Mechanisms by which silencing long-stranded noncoding RNA KCNQ1OT1 alleviates myocardial ischemia/reperfusion injury (MI/RI)-induced cardiac injury via miR-377-3p/HMOX1

Background The key complication of myocardial infarction therapy is myocardial ischemia/reperfusion injury (MI/RI), and there is no effective treatment. The present study elucidates the mechanism of action of lncRNA KCNQ1OT1 in alleviating MI/RI and provides new perspectives and therapeutic targets for cardiac injury-related diseases. Methods An ischemia/reperfusion (I/R) injury model of human adult cardiac myocytes (HACMs) was constructed, and the expression of KCNQ1OT1 and miR-377-3p was determined by RT‒qPCR. The levels of related proteins were detected by western blot analysis. Cell proliferation was detected by a CCK-8 assay, and cell apoptosis and ROS content were determined by flow cytometry. SOD and MDA expression as well as Fe2+ changes were detected by related analysis kits. The target binding relationships between lncRNA KCNQ1OT1 and miR-377-3p as well as between miR-377-3p and heme oxygenase 1 (HMOX1) were verified by a dual-luciferase reporter gene assay. Results Myocardial ischemia‒reperfusion caused oxidative stress in HACMs, resulting in elevated ROS levels, increased Fe2+ levels, decreased cell viability, and increased LDH release (a marker of myocardial injury), and apoptosis. KCNQ1OT1 and HMOX1 were upregulated in I/R-induced myocardial injury, but the level of miR-377-3p was decreased. A dual-luciferase reporter gene assay indicated that lncRNA KCNQ1OT1 targets miR-377-3p and that miR-377-3p targets HMOX1. Inhibition of HMOX1 alleviated miR-377-3p downregulation-induced myocardial injury. Furthermore, lncRNA KCNQ1OT1 promoted the level of HMOX1 by binding to miR-377-3p and aggravated myocardial injury. Conclusion LncRNA KCNQ1OT1 aggravates ischemia‒reperfusion-induced cardiac injury via miR-377-3P/HMOX1.


Introduction
Myocardial ischemia-reperfusion injury (MI/RI) is a key complication of reperfusion therapy for myocardial infarction [1].Although reperfusion improves patient survival by rapidly restoring collateral flow in infarctrelated coronary arteries, it itself triggers myocardial injury [2].Although an increasing number of studies are dedicated to finding new targets to improve the clinical benefit of treatment for this disease, the gene expression and its regulatory mechanism during I/R injury are not completely clear [3].
Oxidative stress (OS) is defined as a disturbance in the pro-/antioxidant balance [4].During I/R injury, oxidative stress plays a key role in the activation of iron death.Irondriven cell death is characterized by iron accumulation and lipid peroxidation [5].Heme oxygenase 1 (HMOX1) activation mediates the release of free iron ions from heme, and plays a key role in ferroptosis-induced ROS [6,7].In addition, it has been reported that some miRNAs have been designated regulators of oxidative stress in the cardiovascular system [8].
Long-stranded noncoding RNA (lncRNA) is a type of nonprotein-coding RNA that is more than 200 nucleotides in length, and lncRNA is involved in chromosome modification, genome modification, transcription inhibition, and activation of related genes in cells [9,10].It has been reported that reverse chain/antisense transcript 1 (KCNQ1OT1) of KCNQ1 is involved in the regulation of the cell cycle, invasion, proliferation, migration, glucose metabolism, and immune escape of cancer cells [11][12][13].Downregulation of lncRNA KCNQ1OT1 protects against MI/RI following acute myocardial infarction [14,15].However, studies on lncRNA KCNQ1OT1 in MI/RI are scarce.
There is an unclear regulatory network between lncRNA KCNQ1OT1 and miR-377-3p in I/R injury.
This study aims to investigate the role and mechanism of lncRNA KCNQ1OT1, miRNA-377-3p and HMOX1 in MI/RI, as well as the way the genes are regulated.

Cell culture
Human adult cardiac myocytes (HACMs) were purchased form Otwo Biotech (Shenzhen, China), cultured in complete DMEM medium (10% fetal bovine serum and 1% streptomycin, Gibco, USA) at 5% CO 2 and 37℃.HACMs were cultured in a hypoxic incubator with an atmosphere of 94% N 2 , 1% O 2 , and 5% CO 2 for 4 h to simulate ischemia.Subsequently, cells were transferred to the aforementioned complete medium.Thus, the H/R model was successfully constructed.

