Notch-Driven Cholangiocarcinogenesis Involves the Hippo Pathway Effector TAZ via METTL3-m6A-YTHDF1.

BACKGROUND AND AIMS: Notch and TAZ are implicated in cholangiocarcinogenesis, but whether and how these oncogenic molecules interact remain unknown. METHODS: The development of CCA was induced by hydrodynamic tail vein (HDTV) injection of oncogenes (NICD/AKT) to the FVB/NJ mice. CCA xenograft was developed by inoculation of human CCA cells into the livers of SCID mice. Tissues and cells were analyzed using qRT-PCR, Western blotting analyses, Immunohistochemistry, ChIP-qPCR and WST-1 cell proliferation Assay. RESULTS: Our experimental findings show that TAZ is indispensable in NICD-driven cholangiocarcinogenesis. Notch activation induces the expression of METTL3 (Methyltransferase like-3) which catalyzes N6-methyladenosine (m6A) modification of TAZ mRNA and that this mechanism plays a central role in the crosstalk between Notch and TAZ in CCA cells. Mechanistically, Notch regulates the expression of METTL3 through the binding of NICD to its downstream transcription factor CSL in the promoter region of METTL3. METTL3 in turn mediates m6A modification of TAZ mRNA which is recognized by the m6A reader YTHDF1 to enhance TAZ protein translation. We observed that inhibition of Notch signaling decreased the protein levels of both MELLT3 and TAZ. Depletion of METTL3 by shRNAs or by the next generation GapmeR antisense oligonucleotides (ASOs) decreased the level of TAZ protein and inhibited the growth of human CCA cells in vitro and in mice. CONCLUSION: This study describes a novel Notch-METTL3-TAZ signaling cascade which is important in CCA development and progression. Our experimental results provide new insight into how the Notch pathway cooperates with TAZ signaling in CCA, and the findings may have important therapeutic implications.

RBM39 Enhances Cholangiocarcinoma Growth Through EZH2-mediated WNT7B/ß-catenin Pathway.

BACKGROUND & AIMS: The RNA-binding motif protein 39 (RBM39) functions as both an RNA-binding protein and a splicing factor in a variety of cancer types. However, the function of RBM39 in cholangiocarcinoma (CCA) remains undefined. In this study, we aimed to investigate the role of RBM39 in CCA and explore its potential as a therapeutic target. METHODS: The expression of RBM39 in CCA was investigated by analyzing human CCA tumor specimens. CRISPR/Cas9 or shRNA-mediated depletion of RBM39 was performed in vitro and in vivo to document the oncogenic role of RBM39 in CCA. The anti-tumor effect of the RBM39 inhibitor, Indisulam, in combination with the EZH2 degrader MS177 was assessed in vitro and in vivo. RESULTS: RBM39 is significantly increased in human CCA tissues and associated with a poor prognosis in patients with CCA. Depletion of RBM39 by CRISPR/Cas9 or shRNA inhibited CCA cell proliferation in vitro and prevented CCA development and tumor growth in mice. Mechanistically, our results showed that depletion of RBM39 suppressed EZH2 expression via disrupting its mRNA splicing. RBM39-regulated EZH2 controls WNT7B/ß-catenin activity. Pharmacological co-targeting of RBM39 (with Indisulam) and EZH2 (with MS177) resulted in a synergistic antitumor effect, both in vitro and in vivo. CONCLUSION: This study discloses a novel RBM39-EZH2-ß-catenin signaling axis that is crucial for CCA growth. Our findings suggest that simultaneous inhibition of RBM39 and EZH2 presents a promising therapeutic strategy for CCA treatment.

BaP/BPDE suppresses homologous recombination repair in human trophoblast cells to induce miscarriage: The roles of lnc-HZ08.

