Role and mechanism of miR‑144‑5p in LPS‑induced macrophages

Zhou,Guozhu; Li,Yuwei; Ni,Jianping; Jiang,Ping; Bao,Zili

doi:10.3892/etm.2019.8218

Role and mechanism of miR‑144‑5p in LPS‑induced macrophages

Authors:
- Guozhu Zhou
- Yuwei Li
- Jianping Ni
- Ping Jiang
- Zili Bao
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Affiliations: Third Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210000, P.R. China, Department of Orthopedics, Suzhou Hospital of Traditional Chinese Medicine, Suzhou, Jiangsu 215000, P.R. China, Department of Orthopedics, Jingjiang Chinese Medicine Hospital, Taizhou, Jiangsu 214500, P.R. China
Published online on: November 19, 2019 https://doi.org/10.3892/etm.2019.8218
Pages: 241-247
Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to explore the possible role of microRNA‑144‑5p (miR‑144‑5p) in rheumatoid arthritis (RA) and the associated mechanism. Following the induction of THP‑1 cell differentiation into macrophages by phorbol ester (100 ng/ml) treatment, an in vitro inflammatory model of RA was established by treating the THP‑1 macrophages with 1 µg/ml lipopolysaccharide (LPS). The level of miR‑144‑5p was subsequently measured using reverse transcription‑quantitative PCR, which found that the expression of miR‑144‑5p was significantly reduced in LPS‑treated THP‑1 macrophages. Bioinformatics analysis and a dual‑luciferase reporter assay were used to predict and confirm TLR2 as a direct target of miR‑144‑5p, respectively. Toll‑like receptor 2 (TLR2) was then validated as a target gene of miR‑144‑5p. The effects of miR‑144‑5p upregulation and TLR2 silencing on LPS‑treated THP‑1 macrophages were then determined by transfection with miR‑144‑5p mimic and TLR2‑siRNA, respectively. Cell viability was subsequently measured using a Cell Counting Kit‑8 assay, whilst the expression of tumor necrosis factor‑α (TNF‑α), interleukin (IL)‑6 and IL‑8 secreted by THP‑1 macrophages was measured using ELISA. Western blotting was performed to measure p65 phosphorylation (p‑p65) in the NK‑κB signaling pathway. It was found that miR‑144‑5p overexpression reduced macrophage cell viability, reduced the expression of TNF‑α, IL‑6 and IL‑8, and reduced the expression of TLR2 and p‑p65 compared with the control group. Likewise, TLR2 silencing also reduced macrophage cell viability and reduced the expression of TNF‑α, IL‑6 and IL‑8 in THP‑1 macrophages. In conclusion, the data from the present study suggested that miR‑144‑5p overexpression reduced THP‑1 macrophage cell viability and inhibited the expression of TNF‑α, IL‑6 and IL‑8 in cells, possibly by inhibiting the expression of TLR2 and suppressing the activation of NK‑κB signaling. Therefore, miR‑144‑5p may serve as a novel therapeutic target for the treatment of RA.