RT-qPCR
Total RNA Extractor (Sangon Biotech) was used to extract total RNA from HACMs.A cDNA synthesis kit (Vazyme, Nanjing, China) was used to reverse transcribe 2 µg of RNA into cDNA, which was diluted 10-fold.The prepared cDNA (1 µl) was used for RT-qPCR.β-actin and U6 were used as references.All primers (Table 1) used in this study were designed with Premier 5.0.The confidence of the PCR results was assessed by the

Detection of apoptosis by flow cytometry
Flow cytometry was used to measure cell apoptosis.After treatment of cells, they were collected in a flow tube, centrifuged at 4 °C, washed with PBS, resuspended, and incubated with Annexin V-fluorescein isothiocyanate (FITC)/propidine iodide (PI).Annexin V-FITC and PI fluorescence was determined by a flow cytometer (BD Biosciences) and FlowJo software (V11).

Reactive oxygen species (ROS) detection
After trypsin digestion, the cell density of each group of cells was adjusted with cell culture medium, and 2 × 10 5 cells were incubated in 6-well plates in a cell culture incubator containing 5% CO 2 for 24 h.After centrifugation, the medium was discarded, and cells were resuspended in 500 µL of D-Hanks Balanced Salt Solution containing DCFH-DA (20 µL) followed by incubation for 30 min (shaken every 5 min) in an incubator protected from light.Subsequently, cells were resuspended with D-Hanks Balanced Salt Solution, and flow cytometry was used to detect the levels of ROS.
In addition, mitochondrial superoxide production was determined by MitoSOX Red (M36008, MAOKANG, China).Cells were incubated with 5 μm MitoSOX ™ in the dark at 37 ℃ for 30 min and imaged by laser scanning confocal at excitation wavelength 510 nm and emission wavelength 580 nm (LSM510, Zeiss, Germany).

CCK-8
A CCK-8 assay was used to measure the proliferation of HACMs.After transfection, log-phase cells were collected, and the concentration of the cell suspension was adjusted by adding 100 µL per well to a 96-well plate (4,000 cells/well).Cells were cultured at 37 °C for 0, 24, and 48 h.After transfection or dosing, 10 µL of CCK-8 reagent was added followed by incubation for 2 h.Finally, an enzyme marker (ELX800, BioTeK, UK) was used to measure the absorbance at 450 nm.Each experiment was repeated 3 times independently.

Dual-luciferase reporter gene
The 3′-UTR of KCNQ1OT1 was inserted into the pGL3 luciferase reporter vector (Promega, USA).Mutations were inserted into the seed region of the miR-377-3p-binding site of the 3′-UTR of KCNQ1OT1 by overlapping extension PCR.Cells were cotransfected with wild-type pGL3-KCNQ1OT1-3′-UTR or mutant KCNQ1OT1-3′-UTR and a scrambled miRNA control or miR-29b-3p mimics using Lipofectamine 2000 (Invitrogen, USA).After 48 h, luciferase activity was determined by the dual-luciferase reporter assay kit (Promega, USA) according to the manufacturer's instructions.HMOX1 was determined by the same method as above.

Fe 2+ testing
Fe 2+ was detected in HACMs using an iron assay kit according to the manufacturer's instructions and previously described methods (Li et al., 2020).

Oxidative stress factor (SOD and MDA) assay
HACMs were lysed with RIPA buffer (Beyotime, China), and the cell lysates were used for quantification of SOD and MDA using commercially available kits (Solarbio, China).

Construction of a myocardial ischemia-reperfusion injury model in mice
The anterior descending branch of the left coronary artery (LAD) was ligated after the mice were anesthetized with 2% isoflurane.A 5-0 Prolene suture was placed at approximately 2 cm around the root of the left anterior descending coronary artery and released 30 min later to allow myocardial ischemia followed by reperfusion.Buprenorphine hydrochloride, as an analgesic (0.05 mg/ kg), was given subcutaneously intraoperatively.

Statistical analysis
Each of the above experiments was performed at least three times.The experimental data were statistically analyzed using GraphPad Prism 8.0, and the results are presented as the mean ± SD. p < 0.05 was considered statistically significant.Student's t test was used to compare the differences between the samples, and one-way ANOVA was used to evaluate the differences among multiple samples.

