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miR-410-3p regulates proliferation and apoptosis of fibroblast-like synoviocytes by targeting YY1 in rheumatoid arthritis
Biomedicine & Pharmacotherapy 119 (2019) 109426
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miR-410-3p regulates proliferation and apoptosis of fibroblast-like synoviocytes by targeting YY1 in rheumatoid arthritis YueJiao Wang, Ting Jiao, WenYi Fu, Shuai Zhao, LiLi Yang, NeiLi Xu, Ning Zhang
T
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Department of Rheumatology and Immunology, Shengjing Hospital of China Medical University, 39 Huaxiang Road, Tiexi District, Shenyang, Liaoning, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rheumatoid arthritis microRNAs Proliferation Apoptosis
In our previous study, miR-410-3p had been confirmed to regulate inflammatory cytokine release in rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs). However, other biological functions of miR-410-3p in RA FLSs still remain unexplored. In the present study, we focused on the effect of miR-410-3p on proliferation, apoptosis, and cell cycle of RA FLSs, and explored the potential underlying mechanism. miR-410-3p mRNA levels in the synovium and FLSs of patients with RA and of healthy controls were quantitated by RT-qPCR. The levels of miR410-3p were reduced in both synovium and FLSs from patients with RA. Next, we focused on the roles of miR410-3p in cell viability, apoptosis, and cell cycle, by transfecting miR-410-3p mimics and inhibitor into RA FLSs, and conducting CCK-8 assay, EdU staining and flow cytometry. Results showed that miR-410-3p up-regulation suppressed proliferation, promoted apoptosis and G1-S phase transition while miR-410-3p down-regulation had opposite effects. YY1 was verified as a direct target gene of miR-410-3p through the luciferase reporter system; YY1 up-regulation was able to rescue the effects of miR-410-3p in RA FLSs. Taken together, our current findings might provide a potential therapeutic target for RA.
1. Introduction
pannus, which distinguishes RA from other inflammatory disorders of the joint [8]. FLSs in synovial hyperplasia secrete more inflammatory cytokines, which in turn exacerbate the inflammation of the joint as a positive feedback loop [9]. Therefore, the central role of FLSs in the progression of RA makes this unique cell type a highly attractive target of research. In recent years, non-coding RNAs (18–25 nucleotides), namely microRNAs, have been identified to regulate post-transcriptional gene expression by binding to the 3′-untranslated region (UTR) [10]. More recently, a growing number of publications have focused on dysregulated miR-410-3p in multiple cancers [11,12] and autoimmune diseases [13]. miR-410-3p has been specifically reported to be decreased in cancer cells, and thought to be involved in several biological processes, including proliferation [14], apoptosis [15], metastasis [16], drug resistance [11] and stemness maintenance [17]. Wu H et al had reported that miR-410-3p acts as a tumor suppressor, and is decreased in breast cancer; it was shown to suppress cell growth, migration, and invasion [18]. Chen L had demonstrated that miR-410-3p overexpression suppressed cell viability in glioma [16]. Our previous study had shown that miR-410-3p regulates inflammatory cytokine release in RA FLSs [19]. However, its influences
Rheumatoid arthritis (RA) is a common chronic autoimmune disease, characterized by the inflammation of joints, subsequent destruction of cartilage, and erosion of the bone [1]. Recent demographic studies have shown it to affect approximately 0.5–1% of the population worldwide [2]. However, the etiology of RA is still poorly understood. Environment, genetics, and epigenetics are all considered to contribute to RA pathogenesis [3–5].With advancement of medical sciences over the past decades, especially in the target-to-target treatment strategy, there has been a dramatic increase in the remission rate of patients with RA [6]. However, a complete remission, in patients with RA, remains an elusive goal. Therefore, the need to investigate better therapy regimens for RA is truly urgent. Fibroblast-like synoviocytes (FLSs), located on the intimal lining of synovium, normally accounts for the support and nourishment of the joint by secreting multiple cytokines and chemokines into the synovial fluid [7]. When an inflammatory condition occurs, such as in RA, FLSs surprisingly express some aggressive phenotypes, similar to tumor cells. They increase in number, resist apoptosis, and become more invasive, eventually leading to hyperplasia of the synovium and formation of
Abbreviations: RA, rheumatoid arthritis; FLSs, fibroblast-like synoviocytes; miR, microRNAs; YY1, Yin Yang 1 transcription factor; RT-qPCR, real-time quantitative PCR; EdU, 5-ethynyl-2′-deoxyuridine; HFLS-RA, human fibroblast-like synoviocytes-RA; HFLS, human fibroblast-like synoviocytes- normal ⁎ Corresponding author. E-mail address: [email protected] (N. Zhang). https://doi.org/10.1016/j.biopha.2019.109426 Received 30 July 2019; Received in revised form 21 August 2019; Accepted 2 September 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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37℃ for 4 h and OD 490 values were measured using a microplate reader. All experiments were repeated 3 times, and average OD value was used for statistical analysis.
on the proliferation, apoptosis, and cell cycle of RA FLSs remains poorly understood. Moreover, based on the prediction by TargetScan, we hypothesized YinYang 1 (YY1) as a potential target gene of miR-410-3p, which is a transcription factor related to proliferation and apoptosis [20]. In this study, we aimed to explore the roles of miR-410-3p in RA FLSs survival and investigate the potential underlying mechanism.
2.5. 5-Ethynyl-2′-deoxyuridine (EdU) assay Prior to the transfection, HFLS-RA was seeded into 96-well plates at the density of 1 × 104 /well. Cell proliferation after transfection at 48 h was analysis with a Cell-Light EdU Apollo567 In Vitro kit (Ribobio, China) according to the manufacturer’s instructions. Briefly, cells per well were exposed to 100 μL fresh medium containing 50 μM EdU for 18 h at 37 ℃. Then, each well was added in 100 μL 4% formaldehyde for 30 min at room temperature, and added in 0.5% Triton X-100 for 10 min. Next, cells were washed with PBS and each well was incubated with 100 μL 1×Apollo reaction cocktail for 30 min. Finally, 100 μL 1×Hoechst 33342 was used to stain DNA for 30 min. Cellular morphology was observed under a fluorescent microscope.