Benzo(a)pyrene (BaP) or benzo (a) pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE) exposure causes trophoblast cell dysfunctions and induces miscarriage, which is generally epigenetically regulated. Homologous recombination (HR) repair of DNA double strand break (DSB) plays a crucial role in maintenance of genetic stability and cell normal functions. However, whether BaP/BPDE might suppress HR repair in human trophoblast cells to induce miscarriage, as well as its epigenetic regulatory mechanism, is largely unclear. In this study, we find that BaP/BPDE suppresses HR repair of DSB in trophoblast cells and eventually induces miscarriage by up-regulating lnc-HZ08. In mechanism, lnc-HZ08 (1) down-regulates the expression levels of FOXA1 (forkhead box A1) and thus suppresses FOXA1-mediated mRNA transcription of BRCA1 (Breast cancer susceptibility gene 1) and CtIP (CtBP-interacting protein), (2) impairs BRCA1 and CtIP protein interactions by competitive binding with CtIP through lnc-HZ08-1 fragment, and also (3) suppresses BRCA1-mediated CtIP ubiquitination without affecting CtIP stability, three of which eventually suppress HR repair in human trophoblast cells. Supplement with murine Ctip could efficiently restore (i.e. increase) HR repair and alleviate miscarriage in BaP-exposed mouse model. Collectively, this study not only reveals the association and causality among BaP/BPDE exposure, the defective HR repair, and miscarriage, but also discovers novel mechanism in lnc-HZ08-regulated BRCA1/CtIP-mediated HR repair, bridging epigenetic regulation and genetic instability and also providing an efficient approach for treatment against BaP/BPDE-induced unexplained miscarriage.

YAP activation in liver macrophages via depletion of MST1/MST2 enhances liver inflammation and fibrosis in MASLD.

Macrophages have been recognized as pivotal players in the progression of MASLD/MASH. However, the molecular mechanisms underlying their multifaceted functions in the disease remain to be further clarified. In the current study, we developed a new mouse model with YAP activation in macrophages to delineate the effect and mechanism of YAP signaling in the pathogenesis of MASLD/MASH. Genetically modified mice, featuring specific depletion of both Mst1 and Mst2 in macrophages/monocytes, were generated and exposed to a high-fat diet for 12 weeks to induce MASLD. Following this period, livers were collected for histopathological examination, and liver non-parenchymal cells were isolated and subjected to various analyses, including single-cell RNA-sequencing, immunofluorescence and immunoblotting and qRT-PCR to investigate the impact of YAP signaling on the progression of MASLD. Our data revealed that Mst1/2 depletion in liver macrophages enhanced liver inflammation and fibrosis in MASLD. Using single-cell RNA-sequencing, we showed that YAP activation via Mst1/2 depletion upregulated the expressions of both pro-inflammatory genes and genes associated with resolution/tissue repair. We observed that YAP activation increases Kupffer cell populations (i.e., Kupffer-2 and Kupffer-3) which are importantly implicated in the pathogenesis of MASLD/MASH. Our data indicate that YAP activation via Mst1/2 deletion enhances both the pro-inflammatory and tissue repairing functions of Kupffer-1 and -2 cells at least in part through C1q. These YAP-regulatory mechanisms control the plasticity of liver macrophages in the context of MASLD/MASH. Our findings provide important evidence supporting the critical regulatory role of YAP signaling in liver macrophage plasticity and the progression of MASLD. Therefore, targeting the Hippo-YAP pathway may present a promising therapeutic strategy for the treatment of MASH.

BaP/BPDE exposure causes human trophoblast cell dysfunctions and induces miscarriage by up-regulating lnc-HZ06-regulated IL1B.

Exposure to environmental BaP or its metabolite BPDE causes trophoblast cell dysfunctions to induce miscarriage (abnormal early embryo loss), which might be generally regulated by lncRNAs. IL1B, a critical inflammatory cytokine, is closely associated with adverse pregnancy outcomes. However, whether IL1B might cause dysfunctions of BaP/BPDE-exposed trophoblast cells to induce miscarriage, as well as its specific epigenetic regulatory mechanisms, is completely unexplored. In this study, we find that BPDE-DNA adducts, trophoblast cell dysfunctions, and miscarriage are closely associated. Moreover, we also identify a novel lnc-HZ06 and IL1B, both of which are highly expressed in BPDE-exposed trophoblast cells, in villous tissues of recurrent miscarriage patients, and in placental tissues of BaP-exposed mice with miscarriage. Both lnc-HZ06 and IL1B suppress trophoblast cell migration/invasion and increase apoptosis. In mechanism, lnc-HZ06 promotes STAT4-mediated IL1B mRNA transcription, enhances IL1B mRNA stability by promoting the formation of METTL3/HuR/IL1B mRNA ternary complex, and finally up-regulates IL1B expression levels. BPDE exposure promotes TBP-mediated lnc-HZ06 transcription, and thus up-regulates IL1B levels. Knockdown of either murine lnc-hz06 (which down-regulates Il1b levels) or murine Il1b could alleviate miscarriage in BaP-exposed mice. Collectively, this study not only discovers novel biological mechanisms and pathogenesis of unexplained miscarriage but also provides novel potential targets for treatment against BaP/BPDE-induced miscarriage.

