YTHDF2-mediated regulations bifurcate BHPF-induced programmed cell deaths.

N6-methyladenosine (m6A) is a critical regulator in the fate of RNA, but whether and how m6A executes its functions in different tissues remains largely obscure. Here we report downregulation of a crucial m6A reader, YTHDF2, leading to tissue-specific programmed cell deaths (PCDs) upon fluorene-9-bisphenol (BHPF) exposure. Currently, Bisphenol A (BPA) substitutes are widely used in plastic manufacturing. Interrogating eight common BPA substitutes, we detected BHPF in 14% serum samples of pregnant participants. In a zebrafish model, BHPF caused tissue-specific PCDs triggering cardiac and vascular defects. Mechanistically, BHPF-mediated downregulation of YTHDF2 reduced YTHDF2-facilitated translation of m6A-gch1 for cardiomyocyte ferroptosis, and decreased YTHDF2-mediated m6A-sting1 decay for caudal vein plexus (CVP) apoptosis. The two distinct YTHDF2-mediated m6A regulations and context-dependent co-expression patterns of gch1/ythdf2 and tnfrsf1a/ythdf2 contributed to YTHDF2-mediated tissue-specific PCDs, uncovering a new layer of PCD regulation. Since BHPF/YTHDF2-medaited PCD defects were also observed in mammals, BHPF exposure represents a potential health threat.

Bisphenol C induces developmental defects in liver and intestine through mTOR signaling in zebrafish (Danio rerio).

Bisphenol A (BPA) was widely used in the plastic products and banned in infant food containers in many countries due to the environmental and biological toxicity. As a common substitute of BPA to manufacture products, Bisphenol C (BPC) is frequently detected in human samples like infants and toddlers' urine, indicating infants and young children are at risk of BPC exposure. However, the understanding of effects of BPC exposure on early development is limited. Herein, we evaluated the early developmental toxicity of BPC and studied the underlying mechanism in a zebrafish model. We found BPC exposure leading to liver and intestinal developmental defects in zebrafish, which occurred via disruption of GPER-AKT-mTOR-RPS6 pathway. Specifically, BPC downregulated phosphorylated and total levels of mTOR, which synergistically reduced the phosphorylation of RPS6, suppressing the translation of genes essential for cell proliferation in liver and intestine such as yap1 and tcf4. Collectively, our results not only observed clear toxicity of BPC during liver and intestinal development but also demonstrated the underlying mechanism of BPC-mediated defects via disrupting the GPER-AKT-mTOR-RPS6 pathway.

m6A modification confers thermal vulnerability to HPV E7 oncotranscripts via reverse regulation of its reader protein IGF2BP1 upon heat stress.

Human papillomavirus (HPV)-induced carcinogenesis critically depends on the viral early protein 7 (E7), making E7 an attractive therapeutic target. Here, we report that the E7 messenger RNA (mRNA)-containing oncotranscript complex can be selectively targeted by heat treatment. In HPV-infected cells, viral E7 mRNA is modified by N6-methyladenosine (m6A) and stabilized by IGF2BP1, a cellular m6A reader. Heat treatment downregulates E7 mRNA and protein by destabilizing IGF2BP1 without the involvement of canonical heat-shock proteins and reverses HPV-associated carcinogenesis in vitro and in vivo. Mechanistically, heat treatment promotes IGF2BP1 aggregation only in the presence of m6A-modified E7 mRNA to form distinct heat-induced m6A E7 mRNA-IGF2BP1 granules, which are resolved by the ubiquitin-proteasome system. Collectively, our results not only show a mutual regulation between m6A RNA and its reader but also provide a heat-treatment-based therapeutic strategy for HPV-associated malignancies by specifically downregulating E7 mRNA-IGF2BP1 oncogenic complex.

Fusobacterium nucleatum reduces METTL3-mediated m6A modification and contributes to colorectal cancer metastasis.

Microbiota-host interactions play critical roles in colorectal cancer (CRC) progression, however, the underlying mechanisms remain elusive. Here, we uncover that Fusobacterium nucleatum (F. nucleatum) induces a dramatic decline of m6A modifications in CRC cells and patient-derived xenograft (PDX) tissues by downregulation of an m6A methyltransferase METTL3, contributing to inducation of CRC aggressiveness. Mechanistically, we characterized forkhead box D3 (FOXD3) as a transcription factor for METTL3. F. nucleatum activates YAP signaling, inhibits FOXD3 expression, and subsequently reduces METTL3 transcription. Downregulation of METTL3 promotes its target kinesin family member 26B (KIF26B) expression by reducing its m6A levels and diminishing YTHDF2-dependent mRNA degradation, which contributes to F. nucleatum-induced CRC metastasis. Moreover, METTL3 expression is negatively correlated with F. nucleatum and KIF26B levels in CRC tissues. A high expression of KIF26B is also significantly correlated with a shorter survival time of CRC patients. Together, our findings provide insights into modulating human m6A epitranscriptome by gut microbiota, and its significance in CRC progression.

