key: cord-0852802-5z0ank96
authors: Li, Pibao; Yao, Yanfen; Ma, Yuezhen; Chen, Yanbin
title: MiR-30a-5p ameliorates LPS-induced inflammatory injury in human A549 cells and mice via targeting RUNX2
date: 2020-11-24
journal: Innate Immun
DOI: 10.1177/1753425920971347
sha: 6862e51ec4280b6d47f05b9845975adee709fabe
doc_id: 852802
cord_uid: 5z0ank96

In this study, we aim to investigate the role of miR-30a-5p in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) using LPS-induced A549 cells and mice. We found cell viability was significantly declined accompanied by cell apoptosis and cell cycle arrest at G0/G1 phase in LPS-treated A549 cells. MiR-30a-5p was down-regulated by LPS treatment and miR-30a-5p significantly protected A549 cells from LPS-induced injury by increasing cell viability, reducing cell apoptosis, promoting cell cycle progression, and inhibiting inflammatory reactions. Dual-luciferase activity demonstrated that RUNX2 was a direct target for miR-30a-5p and its expression was negatively and directly regulated by miR-30a-5p. Over-expression of RUNX2 weakened the inhibitory effect of miR-30a-5p on inflammatory injury. In vivo, over-expression of miR-30a-5p alleviated LPS-induced inflammatory responses and lung injury in LPS-administrated mice. Besides, miR-30a-5p repressed LPS-elevated phosphorylation levels of the signal transducer and activator of transcription 3 (STAT3) and c-Jun N-terminal kinase (JNK), IκBα degradation, and NF-κB p65 phosphorylation. In conclusion, miR-30a-5p ameliorates LPS-induced inflammatory injury in A549 cells and mice via targeting RUNX2 and related signaling pathways, thereby influencing the progression of ARDS.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is caused by indirect or direct lung injury, and clinically characterized by disruption of the alveolar-capillary barrier and dysfunction of gas exchange. 1,2 ALI/ARDS is histologically characterized by neutrophilic infiltration, lung edema, and exacerbated inflammatory response. 3, 4 Unfortunately, despite the current understanding of the pathophysiology and available treatment options, morbidity and mortality rates are still high due to infection diseases, especially the present outbreak of coronavirus disease 2019 . Therefore, it is essential to investigate the molecular mechanisms of ALI/ARDS. MicroRNAs (miRNAs) are a class of conserved, 20-25-nucleotide and non-coding RNAs, which regulate gene expressions, cell differentiation, development, proliferation, and apoptosis. 5 Previous studies have reported that aberrant expression of miRNAs was associated with some inflammation-related diseases, suggesting the potential involvement of miRNAs in ARDS. 6 To date, miR-30a-5p has been widely explored. For instance, miR-30a expression may contribute to protection from hyperoxic lung injury in female neonatal mice. 7 MiR-30a shields cardiac myocytes against injury caused by ischemia/reperfusion. 8, 9 On the other hand, angiotensin II induces podocyte injury by activating the calcium/calcineurin pathway and reducing levels of miR-30 family members. 10 However, the effect of miR-30a-5p on ARDS has not yet been well studied.

Here, cellular and murine models of inflammatory injury were constructed after exposure of LPS. Cell viability, apoptosis, cell cycle, and release of inflammatory cytokines were applied to evaluate inflammatory injury. Further, the functional role of miR-30a-5p and its possible target gene in LPS-induced cell injury was finally explored.

Human lung epithelial cell line A549 and HEK-293T were purchased from BioVector NTCC Inc. (Beijing, China) and grown in RPMI-1640 medium containing 10% FBS (Gibco, Gran Island, NY, USA), penicillin/ streptomycin (100 U/ml; Invitrogen; Carlsbad, CA, USA) at 5% CO 2 and 37 C. A549 cells were subjected to different concentration of LPS (0, 1, 5, 10, and 20 lg/ml) for different time points (12, 24, 48 , and 72 h), and PBS was used as control.

For the investigation of miR-30a-5p on biological behaviors of A549 cells, miR-30a-5p mimics and corresponding negative control (miR-NC) were synthesized by GenePharma (Shanghai, China). The transfection was carried out by using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.). The pcDNA3.1-RUNX2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and transferred into cells using pcDNA3.1 (Invitrogen).