2. Material and methods 2.1. Subjects Cell lines used in the study, human RA FLS (HFLS-RA) and human normal FLS (HFLS), were purchased from Jennio Biotech Co. Ltd (Guangzhou, China). Cell culture conditions were as described in our previous study [19]. Synovial tissues of 7 RA patients and 5 healthy participants were obtained from the Department of Orthopaedics in the Shengjing Hospital of China Medical University. All RA patients met the 1987 ACR criteria for the classification of RA [21]. All patients were informed of the purpose of the research and gave the written consent. The study was approved by the Ethics Committee of the Shengjing Hospital of China Medical University.
2.6. Flow cytometry analysis 48 h prior to the transfection, HFLS-RA was seeded into 6-well plates at the density of 5 × 105 /well. For apoptosis analysis, 5 μL PE Annexin V and 5 μL 7-AAD (BD, Biosciences, USA) were used. For cell cycle analysis, HFLS-RA was fixed with cold ethanol overnight and stained with PI//RNase Staining Buffer (BD, Biosciences, USA). Cells in different phases and apoptosis rate were quantitated.
2.2. Real-time quantitative PCR (RT-qPCR) Total RNA were extracted from cells or synovial tissues using the RNAiso Plus reagent (Takara, Japan) and quantified using spectrophotometry. 1 μg RNA was reversely transcribed into cDNA using a MirXTM miRNA First-Strand Synthesis kit (Takara) for miR-410-3p, and PrimeScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara) for YY1, respectively. The SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara) was used to quantitate. The following primers were synthesized by Sangon Biotech (Shanghai, China): miR-4103p(5′-CGCGAATATAACACAGATGGCCTGT-3′);U6(forward:5′-GGAACG ATACAGAGAAGATTAGC-3′;reverse:5′-TGGAACGCTTCACGAATTT GCG-3′);YY1(forward:5′-AGCCCTTTCAGTGCACGTT-3′;reverse:5′-TCT CCGGTATGGATTCGCAC-3′);GAPDH(forward:5′-GGAGCGAGATCCCTC CAAAAT-3′;reverse:5′-GGCTGTTGTCATACTTCTCATGG-3′).
2.7. Dual luciferase reporter assay The wild-type 3′UTR and mutated 3′UTR of YY1 were inserted into the GV272 vector to construct WT- and MUT-plasimids. 48 h prior to the transfection, HFLS-RA was seeded into 96-well plates at the density of 1 × 104 /well. miR-410-3p mimics or mimics NC and the luciferase vector were co-transfected into HFLS-RA. Both Renilla luciferase activity and Firefly luciferase activity were detected with a DualLuciferase® Reporter Assay System (Promega, USA). 2.8. Western blot assay Equal amounts (30 μg) of protein samples were subjected to SDSPAGE electrophoresis and transferred onto PVDF membranes. After blocking in 5% BSA for 2 h at room temperature, membranes were incubated with primary antibodies (YY1: CST, USA; GAPDH: ZSGB-BIO, China) followed by secondary antibodies (ZSGB-BIO) incubation for 2 h. The protein bands were visualized by ECL and quantitatively analyzed with Image J software, normalized to GAPDH.
2.3. Transfection assay The miR-410-3p mimics (5′-AAUAUAACACAGAUGGCCUGUAGGC CAUCUGUGUUAUAUUUU-3′), inhibitor (5′-ACAGGCCAUCUGUGUUA UAUU-3′) and YY1 overexpressing plasmid were synthesized from GenePharma (Shanghai, China). The experimental set-up consisted of 7 groups: (1) blank (no transfection); (2) mimics negative control (NC); (3) inhibitor NC; (4) mimics; (5) inhibitor; (6) YY1; (7) mimics + YY1. Prior to the transfection, HFLS-RA was seeded into 6-well plates at the density of 5 × 105 /well. HFLS-RA was transfected with lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Brifely, 100 pmoles miR-410-3p-related sequences and 5 μL lipofectamine 3000 were mixed separately with 125 μL Opti-MEM (Invitrogen) and then incubated together at room temperature for 10 min under serum-free conditions to form transfection complexes. The HFLS-RA was washed twice with PBS and the corresponding transfection complexes were added to each well. Complete DMEM media was replaced 6 h later. The transfection efficiency was checked under a fluorescence microscope.
2.9. Statistical analysis Statistical analyses were performed with GraphPad Prism version 7.00 (GraphPad Software, Inc., San Diego, CA, USA). Data from at least three independent experiments were expressed as the mean ± standard deviation. Two-tailed Student’s t-test and one-way ANOVA with the Turkey’s post hoc test were performed to compare differences among groups. P values < 0.05 were considered as statistically significant. 3. Results
2.4. CCK-8 assay
3.1. Decreased miR-410-3p in RA
Prior to the transfection, HFLS-RA was seeded into 96-well plates at the density of 1 × 104 /well. Cell proliferation after transfection at 24, 48 and 72 h was analysis with a CCK-8 kit (Promega, USA) according to the manufacturer’s instructions. Briefly, old medium was replaced with 100 μL fresh medium and 10 μL CCK-8 reagent. Cells were incubated in
In our previous study, we had demonstrated decreased miR-410-3p in RA. Here, we re-evaluated the mRNA levels of miR-410-3p in another group of patients with RA. Data in Fig. 1A revealed that the mRNA levels of miR-410-3p were approximately one fifth compared to that in healthy controls (p = 0.003). We identified its decreased expression in 2
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Fig. 1. Decreased miR-410-3p in RA. Synovial tissue samples were isolated from non-RA controls and RA patients, and miR-410-3p level was determined by real-time RT-PCR. All samples were performed in triplicates for each condition. Data shown were mean ± SD of three independent experiments. (A) Expression of miR-410-3p in 7 RA synovial tissues and 5 healthy control tissues was detected by RT-qPCR. (B) RT-qPCR was utilized to value the expression of miR-410-3p in HFLS and HFLS-RA. Error bar indicates mean values ± SD. ** p < 0.01, **** P < 0.0001.
