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Introduction

Rheumatoid arthritis (RA) is a complex, heterogeneous, progressive and long-term autoimmune disease characterized by symmetrical joint inflammation and bone erosion (1). The global prevalence of RA is 0.3-1.0% in females, which is five times higher than that in males (2). The risk factors for RA include genetic susceptibility, infectious agents, oxidative stress and inflammatory cytokines. RA is characterized by extensive infiltration of immune cells in the synovium, synovial hyperplasia, joint inflammation and excessive pro-inflammatory cytokine production. Oxidative stress caused by increased production of reactive oxygen species (ROS) and inflammatory cytokines contributes to the pathogenesis of RA (3-5).

MicroRNAs (miRNAs or miRs) are non-coding RNAs that are ~22 nucleotides long; they regulate the expression of target genes by binding to the ‘seed region’, 2-8 nucleotides of the 3' untranslated region (3'-UTR), as a post transcriptional regulator of messenger RNA (mRNA). The perfect complementarity between miRNAs and target mRNA sequences may lead to RNA silencing, resulting in reduced protein expression (6-9). A single nucleotide polymorphism (SNP) is a polymorphism of a DNA sequence caused by single nucleotide variation at the genome level. SNPs in the 3'-UTR of the target gene might affect its binding affinity to the corresponding miRNA, altering gene expression, which could change the biological functions of cells and initiate the onset of the diseases (10). The miRNA-related SNPs (miR-SNPs) rs2296135 and rs1131445 in interleukin-15 receptor α (IL-15RA) and IL-16 gene are associated with risk of RA (11,12).

SET domain containing (lysine methyltransferase) 8 (SET8), also known as PR-SET domain-containing protein 7 (PR-SET7) or Lysine methyltransferase 5a (KMT5a), encodes histone H4 lysine 20 monomethyl transferase, which is involved in cell cycle, DNA damage repair and cancer development (13-16). SET8 regulates the host immune response through the miR-30e-3p/FoxO3a/SET8 axis during mycobacterial infection (17). miR-502 regulates SET8 expression via its binding site in the 3'-UTR of SET8 mRNA and SNP rs16917496 at this site is associated with SET8 expression (14-16). Keratin is involved in RA; therefore, anti-keratin antibodies are used for RA diagnosis (18). Keratin 81 (KRT81) is a member of the keratin gene family that encodes hair keratin, which is a key component of the intermediate filaments and maintains mechanical stability and integrity of epithelial cells (19). Moreover, SNP rs3660 in the miRNA binding site of the 3'-UTR of KRT81 mediates the expression of KRT81 (20,21). In previous studies, miR-SNPs have been associated with the risk of RA (11,12); therefore, the present case-control study aimed to assess the association of miR-SNPs with risk of RA.

Materials and methods

Blood collection and DNA extraction

A total of 2 ml blood samples were collected from 82 patients with RA from May to December 2017 at the Department of Immunology and Rheumatology of the Second Hospital of Hebei Medical University (Shijiazhuang, China). Blood samples were also collected from 105 sex- and age- matched healthy controls. The diagnostic standard for RA was based on the 2010 revised standard of the American College of Rheumatology. Inclusion criteria were patients diagnosed as RA who were between 18 and 80 years old (inclusive) who had not yet started drug therapy. Patients <18 or > 80 years old, pregnant and lactating patients and patients with infectious inflammatory diseases were excluded (22). The clinical characteristics of patients with RA, including age, sex, disease activity score (DAS28) and laboratory results, such as anti-cyclic citrullinated peptide antibody (anti-CCP) and C-reactive protein (CRP) levels, erythrocyte sedimentation rate (ESR) and rheumatoid factor (RF) levels, were recorded. Disease activity score was based on DAS28: ≤2.6, clinical remission; >2.6 and ≤3.2, low activity; >3.2 and ≤5.1, moderate activity and >5.1, high activity (23). DNA from blood samples was extracted using a TIANamp Blood Clot DNA Kit (Tiangen) according to the manufacturer's instruction. All procedures were approved by the Human Tissue Research Committee of The Second Hospital of Hebei Medical University (approval no. 2017-P031). All participants provided written informed consent prior to enrollment.

