A novel gene mutation in ZP3 loop region identified in patients with empty follicle syndrome.

The zona pellucida (ZP) is an extracellular matrix surrounding mammalian oocytes. It is composed of three to four glycoproteins, ZP1-ZP4. ZP3 is essential for sperm binding and zona matrix formation. Here, we identified a novel heterozygous mutation (NM_001110354.2:c.502_504delGAG) of ZP3, occurring in a pair of sisters with empty follicle syndrome (EFS). A mouse model with the same mutation was established using the CRISPR/Cas9 gene-editing system. As in the above family, F0 -, F1 -, and F2 -generation female mice with the mutation were all infertile. Further analysis using the Chinese hamster ovary cells (CHO-K1) also showed that this mutation weakens the strength of binding between ZP3 and ZP2, which hinders the assembly of ZP and results in unstable ZP formation. Immunohistochemical analysis using ovarian serial sections in both humans and mice demonstrated that the ZP of preantral follicles was thinner than normal control, or even absent. Our study presents a new gene mutation that leads to EFS, providing new evidence and support for the genetic diagnosis of infertile individuals with similar phenotypes. Our results also show that the loop of ZP3 is not only a linker between two amphiphilic helices but may play a critical role in specifying the correct heterodimerization partner.

Comparative analyses in transcriptome of human granulosa cells and follicular fluid micro-environment between poor ovarian responders with conventional controlled ovarian or mild ovarian stimulations.

BACKGROUND: Both mild and conventional controlled ovarian stimulation are the frequently used protocols for poor ovarian responders. However, there are some debates about which treatment is better. Moreover, little is known about the follicular physiology after the two ovarian stimulation protocols. This study was intended to investigate the features in granulosa cells and follicular fluid micro-environment after the two different ovarian stimulation protocols in poor responders. METHODS: Granulosa cells RNA were sequenced using Illumina Hiseq technology. Specific differently expressed genes and proteins were verified by real-time quantitative PCR and Western blot analysis. Moreover, hormone and cytokine concentrations in the follicular fluid were measured by electrochemiluminescence immunoassay and enzyme-linked immunoabsorbent assay. The correlation between the results of molecular experiments and the laboratory outcomes were analyzed by Spearman correlation analysis. RESULTS: The differentially expressed genes between the two groups were involved in 4 signaling pathways related to the follicular development; three proteins pertinent to the TGF-ß signaling pathway were expressed differently in granulosa cells between the two, and the constituents in the follicular fluid were also different. Further, a correlation between the TGF-ß signaling pathway and the good-quality embryo was observed. CONCLUSIONS: The present study made a comparison for the first time in the transcriptome of human granulosa cells and the follicular fluid micro-environment between poor responders with the conventional controlled ovarian stimulation or the mild ovarian stimulation, showing that the TGF-ß signaling pathway may correlate with the good-quality of embryos in the mild group, which may be instrumental to the choice of optimal management for IVF patients.

Single-cell transcriptome and cell-specific network analysis reveal the reparative effect of neurotrophin-4 in preantral follicles grown in vitro.

BACKGROUND: In-vitro-grow (IVG) of preantral follicles is essential for female fertility preservation, while practical approach for improvement is far from being explored. Studies have indicated that neurotrophin-4 (NT-4) is preferentially expressed in human preantral follicles and may be crucial to preantral follicle growth. METHODS: We observed the location and expression of Tropomyosin-related kinase B (TRKB) in human and mouse ovaries with immunofluorescence and Western blot, and the relation between oocyte maturation and NT-4 level in follicular fluid (FF). Mice model was applied to investigate the effect of NT-4 on preantral follicle IVG. Single-cell RNA sequencing of oocyte combined with cell-specific network analysis was conducted to uncover the underlying mechanism of effect. RESULTS: We reported the dynamic location of TRKB in human and mouse ovaries, and a positive relationship between human oocyte maturation and NT-4 level in FF. Improving effect of NT-4 was observed on mice preantral follicle IVG, including follicle development and oocyte maturation. Transcriptome analysis showed that the reparative effect of NT-4 on oocyte maturation might be mediated by regulation of PI3K-Akt signaling and subsequent organization of F-actin. Suppression of advanced stimulated complement system in granulosa cells might contribute to the improvement. Cell-specific network analysis revealed NT-4 may recover the inflammation damage induced by abnormal lipid metabolism in IVG. CONCLUSIONS: Our data suggest that NT-4 is involved in ovarian physiology and may improve the efficiency of preantral follicle IVG for fertility preservation.

METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6 -methyladenosine-dependent primary MicroRNA processing.

N6 -Methyladenosine (m6 A) modification has been implicated in many biological processes. However, its role in cancer has not been well studied. Here, we demonstrate that m6 A modifications are decreased in hepatocellular carcinoma, especially in metastatic hepatocellular carcinoma, and that methyltransferase-like 14 (METTL14) is the main factor involved in aberrant m6 A modification. Moreover, METTL14 down-regulation acts as an adverse prognosis factor for recurrence-free survival of hepatocellular carcinoma and is significantly associated with tumor metastasis in vitro and in vivo. We confirm that METTL14 interacts with the microprocessor protein DGCR8 and positively modulates the primary microRNA 126 process in an m6 A-dependent manner. Further experiments show that microRNA 126 inhibits the repressing effect of METTL14 in tumor metastasis. CONCLUSION: These studies reveal an important role of METTL14 in tumor metastasis and provide a fresh view on m6 A modification in tumor progression. (Hepatology 2017;65:529-543).

Systemic genome screening identifies the outcome associated focal loss of long noncoding RNA PRAL in hepatocellular carcinoma.

UNLABELLED: Systemic analyses using large-scale genomic profiles have successfully identified cancer-driving somatic copy number variations (SCNVs) loci. However, functions of vast focal SCNVs in "protein-coding gene desert" regions are largely unknown. The integrative analysis of long noncoding RNA (lncRNA) expression profiles with SCNVs in hepatocellular carcinoma (HCC) led us to identify the recurrent deletion of lncRNA-PRAL (p53 regulation-associated lncRNA) on chromosome 17p13.1, whose genomic alterations were significantly associated with reduced survival of HCC patients. We found that lncRNA-PRAL could inhibit HCC growth and induce apoptosis in vivo and in vitro through p53. Subsequent investigations indicated that the three stem-loop motifs at the 5' end of lncRNA-PRAL facilitated the combination of HSP90 and p53 and thus competitively inhibited MDM2-dependent p53 ubiquitination, resulting in enhanced p53 stability. Additionally, in vivo lncRNA-PRAL delivery efficiently reduced intrinsic tumors, indicating its potential therapeutic application. CONCLUSIONS: lncRNA-PRAL, one of the key cancer-driving SCNVs, is a crucial stimulus for HCC growth and may serve as a potential target for antitumor therapy.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. METHODS: We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. RESULTS: ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. CONCLUSION: The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

H19 inhibits RNA polymerase II-mediated transcription by disrupting the hnRNP U-actin complex.

BACKGROUND: H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown. METHODS: We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19. RESULTS: Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5' 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression. CONCLUSIONS: In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U-actin complex. GENERAL SIGNIFICANCE: These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.

Long noncoding RNAs associated with liver regeneration 1 accelerates hepatocyte proliferation during liver regeneration by activating Wnt/ß-catenin signaling.

UNLABELLED: In recent years, long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of biological function. A recent study reported that lncRNAs control cell proliferation in hepatocellular carcinoma (HCC). However, the role of lncRNAs in liver regeneration and the overall mechanisms remain largely unknown. To address this issue, we carried out a genome-wide lncRNA microarray analysis during liver regeneration in mice after 2/3 partial hepatectomy (PH) at various timepoints. The results revealed differential expression of a subset of lncRNAs, notably a specific differentially expressed lncRNA associated with Wnt/ß-catenin signaling during liver regeneration (an lncRNA associated with liver regeneration, termed lncRNA-LALR1). The functions of lncRNA-LALR1 were assessed by silencing and overexpressing this lncRNA in vitro and in vivo. We found that lncRNA-LALR1 enhanced hepatocyte proliferation by promoting progression of the cell cycle in vitro. Furthermore, we showed that lncRNA-LALR1 accelerated mouse hepatocyte proliferation and cell cycle progression during liver regeneration in vivo. Mechanistically, we discovered that lncRNA-LALR1 facilitated cyclin D1 expression through activation of Wnt/ß-catenin signaling by way of suppression of Axin1. In addition, lncRNA-LALR1 inhibited the expression of Axin1 mainly by recruiting CTCF to the AXIN1 promoter region. We also identified a human ortholog RNA of lncRNA-LALR1 (lncRNA-hLALR1) and found that it was expressed in human liver tissues. CONCLUSION: lncRNA-LALR1 promotes cell cycle progression and accelerates hepatocyte proliferation during liver regeneration by activating Wnt/ß-catenin signaling. Pharmacological intervention targeting lncRNA-LALR1 may be therapeutically beneficial in liver failure and liver transplantation by inducing liver regeneration.