HFLS-RA, compared to that in HFLS (p < 0.0001, Fig. 1B). The results are in accordance with our earlier hypothesis that aberrant expression of miR-410-3p is involved in the progression of RA.
(p < 0.0001, Fig. 2A), whereas miR-410-3p inhibitor down-regulated its expression to one fifth (p < 0.0001). We next detected the growth of HFLS-RA after transfection. CCK-8 assay results showed up-regulation of miR-410-3p in HFLS-RA to significantly inhibit proliferation, especially at 24 h (Fig. 2B, p = 0.02) and 48 h (p = 0.009). On the contrary, down-regulation of miR-410-3p promoted HFLS-RA proliferation at 24, 48, and 72 h (p < 0.0001). In addition, we performed EdU staining and observed similar results. As shown in Fig. 2C-D, up-regulation of miR-410-3p showed less EdU
3.2. Effects of miR-410-3p on the biological properties of RA FLSs First, we manipulated the expression of miR-410-3p in HFLS-RA by transient transfection. RT-qPCR data revealed miR-410-3p mimics to remarkably induce its expression to approximately 1400-fold
Fig. 2. Effects of miR-410-3p on the proliferation of RA FLSs. (A) At 48 h after transfection, HFLS-RA cells were collected and the expression of miR-410-3p was measured by RT-qPCR. (B) Effects of miR-410-3p mimics and inhibitor on the proliferation of HFLS-RA at 0 h, 24 h, 48 h and 72 h after transfection were evaluated using CCK-8 assay. (C–D) EdU assay was performed to investigate the proliferation of HFLS-RA at 48 h after transfection (scale bar, 100 μm). Error bar indicates mean value of triplicate experiments ± SD. * p < 0.05, *** p < 0.001, **** p < 0.0001, compared with relative control group. 3
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Fig. 3. Effects of miR-410-3p on the apoptosis and cell cycle of RA FLSs. (A–D) After PE Annexin V and 7-AAD staining, the effects of miR-410-3p mimics and inhibitor on early, late and total apoptosis were detected by flow cytometry. (E–F) Transfected HFLS-RA cells were stained with PI and cell cycle was measured using flow cytometry. Error bar indicates mean value of triplicate experiments ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with relative control group.
RA transfected with YY1-3′ UTR-MUT + mimics and YY1-3′ UTRMUT + mimics NC showed similar relative luciferase activities compared to that in HFLS-RA transfected with YY1-3′ UTR-WT + mimics NC. The results demonstrate that YY1 3′ UTR might be responsible for the function of miR-410-3p in RA FLSs. Subsequently, we measured the mRNA and protein levels of YY1 after transfection in HFLS-RA. Expression of YY1 was strongly decreased in the mimics group, both in mRNA (p = 0.006, Fig. 3C) and protein levels (p = 0.047, Fig. 3D-E), compared to that in mimics NC and blank groups, whereas the inhibitor promoted both the mRNA (p < 0.0001) as well as protein levels of YY1 (p = 0.002). However, no obvious difference was seen across mimics NC, inhibitor NC, and blank groups (p > 0.05). Above data suggested that miR-410-3p targets YY1 3′ UTR in HFLS-RA. Finally, we detected the expression of YY1 in synovial tissues. As shown in Fig. 4F, RT-qPCR analysis revealed the mRNA level of YY1 to be increased in synovial tissues of patients with RA, compared to that in healthy controls (p = 0.005). Similarly, the protein level of YY1 was also increased in synovial tissues of patients with RA, compared to that in healthy controls (p = 0.02, Fig. 4H-I).
positive cell ratio in mimics group (p = 0.014) compared to that in mimics NC group while miR-410-3p inhibition showed the opposite results (p < 0.0001).Taken together, miR-410-3p was seen to inhibit HFLS-RA proliferation. Next, we explored the apoptosis of HFLS-RA. Flow cytometry data shown in Fig. 3A-D, revealed that early (p = 0.0001), late (p = 0.005) and total (p < 0.0001) apoptosis rates of HFLS-RA in mimics group were significantly promoted, while the late (p = 0.002) and total (p = 0.008) apoptosis rates of HFLS-RA in the inhibitor group were induced compared to the respective NC groups. Data demonstrated that miR-410-3p promotes HFLS-RA apoptosis. Finally, we detected the cell cycle of transfected HFLS-RA through flow cytometry analysis. Cells at S phase were increased in miR-410-3p mimics group (p < 0.0001, Fig. 3E-F) compared to that in mimics NC group, whereas the inhibitor group showed the opposite results. Collectively, the results demonstrated that miR-410-3p inhibited proliferation, and promoted apoptosis and G1-S phase transition of HFLSRA. 3.3. miR-410-3p directly targets YY1
3.4. miR-410-3p influences the proliferation and apoptosis of RA FLSs by regulating YY1
To explore the potential target genes of miR-410-3p, we searched the candidates predicted from TargetScan [22] and PicTar. We focused on YY1 and performed the dual luciferase reporter assay to confirm the target relationship. The mutant 3′ UTR of YY1 differed from that of the wild type at 546–553 position of YY1, as predicted by TargetScan for the potential binding site (Fig. 4A). We performed co-transfection in HFLS-RA, and luciferase activities were detected. The relative luciferase activity in HFLS-RA transfected with both YY1-3′ UTR-WT and mimics, was significantly decreased compared to that in YY1-3′ UTR-WT and mimics NC transfected group (p = 0.048, Fig. 4B). Meanwhile, HFLS-
To verify the notion whether YY1 is responsible for the suppressed proliferation and induced apoptosis caused by miR-410-3p, we first manipulated the expression of YY1 in HFLS-RA. RT-qPCR and western blot results, shown in Fig. 5A-C, confirmed the efficiency of transfection of YY1 plasmid in HFLS-RA (p < 0.0001 and p = 0.0003, respectively). As shown in Fig. 5D-H, CCK-8, EdU staining, and flow cytometry analysis demonstrated the up-regulation of YY1 to partially 4
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Fig. 4. miR-410-3p directly targets YY1. (A) TargetScan predicted that YY1 was a target of miR-410-3p. (B) Dual luciferase reporting assay was performed. HFLS-RA cells were co-transfected with miR-410-3p mimics or mimics NC and a luciferase reporter containing either wild type or mutant type of YY1 3′UTR. Relative luciferase activities were detected. (C) The mRNA expression of YY1 was detected after transfection in HFLS-RA by RT-qPCR. (D–E) The protein level expression of YY1 was detected after transfection in HFLS-RA by Western Blot. Error bar indicates mean value of triplicate experiments ± SD. MT, mutant; WT, wild type; NC, negative control; * p < 0.05, ** p < 0.01, **** p < 0.0001, compared with the relative control group.
