Microrna-146 Protects A549 and H1975 Cells from LPS-Induced Apoptosis and Inﬂammation Injury

J Biosci Vol. 42, No. 4, December 2017, pp. 637–645 Ó Indian Academy of Sciences DOI: 10.1007/s12038-017-9715-4 MicroRNA-146 protects A549 and H1975 cells from LPS-induced apoptosis and inflammation injury 1 2 3 1 1 4 QIANG WANG ,DAGANG LI ,YUQUAN HAN ,XIAOQIAN DING ,TAO XU and BINGJIAN TANG * 1Department of Respiratory, The Affiliated Hospital of Qingdao University, Qingdao 266003, China 2Department of Respiratory, East Medical District, Linyi People’s Hospital, Linyi 276000, China 3Department of Emergency, Qingdao Huangdao District People’s Hospital, Qingdao 266400, China 4Department of Respiratory, Linqing People’s Hospital, Linqing 252600, China *Corresponding author (Email, [email protected]) MS received 28 January 2017; accepted 29 September 2017; published online 26 October 2017 Pneumonia is an inflammatory condition affecting the lungs, in which pro-inflammatory cytokines are secreted. It has been shown that microRNA-146 (miR-146) is involved in the regulation of immune and inflammatory responses. The present study explored the protective effects of miR-146 overexpression on lipopolysaccharide (LPS)-mediated injury in A549 and H1975 cells. In this study, A549 and H1975 cells were transfected with miR-146 mimic or inhibitor, and then were subjected with LPS. Thereafter, cell viability, colony formation capacity, apoptosis, the release of proinflammatory factors, Sirt1 expression, and the expression of NF-jB and Notch pathway proteins were respectively assessed. As a result, miR- 146 overexpression exerted protective functions on LPS-damaged A549 and H1975 cells, as evidenced by the increases in cell viability and colony number, the decrease in apoptotic cell rate, as well as the down-regulations of IL-1, IL-6, and TNF- a. Sirt1 can be positively regulated by miR-146. Furthermore, miR-146 overexpression blocked NF-jB and Notch pathways, while these blocking effects were abolished when Sirt1 was silenced. The findings in the current study indicated that miR-146 protected A549 and H1975 cells from LPS-induced apoptosis and inflammation injury. miR-146 exerted protective functions might be via up-regulation of Sirt1 and thereby blocking NF-jB and Notch pathways. Keywords. Apoptosis; inflammatory cytokine; MicroRNA-146; NF-jB; Notch; pneumonia; Sirt1 1. Introduction modulate various cellular processes such as cell prolifera- tion, invasion, and apoptosis (Bartel 2004). Role of miRNAs Pneumonia is inflammation of the lungs, most commonly in the regulation of oncogenes and tumor suppressor genes caused by infection with viruses or bacteria. In the United has been studied in many types of cancer (Ferrando 2005; States, pneumonia is the sixth most common cause of dis- Garofalo et al. 2009; Shenouda and Alahari 2009; Kouhkan ease-related deaths. In addition, pneumonia is the most et al. 2011), including lung cancer (Shen et al. 2011), but common hospital-acquired fatal infections. In developing their role in infectious lung disease such as pneumonia is not countries, pneumonia along with diarrhea is the most com- well studied. Few studies have indicated that miRNAs are mon infectious cause of death in children (Abd-El-Fattah closely related to the development and progression of et al. 2013). During pneumonia, inflammatory cytokines pneumonia; for instance, miR-21, miR-155, and miR-197 such as tumor necrosis factor-alpha (TNF-a), interleukin have been shown to be overexpressed in pneumonia (Lu (IL)-1b, and granulocyte/macrophage colony-stimulating et al. 2009; O’Connell et al. 2007; Abd-El-Fattah et al. factor are released which lead to cell apoptosis, tissue 2013). necrosis, and microvascular dysfunction (Moldoveanu et al. miR-146 is widely expressed in mammalian cells (Schulte 2009). Thus, suppression of inflammatory response and et al. 2013). Studies have indicated that miR-146 could protection of cells from injury are the effective treatment directly combine with interleukin (IL)-1R-associated kinase strategies for pneumonia. (IRAK1) and TNF receptor-associated factor 6 (TRAF6), MicroRNAs (miRNAs) are small non-coding RNAs that and thus participates in regulation of inflammatory responses are *22 nucleotides in length (Slezak-Prochazka et al. (Xie 2013; Lee et al. 2016). Schulte et al showed that miR- 2010). miRNAs act at the post-transcriptional level and 146 targets the messengers of bacterial lipopolysaccharide http://www.ias.ac.in/jbiosci 637 638 Qiang Wang et al. (LPS) to signal transduction components and thereby down- 2.4 Clonogenic survival assay regulates cellular LPS sensitivity (Schulte et al. 2013). Based on these reports, in this study we explored whether The miR-transfected cells were seeded in 6-well plates with miR-146 affected inflammatory response via regulating pro- a density of 19103 cells/well, and were subjected with LPS inflammatory cytokines in pneumonia. In the present study, for 12 h. After another 2 week incubation in normal con- we determined effects of miR-146 on viability, apoptosis and ditions (without LPS stimulation), cells were fixed in 100% inflammation in LPS-treated adenocarcinomic human alve- methanol and stained with 1% crystal violet (Sigma- olar basal epithelial cells (A549 cells) and in LPS-treated Aldrich). Colonies containing more than 50 cells were human lung adenocarcinoma cells (H1975 cells). defined as survivors. 