Context N⁶-methyladenosine (m⁶A) in mRNA is the most abundant and reversible modification. However, the mechanism behind the decrease in m⁶A in patients with type 2 diabetes (T2D) has not yet been thoroughly investigated. Objective To clarify whether glucose is involved in the dynamic regulation of m6A in T2D and to identify a possible underlying mechanism. Methods Liquid chromatography-electrospray ionization-tandem mass spectrometry and qPCR were performed to determine the m⁶A content and the mRNA expression of target genes in 102 T2D patients and 107 controls. An additional 12 patients with normal fasting blood glucose, emergency hyperglycemia or emergency hypoglycemia, as well as HepG2 cells with high-glucose treatment and FTO knockout or overexpression were utilized to confirm the initial observations in patients. Results In patients, the m⁶A content was decreased, and mRNA expression levels of FTO, METTL3, METTL14 and WTAP were increased. Interestingly, the m⁶A content was negatively associated with mRNA expression levels of METTL3, METTL14 and FTO. Moreover, FTO was positively correlated with serum glucose. In HepG2 cells, high glucose upregulated FTO protein, whereas it had no significant effect on METTL3 or METTL14. Additionally, mRNA expression levels of FOXO1, G6PC, and DGAT2 were significantly increased and positively correlated with FTO and serum glucose in patients. Conclusions Our data revealed that, in T2D patients, high-glucose-enhanced FTO mRNA expression resulted in a decrease in m⁶A. The lower m⁶A content might be responsible for the upregulation of methyltransferases. Additionally, FTO induced mRNA expression of FOXO1, G6PC, and DGAT2 and was closely associated with glucose metabolism.

Context: N⁶-methyladenosine (m⁶A) modification plays a fundamental role in the epigenetic regulation of the mammalian transcriptome. m⁶A 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 m⁶A alteration in type 2 diabetes mellitus (T2DM) has not been explored. Objective: The objective of the study was to investigate whether m⁶A content was reduced in T2DM patients and whether m⁶A 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 m⁶A 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 m⁶A 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 m⁶A. 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 m⁶A 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 m⁶A in T2DM, which may further increase the risk of complications of T2DM. Low m⁶A should be investigated further as a novel potential biomarker of T2DM.

BACKGROUND: 5-Methylcytosine (5-mC) is an important epigenetic modification involved in development and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5-hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons, but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored. METHODS: We developed an online trapping/capillary hydrophilic-interaction liquid chromatography (cHILIC)/in-source fragmentation/tandem mass spectrometry system for quantifying 5-mC and 5-hmC in genomic DNA from hepatocellular carcinoma (HCC) tumor tissues and relevant tumor adjacent tissues. A polymer-based hydrophilic monolithic column was prepared and used for the separation of 12 nucleosides by cHILIC coupled with an online trapping system. Limits of detection and quantification, recovery, and imprecision of the method were determined. RESULTS: Limits of detection for 5-mC and 5-hmC were 0.06 and 0.19 fmol, respectively. The imprecision and recovery of the method were determined, with the relative SDs and relative errors being <14.9% and 15.8%, respectively. HCC tumor tissues had a 4- to 5-fold lower 5-hmC content compared to tumor-adjacent tissues. In addition, 5-hmC content highly correlated with tumor stage (tumor-nodes-metastasis, P = 0.0002; Barcelona Clinic liver cancer, P = 0.0003). CONCLUSIONS: The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.

The insulin/insulin-like growth factors (IGFs) have crucial tasks in the growth, differentiation, and proliferation of healthy and pernicious cells. They are involved in coordinated complexes, including receptors, ligands, binding proteins, and proteases. However, the systems can become dysregulated in tumorigenesis. Insulin-like growth factor-binding protein 7 (IGFBP7) is a protein belonging to the IGFBP superfamily (also termed GFBP-related proteins). Numerous studies have provided evidence that IGFBP3 and IGFBP7 are involved in a variety of cancers, including hepatocellular carcinoma (HCC), breast cancer, gastroesophageal cancer, colon cancer, prostate cancer, among many others. Still, very few suggest an interaction between these two molecules. In studying several cancer types in our laboratories, we found that both proteins share some crucial signaling pathways. The objective of this review is to present a comprehensive overview of the relationship between IGFBP7 and cancer, as well as highlighting IGFBP3 crosstalk with IGFBP7 reported in recent studies.

Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N⁶-methyladenosine (m⁶A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m⁶A contents in sperm RNA, and real-time PCR was performed to determine the mRNA expression of demethylase (FTO, ALKBH5), methyltransferase (METTL3, METTL14, WTAP) and an m⁶A-selective-binding protein (YTHDF2). We found that m⁶A content (p = 0.033) and the mRNA expression of METTL3 (p = 0.016) and METTL14 (p = 0.025) in asthenozoospermia patients were significantly higher than those of controls. Increased m⁶A content was a risk factor for asthenozoospermia (odds ratio (OR) 3.229, 95% confidence interval (CI) 1.178 – 8.853, p = 0.023). Moreover, m⁶A content was correlated with the expression of METTL3 (r = 0.303, p = 0.032) and with sperm motility (progressive motility: r = −0.288, p = 0.038; non-progressive motility: r = −0.293, p = 0.037; immotility: r = 0.387, p = 0.005). Our data suggest that increased m⁶A content is a risk factor for asthenozoospermia and affects sperm motility. Methyltransferases, particularly METTL3, play key roles in increasing m⁶A contents in sperm RNA.

BACKGROUND: Hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus (HBV) infection. False-negative results are common in routine serological tests and quantitative real-time PCR because of HBV surface antigen (HBsAg) variation and low HBV copy number. Droplet digital PCR (ddPCR), a next generation digital PCR, is a novel, sensitive, and specific platform that can be used to improve HBV detection. METHODS: A total of 131 HCC cases with different tumor stages and clinical features were initially classified with a serological test as HBsAg positive (n = 107) or negative (n = 24) for HBV infection. Next, DNA templates were prepared from the corresponding formalin-fixed paraffin-embedded (FFPE) tissues to determine HBV copy number by ddPCR. RESULTS: HBV copy numbers, successfully determined for all clinical FFPE tissues (n = 131), ranged from 1.1 to 175.5 copies/μL according to ddPCR. The copy numbers of HBV were positively correlated with tumor-nodes-metastasis (P = 0.008) and Barcelona-Clinic Liver Cancer (P = 0.045) classification. Moreover, serum cholinesterase correlated with hepatitis B viral load (P = 0.006). CONCLUSIONS: HBV infection is a key factor that influences tumorigenesis in HCC by regulating tumor occurrence and development. ddPCR improves the analytical sensitivity and specificity of measurements in nucleic acids at a single-molecule level and is suitable for HBV detection.