Long noncoding RNA MAPKAPK5-AS1 promoted lipopolysaccharide-induced inflammatory damage in the myocardium by sponging microRNA-124-3p/E2F3

Background Myocardial dysfunction caused by sepsis (SIMD) leads to high mortality in critically ill patients. We investigated the function and mechanism of long non-coding RNA MAPKAPK5-AS1 (lncRNA MAPKAPK-AS1) on lipopolysaccharide (LPS)-induced inflammation response in vivo and in vitro. Method Male SD rats were utilized for in vivo experiments. Rat cardiomyocytes (H9C2) were employed for in vitro experiments. Western blotting was employed to measure protein expression, and RT-PCR was performed to measure mRNA expression of inflammation factors. TUNEL and flow cytometry were carried out to evulate cell apoptosis. Result The results showed that the expression of MAPKAPK5-AS1 was increased, while the expression of miR-124-3p was decreased in the inflammatory damage induced by LPS in vivo and in vitro. Knockdown of MAPKAPK5-AS1 reduced LPS-induced cell apoptosis and inflammation response, while overexpression of miR-124-3p weakened the effects of MAPKAPK5-AS1 knockdown on LPS-induced cell apoptosis and inflammation response. Moreover, miR-124-3p was identified as a downstream miRNA of MAPKAPK5-AS1, and E2F3 was a target of miR-214-3p. MAPKAPK5-AS1 knockdown increased the expression of miR-124-3p, while miR-124-3p overexpression reduced the expression of MAPKAPK5-AS1. In addition, miR-124-3p was found to downregulate E2F3 expression in H9C2 cells. Conclusion MAPKAPK5-AS1/miR-124-3p/E2F3 axis regulates LPS-related H9C2 cell apoptosis and inflammatory response.


Introduction
Sepsis-induced myocardial dysfunction (SIMD) is one of the most serious complication in patients with sepsis (Kakihana et al. 2016;Yamaji et al. 2017). Studies have shown that inflammation, oxidative stress and cardiomyocyte apoptosis are the three key factors in the eventually leading to congestive heart failure Tan et al. 2019). inflammatory damage in myocardium injury induced by LPS is an important factor that causes refractory hypotension and sepsis death, which has received increasing attention in research (Gichana et al. 2018).
Long non-coding RNA (lncRNA, > 200 nt) is known to be involved in gene expression regulation, genomic imprinting, transcription activation and interference, and intracellular nuclear transport (Chen et al. 2018;Wu et al. 2016). LncRNA dysregulations are closely related to various human disease (Han 2020). LncRNA MAP kinase-activated protein kinase 5 antisense gene protein 1 (MAPKAPK5-AS1) is a newly discovered lncRNA, which has been reported to play important roles in the development of glioma (Luan et al. 2019) and lung cancer . In our preliminary experiment, we found that MAPKAPK5-AS1 was dysregulated in LPS treated myocardium. Herein, we are interested to investigate the role of MAPKAPK5-AS1 LPS-induced inflammatory damage in the myocardium.
MicroRNAs (miRNAs) are a type of endogenous noncoding single-stranded RNA consisting of about 21 to 25 nucleotides (Ge et al. 2018). They are mainly paired with the non-transcribed region of the 3′ end of the target mRNA to cleave or inhibit target mRNA translation, thereby affecting protein expression (Mirna et al. 2019). During sepsis, some miRNAs expressions are dysregulated, such as miR-155 , miR-135a (Zheng et al. 2017), and miR-125b (Ma et al. 2016). Accumulative evidence suggested that miRNAs can regulate the inflammatory response (Ektesabi et al. 2019;Liu et al. 2017). However, due to the complexity of the occurrence and development of sepsis, more experiments are still needed to confirm the reliability and accuracy of these miRNAs as SIMD biomarkers. In our study, we found that MAP-KAPK5-AS1 has common binding sites with miR-124-3p by bioinformatics ananlysis. We also found that miR-124-3p expression was dysregulated in LPS induced myocardium. Therefore, we will focus on their associations and combined role in LPS-induced inflammatory damage in myocardium.
E2F3 is a group of genes that can encode transcriptional regulators and plays an important role in the transformation process (Weintraub et al. 1992;Nevins 1992). E2F3 is an important positive regulator of the cell cycle, which is an important regulatory factor in the G1 to S phase of the cell cycle (Sundarraj and Kannan 2017). E2F3 is closely related to cell metabolism and inflammation. For example, Y. Liao reported that Rb-independent E2F3 promotes cell proliferation and alters the expression of genes involved in metabolism and inflammations in 2017 (Liao and Du 2017). In our study, we found that miR-124-3p could target E2F3 through bioinformatics analysis. We have studied the interactions among MAPKAPK5-AS1, miR-124-3p and E2F3 in LPS-induced inflammatory damage in myocardium. Our result may provide an effective method for the therapeutic directions of myocardial dysfunction.

