Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine.

N6-methyladenosine (m6A) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific m6A marks are complicated to operate, it is difficult to quantify the methylation level, and they have high false-positive levels. Here, we report a new method for locus-specific m6A detection based on the methylate-sensitive endonuclease activity of MazF and the simultaneous amplification and testing (SAT) method, termed "m6A-MazF-SAT". Mechanically, MazF fails to cleave the A (m6A) CA motif; therefore, the undigested template can be SAT-amplified using specific probes targeting the upstream and downstream of sites of interest. Fluorescent signals of SAT amplification can be detected by real-time PCR, and therefore, they achieve the detection of m6A existence. After the condition optimization, m6A-MazF-SAT can significantly, accurately, and rapidly detect the m6A-modified sites in mRNA, rRNA, and lncRNA at the fmol level, as well as 10% m6A at the fmol level. In addition, m6A-MazF-SAT can quantify the abundance of target m6A in biological samples and can be used for the inhibitor selection of m6A-related enzymes. Together, we offer a new approach to detect locus-specific m6A both qualitatively and quantitatively; it is easy to operate, results can be obtained rapidly, and it has low false-positive levels and high repeatability.

Circulating exosomal small RNAs are promising non-invasive diagnostic biomarkers for gastric cancer.

Dysregulation of small non-coding RNA (ncRNA) is associated with various human diseases including cancer. This study aimed to evaluate the circulating exosomal small RNAs including microRNAs (miRNAs) and P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) as sensitive and specific non-invasive biomarkers for gastric cancer (GC) diagnosis. Serum exosomal small RNA transcriptome was examined using unique molecular identifiers (UMI) small RNA sequencing. Dysregulated miRNAs and piRNAs were verified in 70 GC patients and 60 healthy controls (HC) by reverse transcription quantitative PCR. The expressions of miR-1307-3p, piR-019308, piR-004918 and piR-018569 in serum exosomes were significantly increased in GC group as compared to those in HC group. Moreover, GC patients with metastasis had significantly higher expression levels of piR-004918 and piR-019308 than GC patients without metastasis. The area under the curve (AUC) for miR-1307-3p, piR-019308, piR-004918 and piR-018569 in the GC group was 0.845, 0.820, 0.754 and 0.732, respectively. The combination of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 199 can improve the AUC of miR-1307-3p to 0.902 and piR-019308 to 0.914 for GC diagnosis. In conclusion, our findings indicate that serum exosomal piRNAs are promising non-invasive diagnostic biomarkers for GC patients and potential markers for monitoring metastasis.

Level of N6-Methyladenosine in Peripheral Blood RNA: A Novel Predictive Biomarker for Gastric Cancer.

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.

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: a comparative proteomic analysis.

BACKGROUND: Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study. METHODS: We utilized proteomic study to identify the protein expression changes of Sertoli TM4 cells treated with 10(-8)M and 10(-5)M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis. RESULTS: Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10(-8)M and 10(-5)M BPA, respectively, for 24h. Exposure to 10(-5)M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10(-8)M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages. CONCLUSIONS: Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism. GENERAL SIGNIFICANCE: Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.

Bisphenol A stimulates the epithelial mesenchymal transition of estrogen negative breast cancer cells via FOXA1 signals.

Estrogen receptor negative (ER-) breast cancer are associated with increased risks for metastasis and high rates of recurrence. Our present study revealed that nanomolar bisphenol A (BPA), a typical endocrine disrupting chemical, promoted the in vitro migration and induced mesenchymal transition (EMT) of ER-breast cancer cells. PCR array revealed that BPA can down regulate 12 and up regulate 2 genes involved in regulation of signal transduction and biological pathways of breast cancer. The down regulated genes included FOXA1, which is a key determinant of endocrine response and down regulated by BPA via a time dependent manner. Silencing of FOXA1 by siRNA triggered the EMT of SkBr3 cells. While over expression of FOXA1 abolished BPA induced EMT. Further, 10(-8) M BPA significantly increased the phosphorylation of ERK1/2, p38-MAPK, and Akt in SkBr3 cells, while only PI3K/Akt inhibitor LY294002 attenuated the BPA induced down regulation of FOXA1 and E-Cadherin (E-Cad). Over expression of Akt also suppressed FOXA1 expression in SkBr3 cells. It suggested that PI3K/Akt mediated, at least partially, BPA induced EMT of ER-breast cancer cells. In summary, our data provided the first evidence that BPA can promote the EMT of ER-breast cancer cells through down regulation of FOXA1.