First, our experiments conformed to the policies of Shandong Provincial Third Hospital Animal Ethical and Welfare Committee. In total, 40 female BALB/c mice (7-10-wk-old) were randomly assigned to four groups: PBS (n ¼ 10); LPS alone (n ¼ 10), agomir-30a-5p (n ¼ 10), and agomir-negative control (agomir-NC) (n ¼ 10). The ALI model was induced by a nasal drip with a lethal dose of LPS (10 mg/kg) in mice for 24 h. In control group, mice were initially injected intratracheally with 50 ll of PBS after 4 h. The agomir-30a-5p and agomir-NC were injected into the mice via tail vein for 72 h. The mice were anesthetized with 2% chloral hydrate (0.2 ml/10 g). Lungs were dissected (free of heart and trachea) and placed into Eppendorf tubes. Bronchoalveolar lavage fluid (BALF) was collected by flushing 1 ml ice-cold PBS back and forth three times through a tracheal cannula and then centrifuged at 1000 g at 4 C.

Total RNA including miRNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), and stored at -20 C. For quantification of miRNA, qRT-PCR was performed with an SYBR Green I Master Mix kit (Invitrogen) with the 7300 Real-Time PCR System (Applied Biosystems, USA). Finally, the expression was calculated by 2-DCt method, and GAPDH or U6 was used to normalize the levels.

Cells were collected and lyzed in the lysate (RIPA), where total protein was extracted for further determination of protein content in accordance with the BCA kit. According to results of the detection of protein concentration, 20 ll protein samples were extracted for Western blot assay. A 10% SDS-PAGE separation gel was then prepared. The sample was blocked for 2 h at room temperature with 5% skim milk powder buffer and then incubated with primary Abs overnight at 4 C. Then, the relative protein expression was determined after 1 h of secondary Ab incubation, electrochemiluminescence (ECL) development and photography.

Cell viability assay CCK-8 reagent (Dojindo, Kumamoto, Japan) was employed to detect cell viability. First, cells (5 Â 10 3 / ml per well) were seeded into 96-well plates, and then 10 ll/well of CCK-8 reagent was added to each well at 0, 24, 48, and 72 h, and incubated for 4 h at 37 C with 5% CO 2 . After incubating, a microplate reader (Thermo Fisher Scientific) was used to read the values of OD 450 nm. Experiments were repeated in triplicate.

After 48 h of transfection, cells of each group were collected and washed in pre-cooled PBS. After that, 100 ll binding buffer was supplemented to re-suspend cells, followed by the addition and gentle mixing of 5 ll Annexin V-FITC and 5 ll propidium lodide (PI), in which cells were incubated for another 15 min before flow cytometry.

The cells were trypsinized and washed with PBS, then incubated with RNase A at 37 C for 30 min, and stained with 50 mg/ml PI at 4 C for 30 min. The distribution of cells in different phases of the cell cycle (G0/G1, S, and G2/M) was analyzed using a FACscan (Becton Dickinson, Franklin Lakes, NJ, USA).

The levels of IL-1b, IL-6, and TNF-a in the cell culture medium of different groups were detected using ELISA kits (BD Biosciences) according to the manufacturer's instructions. Optical density values were measured at 450 nm using an ELISA plate reader (Bio-Rad, Hercules, CA, USA).

HEK-293T cells were co-transfected with miR-30a-5p/ miR-NC and pmirGLO-RUNX2-3 0 UTR-wild type (WT) and pmirGLO-RUNX2-3 0 UTR-mutation (MUT) (Promega, Madison, Wisconsin, USA). The cells were lyzed 48 h after transfection. Then, luciferase activity was measured using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer's instructions.

Results are expressed as the mean AE SD of triplicate experiments. Statistical one-way ANOVA was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., Chicago, USA). P < 0.05 was considered statistically significant.

First, A549 cells were exposed to different concentration of LPS. We found that miR-30a-5p exhibited a significantly decreased level following the increase of LPS concentration (Figure 1a ). MiR-30a-5p level was reduced for approximately 50% when A549 was subjected to 10 lg/ml of LPS compared with PBS control group (P < 0.01) (Figure 1a ), which can be applied for subsequent experiments. CCK-8 assay showed that cell viability obviously decreased in the presence of LPS (P < 0.01) (Figure 1b) . Flow cytometry identified that the percentage of apoptotic cells was significantly increased after exposure of 10 lg/ml of LPS (P < 0.01, Figure 1c ). Likewise, cell cycle analysis revealed that LPS caused a dramatic accumulation in G1-phase and reduction in S-phase of A549 cells, whereas PBS control normally accelerated cell cycle progression of A549 cells to S-phase ( Figure 1d) . Further, Western blotting demonstrated that the apoptosis-related proteins, including Bax, cleaved-caspase-3 (C-caspase-3), and cleaved-caspase-9 (C-caspase-9), were markedly increased, while the Bcl-2 was significantly decreased (Figure 1e ). Finally, results from qRT-PCR ( Figure 1f ) and ELISA (Figure 1g ) showed that the expression levels of inflammatory cytokines (IL-1b, IL-6, and TNF-a) obviously increased after incubation with LPS compared with the PBS control group (P < 0.01).

