance of the variance between 2 or more groups was evaluated through Student's t-test or ANOVA. P < 0.05 was regarded as statistical significance.

SOX2-OT Was Upregulated in HCM Cells Induced by
Hypoxia. Previous study has identified several upregulated lncRNAs including SOX2-OT in ischemic heart failure tissues. Additionally, data from GSE66360 (https://www. ncbi.nlm.nih.gov) depicted that SOX2-OT was also upregulated in ischemic cardiomyopathy or myocardial infarction patients' peripheral blood compared with healthy peripheral blood (Figure 1(a)). To further explore the function of SOX2-OT, HCMs were treated with hypoxia for different time to construct the myocardial injury cell model. As shown in Figures 1(b) and 1(c), after treated with hypoxia for 24 or 48 hours, HCMs viability was declined, but the apoptosis rate of HCMs was increased. According to the result of RT-qPCR, SOX-OT displayed the most significant upregulation among these lncRNAs compared with the control group (Figure 1(d)). To sum up, SOX2-OT was upregulated in HCM cells induced by hypoxia.

SOX2-OT Cooperated with miR-27a-3p to Modulate Cellular Processes in HCM Cells.
To explore the specific function of SOX2-OT and miR-27a-3p on cellular processes in HCMs, a wide range of experiments were carried out. To begin with, cell viability was improved by SOX2-OT inhibition and the promoting effect of SOX2-OT silencing were offset by the knockdown of miR-27a-3p (Figure 3(a)). On the contrary, the depletion of miR-27a-3p neutralized the suppressive effect of SOX2-OT attenuation on cell apoptosis and caspase-3 activity (Figures 3(b) and 3(d)). At last, transwell assay revealed that sh-SOX2-OT-mediated the elevation of migrated and invaded cells was reversed by cotransfection of sh-SOX2-OT and anti-miR-27a-3p (Figures 3(e)-3(h)). Taken together, SOX2-OT cooperated with miR-27a-3p to modulate cellular processes in HCM cells.

MiR-27a-3p Inhibited Heart Failure Development by
Modulating TGFβR1 in HCM Cells. Rescue assays were conducted to confirm the miR-27a-3p/TGFβR1 axis in HCM cells. At the beginning, TGFBR1 overexpression inhibited cell viability and reversed the promotive impact of miR-27a-3p mimics on cell viability ( Figure 5(a)). Conversely, TGFBR1 overexpression retarded the inhibitive function of miR-27a-3p mimics on cell apoptosis (Figures 5(b) and 5(c)). e miR-27a-3p mimics induced the augmentation of migrated and invaded cells was repressed by ectopic TFGBR1, which also reduced the number of migrated and invaded cells compared with the mock/pcDNA3.1 group (Figures 5(d)-5(g)). In conclusion, miR-27a-3p inhibited heart failure development by modulating TGFβR1 in HCM cells.

SOX2-OT Facilitated HCM Cells Injury via TGFβR1.
To explore the relationship between SOX2-OT and TGFBR1, HCM cells transfected with different vectors were used in the following assays. According to Figure 6(a), TGFBR1 overexpression countervailed the motivative impact of SOX2-OT suppression on cell viability. Inversely, sh-SOX2-OT-mediated the drop of cell apoptosis rate was neutralized by TGFBR1 overexpression (Figures 6(b) and 6(c)). On the top of that the restraint of cell migration and invasion caused by SOX2-OT inhibition was also abrogated by TGFBR1 overexpression (Figures 6(d)-6(g)). All the experimental results indicated that SOX2-OT facilitated heart failure by modulating TGFβR1 in HCM cells.

Discussions
In recent years, the occurrence of MI has been increasing in China [26]. About 500,000 individuals have been diagnosed with MI each year at least and approximately 2 million are suffering from MI [27]. e typical symptoms of MI were hypotension, shock, arrhythmia, and heart failure [28]. e prognosis of MI was closely related to the size of infarct size and complication [29]. Hence, figuring out more molecules for prognostic marker is necessary. Although lncRNAs lacked the ability to encode protein, a multitude of research studies reported that lncRNAs were supposed to regulate the development of diverse diseases by modifying nucleic acids or proteins, sponging microRNAs,   activating, or inactivating signal molecules [30]. More importantly, emerging reports proposed a competitive endogenous RNA (ceRNA) pattern that lncRNA could competitively combine with miRNA to release mRNA [31,32]. For instance, lncRNA Malat1 contributed to berberine-mediated suppression of HMGB1 to promote poststroke inflammation by sponging miR-181c-5p [33]. Long noncoding RNA GAS5 was proved to limit cell proliferation and fibrosis by sponging miR-221 and regulating SIRT1 levels in diabetic nephropathy [34]. Recently, a few lncRNAs including SOX2-OT were reported to be upregulated in MI [19,35]. Consistent with that, in our study, SOX2-OT presented higher level in HCMs induced by hypoxia relative to the control group. Hereafter, we confirmed that SOX2-OT bound with miR-27a-3p, and SOX2-OT negatively regulated miR-27a-3p levels in HCMs, and vice versa. In function, the   effects of SOX2-OT knockdown on cellular processes (proliferation, apoptosis, migration, and invasion) were reversed by transfection of anti-miR-27a-3p. Transforming growth factor beta receptor 1 (TGFBR1), a key molecule in TGFβ/smad signaling pathway [36], has been widely reported to modulate cellular processes in diseases [37]. For instance, miR-22 regulated the proliferation and differentiation of C2C12 myoblast by targeting TGFBR1 [38]. MicroRNA-98 hampered collagen production and TGF-β1-induced differentiation of cardiac fibroblasts via regulating TGFBR1 [39]. In our research, TGFBR1 was directly targeted by miR-27a-3p. e mRNA and protein level of TGFBR1 was regulated by SOX2-OT/ miR-27a-3p axis. Previous study claimed that TGFBR1  Trans-well assay (migration) exacerbated myocardial cell damage induced by hypoxia via inhibiting NF-κB pathway [40]. Similarly, in our exploration, TGFBR1 facilitated proliferation, migration, invasion, and hindered apoptosis of HCMs to aggravate MI. On the top of that, rescue assays suggested that the TGFBR1 amplification offsets the influences of miR-27a-3p mimics or sh-SOX2-OT on cell proliferation, apoptosis, migration, and invasion. All in all, we confirmed that SOX2-OT exacerbated hypoxia-induced cardiomyocytes injury by regulating miR-27a-3p/TGFβR1 axis for the first time, implying a potential diagnostic or therapeutic target for MI patients. However, this was just the initial investigation of SOX2-OT in MI, and other mechanisms remain to be explored in the future.
Data Availability e data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.