I/R damage cell model establishment and detection
First, we constructed an I/R injury cell model using HACMs.RT-qPCR was used to detect the levels of lncRNA KCNQ1OT1, miR-377-3p, and HMOX1.The constructed I/R injury cell model had high levels of lncRNA KCNQ1OT1 and HMOX1 but miR-377-3p (Fig. 1A-C).Higher levels of ROS were detected in the I/R injury cell model than in the control (Fig. 1D).Mito-SOX Red probe was used to measure mitochondrial ROS levels, the increased mitochondrial ROS levels was observed in H/R group (Fig. 1E).The levels of SOD and MDA were detected using oxidative stress marker assay kits, which demonstrated that the I/R injury cell model expressed low levels of SOD but high levels of MDA (Fig. 1F-G).Next, changes in Fe 2+ were assayed using the iron assay kit, and the results showed significantly higher levels of Fe 2+ in the I/R injury cell model (Fig. 1H).In addition, Western blot analysis demonstrated high levels of the myocardial injury markers (CK and LDH) in the I/R injury cells (Fig. 1I).These results indicated the successful establishment of a cardiomyocyte I/R injury

Effect of LncRNA KCNQ1OT1 on MI/RI
To confirm the biological influence of lncRNA-KCNQ1OT1 in MI/RI injury, a small interfering RNA (siRNA) targeting lncRNA-KCNQ1OT1 was used, and RT-qPCR demonstrated high transfection efficiency of the siRNA (Fig. 2A).After silencing lncRNA-KCNQ1OT1, the expression of lncRNA-KCNQ1OT1 and HMOX1 was significantly decreased, and miR-377-3p was increased in comparison to the H/R group (Fig. 2B-D).Intracellular ROS levels were significantly reduced under si-KCNQ1OT1 (Fig. 2E).MitoSOX Red Fig. 2 Effect of lncRNA KCNQ1OT1 on MI/RI (n = 3).A-D: Detection of the level of lncRNA-KCNQ1OT1, miR-377-3p, and HMOX1 by RT-qPCR.E: Flow cytometry was used to detect ROS levels.F: MitoSOX Red probe was used to measure mitochondrial ROS levels (scale bar = 100 μm).G-H: Oxidative stress marker kits were utilized to evaluate SOD and MDA levels.Compared with the NC group, *** P < 0.001; compared with the H/R+si-NC or H/R + si-KCNQ1OT1 group, # P < 0.05, ## P < 0.01 and ### P < 0.001 probe was used to measure mitochondrial ROS levels, si-KCNQ1OT1 pretreatment attenuated the increase in H/R-induced mitochondrial ROS levels (Fig. 2F).Compared with the H/R group, the MDA content was significantly reduced, but the SOD content was increased in the H/R + si-KCNQ1OT1 group (Fig. 2G-H).These results suggested that knockdowning lncRNA KCNQ1OT1 alleviates MI/RI.

Dual-luciferase reporter gene assay validation of the targeting relationship between mir-377-3p and HMOX1
First, Starbase predicted the target binding site between miR-377-3p and HMOX1 (Fig. 5A).Next, RT-qPCR was performed to detect the level of HMOX1.After miR-377-3p overexpression, the level of HMOX1 was significantly decreased (Fig. 5B).In addition, a dual-luciferase reporter gene assay demonstrated that the miR-377-3p mimic significantly reduced the luciferase activity of the HMOX1 WT reporter but had no impact on the luciferase activity of the HMOX1 MUT reporter (Fig. 5C).These results indicated that miR-377-3p targeted HMOX1 and had a negative regulatory effect.

Animal experimental validation of the influence of lncRNA KCNQ1OT1 on MI/RI via the miR-377-3p/HMOX1 pathway
To further verify the relationship among the effects of lncRNA KCNQ1OT1, miR-377-3p, and HMOX1, experiments were performed using a MI/RI mouse model.The apoptosis rate of atrial tissue cells was significantly decreased after knockdowning HMOX1, miR-388-3p inhibitor promoted the apoptosis and lncRNA KCNQ1OT1 further promoted the apoptosis (Fig. 8A).RT-qPCR analysis demonstrated that the levels of lncRNA KCNQ1OT1 and HMOX1 were increased, but that the levels of miR-377-3p were decreased in atrial tissue with lncRNA KCNQ1OT1 overexpression (Fig. 8B-D).After MI/RI, an increase in ROS and MDA, but a decrease in SOD.ROS and MDA were inhibited after knockdowning HMOX1, miR-388-3p inhibitor promoted the level of ROS and MDA, but inhibited the level of SOD.lncRNA KCNQ1OT1 further promoted the level of ROS and MDA, but inhibited SOD (Fig. 8E-G).The content of Fe 2+ in HACMs was significantly increased after lncRNA KCNQ1OT1 overexpression (Fig. 8H).In addition, compared with the MI/RI + si-HMOX1 + miR-377-3p inhibitor group, CK, LDH and Bax were significantly increased, while Bcl-2 expression was significantly decreased in atrial tissue after overexpressing lncRNA KCNQ1OT1 (Fig. 8I).The in vitro experiments were consistent with the results described above, further validates the mechanism of the lncRNA KCNQ1OT1/miR-377-3p/HMOX1 ceRNA network in MI/RI.