Global crotonylome identifies EP300-regulated ANXA2 crotonylation in cumulus cells as a regulator of oocyte maturation.

Lysine crotonylation (Kcr), a newly discovered post-translational modification, played a crucial role in physiology and disease progression. However, the roles of crotonylation in oocyte meiotic resumption remain elusive. As abnormal cumulus cell development will cause oocyte maturation arrest and female infertility, we report that cumulus cells surrounding human meiotic arrested oocytes showed significantly lower crotonylation, which was associated with decreased EP300 expression and blocked cumulus cell expansion. In cultured human cumulus cells, exogenous crotonylation or EP300 activator promoted cell proliferation and reduced cell apoptosis, whereas EP300 knockdown induced the opposite effect. Transcriptome profiling analysis in human cumulus cells indicated that functions of crotonylation were associated with activation of epidermal growth factor receptor (EGFR) pathway. Importantly, we characterized the Kcr proteomics landscape in cumulus cells by LC-MS/MS analysis, and identified that annexin A2 (ANXA2) was crotonylated in cumulus cells in an EP300-dependent manner. Crotonylation of ANXA2 enhanced the ANXA2-EGFR binding, and then activated the EGFR pathway to affect cumulus cell proliferation and apoptosis. Using mouse oocytes IVM model and EP300 knockout mice, we further confirmed that crotonylation alteration in cumulus cells affected the oocyte maturation. Together, our results indicated that EP300-mediated crotonylation is important for cumulus cells functions and oocyte maturation.

Single-Cell Atlas of Human Ovaries Reveals The Role Of The Pyroptotic Macrophage in Ovarian Aging.

Female fecundity declines in a nonlinear manner with age during the reproductive years, even as ovulatory cycles continue, which reduces female fertility, disrupts metabolic homeostasis, and eventually induces various chronic diseases. Despite this, the aging-related cellular and molecular changes in human ovaries that occur during these reproductive years have not been elucidated. Here, single-cell RNA sequencing (scRNA-seq) of human ovaries is performed from different childbearing ages and reveals that the activation of the pyroptosis pathway increased with age, mainly in macrophages. The enrichment of pyroptotic macrophages leads to a switch from a tissue-resident macrophage (TRM)-involve immunoregulatory microenvironment in young ovaries to a pyroptotic monocyte-derived macrophage (MDM)-involved proinflammatory microenvironment in middle-aged ovaries. This remolded ovarian immuno-microenvironment further promotes stromal cell senescence and accelerated reproductive decline. This hypothesis is validated in a series of cell and animal experiments using GSDMD-KO mice. In conclusion, the work expands the current understanding of the ovarian aging process by illustrating a pyroptotic macrophage-involved immune mechanism, which has important implications for the development of novel strategies to delay senescence and promote reproductive health.

Accessing multiple conical intersections in the 3s and 3p(x) photodissociation of the hydroxymethyl radical.

The photodissociation dynamics of the hydroxymethyl radical (CH2OH, CH2OD, and CD2OD) following excitation to the 3s and 3p(x) Rydberg states is studied using time-sliced velocity map imaging of hydrogen photofragments. Dissociation takes place on the ground potential energy surface reached via conical intersections from the excited states, and formaldehyde and hydrxymethylene are identified as reaction products. The major product, formaldehyde, has a bimodal internal energy distribution. The largest fraction has high kinetic energy (KE), modest rotational excitation, and vibrational excitation mainly in the CO stretch and the CH(D)2 deformations modes (scissors, wag, and rock). The minor fraction has lower KEs and a higher rovibrational excitation that is unresolved. A bimodal internal energy distribution in the formaldehyde fragment has been predicted by Yarkony [J. Chem. Phys. 2005, 122, 084316] for a conical intersection along the O-H bond coordinate. The hydroxymethylene product state distributions depend strongly on the nature of the excited state. In dissociation via the 3s state, the hydroxymethylene products have broad rovibrational state distributions and are produced at low yield. As suggested by Yarkony, they may be produced in the same dissociation events that give rise to low KE formaldehyde. In these events, the bound region of the PES is sampled following the conical intersection along O-H(D). The hydroxymethylene yield is low near its threshold and increases slowly with excitation energy to the 3s state, but its internal energy distribution remains broad and the contributions of the cis- and trans-isomers cannot be resolved. The mechanism changes markedly when exciting to the 3p(x) state. The hydroxymethylene products have less rotational excitation and show separate contributions of cis- and trans-isomers. The trans-isomer is found to be a minor product relative to the higher-energy cis-isomer, as predicted by Yarkony for conical intersections along the C-H coordinate. It appears that the efficiency of dissociation via conical intersections along the O-H and C-H coordinates depends on the initial excited state. While the O-H conical intersection seam (vertical cone) provides an efficient route to the ground state following excitation via the 3s or the 3p(x) Rydberg states, conical intersections along the C-H bond coordinate (tilted cone) are sampled more efficiently via 3p(x) excitation and proceed through different dynamics. The energy separations between formaldehyde and hydroxymethylene and between the cis- and trans-isomers of hydroxymethylene are determined experimentally for all the investigated isotopologs and are in good agreement with theory.