particularly FLSs. Our current study demonstrated that up-regulation of miR-410-3p suppresses proliferation, and promotes apoptosis and G1-S phase transition at the S phase in RA FLSs. To gain insight into the mechanism of miR-410-3p action in RA, we focused on the unique features of microRNAs by which they directly bind to 3′UTR region of the target genes and eventually suppress their expression. In our study, YY1, a cell viability-associated transcription factor, was confirmed to bind to miR410-3p at 3′UTR region and showed a reverse association with miR410-3p in RA FLSs. YY1 overexpression has been identified in multiple kinds of cancers, including breast cancer [26], prostate cancer [27], colorectal cancer [28] and gastric cancer [29]. In addition, YY1 has been reported to be overexpressed in patients with RA and in CIA mice [30]; YY1 deficient mice showed attenuation of inflammation [31], including factors such as tumor necrosis factor-α, IL-1β, IL-6, IL-8 and IL-17, and down-regulated Th17 population [32]. Especially, YY1 was found to be involved in proliferation, migration, and inflammatory cytokine secretion in RA FLSs [33]. We also observed that overexpression of YY1 partially rescued the effects of miR-410-3p on proliferation, apoptosis, and cell cycle of RA FLSs, thus supporting the notion that miR-410-3p functions in RA FLSs, at least partially, by targeting YY1. Taken together, miR-410-3p levels were reduced in RA; it suppressed proliferation, and promoted apoptosis and G1-S phase transition in RA FLSs by targeting YY1. Therefore, our data indicate the possibility of miR-410-3p serving as a potential therapeutic target for RA.
rescue the inhibited proliferation (p < 0.0001) and promoted the total apoptosis rates caused by the mimics (p = 0.038). Furthermore, the G1S phase transition, promoted by miR-410-3p, was also partially restored by co-transfection of YY1 in HFLS-RA (p < 0.0001, Fig. 5I-J). Taken together, our data supported the notion that miR-410-3p inhibits proliferation, and promotes apoptosis and G1-S phase transition by targeting YY1 in HFLS-RA. 4. Discussion Our previous study had provided the first report of miR-410-3p regulating inflammatory cytokine release in RA FLSs [19]. However, other biological functions of miR-410-3p in RA FLSs remained unexplored. In this study, we focused on the effect of miR-410-3p on proliferation, apoptosis, and cell cycle of RA FLSs, and explored the potential underlying mechanism. In accordance with earlier data, we re-established the reduced miR-410-3p levels across a different subgroup of patients with RA, which indicated its potential role in the progression of RA. MicroRNAs are well established to be involved in multiple biological processes of autoimmune disease, especially RA. For instance, miR-20a has been reported to be reduced in lipopolysaccharide (LPS)induced RA FLSs, whereas its up-regulation suppressed IL-6 and CXCL10 release by targeting ASK1, which play a critical role in the TLR4 pathway [23]. Moreover, low levels of miR-146a have been associated with decreased proliferation and increased apoptosis of RA FLSs by regulating TLR4/NF-κB signaling [24]. Dysregulated exosomedelivered microRNAs were explored in patients with RA; miR-6089 was confirmed to be reduced in serum exosomes of patients with RA and to be involved in macrophage proliferation and inflammation [25]. These data suggested aberrantly expressed microRNAs as a novel, promising biomarker in RA that are involved in the pathogenesis of RA by regulating multiple biological processes via targeting different cells,
Funding This work was supported by grants from the Precision Medicine Project of Shengjing Hospital of China Medical University (No. MF55), the Natural Science Foundation of Liaoning province (No. MS0109), 5
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Fig. 5. miR-410-3p influences the proliferation and apoptosis of RA FLSs by regulating YY1. (A–C) At 48 h after transfection, HFLS-RA cells were harvested and the expression of YY1 was confirmed by RT-qPCR and western blot. (D) CCK-8 assay was utilized to measure HFLS-RA cell proliferation among three groups. (E–F) EdU staining assay was performed to measure HFLS-RA cell proliferation among three groups. (G–H) Flow cytometry was performed to detect total apoptosis rate of HFLS-RA among three groups. (I–J) Flow cytometry was performed to measure HFLS-RA cell cycle at 48 h after transfection by PI staining among three groups, including mimics group, mimics + YY1 groups and mimics NC group. Error bar indicates mean value of triplicate experiments ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with relative control group.
and the Project of Shenyang Science and Technology Bureau (No. D267).