Discussion
MI/RI leads to apoptosis and cytonecrosis of cardiomyocytes, and it may even lead to cardiac arrest [2].The mechanisms of MI/RI are complex and involve reperfusion exacerbating cellular ROS production, increasing oxidative stress, and activating downstream transcription factors to aggravate inflammation and accelerate cell death [24,25].However, the specific molecular mechanisms of action are not fully understood.An important regulatory effect of the lncRNA-miRNA-mRNA ceRNA network in MI/RI has been reported [26][27][28].Yingping Liang et al. [29] found that the inflammatory response induced by ischemia-reperfusion injury of human cardiomyocytes is regulated by the lncRNA ROR/miR-124-3p/ TRAF6 axis.Xueying Tong et al. [30] reported that MI/ RI is regulated by lncRNA LSINCT5/miR-222 through the PI3K/AKT pathway.Zhihao Guo et al. [31] also found that MI/RI is alleviated by mitochondrial apoptosis mediated by miR-503-5p/BIRC5, and they reported that this apoptosis is inhibited by lncRNA PART1.The present study found that the lncRNA KCNQ1OT1/miR-377-3p/ HMOX1 ceRNA network has important regulatory roles in MI/RI, and a series of experiments were performed to confirm the specific molecular mechanisms of action and the relationships among their actions.
LncRNAs and miRNAs both have been shown to influence I/R injury.LncRNA KCNQ1OT1 has been reported to promote cardiomyocyte injury by inducing apoptosis, inhibiting cell proliferation, and exacerbating oxidative stress [14].We constructed a siRNA against lncRNA KCNQ1OT1 and detected a decrease in intracellular ROS levels, an increase in SOD levels, and a significant decrease in MDA levels in the si-KCNQ1OT1 group, which further supported the previously reported findings.In previous studies, many miRNAs have been found to act as key regulators of cardiomyocyte activity, proliferation, and apoptosis [32,33].For example, miR-125b reduces myocardial infarct size and inhibits myocardial ischemia-reperfusion injury [34], and miRNA-30c-5p prevents MI/RI by regulating Bach1/Nrf2 [35].In the present study, we found that miR-377-3p was expressed at low levels in the H/R cell model.The miR-377-3p inhibitor significantly increased the contents of ROS and MDA but significantly decreased the contents of SOD compared to the H/R group.Western blot analysis demonstrated an increase in the expression of myocardial injury markers (LDH and CK), a significant decrease in Bcl-2, and an increase in Bax after treatment with miR-377-3p inhibitor.In addition, Fe 2+ levels were significantly increased after miR-377-3p inhibition.Moreover, Fig. 7 LncRNA KCNQ1OT1 targets miR-377-3p to mediate I/R injury via the HMOX1 pathway (n = 3).A: CCK-8 assay was used to detect the proliferation of HACMs.B: Flow cytometry was used to detect HACMs apoptosis.C-E: RT-qPCR was used to detect the expression of lncRNA KCNQ1OT1, miR-377-3p, and HMOX1.F: Flow cytometry was used to measure ROS levels.G: MitoSOX Red probe was used to measure mitochondrial ROS levels (scale bar = 100 μm).H-I: Oxidative stress marker kits were utilized to measure SOD and MDA expression.J: An iron analysis kit was used to detect changes in Fe 2+ .K: Western blot analysis was used to detect the levels of CK, LDH, Bax, and Bcl-2.Compared with the NC group, * P < 0.05, ** P < 0.01 and *** P < 0.001; compared with the H/R group, # P < 0.05, ## P < 0.01 and ### P < 0.001; compared with the H/R + si-HMOX1 group, ^P < 0.05, ^^P < 0.01 and ^^^P < 0.001; compared with the H/R + si-HMOX1 + miR-377-3p inhibitor group, & P < 0.05, && P < 0.01 and &&& P < 0.001 Fig. 8 Animal experimental validation of the influence of lncRNA KCNQ1OT1 on MI/RI via the miR-377-3p/HMOX1 pathway (n = 5).A: Apoptosis was measured by a TUNEL assay (scale bar = 100 μm).B-D: RT-qPCR was used to measure lncRNA KCNQ1OT1, miR-377-3p, and HMOX1 expression.E: An ROS assay kit was utilized to measure ROS levels.F and G: Oxidative stress marker kits were used to evaluate SOD and MDA expression.H: An iron analysis kit was used to detect Fe 2+ changes.I: Western blot analysis was used to detect the levels of CK, LDH, Bax, and Bcl-2.Compared with the control group, * P < 0.05, ** P < 0.01 and *** P < 0.001; compared with the MI/RI group, # P < 0.05, ## P < 0.01 and ### P < 0.001; compared with the MI/RI + si-HMOX1 group, ^P < 0.05, ^^P < 0.01 and ^^^P < 0.001; compared with the MI/RI + si-HMOX1 + miR-377-3p inhibitor group, & P < 0.05, && P < 0.01 and &&& P < 0.001 all of these changes were opposite after overexpression of miR-377-3p.These results confirmed the regulatory effect of miR-377-3p on cardiomyocytes by downregulating the promotion of oxidative stress and MI/RI in HACMs.
It has been reported that lncRNA-targeted miRNAs regulate MI/RI damage, and Yinghao Pei et al. [36] found that lncRNA PEAMIR acts as a competitive endogenetic RNA for miR-29b-3p, which is aggravated by apoptosis and inflammatory responses in MI/RI.Shuang Wang et al. [37] reported that lncRNA MALAT1 induces cardiomyocyte injury via miR-20b.In the present study, we used a dual-luciferase assay to verify the relationship between lncRNA KCNQ1OT1 and miR-377-3p.Compared to the H/R + si-HOMX1 + miR-377-3p inhibitor group, overexpression of KCNQ1OT1 induced cell apoptosis, increased intracellular levels of Fe 2+ , ROS and MDA, decreased SOD levels, and increased the expression of myocardial injury markers (CK and LDH).
Heme oxygenase 1 (HMOX1), as a cell protective enzyme, degrades heme [38].HMOX1 activity provides antioxidant, antiapoptotic, and cytoprotective effects through its catabolic metabolites [39].HMOX1 activation mediates the release of free iron ions from heme, and iron plays a key role in the formation and destruction of ROS [6,7].HMOX1 inhibits oxidative stress [40], but the accumulation of excess iron ions in cardiomyocytes tends to induce iron death [41].Therefore, exploring the molecular mechanisms by which HMOX1 regulates iron and oxidative stress in MI/RI is of great importance for the treatment of heart-related diseases.In the present study, we demonstrated that miR-377-3p targets HMOX1 using a dual-luciferase reporter assay.Compared to the H/R + miR-377-3p inhibitor group, knockdown of HMOX1 inhibited cell apoptosis, decreased intracellular levels of Fe 2+ , ROS and MDA, increased SOD levels, and decreased the expression of myocardial injury markers (CK and LDH).
Finally, animal experiments demonstrated that the miR-377-3p inhibitor reverses the protective effect of HOMX knockdown on MI/RI.Overexpression of lncRNA KCNQ1OT1 further exacerbates MI/RI, including apoptosis, as well as increases the level of oxidative stress and the expression of CK and LDH.The present study provided potential therapeutic strategies for better elucidating the mechanism, clinical prevention, and treatment of MI/RI, thereby laying the experimental foundation for future studies.