Hyperthermia Selectively Destabilizes Oncogenic Fusion Proteins.

The PML/RARα fusion protein is the oncogenic driver in acute promyelocytic leukemia (APL). Although most APL cases are cured by PML/RARα-targeting therapy, relapse and resistance can occur due to drug-resistant mutations. Here we report that thermal stress destabilizes the PML/RARα protein, including clinically identified drug-resistant mutants. AML1/ETO and TEL/AML1 oncofusions show similar heat shock susceptibility. Mechanistically, mild hyperthermia stimulates aggregation of PML/RARα in complex with nuclear receptor corepressors leading to ubiquitin-mediated degradation via the SIAH2 E3 ligase. Hyperthermia and arsenic therapy destabilize PML/RARα via distinct mechanisms and are synergistic in primary patient samples and in vivo, including three refractory APL cases. Collectively, our results suggest that by taking advantage of a biophysical vulnerability of PML/RARα, thermal therapy may improve prognosis in drug-resistant or otherwise refractory APL. These findings serve as a paradigm for therapeutic targeting of fusion oncoprotein-associated cancers by hyperthermia. SIGNIFICANCE: Hyperthermia destabilizes oncofusion proteins including PML/RARα and acts synergistically with standard arsenic therapy in relapsed and refractory APL. The results open up the possibility that heat shock sensitivity may be an easily targetable vulnerability of oncofusion-driven cancers.See related commentary by Wu et al., p. 300.

Tanshinone I, a new EZH2 inhibitor restricts normal and malignant hematopoiesis through upregulation of MMP9 and ABCG2.

Rationale: Tanshinone, a type of diterpenes derived from salvia miltiorrhiza, is a particularly promising herbal medicine compound for the treatment of cancers including acute myeloid leukemia (AML). However, the therapeutic function and the underlying mechanism of Tanshinone in AML are not clear, and the toxic effect of Tanshinone limits its clinical application. Methods: Our work utilizes human leukemia cell lines, zebrafish transgenics and xenograft models to study the cellular and molecular mechanisms of how Tanshinone affects normal and abnormal hematopoiesis. WISH, Sudan Black and O-Dianisidine Staining were used to determine the expression of hematopoietic genes on zebrafish embryos. RNA-seq analysis showed that differential expression genes and enrichment gene signature with Tan I treatment. The surface plasmon resonance (SPR) method was used with a BIAcore T200 (GE Healthcare) to measure the binding affinities of Tan I. In vitro methyltransferase assay was performed to verify Tan I inhibits the histone enzymatic activity of the PRC2 complex. ChIP-qPCR assay was used to determine the H3K27me3 level of EZH2 target genes. Results: We found that Tanshinone I (Tan I), one of the Tanshinones, can inhibit the proliferation of human leukemia cells in vitro and in the xenograft zebrafish model, as well as the normal and malignant definitive hematopoiesis in zebrafish. Mechanistic studies illustrate that Tan I regulates normal and malignant hematopoiesis through direct binding to EZH2, a well-known histone H3K27 methyltransferase, and inhibiting PRC2 enzymatic activity. Furthermore, we identified MMP9 and ABCG2 as two possible downstream genes of Tan I's effects on EZH2. Conclusions: Together, this study confirmed that Tan I is a novel EZH2 inhibitor and suggested MMP9 and ABCG2 as two potential therapeutic targets for myeloid malignant diseases.

The roles of m6A RNA modifiers in human cancer.

Like DNA and proteins, RNA is subject to numerous (over 160) covalent modifications which play critical roles to regulate RNA metabolism. Among these modifications, N-methyladenosine (mA) is the most prevalent RNA methylation on mRNA which occurs on around 25% of transcripts. The recent studies demonstrated that mA participates in many aspects of RNA processing, including splicing, nuclear exporting, translation, stabilization, etc. Therefore, it revealed a new layer of regulatory mechanism for gene expression and has been termed "RNA Epigenetics" or "Epitranscriptomics". RNA mA is regulated and exerts its functions by three groups of "mA RNA modifiers" including mA methyltransferases (writers), mA demethylases (erasers), and mA binding proteins (readers). In this review, we would summarize and discuss the current understandings of the roles of the conventional mA RNA modifiers in human cancers.