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cytokine release in rheumatoid fibroblast-like synoviocytes, Ann. Rheum. Dis. 72 (2013) 1071–1079, https://doi.org/10.1136/annrheumdis-2012-201654. W. Liu, et al., MicroRNA-146a suppresses rheumatoid arthritis fibroblast-like synoviocytes proliferation and inflammatory responses by inhibiting the TLR4/NF-kB signaling, Oncotarget 9 (2018) 23944–23959, https://doi.org/10.18632/ oncotarget.24050. D. Xu, et al., Exosome-encapsulated miR-6089 regulates inflammatory response via targeting TLR4, J. Cell. Physiol. 234 (2019) 1502–1511, https://doi.org/10.1002/ jcp.27014. Q. Zhang, et al., Yin Yang 1 promotes mTORC2-mediated AKT phosphorylation, J. Mol. Cell Biol. 8 (2016) 232–243, https://doi.org/10.1093/jmcb/mjw002. Z. Deng, et al., Yin Yang 1 regulates the transcriptional activity of androgen receptor, Oncogene 28 (2009) 3746–3757, https://doi.org/10.1038/onc.2009.231. W. Tang, et al., The p300/YY1/miR-500a-5p/HDAC2 signalling axis regulates cell proliferation in human colorectal cancer, Nat. Commun. 10 (2019) 663, https://doi. org/10.1038/s41467-018-08225-3. A.M. Wang, et al., Yin Yang 1 is a target of microRNA-34 family and contributes to gastric carcinogenesis, Oncotarget 5 (2014) 5002–5016, https://doi.org/10.18632/ oncotarget.2073. J. Lin, et al., A critical role of transcription factor YY1 in rheumatoid arthritis by regulation of interleukin-6, J. Autoimmun. 77 (2017) 67–75, https://doi.org/10. 1016/j.jaut.2016.10.008. J.E. Kwon, et al., YinYang1 deficiency ameliorates joint inflammation in a murine model of rheumatoid arthritis by modulating Th17 cell activation, Immunol. Lett. 197 (2018) 63–69, https://doi.org/10.1016/j.imlet.2018.03.003. J. Lin, et al., Blocking of YY1 reduce neutrophil infiltration by inhibiting IL-8 production via the PI3K-Akt-mTOR signaling pathway in rheumatoid arthritis, Clin. Exp. Immunol. 195 (2019) 226–236, https://doi.org/10.1111/cei.13218. N. Mu, et al., A novel NF-kappaB/YY1/microRNA-10a regulatory circuit in fibroblast-like synoviocytes regulates inflammation in rheumatoid arthritis, Sci. Rep. 6 (2016) 20059, https://doi.org/10.1038/srep20059.
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miR-410-3p regulates proliferation and apoptosis of fibroblast-like synoviocytes by targeting YY1 in rheumatoid arthritis YueJiao Wang, Ting Jiao, WenYi Fu, Shuai Zhao, LiLi Yang, NeiLi Xu, Ning Zhang
T
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Department of Rheumatology and Immunology, Shengjing Hospital of China Medical University, 39 Huaxiang Road, Tiexi District, Shenyang, Liaoning, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rheumatoid arthritis microRNAs Proliferation Apoptosis
In our previous study, miR-410-3p had been confirmed to regulate inflammatory cytokine release in rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs). However, other biological functions of miR-410-3p in RA FLSs still remain unexplored. In the present study, we focused on the effect of miR-410-3p on proliferation, apoptosis, and cell cycle of RA FLSs, and explored the potential underlying mechanism. miR-410-3p mRNA levels in the synovium and FLSs of patients with RA and of healthy controls were quantitated by RT-qPCR. The levels of miR410-3p were reduced in both synovium and FLSs from patients with RA. Next, we focused on the roles of miR410-3p in cell viability, apoptosis, and cell cycle, by transfecting miR-410-3p mimics and inhibitor into RA FLSs, and conducting CCK-8 assay, EdU staining and flow cytometry. Results showed that miR-410-3p up-regulation suppressed proliferation, promoted apoptosis and G1-S phase transition while miR-410-3p down-regulation had opposite effects. YY1 was verified as a direct target gene of miR-410-3p through the luciferase reporter system; YY1 up-regulation was able to rescue the effects of miR-410-3p in RA FLSs. Taken together, our current findings might provide a potential therapeutic target for RA.
1. Introduction
pannus, which distinguishes RA from other inflammatory disorders of the joint [8]. FLSs in synovial hyperplasia secrete more inflammatory cytokines, which in turn exacerbate the inflammation of the joint as a positive feedback loop [9]. Therefore, the central role of FLSs in the progression of RA makes this unique cell type a highly attractive target of research. In recent years, non-coding RNAs (18–25 nucleotides), namely microRNAs, have been identified to regulate post-transcriptional gene expression by binding to the 3′-untranslated region (UTR) [10]. More recently, a growing number of publications have focused on dysregulated miR-410-3p in multiple cancers [11,12] and autoimmune diseases [13]. miR-410-3p has been specifically reported to be decreased in cancer cells, and thought to be involved in several biological processes, including proliferation [14], apoptosis [15], metastasis [16], drug resistance [11] and stemness maintenance [17]. Wu H et al had reported that miR-410-3p acts as a tumor suppressor, and is decreased in breast cancer; it was shown to suppress cell growth, migration, and invasion [18]. Chen L had demonstrated that miR-410-3p overexpression suppressed cell viability in glioma [16]. Our previous study had shown that miR-410-3p regulates inflammatory cytokine release in RA FLSs [19]. However, its influences
Rheumatoid arthritis (RA) is a common chronic autoimmune disease, characterized by the inflammation of joints, subsequent destruction of cartilage, and erosion of the bone [1]. Recent demographic studies have shown it to affect approximately 0.5–1% of the population worldwide [2]. However, the etiology of RA is still poorly understood. Environment, genetics, and epigenetics are all considered to contribute to RA pathogenesis [3–5].With advancement of medical sciences over the past decades, especially in the target-to-target treatment strategy, there has been a dramatic increase in the remission rate of patients with RA [6]. However, a complete remission, in patients with RA, remains an elusive goal. Therefore, the need to investigate better therapy regimens for RA is truly urgent. Fibroblast-like synoviocytes (FLSs), located on the intimal lining of synovium, normally accounts for the support and nourishment of the joint by secreting multiple cytokines and chemokines into the synovial fluid [7]. When an inflammatory condition occurs, such as in RA, FLSs surprisingly express some aggressive phenotypes, similar to tumor cells. They increase in number, resist apoptosis, and become more invasive, eventually leading to hyperplasia of the synovium and formation of
Abbreviations: RA, rheumatoid arthritis; FLSs, fibroblast-like synoviocytes; miR, microRNAs; YY1, Yin Yang 1 transcription factor; RT-qPCR, real-time quantitative PCR; EdU, 5-ethynyl-2′-deoxyuridine; HFLS-RA, human fibroblast-like synoviocytes-RA; HFLS, human fibroblast-like synoviocytes- normal ⁎ Corresponding author. E-mail address: [email protected] (N. Zhang). https://doi.org/10.1016/j.biopha.2019.109426 Received 30 July 2019; Received in revised form 21 August 2019; Accepted 2 September 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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37℃ for 4 h and OD 490 values were measured using a microplate reader. All experiments were repeated 3 times, and average OD value was used for statistical analysis.
on the proliferation, apoptosis, and cell cycle of RA FLSs remains poorly understood. Moreover, based on the prediction by TargetScan, we hypothesized YinYang 1 (YY1) as a potential target gene of miR-410-3p, which is a transcription factor related to proliferation and apoptosis [20]. In this study, we aimed to explore the roles of miR-410-3p in RA FLSs survival and investigate the potential underlying mechanism.
