Decreased N(6)-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5.
Shen Fan,Huang Wei,Huang Jing-Tao,Xiong Jun,Yang Ying,Wu Ke,Jia Gui-Fang,Chen Jinyun,Feng Yu-Qi,Yuan Bi-Feng,Liu Song-Mei
The Journal of clinical endocrinology and metabolism
CONTEXT:N(6)-methyladenosine (m(6)A) modification plays a fundamental role in the epigenetic regulation of the mammalian transcriptome. m(6)A can be demethylated by fat mass- and obesity-associated (FTO) protein and α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) protein. However, the importance of m(6)A alteration in type 2 diabetes mellitus (T2DM) has not been explored. OBJECTIVE:The objective of the study was to investigate whether m(6)A content was reduced in T2DM patients and whether m(6)A content was correlated with the mRNA expression levels of the FTO and ALKBH5 genes. METHODS:In this case-control study, peripheral blood samples were obtained from 88 T2DM patients and 92 healthy controls. For the diabetic animal model experiment, blood samples were obtained from seven diabetic and eight nondiabetic rats. A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the determination of the m(6)A content in RNA, quantitative real-time PCR was used to examine the mRNA expression levels of the FTO and ALKBH5 genes, and high-resolution melting and DNA sequencing were used to detect FTO single-nucleotide polymorphisms. RESULTS:Our results showed that the m(6)A contents in the RNA from T2DM patients and diabetic rats were significantly lower compared with the control groups (P = 2.6 × 10(-24) for T2DM patients; P = .001 for diabetic rats, respectively), and T2DM can be characterized by the content of m(6)A. The mRNA expression level of FTO was significantly higher in T2DM patients than that of the controls (P = .0007) and was associated with the risk of T2DM (odds ratio 2.797, 95% confidence interval 1.452-5.389, P = .002). Moreover, the m(6)A contents were correlated with FTO mRNA expression. CONCLUSIONS:These data suggest that the increased mRNA expression of FTO could be responsible for the reduction of m(6)A in T2DM, which may further increase the risk of complications of T2DM. Low m(6)A should be investigated further as a novel potential biomarker of T2DM.
10.1210/jc.2014-1893
Elevated N6-Methyladenosine RNA Levels in Peripheral Blood Immune Cells: A Novel Predictive Biomarker and Therapeutic Target for Colorectal Cancer.
Xie Jinye,Huang Zhijian,Jiang Ping,Wu Runan,Jiang Hongbo,Luo Chuanghua,Hong Honghai,Yin Haofan
Frontiers in immunology
Effective biomarkers for the diagnosis of colorectal cancer (CRC) are essential for improving prognosis. Imbalance in regulation of N6-methyladenosine (mA) RNA has been associated with a variety of cancers. However, whether the mA RNA levels of peripheral blood can serve as a diagnostic biomarker for CRC is still unclear. In this research, we found that the mA RNA levels of peripheral blood immune cells were apparently elevated in the CRC group compared with those in the normal controls (NCs) group. Furthermore, the mA levels arose as CRC progressed and metastasized, while these levels decreased after treatment. The area under the curve (AUC) of the mA levels was 0.946, which was significantly higher than the AUCs for carcinoembryonic antigen (CEA; 0.817), carbohydrate antigen 125 (CA125; 0.732), and carbohydrate antigen 19-9 (CA19-9; 0.771). Moreover, the combination of CEA, CA125, and CA19-9 with mA levels improved the AUC to 0.977. Bioinformatics and qRT-PCR analysis further confirmed that the expression of mA modifying regulator IGF2BP2 was markedly elevated in peripheral blood of CRC patients. Gene set variation analysis (GSVA) implied that monocyte was the most abundant mA-modified immune cell type in CRC patients' peripheral blood. Additionally, mA modifications were negatively related to the immune response of monocytes. In conclusion, our results revealed that mA RNA of peripheral blood immune cells was a prospective non-invasive diagnostic biomarker for CRC patients and might provide a valuable therapeutic target.
10.3389/fimmu.2021.760747
Increased levels of N6-methyladenosine in peripheral blood RNA: a perspective diagnostic biomarker and therapeutic target for non-small cell lung cancer.