Triclosan-induced liver and brain injury in zebrafish (Danio rerio) via abnormal expression of miR-125 regulated by PKCα/Nrf2/p53 signaling pathways.

Triclosan (TCS) is widely used in personal care products, and its chronic exposure leads to severely toxic effects in zebrafish (Danio rerio). PKCα, Nrf2 and p53 are three important signaling pathways concerned with cell development. Herein, we speculated on and verified a novel TCS regulatory pathway: (1) TCS acted on GPER (G-protein-coupled estrogen receptor) to activate MAPK/ERK pathway, further resulting in the expression changes of protein kinase C (PKC) family; (2) PKC participated in Nrf2 phosphorylation; (3) The expression of miR-125b was regulated by Nrf2; and (4) The expression changes of related genes in the PKCs-Nrf2-ARE pathway showed the specificity of zebrafish tissue and organ. TCS exposure led to down-regulation of the Nrf2 and phosphorylated Nrf2(Ser40) protein in diencephalon nucleus, stratum marginale and stratum centrale areas in adult zebrafish brain. The phosphorylated Nrf2(Ser40) was mainly expressed in PGz area, while it was not the case for Nrf2. Both Nrf2 and phosphorylated Nrf2 were activated by TCS exposure; however, the changing trend of PKCs was opposite to that of Nrf2 in the liver. Both DAPI staining and Merge images demonstrated that TCS induced oxidative phosphorylation, and phosphorylated Nrf2 is translocated into the nucleus as the transcription factor to regulate gene transcription in liver and brain. Nrf2 over-expression increased accumulation of lipid droplets in yolk, brain and liver, resulting from the upregulation of pri-miR-125b1, pri-miR-125b3, but not pri-miR-125b2. These findings reveal the upstream regulation mechanism of miR-125b for TCS-induced fat-metabolism disorder from the regulatory perspective of the pri-miR-125b promoter region.

Up-stream mechanisms for up-regulation of miR-125b from triclosan exposure to zebrafish (Danio rerio).

Triclosan (TCS) exposure has widely adverse biological effects such as influencing biological reproduction and endocrine disorders. While some studies have addressed TCS-induced expression changes of miRNAs and their related down-stream target genes, no data are available concerning how TCS impairs miRNA expression leading us to study up-stream regulating mechanisms. Four miRNAs (miR-125b, miR-205, miR-142a and miR-203a) showed differential expression between TCS-exposure treatments and the control group; their functions mainly involved fatty acid synthesis and metabolism. TCS exposure led to the up-regulation of mature miR-125b that was concomitant with consistent changes in pri-mir-125b-1 and pri-mir-125b-3 among its 3 pri-mir-125bs. Up-regulation of miR-125b originated from direct shear processes involving the two up-regulated precursors, but not pri-mir-125b2. Increased expression of pri-mir-125b-1 and pri-mir-125b-3 resulted from nfe2l2- and c/ebpα-integration with positive control elements of promoters for the two precursors. The overexpression of transcriptional factors, nfe2l2 and c/ebpα, initiated the promoter activity for the miR-125b precursor. CpG islands and Nfe2l2 were involved in constitutive expression of mir-125b-1 and mir-125b-3. The activities of two promoter regions, -487 to -1bp for pri-mir-125b1 and -1327 to +14bp for pri-mir-125b-3 having binding sites for NFE2 and Nfe2l2/MAF:NFE2, were higher than other regions, further demonstrating that the transcriptional factor Nfe2l2 was involved in the regulation of pri-mir-125b1 and pri-mir-125b-3. TCS's estrogen activity resulted from its effects on GPER, a novel membrane receptor, rather than the classical ERα and ERß. These results explain, to some extent, the up-stream mechanism for miR-125b up-regulation, and also provide a guidance to future mechanistic study on TCS-exposure.

Screening and functional identification of lncRNAs under ß-diketone antibiotic exposure to zebrafish (Danio rerio) using high-throughput sequencing.