2.5. 5-Ethynyl-2′-deoxyuridine (EdU) assay Prior to the transfection, HFLS-RA was seeded into 96-well plates at the density of 1 × 104 /well. Cell proliferation after transfection at 48 h was analysis with a Cell-Light EdU Apollo567 In Vitro kit (Ribobio, China) according to the manufacturer’s instructions. Briefly, cells per well were exposed to 100 μL fresh medium containing 50 μM EdU for 18 h at 37 ℃. Then, each well was added in 100 μL 4% formaldehyde for 30 min at room temperature, and added in 0.5% Triton X-100 for 10 min. Next, cells were washed with PBS and each well was incubated with 100 μL 1×Apollo reaction cocktail for 30 min. Finally, 100 μL 1×Hoechst 33342 was used to stain DNA for 30 min. Cellular morphology was observed under a fluorescent microscope.
2. Material and methods 2.1. Subjects Cell lines used in the study, human RA FLS (HFLS-RA) and human normal FLS (HFLS), were purchased from Jennio Biotech Co. Ltd (Guangzhou, China). Cell culture conditions were as described in our previous study [19]. Synovial tissues of 7 RA patients and 5 healthy participants were obtained from the Department of Orthopaedics in the Shengjing Hospital of China Medical University. All RA patients met the 1987 ACR criteria for the classification of RA [21]. All patients were informed of the purpose of the research and gave the written consent. The study was approved by the Ethics Committee of the Shengjing Hospital of China Medical University.
2.6. Flow cytometry analysis 48 h prior to the transfection, HFLS-RA was seeded into 6-well plates at the density of 5 × 105 /well. For apoptosis analysis, 5 μL PE Annexin V and 5 μL 7-AAD (BD, Biosciences, USA) were used. For cell cycle analysis, HFLS-RA was fixed with cold ethanol overnight and stained with PI//RNase Staining Buffer (BD, Biosciences, USA). Cells in different phases and apoptosis rate were quantitated.
2.2. Real-time quantitative PCR (RT-qPCR) Total RNA were extracted from cells or synovial tissues using the RNAiso Plus reagent (Takara, Japan) and quantified using spectrophotometry. 1 μg RNA was reversely transcribed into cDNA using a MirXTM miRNA First-Strand Synthesis kit (Takara) for miR-410-3p, and PrimeScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara) for YY1, respectively. The SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara) was used to quantitate. The following primers were synthesized by Sangon Biotech (Shanghai, China): miR-4103p(5′-CGCGAATATAACACAGATGGCCTGT-3′);U6(forward:5′-GGAACG ATACAGAGAAGATTAGC-3′;reverse:5′-TGGAACGCTTCACGAATTT GCG-3′);YY1(forward:5′-AGCCCTTTCAGTGCACGTT-3′;reverse:5′-TCT CCGGTATGGATTCGCAC-3′);GAPDH(forward:5′-GGAGCGAGATCCCTC CAAAAT-3′;reverse:5′-GGCTGTTGTCATACTTCTCATGG-3′).
2.7. Dual luciferase reporter assay The wild-type 3′UTR and mutated 3′UTR of YY1 were inserted into the GV272 vector to construct WT- and MUT-plasimids. 48 h prior to the transfection, HFLS-RA was seeded into 96-well plates at the density of 1 × 104 /well. miR-410-3p mimics or mimics NC and the luciferase vector were co-transfected into HFLS-RA. Both Renilla luciferase activity and Firefly luciferase activity were detected with a DualLuciferase® Reporter Assay System (Promega, USA). 2.8. Western blot assay Equal amounts (30 μg) of protein samples were subjected to SDSPAGE electrophoresis and transferred onto PVDF membranes. After blocking in 5% BSA for 2 h at room temperature, membranes were incubated with primary antibodies (YY1: CST, USA; GAPDH: ZSGB-BIO, China) followed by secondary antibodies (ZSGB-BIO) incubation for 2 h. The protein bands were visualized by ECL and quantitatively analyzed with Image J software, normalized to GAPDH.
2.3. Transfection assay The miR-410-3p mimics (5′-AAUAUAACACAGAUGGCCUGUAGGC CAUCUGUGUUAUAUUUU-3′), inhibitor (5′-ACAGGCCAUCUGUGUUA UAUU-3′) and YY1 overexpressing plasmid were synthesized from GenePharma (Shanghai, China). The experimental set-up consisted of 7 groups: (1) blank (no transfection); (2) mimics negative control (NC); (3) inhibitor NC; (4) mimics; (5) inhibitor; (6) YY1; (7) mimics + YY1. Prior to the transfection, HFLS-RA was seeded into 6-well plates at the density of 5 × 105 /well. HFLS-RA was transfected with lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Brifely, 100 pmoles miR-410-3p-related sequences and 5 μL lipofectamine 3000 were mixed separately with 125 μL Opti-MEM (Invitrogen) and then incubated together at room temperature for 10 min under serum-free conditions to form transfection complexes. The HFLS-RA was washed twice with PBS and the corresponding transfection complexes were added to each well. Complete DMEM media was replaced 6 h later. The transfection efficiency was checked under a fluorescence microscope.
