miR-34a in extracellular vesicles from bone marrow mesenchymal stem cells reduces rheumatoid arthritis inflammation via the cyclin I/ATM/ATR/p53 axis

Abstract

Extracellular vesicles (Evs) participate in the development of rheumatoid arthritis (RA), but the mechanisms remain unclear. This study aimed to determine the mechanism by which microRNA-34a (miR-34a) contained in bone marrow mesenchymal stem cell (BM-MSC)-derived Evs functions in RA fibroblast-like synoviocytes (RA-FLSs). BM-MSC-derived Evs and an Evs inhibitor were extracted. A rat model of RA was established. miR-34a gain- and loss-of-function experiments were performed, and the inflammation in rat synovial fluid and tissues was detected. The role of miR-34a in RA-FLSs was also measured in vitro. The target gene of miR-34a was predicted using the online software TargetScan and identified using a dual-luciferase reporter gene assay, and the activation of the ATM/ATR/p53 signalling pathway was assessed. BM-MSC-derived Evs mainly elevated miR-34a expression, which reduced RA inflammation in vivo and inhibited RA-FLS proliferation and resistance to apoptosis in vitro, while inhibited miR-34a expression enhanced RA development. In addition, miR-34a could target cyclin I to activate the ATM/ATR/p53 signalling pathway, thus inhibiting abnormal RA-FLS growth and RA inflammation. Our study showed that miR-34a contained in BM-MSC-derived Evs could reduce RA inflammation by inhibiting the cyclin I/ATM/ATR/p53 signalling pathway.

Keywords: ATM/ATR/p53 signalling pathway; Cyclin I; MicroRNA-34a; extracellular vesicles; inflammation; rheumatoid arthritis.

FIGURE 1

Identification of BM‐MSCs and BM‐MSC‐derived Evs. A, Biomarkers of BM‐MSCs detected by flow cytometry; B, morphological observation of BM‐MSCs, and differentiation ability of BM‐MSCs measured with Alizarin Red staining and Oil Red O staining; C, sizes of Evs measured using nanoparticle tracking analysis; D, Evs‐related proteins evaluated using Western blot analysis, with the cells grown in conditioned medium supplemented with GW4869, an EV inhibitor, as a control; E, Evs morphology and size observed by TEM, with the cells grown in conditioned medium supplemented with GW4869, an EV inhibitor, as a control. BM‐MSCs: bone marrow mesenchymal stem cells; Evs: extracellular vesicles; TEM: transmission electron microscope

FIGURE 2

BM‐MSC‐derived Evs reduce inflammation in RA rats. A, Immunofluorescence of PKH26 for the absorption of PKH26‐labelled EVs by synovium; B, evaluation of RA inflammation in rats; C, the morphology of synovium observed using HE staining; D&E, expression of TNF‐α, IL‐6 and IL‐8 in rat synovial tissues detected using qRT‐PCR (D) and in synovial fluid detected using ELISA (E); n = 3. Compared with the normal group; *P < 0.05, **P < 0.01; compared with the model group; ^# P < 0.05, ^## P < 0.01. The normal group represented the rats injected with saline; the model group represented the rats with RA; the NC group represented the rats injected with BM‐MSC medium containing GW4869; and the EV group represented the RA rats injected with EVs. RA: rheumatoid arthritis

FIGURE 3

BM‐MSC‐derived Evs inhibit RA‐FLS proliferation and inflammation. A, Immunofluorescence of CD90 and Vimentin; B, dose and time determination of Ev treatment measured by MTT assay; C, immunofluorescence of PKH26 for the absorption of PKH26‐labelled EVs by FLSs; D, cell proliferation detected using flow cytometry; E, cell viability detected using MTT assay; F, immunofluorescence of PCNA; G&H, expression of TNF‐α, IL‐6 and IL‐8 in cells detected using qRT‐PCR (G) and ELISA (H); n = 3; compared with the normal group; *P < 0.05, **P < 0.01; compared with the RA‐FLS group; ^# P < 0.05, ^## P < 0.01. FLSs were cultured from the synovial tissues of rats in the normal group, whereas RA‐FLSs were cultured from the synovial tissues of rats in the model group. RA‐FLS‐NC are RA‐FLS cells grown in BM‐MSC medium supplemented with GW4869, whereas RA‐FLS‐Evs were RA‐FLS cells to which Evs were added. FLS: fibroblast‐like synoviocytes