Long non-coding RNAs (lncRNAs) have attracted considerable research interest, but so far no data are available on the roles of lncRNAs and their target genes under chronic ß-diketone antibiotic (DKAs) exposure to zebrafish (Danio rerio). Herein, we identified 1.66, 3.07 and 3.36×104 unique lncRNAs from the 0, 6.25 and 12.5mg/L DKA treatment groups, respectively. In comparison with the control group, the 6.25 and 12.5mg/L treatments led to up-regulation of 2064 and 2479 lncRNAs, and down-regulation of 778 and 954 lncRNAs, respectively. Of these, 44 and 39 lncRNAs in the respective 6.25 and 12.5mg/L treatments displayed significant differential expression. Volcano and Venn diagrams of the differentially expressed lncRNAs were constructed on the basis of the differentially expressed lncRNAs. After analyzing 10 lncRNAs and potential target genes, a complex interaction network was constructed between them. The consistency of 7 target genes (tenm3, smarcc1b, myo9ab, ubr4, hoxb3a, mycbp2 and CR388046.3), co-regulated by 3 lncRNAs (TCONS_00129029, TCONS_00027240 and TCONS_00017790), was observed between their qRT-PCR and transcriptomic sequencing. By in situ hybridization (ISH), abnormal expression of 3 lncRNAs was observed in hepatic and spleen tissues, suggesting that they might be target organs for DKAs. A similar abnormal expression of two immune-related target genes (plk3 and syt10), co-regulated by the 3 identified lncRNAs, was observed in liver and spleen by ISH. Histopathological observations demonstrated hepatic parenchyma vacuolar degeneration and clot formation in hepatic tissues, and uneven distribution of brown metachromatic granules and larger nucleus in spleen tissues resulting from DKA exposure. Overall, DKA exposure led to abnormal expression of some lncRNAs and their potential target genes, and these genes might play a role in immune functions of zebrafish.

Effects of ß-diketone antibiotics on F1-zebrafish (Danio rerio) based on high throughput miRNA sequencing under exposure to parents.

The toxicity of ß-diketone antibiotics (DKAs), a class of ''pseudo-persistent'' environmental pollutants, to F0-zebrafish (Danio rerio) was investigated using 7-dpf F1-zebrafish miRNA sequencing and bioinformatics analyses. Based on relative expression, 47, 134 and 118 of 193 mature miRNAs were differentially expressed between control vs 6.25 mg/L, control vs 12.5 mg/L and 6.25 vs 12.5 mg/L treatments, respectively. Utilizing three databases, 2523 potential target genes were predicted, and they were assigned to 19 high-abundance KEGG pathways and 20 functional categories by COG analysis. Among 11 significantly differential expression and high-abundance miRNAs, the expression levels for 7 miRNAs (miR-144, -124, -499, -125b, -430b, -430c and -152) assessed by qRT-PCR were consistent with those determined by sRNA-seq. A potential network was plotted between 11 miRNAs and their target genes based on differential expression and binding effectiveness. The high degree of connectivity between miRNA-gene pairs suggests that these miRNAs play critical roles in zebrafish development. The expression of miR-124 and miR-499 in whole-mount in situ hybridization was in general agreement with those from qRT-PCR and miRNA-seq and were DKA concentration-dependent. DKA exposure induced severe histopathological changes and damage in F0-zebrafish ovary tissue, as reflected by an increased number of early developmental oocytes, irregular cell distribution, decreased yolk granules, cytoplasmic shrinkage, cell lysis in mature oocytes, and dissolution of internal corona radiata. Chronic DKA exposure affected reproduction of F0-zebrafish and development of F1-zebrafish. These observations demonstrate the toxic effect transfer relation across parent and their offspring, and enhance our understanding of drug-induced diseases.

Immunotoxicity of ß-Diketone Antibiotic Mixtures to Zebrafish (Danio rerio) by Transcriptome Analysis.

Fluoroquinolones and tetracyclines are known as ß-diketone antibiotics (DKAs) because of bearing a diketone group in their molecular structure. DKAs are the most widely used antibiotics to prevent generation of disease in humans and animals and to suppress bacterial growth in aquaculture. In recent years, overuse of DKAs has caused serious environmental risk due to their pseudo-persistence in the environment, even though their half-lives are not long. So far, no reports were concerned with the joint immunotoxicity of DKAs. Herein, we reported on the immunotoxicity of DKAs on zebrafish after a 3-month DKAs exposure using transcriptomic techniques. According to transcriptome sequencing, 10 differentially expressed genes were screened out among the genes related to KEGG pathways with high enrichment. The identified 7 genes showed to be consistent between RNA-seq and qRT-PCR. Due to DKAs exposure, the content or activity for a series of immune-related biomarkers (Complement 3, lysozyme, IgM and AKP) showed the inconsistent changing trends as compared with the control group. Histopathological observations showed that the number of goblet cells increased sharply, the columnar epithelial cells swelled, the nucleus became slender in intestinal villi, and numerous brown metachromatic granules occurred in spleens of DKAs-exposed groups. Overall, both detection of biomarkers and histopathological observation corroborated that chronic DKAs exposure could result in abnormal expression of immune genes and enzymes, and variable levels of damage to immune-related organs. These complex effects of DKAs may lead to zebrafish dysfunction and occurrence of diseases related to the immune system.