Clinical chemistry and laboratory medicine
OBJECTIVES:Due to lack of effective biomarkers for non-small cell lung cancer (NSCLC), many patients are diagnosed at an advanced stage, which leads to poor prognosis. Dysregulation of N6-methyladenosine (mA) RNA contributes significantly to tumorigenesis and tumor progression. However, the diagnostic value of mA RNA status in peripheral blood to screen NSCLC remains unclear. METHODS:Peripheral blood samples from 152 NSCLC patients and 64 normal controls (NCs) were applied to assess the mA RNA levels. Bioinformatics and qRT-PCR analysis were performed to identify the specific immune cells in peripheral blood cells and investigate the mechanism of the alteration of mA RNA levels. RESULTS:Robust elevation of mA RNA levels of peripheral blood cells was exhibited in the NSCLC group. Moreover, the mA levels increased as NSCLC progressed, and reduced after treatment. The mA levels contained area under the curve (AUC) was 0.912, which was remarkably greater than the AUCs for CEA (0.740), CA125 (0.743), SCC (0.654), and Cyfra21-1 (0.730). Furthermore, the combination of these traditional biomarkers with mA levels elevated the AUC to 0.970. Further analysis established that the expression of mA erasers FTO and ALKBH5 were both markedly reduced and negatively correlated with mA levels in peripheral blood of NSCLC. Additionally, GEO database and flow cytometry analysis implied that FTO and ALKBH5 attributes to peripheral CD4 T cells proportion and activated the immune functions of T cells. CONCLUSIONS:These findings unraveled that mA RNA of peripheral blood immune cells was a prospective biomarker for the diagnosis of NSCLC.
10.1515/cclm-2022-1033
Alterations of m6A RNA methylation regulators contribute to autophagy and immune infiltration in primary Sjögren's syndrome.
Frontiers in immunology
N6-methyladenosine (m6A) RNA modification is a new epigenetic regulation mechanism on eukaryotic mRNA. Few autoimmune diseases focused on the role of m6A in their pathogenies, and m6A modulation in the pathological process of primary Sjögren's syndrome (pSS) is still unknown. In this work, three microarray datasets of pSS patients were downloaded from the GEO database: datasets #1 and #2 from the whole peripheral blood (PB) samples, dataset #3 from the labial salivary gland tissue samples, as well as a PB cohort collected from our hospital. Six differentially expressed m6A regulators were identified by comparing the PB dataset #1 of pSS and healthy controls using the Wilcox test and logistic regression analysis. Among them, four (ALKBH5, RBMX, RBM15B, and YTHDF1) were confirmed as down-regulated in PB dataset #2 and in our PB cohort by RT-PCR, and four (ALKBH5, METTL3, RBM15B, and YTHDF1) were confirmed as down-regulated in the dataset #3 of the labial gland tissue. In addition, discrepantly expressed m6A regulators accompanied by diverse immunocytes, including dendritic cells (DCs), T cells, and CD56dim natural killer cells, and among the regulators, ALKBH5 and METTL3 were comprehensively linked with the infiltrated immune cells. Notably, the most enriched autophagy mechanism mediated by m6A was observed in pSS using functional annotation analysis. Ten hub genes were identified using a protein-protein interaction network, and their expression in PB dataset #2 and the expression of three genes (PIK3CA, STAT1, and MAPK3) in the labial gland tissue dataset #3 were confirmed. Our study provides evidence that m6A methylation is widely involved in the immune infiltration and autophagy of pSS, thus contributing to the pathogenesis of this disease and potentially representing a novel therapeutic target.
10.3389/fimmu.2022.949206
N-methyladenosine levels in peripheral blood RNA: a potential diagnostic biomarker for colorectal cancer.
Cancer cell international
BACKGROUND:N-methyladenosine (mA) is dysregulated in various cancers, including colorectal cancer (CRC). Herein, we assess the diagnostic potential of peripheral blood (PB) mA levels in CRC. METHODS:We collected PB from healthy controls (HCs) and patients with CRC, analyzed PB RNA mA levels and the expression of mA-related demethylase genes FTO and ALKBH5, cocultured CRC cells with PB mononuclear cells (PBMCs), and constructed an MC38 cancer model. RESULTS:PB RNA mA levels were higher in the CRC than that in HCs. The area under the curve (AUC) of mA levels (0.886) in the CRC was significantly larger compared with carbohydrate antigen 199 (CA199; 0.666) and carcinoembryonic antigen (CEA; 0.834). The combination of CEA and CA199 with PB RNA mA led to an increase in the AUC (0.935). Compared with HCs, the expression of FTO and ALKBH5 was decreased in the CRC. After coculturing with CRC cells, the PBMCs RNA mA were significantly increased, whereas the expression of FTO and ALKBH5 decreased. Furthermore, mA RNA levels in the PB of MC38 cancer models were upregulated, whereas the expression of FTO and ALKBH5 decreased. CONCLUSIONS:PB RNA mA levels are a potential diagnostic biomarker for patients with CRC.
10.1186/s12935-024-03289-2
Level of N6-Methyladenosine in Peripheral Blood RNA: A Novel Predictive Biomarker for Gastric Cancer.
