Introduction
Primary Sjögren’s syndrome (pSS) is a clinically common autoimmune disorder characterized by lymphocyte infiltration of exocrine glands (primarily the lacrimal and salivary glands) [
1]. Glandular inflammation and tissue impairment eventually give rise to disturbances of secretory and clinical manifestations of dryness, including dry eye and xerostomia [
2‐
4]. The pathogenesis of pSS is still poorly understood, and genetic and epigenetic factors have been considered to affect pSS development [
5]. Existing research found irregular DNA methylation in pSS patients, and a genome-wide DNA methylation study recognized 1977 hypomethylated and 842 hypermethylated differentially methylated positions in pSS patients [
6]. In addition to aberrant DNA methylation, increased miR-146a expression was also observed in peripheral blood mononuclear cells (PBMCs) from pSS patients [
7]. A study showing histone hypoacetylation in pSS patients supports the concept that epigenetic factors contribute to the disease’s pathogenesis [
8]. These studies show that epigenetic modifications promote the pathogenesis of pSS, but whether N6-methyladenosine (m
6A) methylation is involved in epigenetic regulation in pSS with dry eye pathogenesis remains unknown.
m
6A, the methylation modification at the sixth position of adenine bases in RNA, is the most common and evolutionarily conserved mRNA modification in eukaryotes [
9]. m
6A can affect RNA metabolism from some aspects, including mRNA splicing, mRNA stability and translation efficiency. The m
6A modification is dynamically regulated by three groups of enzymes called methyltransferases (writer), demethylases (eraser) and binding proteins (reader) [
10]. Methyltransferase-like 3 (METTL3), as the main RNA methyltransferase, forms a methyltransferase complex with its auxiliary partners methyltransferase-like 14 (METTL14) and Wilms tumor 1-associated protein (WTAP) to catalyze m
6A modification [
11]. It is well known that fat mass and obesity-associated protein (FTO) and alkylation repair homolog protein 5 (ALKBH5) are involved in removing m
6A methylation [
12]. In addition, m
6A methylation is recognized via readers, including YTH and IGF2BP family proteins and affect the degradation and translation of downstream RNA [
13,
14]. Recently, emerging studies have indicated that m
6A modification is connected to some vital biological processes, especially inflammatory and autoimmune responses [
15‐
17]. METTL3-mediated mRNA m
6A methylation promotes dendritic cell activation [
18]; B-cell-specific absence of METTL14 results in the B cell development defect [
19]; WTAP promotes the differentiation of thymocytes [
20]; FTO silencing inhibits macrophage polarization [
21]. Moreover, ALKBH5 deficiency alleviates CD4 + T cell responses [
22]; the deletion of YTHDF1 promotes the cross presentation of tumour antigens [
23]. These results indicated that m
6A may play a complicated role in pSS.
The objective of our research aimed to investigate the potential role of m6A modification in pSS with dry eye.
Materials and methods
Patients and controls
This cross-sectional study enrolled 48 pSS patients with dry eye from the First Affiliated Hospital of Chongqing Medical University from February 1, 2021, to April 1, 2022. These pSS patients accompanying with dry eye were diagnosed with the 2002 US-EU Consensus Group Criteria and the 2017 Dry Eye Workshop II Diagnostic Methodology Report strictly [
24,
25]. The pSS patients were diagnosed by an ophthalmologist and a rheumatologist. The patients diagnosed as any other autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus, severe infection or taking immunomodulatory therapy previously were excluded. As the control group, forty healthy volunteers matched by gender and age were chosen. Approval for the present study was obtained from the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (2020 − 765).
Measurement of serological indicators
Serum samples were routinely tested by the clinical pathology laboratory, including antinuclear antibodies (ANA), anti-SSA autoantibody, anti-SSB autoantibody, rheumatoid factor (RF), immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), complement 3 (C3), complement 4 (C4), C-reaction protein (CRP), and erythrocyte sedimentation rate (ESR).
Ophthalmological evaluation
The dry eye symptoms and signs of pSS patients were examined via ocular surface disease index (OSDI) questionnaires. Patients with a score over 13 were diagnosed with symptomatic dry eye. Tear break-up time (TBUT), corneal fluorescein staining score (CFS), and Schirmer’s test (ST) were determined to examine the tear film. The TBUT and CFS evaluation was conducted in a room with low lighting. Using a fluorescein strip (Liaoning Meizilin Pharmaceutical Co. Ltd., Tianjin, China), fluorescein was applied to the lower conjunctival sac. The subjects were asked to blink, and the time before the first fault shown in the stained tear film was recorded. Under cobalt blue light, the CFS was measured utilizing the Oxford scale [
26]. ST was carried out without topical anesthesia to assess the tear production of each individual. Filter paper (Liaoning Meizilin Pharmaceutical Co. Ltd., Tianjin, China) was applied for five mins. Readings were expressed as wetting millimeters.