Animals
Male SD rats (weighing 250-300 g) were obtained from China-Japan Union Hospital of Jilin University and kept at 24 °C with humidity in a half-day and half-dark environment. The rats had the freedom to eat or drink. The experiment protocol was approved by Animal Usage Board of China-Japan Union Hospital of Jilin University. The rats were classified as: (i) vehicle (n = 5); (ii) LPS administration (5 mg/kg) (n = 5); (iii) LPS administration (10 mg/kg) (n = 5) (iv) LPS (10 mg/kg) plus si-MAP-KAPK5-AS1 treatment (n = 5). PBS was employed as a carrier buffer. Intraperitoneal injection of 5 or 10 mg/ kg LPS (Sigma, Missouri, USA) induced endotoxemia. Rats were injected with lentiviral vectors through the tail vein, and the lentiviral vectors each carried the following plasmids: si-NC, si-MAPKAPK5-AS1. Three days before LPS injection, lentiviral vector injection was performed through the above tail vein (19 × 10 7 TU per rat). Twenty-four hours after endotoxemia, we treated the rats with 100 mg/kg of ketamine and 10 mg/kg of wood diazine. The hearts were obtained.

Immunohistochemical analysis of myocardial
CD68 and TNF-α were immunohistochemically stained. The heart tissues were fixed, embedded in paraffin, dewaxed and rehydrated. After blocking, tissues were treated with anti-rat CD68 or TNF-a (Abcam, UK) at 4 °C for a night. The tissues were treated with Chromogen (DakoCytomation, Denmark) and stained with hematoxylin (Sigma, USA). For morphology, tissues were observed by a microscope (Zeiss, Germany) × 400.

TUNEL
Apoptotic cardiomyocyte and myocardium were detected using a TUNEL kit (Zhongshan Bio., China). Briefly, sections (5 μm) were collected and prepared, deparaffinized and hydrated, and incubated in proteinase K working solution for 30 min at 37 °C. Following washing with PBS twice, they were blocked for 10 min using 3% H 2 O 2 at room temperature. Subsequently, the sections were incubated in 50 µL terminal deoxynucleotide transferase (TdT) buffer (45 µL Equilibration buffer + 1 µL FITC-12-dUTP + 4 µL TdT Enzyme) for 1 h at 37 °C. After being washed in PBS 3 times, nuclei were stained with DAPI, and TUNEL positive cells were observed and photographed using a fluorescence-capable microscope (Leica DMI4000B, Wetzlar, Germany).

Luciferase assays
Sequences of MAPKAPK5-AS1 or E2F3 containing a putative miR-124-3p binding site were amplified by RT-PCR. The sequence was cloned to the pmirGLO luciferase vector (Promega, USA). GeneArtTM (Thermo Fisher Scientific) was employed for miR-1214-3p mutant (MUT) in MAPKAPK5-AS1 or E2F3 sequences. Then, in the presence of miR-124-3p mimics or miR-NC, H9C2 cells had transfection with wild-type (WT) or MUT vector. The luciferase activities were detected by luciferase assays (Promega) and normalized to Renilla.

Pull-down assay
H9C2 cells had transfections to biotin-labeled WT-miR-124-3p WT or MUT-bio-miR-124-3p. After 2 days, H9C2 cells were washed and lysed and incubated with magnetic bead (S3762; Millipore) and BSA (Sigma). The bead had incubation at 4 °C for 3 h and washed. Finally, Trizol was employed to purify the bound RNA and determine its content.