PCR and sequence analysis

SNPs of the miRNA binding sites of SET8 (rs16917496) and KRT81 (rs3660) were genotyped based on the sequences available in the National Center for biotechnology information SNP database (ncbi.nlm.nih.gov/snp/) using a PCR-ligase detection reaction assay that amplified the DNA fragment flanking the miR-SNPs. The probes used for miR-SNPs genotyping were as follows: Probe S1 and S2 located upstream of the SNPs matched alleles of the miR-SNPs with length difference of three base pairs and probe S3 located downstream of SNPs with the complementary sequence. Following ligation with the PCR products using probes S1 + S3 or S2 + S3, SNPs were detected and verified based on length of ligated products. The sequences of primers and probes are listed in Table I. PCR was performed using a DreamTaq Green PCR Master Mix (2X) (K1081) (Shanghai Yisheng Biotechnology Co., Ltd.). The thermocycling conditions were as follows: 5 min denaturation at 95˚C followed by 35 cycles of 20 sec denaturation at 94˚C, 20 sec annealing at 55˚C and 40 sec extension at 72˚C and final extension at 72˚C for 10 min. Ligation was performed using different probes and the ligated products were separated using an ABI PRISM genetic analyzer 3730XL (Applied Biosystems; Thermo Fisher Scientific, Inc.). Polymorphisms were confirmed by repeating the analysis of two DNA strands.

Table I Primers and probes for microRNA-associated SNP genotyping.

Table I

Primers and probes for microRNA-associated SNP genotyping.

Gene	SNP	Primer sequence	Probe sequence
SET8	rs16917496	F: 5'-TCAGCAAATTGGA	S1: 5'-ctgactgaTTGTGGTTTAGCTTTGTATTTAAAC-3'
	(C/T)	AGAGGAT-3’	S2: 5'-ctgactgactgTTGTGGTTTAGCTTTGTATTTAAAT-3'
		R: 5’-GTGGCCTTGCCTG	S3: 5'-AAGGAAATAAACTTGAAAATTATTTGTCATC-3'
		GCAAT-3’
KRT81	rs3660	F: 5'-GGGGATCACACAG	S1: 5'-ctgactgactgaGAGTGGGAGGGGTCTTTCAAAGTGC-3'
	(C/G)	AGAAATGT-3’	S2: 5'-ctgactgactgactgGAGTGGGAGGGGTCTTTCAAAGTGG-3'
		R: 5’-ATCTTTCCTGCCC	S3: 5'-AGGAGAAGTAGCTGAGCACTTGCTCctgactgac-3'
		TGCCTTG-3’

[i] SNP, single nucleotide polymorphism; F, forward; R, reverse; SET, SET domain containing (lysine methyltransferase); KRT, keratin. Probe sequence, the upper case is the internal gene sequence, and the lower case is the sequence artificially added into 5' terminal of S1 and S2 probes to make the two probes different in length.

Western blotting

Western blotting was performed to confirm the association between SET8 expression and SNPs rs16917496. Total protein was isolated from human peripheral blood mononuclear cells using a ReadyPrep protein extraction kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. BCA assay was used to determine protein concentration. A total of 40 µg total protein/lane was loaded in a 10% denaturing polyacrylamide gel for separation and transferred to polyvinylidene difluoride membranes. The membranes were blocked using 5% skimmed milk powder with TBST containing 0.1% Tween-20 for 1 h at room temperature, then incubated at 4˚C overnight with the following primary antibodies: Mouse monoclonal anti-SET8 (Abcam; cat. no. ab3798; 1:500) or anti-β-actin antibody (Santa Cruz Biotechnology, Inc.; cat. no. SC-47778A; 1:20,000). The membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Thermo Fisher Scientific, Inc.; cat. no. 31430, 1:10,000) at room temperature for 2 h. FluorChem HD2 (ProteinSimple) was used to detect signals.