Peripheral blood samples (5 mL) were obtained, and then PBMCs were isolated by using lymphocyte separation medium (LDS1075, TBD, China) within two hours. The isolated PBMCs were divided into several small tubes and then separately preserved in TRIzol reagent (Roche, Switzerland) and RIPA buffer (P0013B, Beyotime, China). The isolated PBMCs samples were stored in liquid nitrogen until use.
RNA isolation and real-time qPCR
Total RNA was isolated from the PBMCs of all participants with TRIzol reagent based on the manufacturer’s protocol. A spectrophotometer (NP80-Touch, Agilent Technologies) was employed to assess the purity and concentration of total RNA. Samples containing 1.0 µg of RNA were purified and synthesized into cDNA using RT Master Mix for qPCR (HY-K0511, MCE, USA). The cDNA was amplified employing SYBR Green qPCR Master Mix (HY-K0522, MCE, USA), and the fluorescent signal was monitored by an Applied Biosystems 7500 System. All primer sequences utilized in this work are displayed in Table
1. The relative expression of m
6A methylation-related genes was normalized to the internal reference and assessed through the 2
−ΔΔCT method.
Table 1
Primers used in this study
GAPDH | GGA GCG AGA TCC CTC CAA AAT | GGC TGT TGT CAT ACT TCT CAT GG |
METTL3 | TTG TCT CCA ACC TTC CGT AGT | CCA GAT CAG AGA GGT GGT GTA G |
METTL14 | AGT GCC GAC AGC ATT GGT G | GGA GCA GAG GTA TCA TAG GAA GC |
WTAP | ACC TCT TCC CAA GAA GGT TCG | GAT CTG TGT ACT TGC CCT CCA |
ALKBH5 | CGC TGC CGC CGA ACC TTA C | GGA TGC CGC TCT TCA CCT TGC |
FTO | CCA GGG TTG GGA TGG GTT CA | CGC TGA CCT GTC CAC CAG AT |
YTHDF1 | AGC ACA GAG CAC GGC AAC AAG | CCA TTG ACG CTG AAG AGC AGG TAG |
YTHDF2 | CAG ACA CAG CCA TTG CCT CCA C | AGA ACC AGC CTG AGA CTG TCC TAC |
Western blot analysis
The preserved protein samples from the PBMCs were isolated with RIPA lysis buffer (P0013B, Beyotime, China) containing a protease inhibitor cocktail (ST507, Beyotime, China), and the content was quantified by the BCA assay (P0012S, Beyotime). Lysates with 10 µg of total protein were isolated on 4–20% SDS‒PAGE gels and subsequently placed onto polyvinylidene fluoride membranes, according to the conventional method. After blocking with fat-free milk for one hour, the membranes were incubated with primary antibody overnight at room temperature. Primary antibodies against GAPDH (ab181602, Abcam, USA), METTL3 (ab195352, Abcam, USA), and YTHDF1 (ab220162, Abcam, USA) were used. Then, the membranes were rinsed with washing buffer three times and incubated for one hour with the secondary antibody (ab97051, Abcam, USA). The protein blots were visualized with an enhanced chemiluminescence kit (P0018FS, Beyotime, China). Protein expression was semi quantitatively analyzed with ImageJ software.
Quantification of RNA m6A
The m6A RNA methylation level was determined by utilizing the EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric, Epigentek, USA) following the manufacturer’s protocols. The relative abundance of m6A was measured and calculated by the absorbance detected by a microplate spectrophotometer (Varioskan Lux, Thermo) at 450 nm.
Statistical analysis
GraphPad Prism 8.0 (GraphPad Software) was used for all statistical analyses. Numerical data with a normal distribution are indicated as the mean ± standard deviation (SD), and differences between the two groups were studied by a two-tailed Student’s t test. Numerical data with skewed distributions are indicated as the median (25th percentile-75th percentile), and differences between the two groups were examined by the Mann–Whitney U test. Categorical data are indicated as percentages and frequencies. The correlation was evaluated by Spearman’s correlation coefficient. A P value < 0.05 was regarded as statistically significant.