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1	Abeles AM and Pillinger MH: The role of the synovial fibroblast in rheumatoid arthritis: Cartilage destruction and the regulation of matrix metalloproteinases. Bull NYU Hosp Jt Dis. 64:20–24. 2006.PubMed/NCBI
2	Mosser DM and Edwards JP: Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 8:958–969. 2008. View Article : Google Scholar : PubMed/NCBI
3	Jeannin P, Paolini L, Adam C and Delneste Y: The roles of CSFs on the functional polarization of tumor-associated macrophages. FEBS J. 285:680–699. 2018. View Article : Google Scholar : PubMed/NCBI
4	Zhou D, Chen L, Yang K, Jiang H, Xu W and Luan J: SOCS molecules: The growing players in macrophage polarization and function. Oncotarget. 8:60710–60722. 2017.PubMed/NCBI
5	Kokkonen H, Söderström I, Rocklöv J, Hallmans G, Lejon K and Rantapää Dahlqvist S: Up-regulation of cytokines and chemokines predates the onset of rheumatoid arthritis. Arthritis Rheum. 62:383–391. 2010.PubMed/NCBI
6	Ha M and Kim VN: Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 15:509–524. 2014. View Article : Google Scholar : PubMed/NCBI
7	Wang Y, Han CC, Cui D, Li Y, Ma Y and Wei W: Is macrophage polarization important in rheumatoid arthritis? Int Immunopharmacol. 50:345–352. 2017. View Article : Google Scholar : PubMed/NCBI
8	Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K and Akira S: Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity. 11:443–451. 1999. View Article : Google Scholar : PubMed/NCBI
9	Park H, Park SG, Kim J, Ko YG and Kim S: Signaling pathways for TNF production induced by human aminoacyl-tRNA synthetase-associating factor, p43. Cytokine. 20:148–153. 2002. View Article : Google Scholar : PubMed/NCBI
10	Elias JA, Reynolds MM, Kotloff RM and Kern JA: Fibroblast interleukin 1 beta: Synergistic stimulation by recombinant interleukin 1 and tumor necrosis factor and posttranscriptional regulation. Proc Natl Acad Sci USA. 86:6171–6175. 1989. View Article : Google Scholar : PubMed/NCBI
11	Krol J, Loedige I and Filipowicz W: The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 11:597–610. 2010. View Article : Google Scholar : PubMed/NCBI
12	Wang Y and Lee CG: MicroRNA and cancer-focus on apoptosis. J Cell Mol Med. 13:12–23. 2009. View Article : Google Scholar : PubMed/NCBI
13	Bueno MJ, Pérez de Castro I and Malumbres M: Control of cell proliferation pathways by microRNAs. Cell Cycle. 7:3143–3148. 2008. View Article : Google Scholar : PubMed/NCBI
14	Yamada Y, Arai T, Kojima S, Sugawara S, Kato M, Okato A, Yamazaki K, Naya Y, Ichikawa T and Seki N: Regulation of antitumor miR-144-5p targets oncogenes: Direct regulation of syndecan-3 and its clinical significance. Cancer Sci. 109:2919–2936. 2018. View Article : Google Scholar : PubMed/NCBI
15	Song L, Peng L, Hua S, Li X, Ma L, Jie J, Chen D, Wang Y and Li D: miR-144-5p enhances the radiosensitivity of non-small-cell lung cancer cells via targeting ATF2. Biomed Res Int. 2018:51094972018. View Article : Google Scholar : PubMed/NCBI
16	Li J, Wang R, Ge Y, Chen D, Wu B and Fang F: Assessment of microRNA-144-5p and its putative targets in inflamed gingiva from chronic periodontitis patients. J Periodontal Res. 54:266–277. 2019. View Article : Google Scholar : PubMed/NCBI
17	Sokol RJ, Hudson G, James NT, Frost IJ and Wales J: Human macrophage development: A morphometric study. J Anat. 151:27–35. 1987.PubMed/NCBI
18	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
19	Agarwal V, Bell GW, Nam J and Bartel DP: Predicting effective microRNA target sites in mammalian mRNAs. Elife. 4:2015. View Article : Google Scholar
20	Li D, Wang X, Lan X, Li Y, Liu L, Yi J, Li J, Sun Q, Wang Y, Li H, et al: Down-regulation of miR-144 elicits proinflammatory cytokine production by targeting toll-like receptor 2 in nonalcoholic steatohepatitis of high-fat-diet-induced metabolic syndrome E3 rats. Mol Cell Endocrinol. 402:1–12. 2015. View Article : Google Scholar : PubMed/NCBI
21	Wang X, Lan X, Liu L, Yi J, Li J, Li Y, Wang M, Li J, Song LM and Li D: MicroRNA 144 negatively regulates Toll-like receptor 2 expression in rat macrophages. Nan Fang Yi Ke Da Xue Xue Bao. 35:319–325. 2015.(In Chinese). PubMed/NCBI
22	Ma Y and Pope RM: The role of macrophages in rheumatoid arthritis. Curr Pharm Des. 11:569–580. 2005. View Article : Google Scholar : PubMed/NCBI
23	Wei ST, Sun YH, Zong SH and Xiang YB: Serum levels of IL-6 and TNF-α may correlate with activity and severity of rheumatoid arthritis. Med Sci Monit. 25:4030–4038. 2015. View Article : Google Scholar