2.9. Statistical analysis Statistical analyses were performed with GraphPad Prism version 7.00 (GraphPad Software, Inc., San Diego, CA, USA). Data from at least three independent experiments were expressed as the mean ± standard deviation. Two-tailed Student’s t-test and one-way ANOVA with the Turkey’s post hoc test were performed to compare differences among groups. P values < 0.05 were considered as statistically significant. 3. Results
2.4. CCK-8 assay
3.1. Decreased miR-410-3p in RA
Prior to the transfection, HFLS-RA was seeded into 96-well plates at the density of 1 × 104 /well. Cell proliferation after transfection at 24, 48 and 72 h was analysis with a CCK-8 kit (Promega, USA) according to the manufacturer’s instructions. Briefly, old medium was replaced with 100 μL fresh medium and 10 μL CCK-8 reagent. Cells were incubated in
In our previous study, we had demonstrated decreased miR-410-3p in RA. Here, we re-evaluated the mRNA levels of miR-410-3p in another group of patients with RA. Data in Fig. 1A revealed that the mRNA levels of miR-410-3p were approximately one fifth compared to that in healthy controls (p = 0.003). We identified its decreased expression in 2
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Fig. 1. Decreased miR-410-3p in RA. Synovial tissue samples were isolated from non-RA controls and RA patients, and miR-410-3p level was determined by real-time RT-PCR. All samples were performed in triplicates for each condition. Data shown were mean ± SD of three independent experiments. (A) Expression of miR-410-3p in 7 RA synovial tissues and 5 healthy control tissues was detected by RT-qPCR. (B) RT-qPCR was utilized to value the expression of miR-410-3p in HFLS and HFLS-RA. Error bar indicates mean values ± SD. ** p < 0.01, **** P < 0.0001.
HFLS-RA, compared to that in HFLS (p < 0.0001, Fig. 1B). The results are in accordance with our earlier hypothesis that aberrant expression of miR-410-3p is involved in the progression of RA.
(p < 0.0001, Fig. 2A), whereas miR-410-3p inhibitor down-regulated its expression to one fifth (p < 0.0001). We next detected the growth of HFLS-RA after transfection. CCK-8 assay results showed up-regulation of miR-410-3p in HFLS-RA to significantly inhibit proliferation, especially at 24 h (Fig. 2B, p = 0.02) and 48 h (p = 0.009). On the contrary, down-regulation of miR-410-3p promoted HFLS-RA proliferation at 24, 48, and 72 h (p < 0.0001). In addition, we performed EdU staining and observed similar results. As shown in Fig. 2C-D, up-regulation of miR-410-3p showed less EdU
3.2. Effects of miR-410-3p on the biological properties of RA FLSs First, we manipulated the expression of miR-410-3p in HFLS-RA by transient transfection. RT-qPCR data revealed miR-410-3p mimics to remarkably induce its expression to approximately 1400-fold
Fig. 2. Effects of miR-410-3p on the proliferation of RA FLSs. (A) At 48 h after transfection, HFLS-RA cells were collected and the expression of miR-410-3p was measured by RT-qPCR. (B) Effects of miR-410-3p mimics and inhibitor on the proliferation of HFLS-RA at 0 h, 24 h, 48 h and 72 h after transfection were evaluated using CCK-8 assay. (C–D) EdU assay was performed to investigate the proliferation of HFLS-RA at 48 h after transfection (scale bar, 100 μm). Error bar indicates mean value of triplicate experiments ± SD. * p < 0.05, *** p < 0.001, **** p < 0.0001, compared with relative control group. 3
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Fig. 3. Effects of miR-410-3p on the apoptosis and cell cycle of RA FLSs. (A–D) After PE Annexin V and 7-AAD staining, the effects of miR-410-3p mimics and inhibitor on early, late and total apoptosis were detected by flow cytometry. (E–F) Transfected HFLS-RA cells were stained with PI and cell cycle was measured using flow cytometry. Error bar indicates mean value of triplicate experiments ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with relative control group.
RA transfected with YY1-3′ UTR-MUT + mimics and YY1-3′ UTRMUT + mimics NC showed similar relative luciferase activities compared to that in HFLS-RA transfected with YY1-3′ UTR-WT + mimics NC. The results demonstrate that YY1 3′ UTR might be responsible for the function of miR-410-3p in RA FLSs. Subsequently, we measured the mRNA and protein levels of YY1 after transfection in HFLS-RA. Expression of YY1 was strongly decreased in the mimics group, both in mRNA (p = 0.006, Fig. 3C) and protein levels (p = 0.047, Fig. 3D-E), compared to that in mimics NC and blank groups, whereas the inhibitor promoted both the mRNA (p < 0.0001) as well as protein levels of YY1 (p = 0.002). However, no obvious difference was seen across mimics NC, inhibitor NC, and blank groups (p > 0.05). Above data suggested that miR-410-3p targets YY1 3′ UTR in HFLS-RA. Finally, we detected the expression of YY1 in synovial tissues. As shown in Fig. 4F, RT-qPCR analysis revealed the mRNA level of YY1 to be increased in synovial tissues of patients with RA, compared to that in healthy controls (p = 0.005). Similarly, the protein level of YY1 was also increased in synovial tissues of patients with RA, compared to that in healthy controls (p = 0.02, Fig. 4H-I).