Fig. 1
Fig. 1 The establishment and detection of I/R damage cell model (n = 3).A-C: Detection of the expression of lncRNA KCNQ1OT1, miR-377-3p and HMOX1 by RT-qPCR.D: Flow cytometry was used to detect ROS levels.E: MitoSOX Red probe was used to measure mitochondrial ROS levels (scale bar = 100 μm).F-G: Oxidative stress marker kits were utilized to evaluate SOD and MDA levels.H: Iron analysis kits were used to measure Fe 2+ levels.I: CK and LDH expression levels were determined by western bolt.Compared with the NC group, ** P < 0.01 and *** P < 0.001

Fig. 4
Fig.4 Effect of miR-377-3p on MI/RI (n = 3).A: CCK-8 assay was used to detect cell viability.B: Detection of cell apoptosis by flow cytometry.C-E: Detection of the level of lncRNA-KCNQ1OT1, miR-377-3p, and HMOX1 by RT-qPCR.F: Flow cytometry analysis of ROS levels.G-H: Oxidative stress marker kits were used to measure SOD and MDA levels.I: MitoSOX Red probe was used to measure mitochondrial ROS levels (scale bar = 100 μm).J: Iron analysis kits were used to measure Fe 2+ levels.K: CK, LDH, Bax and Bcl-2 expression levels were determined by western bolt.Compared with the NC group, ** P < 0.01, and *** P < 0.001; compared with the H/R group, # P < 0.05, ## P < 0.01 and ### P < 0.001