FIGURE 4

BM‐MSC‐derived Evs mainly elevate miR‐34a expression. A, volcano plots of miRNAs whose expression changed after Evs treatment; B, miR‐34a, miR‐1180 and miR123b expression in cells after Evs treatment detected using qRT‐PCR, * compared with the RA‐FLS or model group; C, miR‐34a expression in BM‐MSCs after sphingomyelinase inhibitor (GW4869) treatment, * compared with the control group; D, miR‐34a expression in BM‐MSCs detected using qRT‐PCR, * compared with the NC group; E, miR‐34a expression in Evs measured using qRT‐PCR, * compared with the Evs‐NC group; F, miR‐34a expression in RA‐FLS after different Evs treatment measured using qRT‐PCR, *compared to RA‐FLSs, #compared with RA‐FLS‐Evs; n = 3; *P < 0.05, **P < 0.01, *** or ^### P < 0.001. miR‐In: miR‐34a inhibitor; Evs‐In: Evs transfected with miR‐34a inhibitor

FIGURE 5

miR‐34a suppresses inflammation in RA rats. A, immunofluorescence of PKH26 in synovial tissues; B, miR‐34a expression in synovial tissues detected using qRT‐PCR; C, evaluation of RA inflammation in rats; D, inflammation detected using HE staining; E&F, expression of TNF‐α, IL‐6 and IL‐8 in rat synovial tissues detected using qRT‐PCR (E) and synovial fluid detected using ELISA (F); G, caspase‐3 expression measured using immunohistochemistry; n = 3; *compared with the NC group; *P < 0.05, **P < 0.01

FIGURE 6

miR‐34a inhibits RA‐FLS proliferation and promotes RA‐FLS apoptosis. A, Immunohistochemistry of PKH26 in RA‐FLSs; B, miR‐34a expression in RA‐FLSs detected using qRT‐PCR; C, cell proliferation detected using flow cytometry; D, cell viability detected using MTT assay; E, immunofluorescence of PCNA; F, flow cytometry used to detect apoptosis; G, immunofluorescence of cytochrome C; H, expression of Bax, PUMA and cleaved PARP detected using Western blot analysis, *compared with the blank group; n = 3; *P < 0.05, **P < 0.01; compared with the Evs group, #P < 0.05, ## P < 0.01, ###P < 0.001

FIGURE 7

miR‐34a targets cyclin I and activates the ATM/ATR/p53 signalling pathway. A, Prediction of the target gene of miR‐34a by TargetScan; B, identification of miR‐34a binding cyclin I using a dual‐luciferase reporter gene assay; C&D, mRNA and protein expression of cyclin I in cells detected using qRT‐PCR (C) and Western blot analysis (D); E&F, ATM, ATR and p53 expression in the synovial tissues of the RA rats; G&H, ATM, ATR and p53 expression in FLSs; *compared with the NC group, *P < 0.05, **P < 0.01; n = 3. ATM: ataxia‐telangiectasia mutated; ATR: ataxia‐telangiectasia and Rad3‐related

FIGURE 8

Cyclin I inactivates the ATM/ATR/p53 signalling pathway. A, Expression of ATM, ATR and p53 in RA‐FLSs after Tenovin‐6 treatment; B, RA‐FLS viability after Tenovin‐6 treatment detected with MTT assay; C, RA‐FLS proliferation detected using flow cytometry; D, immunofluorescence of PCNA; E, FLS apoptosis detected using flow cytometry; F, cell cycle distribution evaluated using flow cytometry; G, immunofluorescence of cytochrome C; H, apoptosis‐related protein detected using Western blot analysis; n = 3; *compared with the Evs‐IN group; *P < 0.05, **P < 0.01