Pyroptosis orchestrates immune responses in endometriosis.

Endometriosis is a refractory and recurrent gynecological condition which affects about 10 % of reproductive-age women. The dysfunctional immune system is a well-established element in disease pathogenesis. Pyroptosis, a novel form of inflammatory cell death, has been revealed to be strongly connected with immune responses in tumors. Nevertheless, its relationship with microenvironment characteristics and clinical features in endometriosis is unclear. Here, we performed bioinformatics analysis on published data in humans and revealed a significant but neglected role of pyroptosis in endometriosis. Samples with higher PyrScores were generally accompanied with more aggressive disease features, such as EMT, angiogenesis and immune disorders. We further confirmed in animal models that pyroptosis exacerbated immune dysfunction by recruiting activated immune cell including macrophages, DC, neutrophils, CD8+ Tcm and Tregs with unregulated CCL2, CCL3, CXCL2 and CXCL3. Collectively, pyroptosis is a distinctive feature of endometriosis. Our work provides insights into further studies targeting pyroptosis for molecular typing and individualized precise therapy.

Multi-Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging.

Abnormal resumption of meiosis and decreased oocyte quality are hallmarks of maternal aging. Transcriptional silencing makes translational control an urgent task during meiosis resumption in maternal aging. However, insights into aging-related translational characteristics and underlying mechanisms are limited. Here, using multi-omics analysis of oocytes, it is found that translatomics during aging is related to changes in the proteome and reveals decreased translational efficiency with aging phenotypes in mouse oocytes. Translational efficiency decrease is associated with the N6-methyladenosine (m6A) modification of transcripts. It is further clarified that m6A reader YTHDF3 is significantly decreased in aged oocytes, inhibiting oocyte meiotic maturation. YTHDF3 intervention perturbs the translatome of oocytes and suppress the translational efficiency of aging-associated maternal factors, such as Hells, to affect the oocyte maturation. Moreover, the translational landscape is profiled in human oocyte aging, and the similar translational changes of epigenetic modifications regulators between human and mice oocyte aging are observed. In particular, due to the translational silence of YTHDF3 in human oocytes, translation activity is not associated with m6A modification, but alternative splicing factor SRSF6. Together, the findings profile the specific translational landscapes during oocyte aging in mice and humans, and uncover non-conservative regulators on translation control in meiosis resumption and maternal aging.

Regenerative Effects of Locally or Intra-Arterially Administered BMSCs on the Thin Endometrium.

Stem cell-based therapy plays a pivotal role in the regeneration of damaged endometrium. Previous studies have demonstrated the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) through diverse administration ways. However, the homing, survival, and differentiation potential of these differently administered BMSCs are poorly defined, and the best route of administration is not well-defined. Herein, we aim to compare the engraftment, retaining time, and therapeutic efficiency of differently administered BMSCs. To achieve this, GFP/Luc-labeled BMSCs administered in two modes were assessed in a thin endometrium rat model: either into the damaged horns directly or through the ipsilateral iliac artery. The retaining time and hemi-quantitative distribution were evaluated by in vivo bioluminescence imaging and immunohistological analysis. Locally administered BMSCs were strongly detected in the abdomen at the first 4 days post treatment but underwent a rapid decrease in luminescent signal afterward and were rarely found 28 days after treatment. In contrast, the retaining time of BMSCs injected through the iliac artery was longer, reflected by more GFP-positive cells detected in the uterine section 28 days post treatment. Differentiation toward endometrial stromal cells was observed. Both routes of administration contributed to the restoration of the damaged endometrium, showing a comparable increase in the endometrial thickness and a decrease in fibrosis. However, more importantly, higher expression of LIF and VEGF, better recruitment, and longer retainment were found in the intra-arterial administration, contributing to the establishment of the optimal administration mode in clinical practice.