The RNA m6A reader IGF2BP3 regulates NFAT1/IRF1 axis-mediated anti-tumor activity in gastric cancer.

N6-methyladenosine (m6A) and its associated reader protein insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) are involved in tumor initiation and progression via regulating RNA metabolism. This study aims to investigate the biological function and clinical significance of IGF2BP3 in gastric cancer (GC). The clinical significance of IGF2BP3 was evaluated using tumor related databases and clinical tissues. The biological role and molecular mechanism of IGF2BP3 in GC progression were investigated by multi-omics analysis including Ribosome sequence (Ribo-seq), RNA sequence (RNA-seq) and m6A sequence (m6A-seq) combined with gain- and loss- of function experiments. IGF2BP3 expression is significantly elevated in GC tissues and associated with poor prognosis of GC patients. Knockdown of IGF2BP3 significantly weakens the migration and clonogenic ability, promotes the apoptosis, inhibits translation, and suppresses in vitro growth and progression of GC cells. Mechanistically, IGF2BP3 regulates the mRNA stability and translation of the nuclear factor of activated T cells 1(NFAT1) in a m6A dependent manner. Then NFAT1 induced by IGF2BP3 acts as a transcription factor (TF) to negatively regulates the promoter activities of interferon regulatory factor 1 (IRF1) to inhibit its expression. Inhibition of IGF2BP3-induced expression of IRF1 activates interferon (IFN) signaling pathway and then exerts its anti-tumor effect. Elevated IGF2BP3 promotes in vivo and in vitro GC progression via regulation of NFAT1/IRF1 pathways. Targeted inhibition of IGF2BP3 might be a potential therapeutic approach for GC treatment.

RNA m6 A methylation in cancer.

N6 -methyladenosine (m6 A) is one of the most abundant internal modifications in eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). It is a reversible and dynamic RNA modification that has been observed in both internal coding segments and untranslated regions. Studies indicate that m6 A modifications play important roles in translation, RNA splicing, export, degradation and ncRNA processing control. In this review, we focus on the profiles and biological functions of RNA m6 A methylation on both mRNAs and ncRNAs. The dynamic modification of m6 A and its potential roles in cancer development are discussed. Moreover, we discuss the possibility of m6 A modifications serving as potential biomarkers for cancer diagnosis and targets for therapy.

Erratum: N6-methyladenosine-induced ERRγ triggers chemoresistance of cancer cells through upregulation of ABCB1 and metabolic reprogramming: Erratum.

[This corrects the article DOI: 10.7150/thno.40144.].

N6-Methyladenosine Promotes Translation of VEGFA to Accelerate Angiogenesis in Lung Cancer.