2. Materials and methods 2.5 Apoptosis assay Cell apoptosis assay was performed to identify and quantify 2.1 Cell culture and LPS treatment the apoptotic cells using Annexin V-FITC/PI apoptosis H1975 and A549 cell lines were provided by the Institute of detection kit (Beijing Biosea Biotechnology, Beijing, Cell Biology (Shanghai, China). H1975 and A549 cells were China). The miR-transfected cells were seeded in 6-well 9 5 maintained in Dulbecco’s Modified Eagle’s Medium plates with a density of 1 10 cells/well, and were subjected with LPS for 12 h. Treated cells were washed twice with (DMEM) supplemented with 10% fetal bovine serum and l penicillin/streptomycin (100 U/mL and 100 mg/mL, cold PBS and resuspended in 200 L binding buffer containing 10 lL Annexin V and 5 lL PI. The adherent and respectively) and incubated at 37°C, 5% CO2. The cells were treated with different concentrations (5, 10, and 15 lg/mL) floating cells were combined and treated according to the of LPS under serum-free conditions for 12 h. manufacturer’s instruction and measured with flow cytometer (Beckman Coulter, USA) to differentiate apoptotic cells (Annexin-V positive and PI-negative) from necrotic cells (Annexin-V and PI-positive). 2.2 Cell transfection 2.6 Quantitative reverse transcription polymerase miR-146 mimics, negative control (NC), and miR-146 chain reaction (qRT-PCR) inhibitor, as well as Sirt1 specific targeted siRNA (siRNA) were all purchased from GenePharma (Shanghai, China). Total RNA was isolated from transfected cells using Trizol Cell transfection was performed using HiPerFect transfec- reagent (Invitrogen) and treated with DNaseI (Promega). tion reagent according to the manufacturer’s instructions Reverse transcription was performed using Multiscribe RT (Qiagen, Germany). After 48 h of transfection, transfected kit (Applied Biosystems) and random hexamers or oligo cells were collected for use in the further investigations. (dT). The reverse transcription conditions were 10 min at 25°C, 30 min at 48°C, and a final step of 5 min at 95°C. For miR-146, the forward primer was 50-GGGTGA- 2.3 MTT assay GAACTGAATTCCA-30 and the reverse primer was 50- CAGTGCGTGTCGTGGAGT-30. For Sirt1, the forward Cell viability was determined using a 3-(4,5-dimethylthia- primer was 50-TGCTGGCCTAATAGAGTGGCA-30 and the zol-2-yl)-2 5-diphenyl-2Htetrazolium bromide (MTT) col- reverse primer was 50-CTCAGCGCCATGGAAAATGT-30. orimetric assay. In brief, the miR-transfected H1975 and For glyceraldehyde 3-phosphate dehydrogenase (GAPDH), A549 cells were seeded in 96-well plates with a density of the forward primer was 50-GCACCGTCAAGGCTGA- 19103 cells/well, and cells were then subjected with LPS for GAAC-30 and the reverse primer was 50-TGGTGAA- 12 h. After twice washing with phosphate buffer saline GACGCCAGTGGA-30. (PBS), 10 lL MTT solution (Sigma-Aldrich, St. Louis, MO, USA) with a final concentration of 5 mg/mL was added into each well. The plates were allowed for culturing at 37°C for 2.7 Western blot analysis 4 h, and then DMSO of 150 lL was added. The plates were placed in a microplate reader (Bio-Rad Laboratories, Her- The proteins were extracted using radioimmunoprecipitation cules, CA, USA), and the absorbance was measured after the assay (RIPA) lysis buffer (Beyotime Biotechnology, plates were shaken for 10 min. Each experiment was per- Shanghai, China) supplemented with protease inhibitors formed three times. (Roche, Guangzhou, China). The proteins were quantified Role of miR-146 in pneumonia 639 using BCATM Protein Assay Kit (Pierce, Appleton, WI, H1975 cells were successfully overexpressed and suppressed USA). The western blot system was established using a Bio- by transfection. Rad Bis-Tris Gel system according to the manufacturer’s instructions. Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1000. Primary antibody 3.3 Expression of miR-146 and cell growth was incubated with the membrane at 4 °C overnight, fol- lowed by wash and incubation with secondary antibody To test whether miR-146 can affect the response of A549 marked by horseradish peroxidase for 1 h at room temper- and H1975 cells to LPS, we measured the effects of miR-146 ature. After rinsing, polyvinylidene difluoride (PVDF) on viability of LPS-treated A549 and H1975 cells using membrane-carried blots and antibodies were transferred into MTT assay at 12, 24, 36, and 48 h (figure 3A–B). We found Bio-Rad ChemiDocTM XRS system, and then 200 lL that overexpression of miR-146 enhanced cell viability Immobilon Western Chemiluminescent HRP Substrate compared with the negative control (NC) group. In contrast, (Millipore, MA, USA) was added to cover the membrane miR-146 suppression decreased cell viability.

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