Transcriptomic analysis shows that surgical treatment is likely to influence the endometrial receptivity of patients with stage III/IV endometriosis.

Background: Endometriosis negatively affects fertility, and it is a common disease in assisted reproductive practice. Surgical removal of endometriotic lesions is widely carried out to relieve symptoms and promote fertility. But it is not intensively investigated what changes in the secretory eutopic endometrium of patients with endometriosis after surgery. Methods: Eighteen patients with stage III/IV endometriosis were included in the study, and they were divided into the untreated group and the treated group (6 vs. 12). Basic clinical data were compared, and transcriptomic data of the secretory eutopic endometrium were analyzed with DESeq2, Cytoscape, ClueGO, CluePedia, and Gene Set Enrichment Analysis (GSEA). CIBERSORT was used to calculate the relative abundance of 22 immune cells in the samples. Results: We determined 346 differentially expressed genes (DEGs) using DESeq2. These DEGs were used to enrich seven Gene Ontology terms including three associated with immune processes and one correlated to prostaglandin using ClueGO and CluePedia. GSEA enriched 28 Gene Ontology terms in the treated group mainly associated with immune and blood pressure regulation process. Compared to the untreated group, the relative abundance of resting CD4+ memory T cells [0.218 (0.069, 0.334) vs. 0.332 (0.181, 0.429), P = 0.022] and the even less abundant memory B cells [0.001 (0.000, 0.083) vs. 0.033 (0.007, 0.057), P = 0.049] are significantly decreased in the treated group. Conclusion: Surgical treatment of stage III/IV endometriosis influences some genes and biological processes related to endometrial receptivity, but more evidence is needed.

Gut microbiota dysbiosis-derived macrophage pyroptosis causes polycystic ovary syndrome via steroidogenesis disturbance and apoptosis of granulosa cells.

Gut microbiota dysbiosis is critical in the etiology of polycystic ovary syndrome (PCOS). However, the mechanisms of gut microbiota in PCOS pathogenesis have not been fully elucidated. We aimed to explore the role of gut microbiota-derived macrophage pyroptosis in PCOS. This study conducted dehydroepiandrosterone (DHEA) induced PCOS mice model, 16S rDNA sequencing, western blot, genetic knocking out, transcriptome and translatome profiling, et al. to evaluate the underlying mechanisms. 16S rDNA sequencing showed reduced gut Akkermansia and elevated gram-negative bacteria (Desulfovibrio and Burkholderia) abundances in DHEA induced PCOS mice, which was accompanied by increased serum lipopolysaccharide (LPS). LPS could induce macrophage pyroptosis in mice ovaries, also activated in PCOS. Gasdermin D (GSDMD) is the final executor of macrophage pyroptosis. We demonstrated that Gsdmd knockout in mice could dramatically ameliorate PCOS. Mechanistically, transcriptome and translatome profiling revealed that macrophage pyroptosis disrupted estrogen production and promoted apoptosis of granulosa cells. Interferon (IFN)-γ, which was elevated in PCOS mice serum and ovaries, enhanced macrophage pyroptosis and exacerbated its effect on estrogen receptor in granulosa cells. Inspiringly, we identified that disulfiram and metformin could augment gut Akkermansia abundance, reduce serum IFN-γ level, inhibit macrophage pyroptosis in ovaries, therefore ameliorating PCOS. Collectively, this study emphasizes that macrophage pyroptosis, which was induced by gut microbiota dysbiosis and enhanced by IFN-γ, plays a key role in PCOS pathogenesis through estrogen synthesis dysfunction and apoptosis of granulosa cells. Disulfiram and metformin, which enhanced gut Akkermansia abundance and suppressed macrophage pyroptosis, may be considered as potential therapeutic strategies for PCOS.

NAT10 Maintains OGA mRNA Stability Through ac4C Modification in Regulating Oocyte Maturation.