Determination of ROS levels

BBOXiProbe® serum active oxygen detection kit (BestBio Technology) was used to determine total serum ROS levels. Briefly, blood was centrifuged at 1,000 x g for 5 min at 25˚C to obtain serum, and 100 µl serum with 10 µl O12 probe were incubated at 37˚C for 30 min. ROS levels were measured using a fluorescence microplate reader (BioTek Instruments, Inc.) at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

Determination of cytokine levels

Human TH1/TH2 panel (8-plex) in a filter plate V02 (BioLegend, Inc.) was used to measure the levels of IL-5, 13, 2, 6, 10 and 4, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). A total of 25 µl serum sample (two-fold diluted with assay buffer) was incubated for 30 min at 25˚C with 25 µl Human Th Panel Detection antibody (BioLegend, Inc.; cat. no. 741041, stock solution), mixed with 25 µl Capture beads (BioLegend) for 1 h in the dark at room temperature and shaken using an oscillator at ~800 rpm for 1 h at 25˚C. The sample was supplemented with 25 µl streptavidin-phycoerythrin and shaken using an oscillator at ~800 rpm in the dark for 30 min at room temperature. The PE fluorescence signal of the analyte-specific bead region was quantitatively analyzed using MACSQuant analyzer 10 (Miltenyi Biotec GmbH) and the analyte concentration was determined using LEGENDplex™ software V8.0 (BioLegend, Inc.).

Statistical analysis

Unpaired student's t test was used for the analysis of continuous variables of independent samples. Data are presented as the mean and standard deviation. The independent experiments were repeated three times. Wilcoxon rank-sum test was used if the assumption of normality was incomplete. χ2 or Fisher's exact test was used to evaluate categorical variables in the contingency tables. All statistical analysis was performed using SPSS software (version 25.0; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

SET8 genotype is associated with risk of RA

A total of 82 patients with RA were compared with 105 sex- and age- matched healthy controls. The clinical characteristics of patients and the control group are listed in Table II.

Table II Clinical characteristics of patients with rheumatoid arthritis and controls.

Table II

Clinical characteristics of patients with rheumatoid arthritis and controls.

Characteristic	Patients (n=82)	Controls (n=105)	t/χ2	P-value
Mean age, years	53.71±11.59	52.02±8.49	1.11	0.27
Sex, male/female	16/66	24/81	0.31	0.58
Other immunological disease, yes/no	37/45	NA	NA	NA
Anti-CCP, +/-	53/29	NA	NA	NA
CRP, +/-	61/21	NA	NA	NA
ESR, +/-	70/12	NA	NA	NA
RF, +/-	61/21	NA	NA	NA
DAS28
≤2.6	2	NA	NA	NA
2.6<DAS28≤3.2	3	NA	NA	NA
3.2<DAS28≤5.1	18	NA	NA	NA
>5.1	59	NA	NA	NA

[i] NA, not applicable; CCP, cyclic citrullinated peptide; CRP, C reactive protein; ESR, erythrocyte sedimentation rate; RF, rheumatoid factor; DAS28, disease activity score in 28 joints.

SNPs in the miRNA binding sites of SET8 (rs16917496) and KRT81 (rs3660) in patients with RA and healthy controls were genotyped to evaluate their impact on the risk of RA. There was no significant difference in the genotype distribution frequency of SNP rs3660 in KRT81 (P=0.091). For SNP rs16917496 in SET8, the frequencies of genotypes CC and CT + TT were 10.98 and 89.02 in patients with RA and 2.86 and 97.14% in controls, respectively. The CC genotype was associated with a 4.192-fold increased risk of RA compared with that of CT + TT (odds ratio, 4.192; 95% CI: 1.097-16.021; P=0.025; Table III). The CC genotype of SET8 was also associated with lower protein expression than TT (Fig. 1). These data implied that SNP rs16917496 was associated with SET8 expression and regulated RA onset.

Figure 1 Expression of SET8 protein in patients with TT and CC genotype of rheumatoid arthritis. SET8, SET domain containing (lysine methyltransferase) 8.

Table III Frequency distribution of SNP sites in patients with rheumatoid arthritis and controls.

Table III

Frequency distribution of SNP sites in patients with rheumatoid arthritis and controls.