RT-qPCR
RNA was extracted by TRIzol (Takara, Japan), and had reverse transcription to cDNA. RT-PCR was performed by ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, California, USA) using the following reaction conditions. 40 cycles of 95 °C for 30 s, 62 °C for 45 s and 72 °C for 90 s; 72 °C for 10 min. GAPDH was internal control for LncRNA MAPKAPK5-AS1, and U6 was internal control for miR-124-3p. The expressions were calculated by 2 −ΔΔCT . Primers utilized in this research were:

Cell counting Kit-8 assay
The cells were seeded at a density of 5000 cells per well in a 96-well plate (Beyotime). After the quantitative treatment, a CCK-8 solution (Bioswamp, Wuhan, China) was added to the culture medium according to the instructions, and the plate was incubated at 37 °C in 5% CO 2 for 1 h in the dark. Quantify absorbance at 450 nm using a microplate reader (Bio-Rad, Sunnyvale, CA).

Data methods
All measurements were performed in triplicate. All the results were expressed as mean ± standard deviations. Student's t-test was used for comparison of the two, one-way ANOVA was used for single factor comparison of multiple groups. P < 0.05 was considered statistically significant.

MAPKAPK5-AS1 is upregulated, while miR-124-3p is downregulated in myocardial injury induced by LPS
To evaluate the expression of MAPKAPK5-AS1 and miR-124-3p in myocardial injury induced by LPS, we conducted TUNEL, CD68, and TNFa immunochemical staining, as well as in vivo RT-PCR. Twenty-four hours after LPS treatment, the number of TUNEL-positive cells, CD68 and TNFa-positive cells was increased in a dose-dependent manner (Fig. 1A-C). Moreover, LPS treatment was found to reduce the cardiac function in a dose-dependent manner. In addition, As shown in Fig. 1D and E, LPS treatment reduced the cardiac function in a dose-dependent manner. Furthermore, the expression of MAPKAPK5-AS1 increased while the expression of miR-124-3p decreased (Fig. 1F, G). Overall, we found that MAPKAPK5-AS1 was upregulated and miR-124-3p was downregulated in LPS-related myocardial injury.

Down-regulation of MAPKAPK5-AS1 attenuated H9C2 cell apoptosis and inflammation response induced by LPS
In order to explore the role of MAPKAPK5-AS1 in LPSinduced inflammatory damage, rats were pre-injected with a lentiviral vector with si-MAPKAPK5-AS1 3 days before LPS injection. The expression of MAPKAPK5-AS1 in cardiac tissue is shown in Fig. 2A. Compared with the LPS (10 mg/kg) group, si-MAPKAPK5-AS1 reduced TUNEL-, CD68, and TNFa positive cells (Fig. 2B, C). Western blot analysis of caspases3, PARP, and Bax/ Bcl2 showed that si-MAPKAPK5-AS1 reduced myocardial cell apoptosis (Fig. 2D). LPS-induced inflammatory cytokines are attenuated by si-MAPKAPK5-AS1 in cardiac tissue (Fig. 2E). It was confirmed that MAPKAPK5-AS1 knockdown attenuated H9C2 cells apoptosis and inflammation response induced by LPS.

miR-124-3p mediated the effects of MAPKAPK5-AS1 on LPS-induced H9C2 cell apoptosis and inflammation response
Then, we aimed to identify whether miR-124-3p could mediate the effects of MAPKAPK5-AS1 on LPS-related H9C2 cell death and inflammations. Flow cytometry and Western blotting were utilized to confirm LPS-related cell death. 1 ug/ml LPS-treated H9C2 cells for 24 h can increase cell death, while pc-MAPKAPK5-AS1 further enhanced LPS-related cell death, and co-transfection with miR-124-3p mimics can attenuate this effect ( Fig. 5B and C). Western blotting of inflammatory cytokine protein expression showed that pc-MAPKAPK5-AS1 enhanced LPS-induced inflammation response and cotransfection with miR-124-3p mimics attenuated this effect (Fig. 5D). Overall, our experiments identified that miR-124-3p mediated the effects of MAPKAPK5-AS1 on LPS-induced H9C2 cell apoptosis and inflammation response.