In vitro maturation (IVM) refers to the process of developing immature oocytes into the mature in vitro under the microenvironment analogous to follicle fluid. It is an important technique for patients with polycystic ovary syndrome and, especially, those young patients with the need of fertility preservation. However, as the mechanisms of oocyte maturation have not been fully understood yet, the cultivation efficiency of IVM is not satisfactory. It was confirmed in our previous study that oocyte maturation was impaired after N-acetyltransferase 10 (NAT10) knockdown (KD). In the present study, we further explored the transcriptome alteration of NAT10-depleted oocytes and found that O-GlcNAcase(OGA) was an important target gene for NAT10-mediated ac4C modification in oocyte maturation. NAT10 might regulate OGA stability and expression by suppressing its degradation. To find out whether the influence of NAT10-mediated ac4C on oocyte maturation was mediated by OGA, we further explored the role of OGA in IVM. After knocking down OGA of oocytes, oocyte maturation was inhibited. In addition, as oocytes matured, OGA expression increased and, conversely, O-linked N-acetylglucosamine (O-GlcNAc) level decreased. On the basis of NAT10 KD transcriptome and OGA KD transcriptome data, NAT10-mediated ac4C modification of OGA might play a role through G protein-coupled receptors, molecular transduction, nucleosome DNA binding, and other mechanisms in oocyte maturation. Rsph6a, Gm7788, Gm41780, Trpc7, Gm29036, and Gm47144 were potential downstream genes. In conclusion, NAT10 maintained the stability of OGA transcript by ac4C modification on it, thus positively regulating IVM. Moreover, our study revealed the regulation mechanisms of oocytes maturation and provided reference for improving IVM outcomes. At the same time, the interaction between mRNA ac4C modification and protein O-GlcNAc modification was found for the first time, which enriched the regulation network of oocyte maturation.

Single-cell transcriptome and translatome dual-omics reveals potential mechanisms of human oocyte maturation.

The combined use of transcriptome and translatome as indicators of gene expression profiles is usually more accurate than the use of transcriptomes alone, especially in cell types governed by translational regulation, such as mammalian oocytes. Here, we developed a dual-omics methodology that includes both transcriptome and translatome sequencing (T&T-seq) of single-cell oocyte samples, and we used it to characterize the transcriptomes and translatomes during mouse and human oocyte maturation. T&T-seq analysis revealed distinct translational expression patterns between mouse and human oocytes and delineated a sequential gene expression regulation from the cytoplasm to the nucleus during human oocyte maturation. By these means, we also identified a functional role of OOSP2 inducing factor in human oocyte maturation, as human recombinant OOSP2 induced in vitro maturation of human oocytes, which was blocked by anti-OOSP2. Single-oocyte T&T-seq analyses further elucidated that OOSP2 induces specific signaling pathways, including small GTPases, through translational regulation.

Folic acid supplementation acts as a chemopreventive factor in tumorigenesis of hepatocellular carcinoma by inducing H3K9Me2-dependent transcriptional repression of LCN2.

The effects and mechanisms of folic acid (FA) as a chemopreventive agent for tumorigenesis of hepatocellular carcinoma (HCC) remain unclear. In this study, the QSG-7701, a human normal liver cell line, was cultured in different FA levels (High, Normal or No) for 6 months. Then, the biological characteristics, the expression of main stem cell-like genes or epithelial-mesenchymal transition (EMT) related genes and the tumorigenicity in vivo of cells cultured in different treatment groups were detected. Our results showed that No FA improved the malignant transformation of cells but High FA depressed the malignant transformation. Meanwhile, cells in different treatment groups were mapped by transcriptome sequencing. Then the relativity of increased LCN2 and decreased FA level was identified and confirmed in vitro and vivo. We also revealed that intracellular control of LCN2 would recover the effects of FA on cell proliferation, cell cycle and tumor formation in vitro and vivo. Finally, our studies displayed that increased FA level induced the down-regulation of LCN2 not by DNA hypermethylation of LCN2 promoter but by promoting the level of histone H3 lysine 9 di-methylation (H3K9Me2) in LCN2 promoter. In conclusion, our studies disclosed the chemopreventive effect of FA supplementation on hepatocarcinogenesis, which partial attributed to the inhibition of LCN2 by regulating histone methylation in promoter. Our results provide a potential mechanism of the chemoprevention of FA supplementation on tumorigenesis of HCC and may be helpful in developing treatment target against HCC.

NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro.

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.