Decreased ALKBH5, FTO, and YTHDF2 in Peripheral Blood Are as Risk Factors for Rheumatoid Arthritis.

ALKBH5 (alkylation repair homolog protein 5), FTO (fat mass and obesity-associated protein), and RNA N6-methyladenosine (m6A) demethylase, are essential for the m6A mRNA modification. YTHDF2 (YT521-B homology domains 2) called m6A "readers" can recognize m6A modification. As the key enzymes of m6A methylation modification, ALKBH5, FTO, and YTHDF2 have been implicated in many diseases. However, little is known about the role of ALKBH5, FTO, and YTHDF2 in rheumatoid arthritis (RA). We measured the mRNA expression of ALKBH5, FTO, and YTHDF2 in RA patients and controls by quantitative real-time polymerase chain reaction, and the global m6A content was detected by an ELISA-like format. The mRNA expression of ALKBH5, FTO, and YTHDF2 in RA patients was further analyzed to investigate its correlations with disease activity. And, multivariate analysis (logistic regression) was used to analyze the risk factors. The mRNA expression of ALKBH5, FTO, and YTHDF2 in RA patients was significantly decreased compared to controls. The mRNA expression of ALKBH5 was significantly increased in RA patients that received regular treatment. The mRNA expression of FTO was associated with disease activity score 28 (DAS28), complement 3 (C3), immunoglobulin G (IgG), and lymphocyte-to-monocyte ratio (LMR), some common markers for RA disease activity. The mRNA expression of YTHDF2 was associated with RBC, L%, N%, NLR, and LMR. Furthermore, logistic regression analysis revealed that decreased expression of ALKBH5, FTO, and YTHDF2 in peripheral blood was a risk factor for RA. Moreover, the peripheral blood global m6A content was significantly increased in patients with RA compared to CON, and increased m6A contents negatively correlated with decreased mRNA expression of FTO. In conclusion, this study firstly demonstrates the critical role of ALKBH5, FTO, and YTHDF2 in RA, which provides novel insights into recognizing the pathogenesis of RA and a promising biomarker for RA.

Luo Q ，Gao Y ，Zhang L ，Rao J ，Guo Y ，Huang Z ，Li J ... -  《-》

Decreased Peripheral Blood ALKBH5 Correlates with Markers of Autoimmune Response in Systemic Lupus Erythematosus.

Although it has been proved that the epigenetic modification of DNA and histones is involved in the pathogenesis of systemic lupus erythematosus (SLE), there is no study to explore whether the modification of N6-methyladenosine (m6A) in RNA is involved. In this study, the mRNA levels of m6A "writers" (METTL3, MTEEL14, and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in peripheral blood were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results demonstrated that the mRNA levels of METTL3, WTAP, FTO, ALKBH5, and YTHDF2 in peripheral blood from SLE patients were significantly decreased. The levels of ALKBH5 mRNA in SLE patients were associated with anti-dsDNA, antinucleosome, rash, and ulceration. Multivariate logistic regression analysis showed that the level of ALKBH5 mRNA in peripheral blood is a risk factor of SLE (P < 0.001). Moreover, our results suggested that there was a positive correlation between m6A"writers" (METTL3 and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in SLE patients. This study suggests that the mRNA level of ALKBH5 in peripheral blood may be involved in the pathogenesis of SLE.

Luo Q ，Fu B ，Zhang L ，Guo Y ，Huang Z ，Li J ... -  《-》

The study of METTL14, ALKBH5, and YTHDF2 in peripheral blood mononuclear cells from systemic lupus erythematosus.

This study was aimed to explore the mRNA expression of m6A "writers" (METTL3, MTEEL14, and WTAP), "erasers" (FTO and ALKBH5), and "readers" (YTHDF2) in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and investigate the relation between their expressions with clinical features. In all, 54 SLE patients and 42 healthy controls (HC) were included in the current study. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to investigate the mRNA expression of m6A "writers," "erasers," and "readers" in PBMCs from SLE patients and HC. Decreased mRNA expression of MTEEL14, ALKBH5, and YTHDF2 was observed in SLE patients compared with those in HC (p < .001). The decreased mRNA expression of METTL14 was associated with white blood cell count (WBC) and monocyte count (M), this decreased mRNA expression of ALKBH5 was associated with C-reactive protein (CRP), neutrophil percentage (N%), lymphocyte percentage (L%), neutrophil-lymphocyte ratio (NLR), complement 3 (C3), and fever, and the decreased mRNA expression of YTHDF2 was associated with L%, NLR, C3, and fever. In addition, there was a positive correlation between mRNA expression of METTL14, ALKBH5, and YTHDF2 in PBMCs from SLE patients. Importantly, logistic regression analysis revealed that decreased mRNA expression of YTHDF2 was a risk factor for SLE. Taken all together, our findings suggested decreased YTHDF2 that was associated with disease activity may play an important role in the pathogenesis of SLE, METTL14 and ALKBH5 may be concomitantly decreased.