The novel lnc-HZ12 suppresses autophagy degradation of BBC3 by preventing its interactions with HSPA8 to induce trophoblast cell apoptosis.

Abnormal expression of long non-coding RNAs (lncRNAs) is associated with the dysfunctions of human trophoblast cells and the occurrence of miscarriage (abnormal early embryo loss). BBC3/PUMA (BCL2 binding component 3) plays significant roles in regulation of cell apoptosis. However, whether specific lncRNAs might regulate BBC3 in trophoblast cells and further induce apoptosis and miscarriage remains completely unclear. Through screening, we identified a novel lnc-HZ12, which was significantly highly expressed in villous tissues of recurrent miscarriage (RM) patients relative to their healthy control (HC) group. Lnc-HZ12 suppressed chaperone-mediated autophagy (CMA) degradation of BBC3, promoted trophoblast cell apoptosis, and was associated with miscarriage. In mechanism, lnc-HZ12 downregulated the expression levels of chaperone molecules HSPA8 and LAMP2A in trophoblast cells. Meanwhile, lnc-HZ12 (mainly lnc-HZ12-SO2 region in F2 fragment) and HSPA8 competitively bound with the 169RVLYNL174 patch on BBC3, which prevented BBC3 from interactions with HSPA8 and impaired the formation of BBC3-HSPA8-LAMP2A complex for CMA degradation of BBC3. Thus, lnc-HZ12 upregulated the BBC3-CASP9-CASP3 pathway and induced trophoblast cell apoptosis. In villous tissues, lnc-HZ12 was highly expressed, CMA degradation of BBC3 was suppressed, and the apoptosis levels were higher in RM vs HC villous tissues, all of which were associated with miscarriage. Interestingly, knockdown of murine Bbc3 could efficiently suppress placental apoptosis and alleviate miscarriage in a mouse miscarriage model. Taken together, our results indicated that lnc-HZ12 and BBC3 played important roles in trophoblast cell apoptosis and miscarriage and might act as attractive targets for miscarriage treatment.Abbreviation: 7-AAD: 7-aminoactinomycin D; BaP: benzopyrene; BBC3/PUMA: BCL2 binding component 3; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: healthy control; HSPA8: heat shock protein family A (Hsp70) member 8; IP: immunoprecipitation; LAMP2A: lysosomal associated membrane protein 2; LncRNA: long non-coding RNA; mRNA: messenger RNA; MT: mutant-type; NC: negative control; NSO: nonspecific oligonucleotide; PARP1: poly(ADP-ribose) polymerase 1; RIP: RNA immunoprecipitation; RM: recurrent miscarriage; TBP: TATA-box binding protein; WT: wild-type.

Copper exposure induces trophoblast cell cuproptosis by up-regulating lnc-HZ11.

Copper pollution has attracted global environmental concern. Widespread Cu pollution results in excessive Cu accumulation in human. Epidemiological studies and animal experiments revealed that Cu exposure might have reproductive toxicity. Cuproptosis is a recently reported Cu-dependent and programmed cell death pattern. However, the mechanism by which copper exposure might cause cell cuproptosis is largely unknown. We chose trophoblast cells as cell model and found that copper exposure causes trophoblast cell cuproptosis. In mechanism, copper exposure up-regulates lnc-HZ11 expression levels, which increases intracellular Cu2+ levels and causes trophoblast cell cuproptosis. Knockdown of lnc-HZ11 efficiently reduces intracellular Cu2+ levels and alleviate trophoblast cell cuproptosis, which could be further alleviated by co-treatment with DC or TEPA. These results discover novel toxicological effects of copper exposure and also provide potential target for protection trophoblast cells from cuproptosis in the presence of excessive copper exposure.

Research progress on the structure, function, and use of angiogenin in malignant tumours.

Angiogenin (ANG) is a specialised secreted ribonuclease, also known as RNase5, that is widely expressed in vertebrates. ANG dysregulation is closely associated with the development of breast, nasopharyngeal, and lung cancers. In recent years, studies have found that ANG not only induces neovascularisation by activating endothelial cells, but also plays a regulatory role in the plasticity of cancer cells. Cellular plasticity plays pivotal roles in cancer initiation, progression, migration, therapeutic resistance, and relapse. Therefore, it is a promising biomarker for cancer diagnosis, prognostic evaluation, and therapy. This review summarises the current knowledge regarding the roles and clinical applications of ANG in cancer development and progression.