Ge Lichen,Zhang Nan,Chen Zhuojia,Song Jiaxi,Wu Yingmin,Li Zhuoling,Chen Feng,Wu Jia,Li Dandan,Li Jiexin,Wang Cheng,Wang Hongsheng,Wang Junjun
Clinical chemistry
BACKGROUND:Dysregulation of N6-methyladenosine (m6A) is associated with various human diseases including cancer. This study aimed to evaluate the level of m6A as a biomarker for gastric cancer (GC) diagnosis. METHODS:Peripheral blood samples were collected from 100 GC patients, 30 benign gastric disease (BGD) patients, and 75 healthy controls (HCs). Levels of m6A in total RNA and expression of m6A-related proteins were analyzed. RESULTS:The m6A levels in peripheral blood RNA were significantly increased in the GC group compared with those in the BGD or HC groups; moreover, levels increased with the progression and metastasis of GC and decreased in GC patients after surgery. The area under the curve (AUC) for m6A in the GC group was 0.929 (95% CI, 0.88-0.96), which is markedly greater than the AUCs for carcinoembryonic antigen (CEA; 0.694) and carbohydrate antigen 199 (CA199; 0.603). The combination of CEA and CA199 with m6A improved the AUC to 0.955 (95% CI, 0.91-0.98). The expressions of m6A demethylases ALKBH5 and FTO were significantly downregulated in the GC group compared with the HC group. Coculture with GC cells increased the m6A of RNA in promyelocytic (HL-60) and monocytic (THP-1) leukemia cells and nontumorigenic human peripheral blood B lymphocyte cells (PENG-EBV). Furthermore, a xenograft model enhanced the m6A in peripheral blood RNA of mice. Accordingly, expressions of ALKBH5 and FTO were decreased both in vitro and in vivo. CONCLUSIONS:Level of m6A in peripheral blood RNA is a promising noninvasive diagnostic biomarker for GC patients.
10.1093/clinchem/hvz004
Upregulated N6-Methyladenosine RNA in Peripheral Blood: Potential Diagnostic Biomarker for Breast Cancer.
Xiao Han,Fan Xiaobo,Zhang Rui,Wu Guoqiu
Cancer research and treatment
PURPOSE:An effective biomarker for the diagnosis of breast cancer (BC) and benign breast diseases (BBD) is crucial for improving the prognosis. We investigated whether N6-methyladenosine (m6A) can be a diagnostic biomarker of BC. Materials and Methods:We detected the contents of peripheral blood m6A in 62 patients with BC, 41 patients with BBD, and 41 normal controls (NCs) using the colorimetric method. The relative expression of the m6A regulated genes methyltransferase-like 14 (METTL14) and fat mass and obesity-associated (FTO) was analyzed using quantitative real-time polymerase chain reaction. RESULTS:m6A in peripheral blood RNA was significantly higher in patients with BC than that in patients with BBD (p < 0.001) or the NCs (p < 0.001). m6A was closely associated with the disease stage (from stage 0 to stage I-IV, p=0.003). The receiver operating characteristic curve of m6A contained an area under the curve (AUC) value of 0.887 in BC, which was greater than that of carcinoembryonic antigen (CEA) or carbohydrate antigen 153 (CA153). The combination of m6A, CEA, and CA153 improved the AUC to 0.914. The upregulated and downregulated mRNA expression of METTL14 and FTO, respectively, might contribute to the increase of m6A in patients with BC. m6A combined with METTL14 and FTO improved the AUC to 0.929 with a specificity of 97.4% in the peripheral blood of patients with BC. CONCLUSION:The peripheral blood RNA of m6A might be a valuable biomarker for the diagnosis of BC.
10.4143/crt.2020.870
Peripheral Blood Leukocyte N6-methyladenosine is a Noninvasive Biomarker for Non-small-cell Lung Carcinoma.
Pei Yuqing,Lou Xiaoying,Li Kexin,Xu Xiaotian,Guo Ye,Xu Danfei,Yang Zhenxi,Xu Dongsheng,Cui Wei,Zhang Donghong
OncoTargets and therapy
Background:N6-methyladenosine (m6A) triggers a new layer of epi-transcription. However, the potential noninvasive screening and diagnostic value of peripheral blood m6A for cancer are still unknown. Here, we intend to investigate whether leukocyte m6A can be a novel biomarker for non-small-cell lung cancer (NSCLC). Materials and Methods:Peripheral blood was collected from 119 NSCLC patients and 74 age-matched healthy controls. Total RNA was isolated from leukocytes for m6A measurement, and clinical information of participants was reviewed. The sensitivity, specificity, and area under the curve (AUC) of m6A for cancer diagnosis were evaluated by the receiver-operating characteristic (ROC) curve analysis. Flow cytometry and the Human Protein Atlas (HPA) database were used to characterize m6A in leukocyte differentials. Pearson's correlation was applied to indicate the relationship between m6A level and hematology variables. qPCR and bioinformatic analysis were used to identity the expression of m6A regulators in leukocyte. Results:Leukocyte m6A was significantly elevated in 119 NSCLC patients compared with 74 healthy controls (<0.001). We did not find significant association between m6A and age or gender. Elevated m6A level in NSCLC was associated with tumor stage (<0.05) and tumor differentiation (<0.05), and was significantly reduced after surgery (<0.01). ROC curve analysis revealed that leukocyte m6A could significantly discriminate patients with lung adenocarcinoma (LUAD) (AUC=0.736, <0.001) and lung squamous cell carcinoma (LUSC) (AUC=0.963, <0.001) from healthy individuals. m6A displayed superior sensitivity (100%) and specificity (85.7%) for LUSC than squamous cell carcinoma (SCC) antigen and cytokeratin fragment 211 (Cyfra211). Flow cytometry analysis showed m6A modification was mainly localized on T cells and monocytes among leukocyte differentials. Leukocyte m6A was positively correlated with the number of lymphocytes and negatively correlated with monocytes in NSCLC but not in healthy controls. qPCR and bioinformatic analysis showed that elevated leukocyte m6A in NSCLC was caused by upregulated methyltransferase complex and downregulated and . Conclusion:Leukocyte m6A represents a potential noninvasive biomarker for NSCLC screening, monitoring and diagnosis.