positive cell ratio in mimics group (p = 0.014) compared to that in mimics NC group while miR-410-3p inhibition showed the opposite results (p < 0.0001).Taken together, miR-410-3p was seen to inhibit HFLS-RA proliferation. Next, we explored the apoptosis of HFLS-RA. Flow cytometry data shown in Fig. 3A-D, revealed that early (p = 0.0001), late (p = 0.005) and total (p < 0.0001) apoptosis rates of HFLS-RA in mimics group were significantly promoted, while the late (p = 0.002) and total (p = 0.008) apoptosis rates of HFLS-RA in the inhibitor group were induced compared to the respective NC groups. Data demonstrated that miR-410-3p promotes HFLS-RA apoptosis. Finally, we detected the cell cycle of transfected HFLS-RA through flow cytometry analysis. Cells at S phase were increased in miR-410-3p mimics group (p < 0.0001, Fig. 3E-F) compared to that in mimics NC group, whereas the inhibitor group showed the opposite results. Collectively, the results demonstrated that miR-410-3p inhibited proliferation, and promoted apoptosis and G1-S phase transition of HFLSRA. 3.3. miR-410-3p directly targets YY1
3.4. miR-410-3p influences the proliferation and apoptosis of RA FLSs by regulating YY1
To explore the potential target genes of miR-410-3p, we searched the candidates predicted from TargetScan [22] and PicTar. We focused on YY1 and performed the dual luciferase reporter assay to confirm the target relationship. The mutant 3′ UTR of YY1 differed from that of the wild type at 546–553 position of YY1, as predicted by TargetScan for the potential binding site (Fig. 4A). We performed co-transfection in HFLS-RA, and luciferase activities were detected. The relative luciferase activity in HFLS-RA transfected with both YY1-3′ UTR-WT and mimics, was significantly decreased compared to that in YY1-3′ UTR-WT and mimics NC transfected group (p = 0.048, Fig. 4B). Meanwhile, HFLS-
To verify the notion whether YY1 is responsible for the suppressed proliferation and induced apoptosis caused by miR-410-3p, we first manipulated the expression of YY1 in HFLS-RA. RT-qPCR and western blot results, shown in Fig. 5A-C, confirmed the efficiency of transfection of YY1 plasmid in HFLS-RA (p < 0.0001 and p = 0.0003, respectively). As shown in Fig. 5D-H, CCK-8, EdU staining, and flow cytometry analysis demonstrated the up-regulation of YY1 to partially 4
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Fig. 4. miR-410-3p directly targets YY1. (A) TargetScan predicted that YY1 was a target of miR-410-3p. (B) Dual luciferase reporting assay was performed. HFLS-RA cells were co-transfected with miR-410-3p mimics or mimics NC and a luciferase reporter containing either wild type or mutant type of YY1 3′UTR. Relative luciferase activities were detected. (C) The mRNA expression of YY1 was detected after transfection in HFLS-RA by RT-qPCR. (D–E) The protein level expression of YY1 was detected after transfection in HFLS-RA by Western Blot. Error bar indicates mean value of triplicate experiments ± SD. MT, mutant; WT, wild type; NC, negative control; * p < 0.05, ** p < 0.01, **** p < 0.0001, compared with the relative control group.
particularly FLSs. Our current study demonstrated that up-regulation of miR-410-3p suppresses proliferation, and promotes apoptosis and G1-S phase transition at the S phase in RA FLSs. To gain insight into the mechanism of miR-410-3p action in RA, we focused on the unique features of microRNAs by which they directly bind to 3′UTR region of the target genes and eventually suppress their expression. In our study, YY1, a cell viability-associated transcription factor, was confirmed to bind to miR410-3p at 3′UTR region and showed a reverse association with miR410-3p in RA FLSs. YY1 overexpression has been identified in multiple kinds of cancers, including breast cancer [26], prostate cancer [27], colorectal cancer [28] and gastric cancer [29]. In addition, YY1 has been reported to be overexpressed in patients with RA and in CIA mice [30]; YY1 deficient mice showed attenuation of inflammation [31], including factors such as tumor necrosis factor-α, IL-1β, IL-6, IL-8 and IL-17, and down-regulated Th17 population [32]. Especially, YY1 was found to be involved in proliferation, migration, and inflammatory cytokine secretion in RA FLSs [33]. We also observed that overexpression of YY1 partially rescued the effects of miR-410-3p on proliferation, apoptosis, and cell cycle of RA FLSs, thus supporting the notion that miR-410-3p functions in RA FLSs, at least partially, by targeting YY1. Taken together, miR-410-3p levels were reduced in RA; it suppressed proliferation, and promoted apoptosis and G1-S phase transition in RA FLSs by targeting YY1. Therefore, our data indicate the possibility of miR-410-3p serving as a potential therapeutic target for RA.
rescue the inhibited proliferation (p < 0.0001) and promoted the total apoptosis rates caused by the mimics (p = 0.038). Furthermore, the G1S phase transition, promoted by miR-410-3p, was also partially restored by co-transfection of YY1 in HFLS-RA (p < 0.0001, Fig. 5I-J). Taken together, our data supported the notion that miR-410-3p inhibits proliferation, and promotes apoptosis and G1-S phase transition by targeting YY1 in HFLS-RA. 4. Discussion Our previous study had provided the first report of miR-410-3p regulating inflammatory cytokine release in RA FLSs [19]. However, other biological functions of miR-410-3p in RA FLSs remained unexplored. In this study, we focused on the effect of miR-410-3p on proliferation, apoptosis, and cell cycle of RA FLSs, and explored the potential underlying mechanism. In accordance with earlier data, we re-established the reduced miR-410-3p levels across a different subgroup of patients with RA, which indicated its potential role in the progression of RA. MicroRNAs are well established to be involved in multiple biological processes of autoimmune disease, especially RA. For instance, miR-20a has been reported to be reduced in lipopolysaccharide (LPS)induced RA FLSs, whereas its up-regulation suppressed IL-6 and CXCL10 release by targeting ASK1, which play a critical role in the TLR4 pathway [23]. Moreover, low levels of miR-146a have been associated with decreased proliferation and increased apoptosis of RA FLSs by regulating TLR4/NF-κB signaling [24]. Dysregulated exosomedelivered microRNAs were explored in patients with RA; miR-6089 was confirmed to be reduced in serum exosomes of patients with RA and to be involved in macrophage proliferation and inflammation [25]. These data suggested aberrantly expressed microRNAs as a novel, promising biomarker in RA that are involved in the pathogenesis of RA by regulating multiple biological processes via targeting different cells,
Funding This work was supported by grants from the Precision Medicine Project of Shengjing Hospital of China Medical University (No. MF55), the Natural Science Foundation of Liaoning province (No. MS0109), 5
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Fig. 5. miR-410-3p influences the proliferation and apoptosis of RA FLSs by regulating YY1. (A–C) At 48 h after transfection, HFLS-RA cells were harvested and the expression of YY1 was confirmed by RT-qPCR and western blot. (D) CCK-8 assay was utilized to measure HFLS-RA cell proliferation among three groups. (E–F) EdU staining assay was performed to measure HFLS-RA cell proliferation among three groups. (G–H) Flow cytometry was performed to detect total apoptosis rate of HFLS-RA among three groups. (I–J) Flow cytometry was performed to measure HFLS-RA cell cycle at 48 h after transfection by PI staining among three groups, including mimics group, mimics + YY1 groups and mimics NC group. Error bar indicates mean value of triplicate experiments ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with relative control group.
and the Project of Shenyang Science and Technology Bureau (No. D267).
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