BaP/BPDE suppresses human trophoblast cell migration/invasion and induces unexplained miscarriage by up-regulating a novel lnc-HZ11 in extracellular vesicles: An intercellular study.

Extracellular vesicles (EVs) mediate the intercellular crosstalk by transferring functional cargoes. Recently, we have discovered that BaP/BPDE exposure suppresses trophoblast cell migration/invasion and induces miscarriage, which are also regulate by lncRNAs at intracelluar levels. However, the EVs-mediated intercellular regulatory mechanisms are completely unexplored. Specifically, whether EVs might transfer BPDE-induced toxic lncRNA to fresh recipient trophoblast cells and suppress their migration/invasion to further induce miscarriage is completely unknown. In this study, we find that BPDE exposure up-regulates a novel lnc-HZ11, which suppresses EGR1/NF-κB/CXCL12 pathway and migration/invasion of trophoblast cells. Intercellular studies show that EV-HZ11 (lnc-HZ11 in EVs), which is highly expressed in BPDE-exposed donor cells, suppresses EGR1/NF-κB/CXCL12 pathway and migration/invasion in recipient cells by transferring lnc-HZ11 through EVs. Analysis of villous tissues collected from UM (unexplained miscarriage) patients and HC (healthy control) group shows that the levels of BPDE-DNA adducts, lnc-HZ11 or EV-lnc-HZ11, and EGR1/NF-κB/CXCL12 pathway are all associated with miscarriage. Mouse assays show that BaP exposure up-regulates the levels of lnc-Hz11 or EV-Hz11, suppresses Egr1/Nf-κb/Cxcl12 pathway, and eventually induces miscarriage. Knockdown of lnc-Hz11 by injecting EV-AS-Hz11 could effectively alleviate miscarriage in BaP-exposed mice. Furthermore, EV-HZ11 in serum samples could well predict the risk of miscarriage. Collectively, this study not only discovers EVs-HZ11-mediated intercellular mechanisms that BaP/BPDE suppresses trophoblast cell migration/invasion and induces miscarriage but also provides new approach for treatment against unexplained miscarriage through EV-HZ11.

Environmental BaP/BPDE suppressed trophoblast cell invasion/migration and induced miscarriage by down-regulating lnc-HZ01/MEST/VIM axis.

Environmental benzo(a)pyrene (BaP) and itsmetabolite benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE), classic endocrine disrupting chemical and persistent organic pollutant, could cause miscarriage. However, the detailed mechanisms are still largely unclear and should be further explored. In this study, we discovered that exposure of trophoblast cells with BPDE could suppressed cell invasion/migration by inhibiting MEST/VIM (Vimentin) pathway. Moreover, BPDE exposure also increased lnc-HZ01 expression level, which further inhibited MEST/VIM pathway and then suppressed invasion/migration. Knockdown of lnc-HZ01 or overexpression of MEST could efficiently rescue invasion/migration of BPDE-exposed Swan 71 cells. Furthermore, lnc-HZ01 was highly expressed and MEST/VIM were lowly expressed in recurrent miscarriage (RM) villous tissues compared with healthy control (HC) group. Finally, we also found that BaP exposure inhibited murine Mest/Vim pathway in placental tissues and induced miscarriage in BaP-exposed mice. Therefore, the regulatory mechanisms were similar in BPDE-exposed human trophoblast cells, RM villous tissues, and placental tissues of BaP-exposed mice with miscarriage, building a bridge to connect BaP/BPDE exposure, invasion/migration, and miscarriage. This study provided novel insights in the toxicological effects and molecular mechanisms of BaP/BPDE-induced miscarriage, which is helpful for better elucidating the toxicological risks of BaP/BPDE on female reproduction.

The landscape of allelic expression and DNA methylation at the bovine SGCE/PEG10 locus.