24	Korani S, Kazemi B, Haghighi A, Nikpoor AR and Bandehpour M: The effect of human recombinant tumor necrosis factor receptor-2 on reducing inflammatory of collagen -induced arthritis in Balb/c Mice. Iran J Biotechnol. 17:e21532019. View Article : Google Scholar : PubMed/NCBI
25	Vasanthi P, Nalini G and Rajasekhar G: Role of tumor necrosis factor-alpha in rheumatoid arthritis: A review. APLAR J Rheumatol. 10:270–274. 2007. View Article : Google Scholar
26	Edrees AF, Misra SN and Abodou NI: Anti-tumor necrosis factor (TNF) therapy in rheumatoid arthritis: Correlation of TNF-alpha serum level with clinical response and benefit from changing dose or frequency of infliximab infusions. Clin Exp Rheumatol. 23:469–474. 2005.PubMed/NCBI
27	Venkatesha SH, Dudics S, Acharya B and Moudgil KD: Cytokine-modulating strategies and newer cytokine targets for arthritis therapy. Int J Mol Sci. 16:887–906. 2014. View Article : Google Scholar : PubMed/NCBI
28	Doyle MK, Rahman MU, Frederick B, Birbara CA, de Vries D, Toedter G, Wu X, Chen D, Ranganath VK, Westerman ME and Furst DE: Effects of subcutaneous and intravenous golimumab on inflammatory biomarkers in patients with rheumatoid arthritis: Results of a phase 1, randomized, open-label trial. Rheumatology (Oxford). 52:1214–1219. 2013. View Article : Google Scholar : PubMed/NCBI
29	Huang RY, Wu JQ, Liu ZH and Sun SL: MicroRNAs in rheumatoid arthritis: What is the latest with regards to diagnostics? Expert Rev Mol Diagn. 19:363–366. 2019. View Article : Google Scholar : PubMed/NCBI
30	Liu C, Pan A, Chen X, Tu J, Xia X and Sun L: MiR-5571-3p and miR-135b-5p, derived from analyses of microRNA profile sequencing, correlate with increased disease risk and activity of rheumatoid arthritis. Clin Rheumatol. 38:1753–1765. 2019. View Article : Google Scholar : PubMed/NCBI
31	Gao J, Kong R, Zhou X, Ji L, Zhang J and Zhao D: Correction to: MiRNA-126 expression inhibits IL-23R mediated TNF-α or IFN-γ production in fibroblast-like synoviocytes in a mice model of collagen-induced rheumatoid arthritis. Apoptosis. 24:3822019. View Article : Google Scholar : PubMed/NCBI
32	Yu FY, Xie CQ, Jiang CL, Sun JT, Feng HC, Li C and Huang XW: MiR-92a inhibits fibroblast-like synoviocyte proliferation and migration in rheumatoid arthritis by targeting AKT2. J Biosci. 43:911–919. 2018. View Article : Google Scholar : PubMed/NCBI
33	McGarry T, Biniecka M, Gao W, Cluxton D, Canavan M, Wade S, Wade S, Gallagher L, Orr C, Veale DJ and Fearon U: Resolution of TLR2-induced inflammation through manipulation of metabolic pathways in Rheumatoid Arthritis. Sci Rep. 7:431652017. View Article : Google Scholar : PubMed/NCBI
34	Arjumand S, Shahzad M, Shabbir A and Yousaf MZ: Thymoquinone attenuates rheumatoid arthritis by downregulating TLR2, TLR4, TNF-α, IL-1, and NFκB expression levels. Biomed Pharmacother. 111:958–963. 2019. View Article : Google Scholar : PubMed/NCBI
35	Quero L, Hanser E, Manigold T, Tiaden AN and Kyburz D: TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype. Arthritis Res Ther. 19:2452017. View Article : Google Scholar : PubMed/NCBI
36	McGarry T, Veale DJ, Gao W, Orr C, Fearon U and Connolly M: Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis. Arthritis Res Ther. 17:1532015. View Article : Google Scholar : PubMed/NCBI
37	Xie J, Li Q, Zhu XH, Gao Y and Zhao WH: IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes. Biochem Biophys Res Commun. 513:820–826. 2019. View Article : Google Scholar : PubMed/NCBI
38	Robertson RC, Guihéneuf F, Bahar B, Schmid M, Stengel DB, Fitzgerald GF, Ross RP and Stanton C: The anti-inflammatory effect of algae-derived lipid extracts on lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Mar Drugs. 13:5402–5424. 2015. View Article : Google Scholar : PubMed/NCBI
39	Roman-Blas JA and Jimenez SA: NF-kappaB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis. Osteoarthritis Cartilage. 14:839–848. 2006. View Article : Google Scholar : PubMed/NCBI
40	Aravilli RK, Vikram SL and Kohila V: Phytochemicals as potential antidotes for targeting NF-κB in rheumatoid arthritis. 3 Biotech. 7:2532017. View Article : Google Scholar : PubMed/NCBI
41	Zhang HJ, Wei QF, Wang SJ, Zhang HJ, Zhang XY, Geng Q, Cui YH and Wang XH: LncRNA HOTAIR alleviates rheumatoid arthritis by targeting miR-138 and inactivating NF-κB pathway. Int Immunopharmacol. 50:283–290. 2017. View Article : Google Scholar : PubMed/NCBI
42	Luo X, Xiao B and Xiao Z: Anti-inflammatory activity of adenosine 5′-trisphosphate in lipopolysaccharide-stimulated human umbilical vein endothelial cells through negative regulation of toll-like receptor MyD88 signaling. DNA Cell Biol. 2019. View Article : Google Scholar