Angiogenesis is hijacked by cancer to support tumor growth. RNA modifications such as N6-methyladenosine (m6A) can regulate several aspects of cancer, including angiogenesis. Here, we find that m6A triggers angiogenesis in lung cancer by upregulating VEGFA, a central regulator of neovasculature and blood vessel growth. m6A-sequencing and functional studies confirmed that m6A modification of the 5'UTR (untranslated region) of VEGFA positively regulates its translation. Specifically, methylation of a 5'UTR internal ribosome entry site (IRES) recruited the YTHDC2/eIF4GI complex to trigger cap-independent translation initiation. Intriguingly, the m6A methylation site A856 of the 5'UTR was located within the conserved upstream open reading frame (uORF) of VEGFA IRES-A, which overcomes uORF-mediated translation suppression while facilitating G-quadruplex-induced translation of VEGFA. Targeted specific demethylation of VEGFA m6A significantly decreased expression of VEGFA and reduced lung cancer cell-driven angiogenesis. In vivo and clinical data confirmed the positive effects of m6A modification of VEGFA on angiogenesis and tumor growth of lung cancer. This study not only reveals that the m6A/VEGFA axis is a potential target for lung cancer therapy but also expands our understanding of the impact of m6A modification of IRES in the 5'UTR of mRNA on translation regulation. SIGNIFICANCE: Methylation of the 5'UTR IRES of VEGFA mRNA increases cap-independent translation via recruitment of the YTHDC2/eIF4GI complex, which stimulates angiogenesis to promote lung tumor growth.

5'-tRF-GlyGCC: a tRNA-derived small RNA as a novel biomarker for colorectal cancer diagnosis.

BACKGROUND: tRNA-derived small RNAs (tDRs), which are widely distributed in human tissues including blood and urine, play an important role in the progression of cancer. However, the expression of tDRs in colorectal cancer (CRC) plasma and their potential diagnostic values have not been systematically explored. METHODS: The expression profiles of tDRs in plasma of CRC and health controls (HCs) are investigated by small RNA sequencing. The level and diagnostic value of 5'-tRF-GlyGCC are evaluated by quantitative PCR in plasma samples from 105 CRC patients and 90 HCs. The mechanisms responsible for biogenesis of 5'-tRF-GlyGCC are checked by in vitro and in vivo models. RESULTS: 5'-tRF-GlyGCC is dramatically increased in plasma of CRC patients compared to that of HCs. The area under curve (AUC) for 5'-tRF-GlyGCC in CRC group is 0.882. The combination of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) with 5'-tRF-GlyGCC improves the AUC to 0.926. Consistently, the expression levels of 5'-tRF-GlyGCC in CRC cells and xenograft tissues are significantly greater than that in their corresponding controls. Blood cells co-cultured with CRC cells or mice xenografted with CRC tumors show increased levels of 5'-tRF-GlyGCC. In addition, we find that the increased expression of 5'-tRF-GlyGCC is dependent on the upregulation of AlkB homolog 3 (ALKBH3), a tRNA demethylase which can promote tRNA cleaving to generate tDRs. CONCLUSIONS: The level of 5'-tRF-GlyGCC in plasma is a promising diagnostic biomarker for CRC diagnosis.

N6 -Methyladenosine Regulates mRNA Stability and Translation Efficiency of KRT7 to Promote Breast Cancer Lung Metastasis.

The roles of RNA modification during organ metastasis of cancer cells are not known. Here we established breast cancer lung metastasis cells by three rounds of selection of lung metastatic subpopulations in vivo and designated them as BCLMF3 cells. In these cells, mRNA N6 -methyladenosine (m6A) and methyltransferase METTL3 were increased, while the demethylase FTO was decreased. Epi-transcriptome and transcriptome analyses together with functional studies identified keratin 7 (KRT7) as a key effector for m6A-induced breast cancer lung metastasis. Specifically, increased METTL3 methylated KRT7-AS at A877 to increase the stability of a KRT7-AS/KRT7 mRNA duplex via IGF2BP1/HuR complexes. Furthermore, YTHDF1/eEF-1 was involved in FTO-regulated translational elongation of KRT7 mRNA, with methylated A950 in KRT7 exon 6 as the key site for methylation. In vivo and clinical studies confirmed the essential roles of KRT7, KRT7-AS, and METTL3 for lung metastasis and clinical progression of breast cancer. Collectively, m6A promotes breast cancer lung metastasis by increasing the stability of a KRT7-AS/KRT7 mRNA duplex and translation of KRT7. SIGNIFICANCE: This study suggests that N6 -methyladenosine is a key driver and potential therapeutic target in breast cancer metastasis.