Genomic imprinting is an epigenetic regulation in mammals in which a small subset of genes is monoallelically expressed dependent on their parental origin. A large imprinted domain, SGCE/PEG10 locus, is located on human chromosome 7q21s and mouse proximal chromosome 6. However, genomic imprinting of bovine SGCE/PEG10 cluster has not been systematically studied. In this study, we investigated allele expression of 14 genes of the SGCE/PEG10 locus in bovine somatic tissues and term placenta using a single nucleotide polymorphism (SNP)-based sequencing method. In addition to SGCE and PEG10, two conserved paternally expressed genes in human and mice, five other genes (TFPI2, GNG11, ASB4, PON1, and PON3) were paternally expressed. Three genes, BET1, COL1A2, and CASD1, exhibited tissue-specific monoallelic expression. CALCR showed monoallelic expression in tissues but biallelic expression in the placenta. Three genes, GNGT1, PPP1R9A, and PON2, showed biallelic expression in cattle. Five differentially methylated regions (DMRs) were found to be associated with the allelic expression of TFPI2, COL1A2, SGCE/PEG10, PON3, and ASB4 genes, respectively. The SGCE/PEG10 DMR is a maternally hypermethylated germline DMR, but TFPI2, COL1A2, PON3, and ASB4 DMRs are secondary DMRs. In summary, we identified five novel bovine imprinted genes (GNG11, BET1, COL1A2, CASD1, and PON1) and four secondary DMRs at the SGCE/PEG10 locus.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy. METHODS: The expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA. RESULTS: We identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth. CONCLUSIONS: This study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Environmental copper exposure, placental cuproptosis, and miscarriage.

Copper pollution has become global environmental concern. Widespread Cu pollution results in excessive Cu exposure in human. Epidemiological studies and animal experiments revealed that Cu exposure might have reproductive toxicity. Cuproptosis is a newly reported Cu-dependent and programmed cell death formTsvetkov et al., 2022. However, whether copper exposure at real environmental exposure dose might cause placental cuproptosis and induce miscarriage was completely unexplored. In this study, we found that Cu exposure during pregnancy induced miscarriage or complete pregnancy loss by inducing placenta cuproptosis in CuCl2-exposed pregnant mice. Notably, Cu exposure at 1.3 mg/kg/d (a real environmental exposure dose) was enough to cause placenta cuproptosis. CuCl2 exposure disrupts the TCA cycle, causes proteotoxic stress, increases Cu2+ ion import/decreases Cu2+ export, and results in the loss of Fe-S cluster proteins in mouse placenta, which induces placenta cuproptosis. Moreover, we also identified that Cu exposure down-regulates the expression levels of mmu-miR-3473b, which interacts with Dlst or Rtel1 mRNA and simultaneously positively regulates Dlst or Rtel1 expression, thereby disrupting the TCA cycle and resulting in the loss of Fe-S cluster proteins, and thus epigenetically regulates placental cuproptosis. Treatment with TTM (a cuproptosis inhibitor) suppressed placental cuproptosis and alleviated miscarriage in CuCl2-exposed mice. This work provides novel reproductive toxicity of Cu exposure in miscarriage or complete pregnancy loss by causing placental cuproptosis. This study also provides new ways for further studies on other toxicological effects of Cu and proposes a new approach for protection against Cu-induced reproductive diseases.

A novel imprinted locus on bovine chromosome 18 homologous with human chromosome 16q24.1.

Genomic imprinting is an epigenetic regulation mechanism in mammals resulting in the parentally dependent monoallelic expression of genes. Imprinting disorders in humans are associated with several congenital syndromes and cancers and remain the focus of many medical studies. Cattle is a better model organism for investigating human embryo development than mice. Imprinted genes usually cluster on chromosomes and are regulated by different methylation regions (DMRs) located in imprinting control regions that control gene expression in cis. There is an imprinted locus on human chromosome 16q24.1 associated with congenital lethal developmental lung disease in newborns. However, genomic imprinting on bovine chromosome 18, which is homologous with human chromosome 16 has not been systematically studied. The aim of this study was to analyze the allelic expressions of eight genes (CDH13, ATP2C2, TLDC1, COTL1, CRISPLD2, ZDHHC7, KIAA0513, and GSE1) on bovine chromosome 18 and to search the DMRs associated gene allelic expression. Three transcript variants of the ZDHHC7 gene (X1, X2, and X5) showed maternal imprinting in bovine placentas. In addition, the monoallelic expression of X2 and X5 was tissue-specific. Five transcripts of the KIAA0513 gene showed tissue- and isoform-specific monoallelic expression. The CDH13, ATP2C2, and TLDC1 genes exhibited tissue-specific imprinting, however, COTL1, CRISLPLD2, and GSE1 escaped imprinting. Four DMRs, established after fertilization, were found in this region. Two DMRs were located between the ZDHHC7 and KIAA0513 genes, and two were in exon 1 of the CDH13 and ATP2C2 genes, respectively. The results from this study support future studies on the molecular mechanism to regulate the imprinting of candidate genes on bovine chromosome 18.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage.