10.2147/OTT.S267344
Expression and Clinical Significance of the m6A RNA-Binding Proteins in Peripheral Blood Mononuclear Cells From New-Onset Ankylosing Spondylitis.
Frontiers in medicine
This study has focused on determining the association of m6A methyltransferase [methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1-associating protein (WTAP)], demethylase [fat mass and obesity-associated protein (FTO) and alkylation repair homolog protein 5 (ALKBH5)], RNA-binding proteins [YT521-B homology domains 2 (YTHDF2)], and ankylosing spondylitis (AS). A total of 154 specimens, containing 79 patients with new-onset AS and 75 healthy controls (HCs), participated in the study. The mRNA expressions of these m6A methyltransferase, demethylase, and RNA-binding protein in peripheral blood mononuclear cells (PBMCs) were detected by quantitative real-time PCR (qRT-PCR). The data showed that the mRNA expressions of and in PBMC from patients with new-onset AS were significantly decreased, and there was a positive correlation between RNA-binding proteins () and demethylase () in patients with new-onset AS. Logistic regression analysis demonstrated that the expression of mRNA in PBMC is a risk factor of AS. Receiver operating characteristic (ROC) analysis of the area under the curve (AUC) for mRNA in new-onset AS and HC was 0.692, with a cutoff value of <0.8724, a sensitivity of 67%, and a specificity of 63%. Moreover, we constructed a novel predictive model based on a combination of mRNA and systemic immune-inflammation index (SII) for AS diagnosis (AUC = 0.865, sensitivity = 79.45%, specificity = 84.00%), and the predictive model correlated with the activity and severity of AS. This study indicates that the mRNA expression of in PBMC may be involved in AS pathogenesis and a predictive model based on a combination of mRNA and SII acts as a marker for diagnosis and progression of diseases.
10.3389/fmed.2022.922219
Expression and clinical significance of the m6A reader in peripheral blood mononuclear cells from rheumatoid arthritis patients.
Journal of immunotoxicology
As an important m6A reader, the YT521-B homology domain family 2 (YTHDF2) has been shown to regulate mRNA degradation and translation, and to be involved in inflammation. However, little is known about the role of YTHDF2 in the autoimmune-based inflammatory disease rheumatoid arthritis (RA). To begin to ascertain any role for this reader, 74 RA patients and 63 healthy controls (HC) were recruited for this study. Blood was collected from each subject and peripheral blood mononuclear cells (PBMC) isolated. Thereafter, mRNA expression of , interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α in the cells was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The harvested blood was also assessed for a variety of parameters, including levels of C-reactive protein (CRP), erythrocyte sedimentation rates (ESR), white blood cell counts (WBC), neutrophils counts (N)/neutrophils percentages (N%), and neutrophil:lymphocyte ratios (NLR) - each markers of inflammation during RA. The results showed that mRNA expression in RA patient PBMC was decreased significantly vs that in healthy control subject cells. Further, mRNA expression in RA patient PBMC negatively-correlated with ESR, CRP levels, WBC counts, as well as neutrophils counts, percentages, and NLR values. In addition, it was seen that mRNA expression in RA patient PBMC was associated with host serum RF levels and treatment. Moreover, it was found that mRNA expression of IL-1β, IL-6, IL-8, and TNFα was increased in PBMC from RA patients relative to in control subject cells; however, only the increased IL-1β expression was seen to be negatively-correlated with decreased mRNA expression. In conclusion, the present study illustrated that expression might have some regulatory role in the underlying mechanisms associated with the autoimmune disease RA and that this m6A reader could at some point represent a potential target for regulating inflammatory responses that occur during RA.
10.1080/1547691X.2022.2067916