MiR-30a-5p alleviates LPS-induced cell apoptosis and inflammatory injury miR-30a-5p mimics were transfected into A549 cells to augment the expression of miR-30a-5p. The transfection efficacy was measured by qRT-PCR, and the results showed a significantly increased expression of miR-30a-5p (Figure 2a) . Then, miR-30a-5p overexpression significantly improved cell viability and decreased cell apoptosis which was induced by LPS (P < 0.05, Figure 2b, c) , whereas miR-30a-5p did not change cell viability and apoptosis of A549 cells without LPS treatment (P > 0.10, Figure 2b , c). In the presence of LPS, flow cytometry analysis showed that miR-30a-5p dramatically increased the cell populations in S and G2/M phase and reduced the cell populations in G0/G1 phase (Figure 2d) , indicating that miR-30a-5p reverses LPS-inhibited cell cycle progression. Besides, apoptosis-related proteins mentioned above were measured by Western blotting. Our findings revealed that the decreased expression of Bcl-2 and increased expressions of Bax, C-caspase-3, and C-caspase-9 by LPS were partially reversed by miR-30a-5p (Figure 2e, f) . Subsequently, the impact of miR-30a-5p on the expressions of IL-1b, IL-6, and TNF-a in LPStreated A549 cells was further investigated using qRT-PCR and ELISA. As illustrated in Figure 2g and 2h, miR-30a-5p mimics effectively decreased the expressions of IL-1b, IL-6, and TNF-a in A549 cells compared with miR-NC in the presence of LPS. When A549 cells were subjected to PBS treatment, miR-30a-5p mimics showed no significant differences from miR-NC in cell cycle, apoptosis-related proteins, and production of inflammatory cytokines (P > 0.10). These findings indicated that miR-30a-5p protects against LPS-induced cell apoptosis and inflammatory injury.

In our previous study, we had demonstrated that LPS successfully induced an ALI model of mice using pathological detection. 11 In the present study, we examined the expression of miR-30a-5p in the ALI mouse model. The RT-qPCR assay demonstrated that the levels of miR-30a-5p in the BALF, peripheral blood, and splenocytes as well as lung tissues were significantly reduced (P < 0.01, Figure 3a ) in contrast to PBS control, suggesting that miR-30a-5p may be required for LPS-induced lung inflammation. For gain-of-function assay, the mice were administrated with agomiR-30a-5p or agomir-NC by tail intravenous injection at 24 h prior to LPS stimulation. We found that miR-30a-5p mimics partially restored the level of miR-30a-5p in the BALF, peripheral blood, splenocytes, and lung tissues (Figure 3a) . Then, miR-30a-5p impaired LPSincreased total white blood cell and neutrophils count (Figure 3b ) as well as levels of inflammatory cytokines (Figure 3c ) in the BALF. In addition, miR-30a-5p markedly reduced the elevated concentrations of total protein, albumin, and IgM in the BALF (P < 0.01, Figure 3d -f). These observations highlighted the involvement of miR-30a-5p in LPS-induced lung injury of mice.

Using the TargetScan online prediction database, we found RUNX2 was a potential target gene of miR-30a-5p. The sequences of 3 0 -UTR of RUNX2 and miR-30a-5p are presented in Figure 4a . Then we found that miR-30a-5p over-expression remarkably inhibited RUNX2 protein expression in HEK-293T cells co-transfected with miR-30a-5p mimics and RUNX2 plasmids with WT 3 0 -UTR rather than MUT 3 0 -UTR (P < 0.01; Figure 4b ). Subsequently, the dual-luciferase reporter assay showed that miR-30a-5p over-expression obviously reduced the luciferase activity of the reporters containing the WT 3 0 -UTR of RUNX2 compared with mimics control (Figure 4c) , whereas no significant change in the luciferase activity was observed in those containing the MUT 3 0 -UTR of RUNX2 (Figure 4d ). These results demonstrated the direct binding of miR-30a-5p with 3 0 -UTR of RUNX2.