BACKGROUND: With rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored. RESULTS: In this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model. CONCLUSIONS: Exposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.

RNASeq Analysis for Accurate Identification of Fusion Partners in Tumor Specific Translocations Detected by Standard FISH Probes in Hematologic Malignancies.

Background: Fluorescence labeled DNA probes and in situ hybridization methods had shorter turn round time for results revolutionized their clinical application. Signals obtained from these probes are highly specific, yet they can produce fusion signals not necessarily representing fusion of actual genes due to other genes included in the probe design. In this study we evaluated discordance between cytogenetic, FISH and RNAseq results in 3 different patients with hematologic malignancies and illustrated the need to perform next generation sequencing (NGS) or RNASeq to accurately interpret FISH results. Methods: Bone marrow or peripheral blood karyotypes and FISH were performed to detect recurring translocations associated with hematologic malignancies in clinical samples routinely referred to our clinical cytogenetics laboratory. When required, NGS was performed on DNA and RNA libraries to detect somatic alterations and gene fusions in some of these specimens. Discordance in results between these methods is further evaluated. Results: For a patient with plasma cell leukemia standard FGFR3 / IGH dual fusion FISH assay detected fusion that was interpreted as FGFR3-positive leukemia, whereas NGS/RNASeq detected NSD2::IGH. For a pediatric acute lymphoblastic leukemia patient, a genetic diagnosis of PDGFRB-positive ALL was rendered because the PDGFRB break-apart probe detected clonal rearrangement, whereas NGS detected MEF2D::CSF1R. A MYC-positive B-prolymphocytic leukemia was rendered for another patient with a cytogenetically identified t(8;14) and MYC::IGH by FISH, whereas NGS detected a novel PVT1::RCOR1 not previously reported. Conclusions: These are 3 cases in a series of several other concordant results, nevertheless, elucidate limitations when interpreting FISH results in clinical applications, particularly when other genes are included in probe design. In addition, when the observed FISH signals are atypical, this study illustrates the necessity to perform complementary laboratory assays, such as NGS and/or RNASeq, to accurately identify fusion genes in tumorigenic translocations.

Hypoxia causes trophoblast cell ferroptosis to induce miscarriage through lnc-HZ06/HIF1α-SUMO/NCOA4 axis.

Defects of human trophoblast cells may induce miscarriage (abnormal early embryo loss), which is generally regulated by lncRNAs. Ferroptosis is a newly identified iron-dependent programmed cell death. Hypoxia is an important and unavoidable feature in mammalian cells. However, whether hypoxia might induce trophoblast cell ferroptosis and then induce miscarriage, as well as regulated by a lncRNA, was completely unknown. In this work, we discovered at the first time that hypoxia could result in ferroptosis of human trophoblast cells and then induce miscarriage. We also identified a novel lncRNA (lnc-HZ06) that simultaneously regulated hypoxia (indicated by HIF1α protein), ferroptosis, and miscarriage. In mechanism, HIF1α-SUMO, instead of HIF1α itself, primarily acted as a transcription factor to promote the transcription of NCOA4 (ferroptosis indicator) in hypoxic trophoblast cells. Lnc-HZ06 promoted the SUMOylation of HIF1α by suppressing SENP1-mediated deSUMOylation. HIF1α-SUMO also acted as a transcription factor to promote lnc-HZ06 transcription. Thus, both lnc-HZ06 and HIF1α-SUMO formed a positive auto-regulatory feedback loop. This loop was up-regulated in hypoxic trophoblast cells, in RM villous tissues, and in placental tissues of hypoxia-treated mice, which further induced ferroptosis and miscarriage by up-regulating HIF1α-SUMO-mediated NCOA4 transcription. Furthermore, knockdown of either murine lnc-hz06 or Ncoa4 could efficiently suppress ferroptosis and alleviate miscarriage in hypoxic mouse model. Taken together, this study provided new insights in understanding the regulatory roles of lnc-HZ06/HIF1α-SUMO/NCOA4 axis among hypoxia, ferroptosis, and miscarriage, and also offered an effective approach for treatment against miscarriage.