Gene (SNP)	Genotype	Patients (n=82)	Controls (n=105)	χ2	P-value	OR	95%CI
SET8	CC	9	3	5.053	0.025	4.192	1.097-16.021
(rs16917496)	CT + TT	73	102
KRT81	CC	56	59	2.848	0.091	1.679	0.918-3.072
(rs3660)	CG + GG	26	46

[i] SNP, single nucleotide polymorphism; SET8, SET domain containing (lysine methyltransferase) 8; KRT81, keratin 81.

Association of SET8 genotype with ROS and cytokines levels

In previous studies, increased ROS levels have been reported in patients with RA (24,25); therefore, the present study investigated the relationship between SNP rs16917496 and ROS levels in patients with RA. ROS levels were higher in the CC genotype carriers (1011.500±536.426 vs. 548.616±190.508, P=0.032, Fig. 2) than in the CT + TT genotype carriers. CC genotype was significantly associated with lower IL-10 levels (P<0.001; Fig. 2). These data suggested that SNPs regulated RA development by mediating ROS production and cytokine expression. There was no significant association between SNP rs16917496 and clinical characteristics of the participants, including age, sex, levels of anti-CCP and CRP, ESR, RF levels and DAS28 (data not shown).

Figure 2 Association between rs16917496 genotype and ROS and cytokine levels. ROS, reactive oxygen species.

Discussion

miRNAs serve important roles in the post-transcriptional regulation by controlling mRNA translation (26,27). SNPs in the miRNA binding sites can alter miRNA-mRNA binding affinity, which alters expression of the corresponding genes, thereby affecting cell proliferation, apoptosis and development. This process is associated with susceptibility to diseases, including cancer and autoimmune diseases (20,28,29). Here, SNP rs16917496 in the miR-502 binding site of the 3'-UTR of SET8 mRNA was associated with the occurrence of RA; low SET8 and higher ROS levels and decreased IL-10 expression were associated with CC genotype and increased risk of RA.

Consistent with the results of a previous study, the present study elucidated that the CC genotype of SET8 was associated with low SET8 expression (30). SET8 overexpression inhibits high glucose-mediated ROS accumulation in human umbilical vein endothelial cells (31) and a decrease in SET8 levels could enhance intracellular ROS levels. Oxidative stress is involved in the development of angiogenesis, synovial proliferation and inflammatory infiltration in RA (32,33). ROS can damage cartilage and extracellular matrix components by promoting the formation of rheumatoid factors and reducing the synthesis of collagen and proteoglycans in the pathogenesis of RA (34). Moreover, ROS oxidize lipids, nucleic acids and proteins to accelerate RA development (33). Therefore, functional analysis is required to assess the impact of enhanced ROS production induced by low SET8 levels during RA onset.

IL-10 inhibits expression of inflammatory factors, such as TNF-α, IL-17 and IL-1 β via the NF-κB signaling pathway (35,36). Moreover, IL-10 overexpression in bone marrow mesenchymal stem cells significantly alleviates RA symptoms by decreasing proliferation of synovium and promotes the repair of articular cartilage in collagen-induced arthritis (CIA) rats (36). It also decreases synovial inflammation, bone destruction and articular cartilage damage in CIA rats (37). The underlying mechanisms for RA risk-associated SNPs and IL-10, and that between SET8 and IL-10, should be evaluated.

The present findings suggested that the SNPs in the miR-502 binding site of the 3'-UTR of SET8 mRNA were predictors of RA risk and may regulate RA pathogenesis by mediating expression of SET8 to regulate ROS and IL-10 levels.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the Key Science and Technology Research Program from Health Commission of Hebei Province (grant no. 20221248).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

SZ designed the experiments. YZ and ZS collected tissue specimens. XZ and JZ performed the experiments. CP and SZ interpreted the data and drafted the manuscript. SZ and CP confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The present study involving human participants was approved by The Human Tissue Research Committee at the Second Hospital of Hebei Medical University (approval no. 2017-P031).

Patient consent for publication

All subjects provided written informed consent to participate in this study.

Competing interests

The authors declare that they have no competing interests.

References