To confirm whether miR-30a-5p inhibited inflammatory response by regulating RUNX2 expression, we transfected the RUNX2 plasmids lacking its 3 0 -UTR into A549 cells with miR-30a-5p mimics. Western blotting results showed that endogenous RUNX2 protein was dramatically increased in A549 cells transfected with RUNX2 plasmids lacking 3 0 -UTR, as compared with those transfected with the vector control ( Figure  5a) . Moreover, miR-30a-5p-alleviated cell viability, -suppressed cell cycle, and -induced cell cycle progression was prevented when RUNX2 was up-regulated (Figure 5b-d) . As expected, RUNX2 over-expression induced up-regulation of Bax, C-caspase-3, and C-caspase-9 (P < 0.01; Figure 5e ), and secretion of inflammatory mRNAs (P < 0.01; Figure 5f ) and cytokines (P < 0.01; Figure 5g ). These results suggested RUNX2 is indeed involved in miR-30a-5p-mediated cell injury.

To assess the modulation of the signaling cascade related to LPS-induced ALI/ARDS, we determined the putative signal transduction pathways involved in the inflammatory reactions. Western blotting results revealed that LPS treatment (10 lg/ml) significantly promoted STAT3 and JNK phosphorylation, and induced IjBa degradation and NF-jB p65 phosphorylation. On the other hand, miR-30a-5p obviously repressed STAT3 and JNK phosphorylation increased (Figure 6a) , and significantly blocked the degradation of IjBa by decreasing the phosphorylation level of IjBa to reduce activation of NF-jB characterized by decreased p65 phosphorylation level (Figure 6b, c) . At the same time, in the absence of LPS, the above signaling pathway molecules in miR-30a-5p and miR-NC group were not markedly changed (P > 0.10, Figure 6 ). These results suggested that miR-30a-5p reduces the inflammatory response of A549 cells to LPS stimulation by inhibiting the phosphorylation levels of STAT3, JNK, p65, and IjBa.

Accumulating studies demonstrate that miRNAs exert essential roles in the initiation and progression of various diseases. 12, 13 It is worth noting that numerous studies have identified that the level of miR-30a is decreased in varieties of tumor tissues. However, its role in ALI/ARDS was not well elucidated. In the present study, LPS-induced inflammatory injury of A549 cells and mice were used to simulate ALI/ARDS, and then the level and role of miR-30a-5p were investigated. In vitro assays revealed the expression of miR-30a-5p and A549 cell viability were effectively suppressed by LPS, whereas apoptosis and inflammatory cytokines were obviously increased, indicating that LPS induced inflammatory injury of A549 cells. In vivo assays showed that the level of miR-30a-5p in the BALF, peripheral blood, splenocytes, and lung tissues was significantly reduced by LPS treatment, suggesting that LPS induced inflammatory injury of lung tissue in mice. Further experiments may be applied to evaluate the effects of miR-30a-5p over-expression using transfection assay. We demonstrated that over-expression of miR-30a-5p improved cell viability and accelerated the cell cycle, and decreased apoptosis and the production of inflammatory cytokines, suggesting that miR-30a-5p could inhibit the inflammation injury. miRNAs are generally recognized as regulating gene expression post-transcriptionally by inhibiting translation or inducing target mRNA degradation. 14, 15 In this study, miR-30a-5p targeted the 3 0 -UTR of RUNX2 mRNA to reduce the expression of RUNX2 protein, which suggested that miR-30a-5p suppressed inflammatory injury by repressing translation of RUNX2 mRNA.