Discussion
SIMD is a life-threatening dysfunction caused by a host's dysregulated response to infection, which is a common clinical critical illness and a serious public health problem worldwide (Cheng 2019). However, the underlying pathogenesis of SIMD remains unknown. SIMD Each column represents the mean ± SEM. Statistical differences were evaluated with Student's t-test or One-way ANOVA followed by Tukey's post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3 can trigger a series of complex and interrelated pathophysiological processes, such as genetic polymorphisms, immune dysfunction, coagulation disorders and tissue damage (Kim et al. 2019), but their related molecular mechanisms need to be further studied (Jain et al. 2018). In this study, we found that MAPKAPK5-AS1 was upregulated, and miR-124-3p was downregulated in myocardial injuries induced by LPS, and MAPKAPK5-AS1 promoted LPS induced inflammatory damage in the myocardium by sponging microRNA-124-3p/E2F3. It has been reported that lncRNA plays an important role in LPS-induced inflammatory response disorder. For example, lncRNA PVT1 induces an increase in TNF-α, IL-6, and IL-1β release, and promotes inflammation by regulating TNF-α and JNK/NF-κB signaling pathways in sepsis (Huang et al. 2017). Fang et al. find that lncRNA H19 reduces the expression of miR-874, downregulates the secretion of inflammatory factors, and restores LPSinduced inflammatory response disorder and myocardial dysfunction in sepsis mice (Fang et al. 2018). NEAT1 in circulating blood is associated with increased disease risk, ascending severity of the disease, poor prognosis, and rising expression of inflammatory factors in sepsis patients (Huang et al. 2018). MAPKAPK5-AS1 is located on chromosome 12q24.12 and has been demonstrated to act as an oncogenic molecule in many kind of cancers, such as hepatocellular carcinoma, thyroid cancer and colorectal cancer et.al (Yang et al. 2020;Zhou et al. 2020). In our study, we investigated the role of MAPKAPK5-AS1 in inflammatory damage induced by LPS, and we found that MAPKAPK5-AS1 knockdown could reduce the increased number of TUNEL-, CD68 and TNFa positive cells and H9C2 cell apoptosis induced by LPS. Moreover, MAPKAPK5-AS1 knockdown attenuated LPS-induced inflammatory response. These results suggest that MAPKAPK5-AS1 knockdown attenuated H9C2 cell apoptosis and inflammation response in myocardial injuries induced by LPS.
Previous studies have pointed out that lncRNA could act as the sponge of miRNA and regulate the activities of HCMs (Piccoli et al. 2015;Kataoka and Wang 2014) and SIMD (Chen et al. 2018;Fang et al. 2018). Based on the bioinformatics results from Starbase, dual-luciferase reporter gene assay, and RNA pull-down assay, we found that miR-124-3p was a target of MAPKAPK5-AS1. Besides, we also found LPS induced cell death, MAP-KAPK5-AS1 overexpression further enhanced cell death, but co-transfection with miR-124-3p mimics can attenuate this effect. Moreover, MAPKAPK5-AS1 overexpression enhanced LPS-induced inflammation reponse, but co-transfection with miR-124-3p mimics attenuated this effect. These results demonstrated for the first time that miR-124-3p could mediate the effects of MAPKAPK5-AS1 on LPS-induced H9C2 cell apoptosis and inflammation response.
It has been reported that E2F3 plays an important role in the development of HCMs (King et al. 2008), and miRNAs are found to be involved in regulate cardiomyocytes cell cycle re-entry (miR-128) (Huang, et al. 2013) or protect cardiomyocytes by inhibiting or targeting E2F3 (miR-210) (Bian et al. 2018). Wang et al. found that miR-124-3p could bind with E2F3 to regulate the properties of Osteosarcoma cells . In this study, using bioinformations analysis and dualluciferase reporter gene assay, we also found that E2F3 was a target gene of miR-124-3p. It is consistent with the previous study conducted by Wang et al. (Wang et al. 2019). Moreover, MAPKAPK5-AS1 was found to be able to regulate the expression of E2F3 in H9C2 cells and rat heart tissue. Taken together, our study firstly proposed that miR-124-3p/ E2F3 axis mediated the effect of MAPKAPK5-AS1 on LPS-induced inflammatory response. Additionally, it's known that there are a series of miRNAs that have multiple target genes, and may participate in different pathogenic processes by regulating one or more target gene(s). For miR-124-3p, there are also different targets that have been studied. Whether one or more other target genes mediated the role of miR-124-3p in LPS-induced inflammatory response needed to be further investigated. Meanwhile, more details involved in the effects of miR-124-3p on LPS-induced inflammatory response should be well studied in the future.

Conclusion
In summary, our study revealed a novel regulatory model of MAPKAPK5-AS1/miR-124-3p/E2F3 axis in the progression of H9C2 cell apoptosis and inflammatory response induced by LPS.