N6-methyladenosine-induced ERRγ triggers chemoresistance of cancer cells through upregulation of ABCB1 and metabolic reprogramming.

Background: Drug resistance severely reduces treatment efficiency of chemotherapy and leads to poor prognosis. However, regulatory factors of chemoresistant cancer cells are largely unknown. Methods: The expression of estrogen receptor related receptors (ERRs) in chemoresistant cancer cells are checked. The roles of ERRγ in chemoresistance are confirmed by in vitro and in vivo studies. The mechanisms responsible for ERRγ-regulated expression of ABCB1 and CPT1B are investigated. Results: The expression of ERRγ is upregulated in chemoresistant cancer cells. Targeted inhibition of ERRγ restores the chemosensitivity. ERRγ can directly bind to the promoter of ABCB1 to increase its transcription. An elevated interaction between ERRγ and p65 in chemoresistant cells further strengthens transcription of ABCB1. Further, ERRγ can increase the fatty acid oxidation (FAO) in chemoresistant cells via regulation of CPT1B, the rate-limiting enzyme of FAO. The upregulated ERRγ in chemoresistant cancer cells might be due to increased levels of N6-methyladenosine (m6A) can trigger the splicing of precursor ESRRG mRNA. Conclusions: m6A induced ERRγ confers chemoresistance of cancer cells through upregulation of ABCB1 and CPT1B.

Signals involved in the effects of bisphenol A (BPA) on proliferation and motility of Leydig cells: a comparative proteomic analysis.

Recent studies indicated that bisphenol A (BPA) can disrupt spermatogenesis and then cause male infertility. The present study revealed that BPA greater than 10-6 M inhibited the proliferation of Leydig TM3 cells via a concentration dependent manner. The proteomic study revealed that 50 proteins were modulated in TM3 cells following exposure to BPA, which was relevant to structure, motility, cell metabolism, protein and nucleotide processing, and cell proliferation. Furthermore, BPA increased the in vitro migration and invasion of Leydig TM3 cells, which might be due to the BPA's modulation of proteins related to cell structure and motility such as actin and heat shock protein (HSP). Silencing of galectin-1, which was up regulated by BPA, significantly abolished the BPA-induced migration of TM3 cells. BPA treatment obviously increased the phosphorylation of ERK1/2 and Akt, while only PD98509 (ERK1/2 inhibitor) significantly attenuated BPA induced up regulation of galectin-1. Furthermore, PD98509 also reversed BPA induced migration of TM3 cells. Our study demonstrated that xenoestrogen BPA at micromolar or greater concentrations can modulate protein profiles, inhibit cell proliferation, and promote the in vitro migration and invasion of Leydig TM3 cells. It provided new insight into the mechanisms responsible for BPA induced male infertility.

Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor α/ß in low doses of bisphenol A promoting growth of Sertoli TM4 cells.

Sertoli cells play a pivotal role in supporting proliferation of germ cells and differentiation during spermatogenesis in mammals. Nanomolar concentrations of Bisphenol A (BPA) can significantly stimulate the proliferation of mouse immature Sertoli (TM4) cells. However, mechanisms by which BPA caused these effects were still unclear. In the present study, an inverse U-shaped curve was observed when treating TM4 cells with increasing doses of BPA: 1 to 10nM BPA significantly stimulated the proliferation of TM4 cells and increased the proportion of cells in S phase; >1 µM BPA caused lesser proliferation of cells. Exposure of TM4 cells to G15 or ICI 182,780, which are specific antagonists of GPR30 and estrogen receptor α/ß (ERα/ß), respectively, abolished BPA-induced proliferation of cells, which suggests that both GPR30 and ERα/ß were involved in the observed effects of BPA. Furthermore, exposure to BPA caused rapid (5 min) activation of ERK1/2 via both GPR30 and ERα/ß. Blocking the GPR30/EGFR signal transduction pathway by antagonists suppressed both phosphorylation of ERK and BPA-induced cell proliferation. BPA up-regulated mRNA and protein expression of GPR30 in a concentration-dependent manner. In summary, the results reported here indicated that activating ERK1/2 through GPR30 and ERα/ß is involved in low doses of BPA that promoted growth of Sertoli TM4 cells. The GPR30/EGFR/ERK signal is the downstream transduction pathway in BPA-induced proliferation of TM4 Sertoli cells.