RUNX2 has been reported as a multifunctional transcription factor for osteoblast differentiation in previous studies, which exerts its effect on mesenchymal stem cell chondrogenic differentiation. [16] [17] [18] [19] As reported by recent studies, the expression of RUNX2 in Schwann cells and axons increased after sciatic nerve crush. 16 RUNX2 over-expression accelerates progression of post-traumatic osteoarthritis in adult mice. 17 miR-203 affects traumatic heterotopic ossification via reducing RUNX2 expression. 18 CTRP13 attenuates vascular calcification through regulation of RUNX2 expression. 19 Here, RUNX2 was a target gene of miR-30a-5p, and reversed miR-30a-5p-mediated inhibition effects, indicating that miR-30a-5p ameliorates LPS-induced inflammatory injury in human A549 cells via targeting RUNX2. Taking all results together, inhibition of RUNX2 combined with miR-30a-5p transfection may be a feasible therapeutic approach for ALI/ ARDS. NF-jB transcription factors and the signaling pathways are central coordinators in innate and adaptive immune responses. STAT3 regulates the expression of a variety of genes in response to cellular stress, and thus plays a key role in cell growth and apoptosis. 20 NF-jB is activated during the inflammatory response to LPS, and is rapidly translocated into the nucleus after its detachment from IjBa and binds to the target gene jB locus to induce transcription of the target gene. 20 Apart from NF-jB, the upstream signaling molecule JNK is activated by LPS and has been demonstrated to play a key role in NF-jB activation. 21 In the present study, we explored the signaling-based mechanisms of the anti-inflammatory effects of miR-30a-5p in A549 cells. Interestingly, we found that miR-30a-5p attenuated LPS-induced phosphorylation of STAT3 and JNK, and further affected the nuclear translocation of LPS-induced NF-jB p65 in A549 cells.

In conclusion, this is the first study to demonstrate that miR-30a-5p regulated inflammatory reactions in LPS-induced ALI/ARDS via modulating RUNX2 expression and inflammatory signaling pathways, promoting new insights into the mechanisms and investigation of therapeutic strategies for patients with ALI/ ARDS.

All experimental animal procedures in this study conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Shandong Provincial Third Hospital.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Medical and Health Figure 6 . Effects of miR-30a-5p on LPS-induced activation of STAT3, JNK and NF-jB pathways. Cells were plated at a density of 5 Â 10 6 cells/dish in 60 mm culture dishes and treated with 10 lg/ml LPS for 72 h. After preparation of the total protein, the phosphorylated and total forms of STAT3 (a), JNK (a), p65 (b) and IjBa (b) were measured by Western blotting. (c) Quantitative analysis was carried out according to the ratio between target protein and GAPDH levels from three different experiments with densitometry. # P > 0.10, vs. PBS þ miR-NC; *P < 0.01, vs. LPS þ miR-NC; using repeated measures ANOVA test.

Prognostic and pathogenetic value of combining clinical and biochemical indices in patients with acute lung injury

Comparison of optimal positive end-expiratory pressure and recruitment maneuvers during lung-protective mechanical ventilation in patients with acute lung injury/acute respiratory distress syndrome

Lungs of patients with acute respiratory distress syndrome show diffuse inflammation in normally aerated regions: A [18F]-fluoro-2-deoxy-D-glucose PET/CT study

The role of glutathione-S-transferase polymorphisms on clinical outcome of ALI/ARDS patient treated with N-acetylcysteine

Integrating microRNAs into a system biology approach to acute lung injury

Circulating miRNAs and miRNA shuttles as biomarkers: Perspective trajectories of healthy and unhealthy aging

Role of HIF-1a-miR30a-Snai1 axis in neonatal hyperoxic lung injury

Salvianolic acid B induced upregulation of miR-30a protects cardiac myocytes from ischemia/reperfusion injury

miR-30 family microRNAs regulate myogenic differentiation and provide negative feedback on the microRNA pathway

Angiotensin II induces calcium/calcineurin signaling and podocyte injury by downregulating microRNA-30 family members

MiR-150 attenuates LPSinduced acute lung injury via targeting AKT3

Hypoxia-sensitive LINC01436 is regulated by E2F6 and acts as an oncogene by targeting miR-30a-3p in non-small cell lung cancer

MiR-30a: A novel biomarker and potential therapeutic target for cancer

MicroRNA dysregulation and multi-targeted therapy for cancer treatment

Role of microRNAs in alcohol-induced liver disorders and nonalcoholic fatty liver disease

Runx2 was correlated with neurite outgrowth and Schwann cell differentiation, migration after sciatic nerve crush

Chondrocytespecific RUNX2 overexpression accelerates posttraumatic osteoarthritis progression in adult mice

miR-203 inhibits the traumatic heterotopic ossification by targeting Runx2

CTRP13 attenuates vascular calcification by regulating Runx2

Visfatin induces inflammation and insulin resistance via the NF-jB and STAT3 signaling pathways in hepatocytes

Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-jB through Akt and p38 inhibition in RAW 264.7 macrophage cells

Yanbin Chen https://orcid.org/0000-0001-8678-5934