Luo Q ，Rao J ，Zhang L ，Fu B ，Guo Y ，Huang Z ，Li J ... -  《Molecular Genetics & Genomic Medicine》

The potential role of RNA N6-methyladenosine in primary Sjögren's syndrome.

The pathogenesis of primary Sjögren's syndrome (pSS) remains incompletely understood. The N6-methyladenosine (m6A) RNA modification, the most abundant internal transcript modification, has close associations with multiple diseases. This study aimed to investigate the role of m6A in patients with pSS. This study enrolled 44 patients with pSS, 50 age- and gender-matched healthy controls (HCs), and 11 age- and gender-matched patients with non-SS sicca. We detected the messenger RNA (mRNA) levels of m6A elements (including METTL3, WTAP, RBM15, ALKBH5, FTO, YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2), ISG15, and USP18 in peripheral blood mononuclear cells (PBMCs) from patients with pSS, patients with non-SS sicca, and HCs. The clinical characteristics and laboratory findings of patients with pSS and patients with non-SS sicca were also collected. We used binary logistic regression to determine if m6A elements were risk factors for pSS. The mRNA levels of m6A writers (METTL3 and RBM15), erasers (ALKBH5 and FTO), and readers (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) were all significantly higher in PBMCs from patients with pSS than in HCs. The mRNA levels of m6A writers (METTL3 and WTAP) and readers (YTHDF2, YTHDF3, and YTHDC2) were lower in PBMCs from patients with pSS compared to patients with non-SS sicca. The expression of METTL3, RBM15, FTO, YTHDF1, YTHDF2, YTHDC1, and YTHDC2 was positively correlated with the level of C-reactive protein (CRP) of patients with pSS. The mRNA level of YTHDF1 in PBMCs from patients with pSS was negatively correlated with the EULAR Sjögren's syndrome disease activity index (ESSDAI) score. In patients with pSS, FTO, YTHDC1, and YTHDC2 were also related to white blood cells (WBCs), neutrophils, lymphocytes, and monocytes. Increased mRNA level of ALKBH5 in PBMCs was a risk factor for pSS, as determined by binary logistic regression analysis. The mRNA level of ISG15 was positively correlated with that of FTO, YTHDF2, YTHDF3, and YTHDC2 in patients with pSS. Compared with HCs, the expression of METTL3, RBM15, ALKBH5, FTO, YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 was considerably higher in PBMCs from patients with pSS. In comparison with patients with non-SS sicca, the expression of METTL3, WTAP, YTHDF2, YTHDF3, and YTHDC2 was reduced in PBMCs from patients with pSS. The m6A elements correlating with clinical variables may indicate the disease activity and inflammation status of pSS. Elevated expression of ALKBH5 was a risk factor for pSS. The dynamic process of m6A modification is active in pSS. m6A elements (FTO, YTHDF2, YTHDF3, or YTHDC2) might target ISG15, stimulate the expression of ISG15, and activate the type I IFN signaling pathway, playing an active role in initiating the autoimmunity in pSS.

Xiao Q ，Wu X ，Deng C ，Zhao L ，Peng L ，Zhou J ，Zhang W ，Zhao Y ，Fei Y ... -  《Frontiers in Medicine》

Increased N6-methyladenosine causes infertility is associated with FTO expression.

The N6-methyladenosine (m6A) modification plays a central role in epigenetic regulation of the mammalian transcriptome. m6A can be demethylated by the fat mass- and obesity-associated (FTO) protein and the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) protein. Much less is known about that whether m6A content is involved in POI (premature ovarian insufficiency) disease. In this case-controlled study, 69 POI and 53 tubal occlusion patients were recruited from the reproduction centers in our hospital. For the POI animal model experiment, ovarian tissue was obtained from ten POI and nine healthy mice. An m6A test kit was developed to determine the m6A content in the RNA, and qPCR and western blot were used to examine the mRNA and protein expression levels of FTO and ALKBH5. FACS was used to measure the levels of proliferation and apoptosis, and siRNA was used to establish FTO and ALKBH5 knockdown cell lines. Our results showed that the m6A content in the RNA from POI patients and POI mice was significantly higher than control groups and that POI was characterized by the content of m6A. The mRNA and protein expression levels of FTO were significantly lower in the POI patients than control group and were associated with a risk of POI. These data suggest that the decreased mRNA and protein expression levels of FTO may be responsible for the increase in m6A in POI, which may further increase the risk of complications of POI. High m6A should be investigated further as a novel potential biomarker of POI.

Ding C ，Zou Q ，Ding J ，Ling M ，Wang W ，Li H ，Huang B ... -  《-》