In vitro estimation of exposure of Hong Kong residents to mercury and methylmercury via consumption of market fishes.

In order to evaluate effects of exposure to mercury (tHg) and methylmercury (MeHg) of Hong Kong residents via consumption of fish, total and bioaccessible concentrations of tHg and MeHg were measured in 10 freshwater and 10 marine fishes collected from markets in Hong Kong. Concentrations of tHg and MeHg in fishes ranged from 27.2 to 311ngg(-1) (median 88.9ngg(-1)) and ND to 116ngg(-1) (median 45.0ngg(-1)), respectively. Concentrations of MeHg in marine fishes (64.4±28.5ngg(-1)) were significantly greater than those in freshwater fishes (40.3±26.0ngg(-1)). Bioaccessibility tHg and MeHg was predicted for edible flesh of twenty fishes by use of an in vitro gastrointestinal assay. Bioaccessibilities of tHg and MeHg ranged from 21.4 to 51.7% (mean 37.4%) and 19.5 to 59.2% (mean 43.7%), respectively. Based on total concentrations, diets of 36% of adults and 51% of children exceeded the reference dose (RfD, 100ngkg(-1)bodymass(bm)d(-1)) for MeHg, but when bioaccessibility was considered, consumption of local market fish would not result in an EDIbio exceeded the RfD of MeHg for Hong Kong adults. These contradictory results suggested that risk assessments based on total concentrations would overestimate exposure because not all of contaminants consumed are bioaccessible. Furthermore, 9% of children had EDIbio for MeHg that exceeded the RfD, which suggests that more attention should be paid to consumption of local fish on health and development of children in Hong Kong.

Hydroxylated and methoxylated polybrominated diphenyl ethers in blood plasma of humans in Hong Kong.

Hydroxylated (OH-) and methoxylated (MeO-) polybrominated diphenyl ethers (PBDE) are suspected endocrine disruptors. Little is known about the accumulation or sources of these chemicals in tissues of humans, particularly those residing in Hong Kong, which is one of the most densely populated cities in the world. Seven MeO-BDEs, fifteen OH-BDEs and three bromophenols (BRPs) were analyzed in blood plasma of 116 humans that had been collected by the Hong Kong Red Cross. Total concentrations of MeO-BDEs, OH-BDEs and BRPs ranged from 3.8×10² to 52×10³ pg g⁻¹ lipid (median 4.5×10³ pg g⁻¹), 5.3 to 4.9×10² pg g⁻¹ lipid (81 pg g⁻¹) and ND to 1.1×10² pg g⁻¹ lipid (3.7 pg g⁻¹), respectively. 3-MeO-BDE-47, 6-OH-BDE-47 and 2, 4, 5-TBP were the predominant MeO-BDEs, OH-BDEs and BRPs, respectively. These results are consistent with accumulation of MeO-BDEs, OH-BDEs and BRPs in human plasma being primarily from natural products and inter-conversion of natural products. Coefficients of determination for some pairs of congeners such as 3-OH-BDE-100 and 6-OH-BDE-47, 6-OH-BDE-85 and 5'-OH-BDE-99, and 2, 4-DBP and 6-OH-BDE-85, were near 1.0, which is consistent with them having common sources. Patterns of relative concentrations of the target analytes were similar in the diet, particularly fish, as in blood plasma of humans, which suggests that the diet and particularly seafood might be a source of these compounds and PBDEs. Furthermore, biotransformation of natural chemicals such as OH-BDEs to BRPs might be the primary route of their elimination from humans.