Discussion
pSS is a chronic inflammatory autoimmune disease and is characterized by exocrine gland impairment, such as in the salivary and lacrimal glands, which could result in dry mouth and eye [
1]. m
6A, as the most abundant modification in mRNA, is receiving increasing attention and has been found to function in viral infections and some autoimmune diseases in recent years [
27,
28]. The potential role of m
6A modification in pSS patients with dry eye remains largely unknown. Therefore, our work aimed to examine the levels of m
6A modification and m
6A-related regulator expression in the lymphocytes of pSS patients with dry eye and analyze their correlation with clinical characteristics. Our findings revealed that the expression level of the m
6A modification and the mRNA and protein expression of METTL3 and YTHDF1 were all increased in pSS patients with dry eye. Moreover, the m
6A level was positively correlated with METTL3 in pSS patients with dry eye. The correlation analyses indicated that m
6A and METTL3 expression were correlated with anti-SSB antibody, IgG, complement, CFS, and ST. These results suggest a complicated role of m
6A modifications in pSS with dry eye.
An increasing number of experiments have stated that the dysregulation of global m
6A abundance and the aberrant expression of m
6A regulators might be associated with various autoimmune disorders. Wang et al. [
29] found that METTL3 was elevated in PBMCs from RA patients. In vitro experiments showed that METTL3 upregulation in macrophages increased the overall m
6A content and that METTL3-related m
6A modification was correlated with the secretion of inflammatory factors. Song et al. [
30] revealed that METTL3 mutations might be a pivotal susceptibility factor for autoimmune thyroid disease. These findings indicated that aberrant m
6A modification is a new regulatory mechanism in autoimmune disease. Recently, Cheng and her colleagues reported the downregulated expression of RNA-binding motif protein X-linked, ALKBH5, YTH domain-containing protein 2, and YTHDF1 in the peripheral blood samples of pSS patients [
31]. The reasons for this conflicting finding might be the distinctions in the disease severity of selected patients and the investigated cell type.
The US-EU Consensus Group has suggested that one of the criteria for the diagnosis of pSS is the occurrence of anti-SSB or anti-SSA autoantibodies [
24]. Previous results indicated that the anti-SSB antibody displays relatively better specificity for diagnosis [
1]. The association of the expression of m
6A and METTL3 with anti-SSB antibodies (not anti‐SSA antibodies) might be due to the better specificity of anti-SSB antibodies. These findings further revealed that m
6A modification might contribute to the pathogenesis of pSS with dry eye. Excessive immunological and inflammatory responses are crucial features of pSS, which can be evaluated by the expression levels of IgG and complement C3 and C4 [
32,
33]. Wang et al. [
29] revealed that increased levels of METTL3 correlated with CRP and ESR in rheumatoid arthritis, which is similar to our findings. Our results indicated that the elevated expression of m
6A and METTL3 correlated with serological immune indicators in pSS patients with dry eye. Huang et al. [
19] reported that m
6A-deficient mice exhibited the impairment of B cells activation and autoantibodies secretion, we speculate that aberrant m
6A modification may lead to the excessive secretion of anti-SSB antibodies and IgG in B cells and the release of complements in pSS. m
6A and METTL3 levels might be used as potential laboratory parameters to evaluate the systemic immune condition of pSS patients in clinical practice. Moreover, m
6A methylation could be a potential candidate for the epigenetic-based treatment of pSS.
Previous studies have focused on the correlation between ocular manifestations of autoimmune diseases and aberrant m
6A modification. Zhu et al. [
34] reported that m
6A expression was significantly increased in extraocular muscle samples from Graves’ ophthalmopathy patients, which suggests that dysregulated m
6A regulators may lead to the upregulated expression of genes related to the immune response and inflammatory process, thereby leading to ocular autoimmune diseases. We observed a significant correlation of m
6A and METTL3 expression with certain signs of dry eye when assessing tear secretion and ocular surface damage, which indicates that aberrant m
6A modification may contribute to the pathogenesis of dry eye in pSS.
However, there are a few limitations of our work. First, our work included only patients from the Department of Ophthalmology, which might lead to selection bias. Further studies could enroll pSS patients from other clinical departments and compare the correlation of m6A expression with other clinical features of pSS. Second, although increased METTL3 and m6A levels influenced the immune response in pSS, the precise regulatory mechanism is unknown. Our results suggest that aberrant m6A modification and METTL3 expression are likely to contribute to pSS pathogenesis, but further in vivo experimental studies are needed. Third, although PBMCs can characterize some disease, the data will be stronger if performed in conjunctiva impression cytology to reflect ocular changes of pSS patients.
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