Xiaoqin Yin

Backgrounds: Pediatric-onset primary ovarian insufficiency (POI) presents distinct clinical challenges, including delayed puberty or growth retardation. Previously, we reported a Chinese family with POI harboring compound heterozygous variants (p.C242R and p.S445*) in MCM8 gene, a critical factor for DNA repair and gonadal homeostasis. Objective: This study describes clinical findings of pediatric-onset POI patients with compound heterozygous MCM8 variants and investigates the in vitro functional consequences of an artificial double-mutant MCM8 construct harboring both p.C242R and p.S445*. Using this overexpression model, we assessed DNA damage response, protein interactions, and transcriptomic changes associated with the double-mutant MCM8. Methods: HeLa cell models were used to overexpress double-mutant (p.C242R and p.S445*) or wild-type MCM8. Functional consequences were evaluated using immunofluorescence staining, flow cytometry, and western blotting to assess DNA damage repair, cell cycle and apoptosis. Co-immunoprecipitation coupled with mass spectrometry (CoIP–MS/MS) was used to screen the disrupted protein interactome. Cut&Tag and mRNA-seq analyses were used to examine chromatin-binding-associated changes and transcriptomic alterations. Results: Ovarian tissues from POI patients exhibited reduced MCM8 expression and increased apoptosis. Cells expressing the artificial double-mutant MCM8 construct showed delayed resolution of DNA damage, as indicated by persistent γ-H2AX accumulation, and altered recruitment of DMC1 and RAD51. These changes were accompanied by S-phase accumulation and increased apoptosis. CoIP–MS/MS analysis identified altered interaction between double-mutant MCM8 and MCM6, suggesting possible changes in replication-associated protein interactions. Integrated CUT&Tag and RNA-seq analyses identified MCM8-associated chromatin-binding changes and transcriptomic alterations involving candidate genes enriched in ovarian survival and metabolic pathways, including the PI3K/AKT signaling pathway. Conclusions: These specific MCM8 variants are clinically associated with pediatric-onset POI. In vitro , expression of double-mutant MCM8 construct was associated with impaired DNA damage resolution, altered MCM8-associated protein interactions, transcriptomic alterations, cell-cycle disturbance, and increased apoptosis. These findings provide cellular observations that may help generate hypotheses regarding how MCM8 dysfunction could contribute to ovarian insufficiency.

Background: NR5A1 encodes a transcription factor essential for adrenal and gonadal development. Gene variants are a known cause of heterogeneous 46,XY disorders of sex development (DSD), but the mechanisms underlying the phenotypic variability remain unclear. We investigated how different NR5A1 variants affect downstream gene regulation and contribute to DSD pathogenesis. Methods: We analyzed four naturally occurring NR5A1 variants identified in patients with 46,XY DSD—two novel (p.Cys65Ser, p.His310Arg) and two previously reported (p.Cys30Ser, p.Gln329*). We performed protein and transcriptomic analyses to characterize variant effects and identify dysregulated and candidate target genes, validated by qPCR and luciferase assays. Transcriptomic and CUT&Tag analyses focused on the p.Gln329* truncating variant. Results: All four variants occurred at conserved residues and resulted in reduced NR5A1 protein expression and impaired nuclear localization upon transfection in HEK293T cells. Transcriptomic analysis using the p.Gln329* variant revealed broad downregulation of genes involved in steroidogenesis, including CYP11A1 , STAR , and CYP17A1 . Notably, AMHR2 and STARD8 were significantly downregulated and showed reduced CUT&Tag signal in variant-transfected cells. Promoter assays confirmed that all variants diminished CYP11A1 and AMHR2 promoter activity. Only the p.Gln329* variant affected STARD8 promoter activity. Conclusions: These findings indicate that NR5A1 variants impair protein expression and localization, leading to transcriptional dysregulation of genes involved in steroid hormone biosynthesis and sexual development. Based on analysis of the p.Gln329* truncating variant, AMHR2 and STARD8 are strong candidate novel downstream targets of NR5A1, offering further insight into the mechanisms driving 46,XY DSD.

Background: Kallmann syndrome (KS) is a genetic disorder characterized by impaired reproductive system and olfactory development. This study aimed to identify a novel variant of SEMA3A in a KS patient and explore its potential pathogenic mechanism. Methods: A gene panel was used to identify potential pathogenic mutations. Wild‐type and mutant SEMA3A overexpression plasmids were constructed. Western blotting, RNA sequencing, and cell migration were performed to assess the effects of SEMA3A gene variations on GnRH neuronal migration. Results: A novel heterozygous mutation in the SEMA3A gene (NM_006080.3: exon 6–9 deletion) was identified in the proband, as well as in his father and sister. The spatial structure of the SEMA3A mutant protein was relatively looser. In vitro experiments revealed that SEMA3A mutation reduced SEMA3A expression and inhibited GnRH neuronal migration. RNAseq analysis revealed that the expression of 76 genes was upregulated and that of 104 genes was downregulated after SEMA3A mutation. The altered gene clusters were enriched mainly in cell migration, male gonad development, motor proteins, and neuron synapses. Conclusions: In this study, we identified a novel variant of SEMA3A in a KS patient and verified its function. These findings expand the mutation spectrum of the SEMA3A gene and offer a theoretical basis for the clinical diagnosis of KS.

Objective: Phenotypic switching of vascular smooth muscle cells (VSMCs) in the corpus spongiosum may contribute to abnormal urethral development in hypospadias, but the underlying molecular regulators remain unclear. This study aimed to identify hub genes associated with VSMCs phenotypic switching in the corpus spongiosum using RNA sequencing and Weighted Gene Co-expression Network Analysis (WGCNA), and to functionally characterize the top candidate gene CDKN2B. Methods: Corpus spongiosum tissue samples were collected from seven patients with proximal hypospadias and five patients with urethral stricture (control group). The expression of the VSMCs contractile markers Calponin 1 and α-SMA, and the secretory marker OPN, was evaluated by qRT-PCR and Western blotting to assess VSMCs phenotypic state. RNA sequencing and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to identify hub genes, which were then validated by qRT-PCR. Primary VSMCs were isolated from corpus spongiosum tissue and transduced with lentiviral vectors to either suppress or overexpress CDKN2B. Changes in VSMC marker expression and in key signaling pathways associated with phenotypic switching—specifically TGF/Smad and SRF/MYOCD—were analyzed using qRT-PCR and Western blotting. Results: In hypospadias tissue, the decreased expression of α-SMA and Calponin 1, together with increased OPN, indicated a shift in VSMCs from a contractile to a secretory phenotype. RNA-seq and WGCNA identified 11 differentially expressed genes, among which CDKN2B showed a marked downregulation in hypospadias samples. In control VSMCs, CDKN2B inhibition led to reduced α-SMA and Calponin 1, elevated OPN, and suppressed activity of TGF/Smad and SRF/MYOCD signaling. Conversely, CDKN2B overexpression in VSMCs from hypospadias samples restored α-SMA and Calponin 1 expression, decreased OPN, and enhanced TGF/Smad and SRF/MYOCD pathway activation. Conclusions: VSMCs in the corpus spongiosum surrounding the urethral plate in hypospadias undergo a transition from a contractile to a secretory phenotype. CDKN2B emerges from unbiased transcriptomic screening as a key hub gene and functions as a critical regulator of this process, maintaining the contractile phenotype by modulating canonical TGF/Smad and SRF/MYOCD signaling. The CDKN2B–TGF/Smad axis may represent a central pathway linking VSMC phenotypic switching to abnormal vascular remodeling in hypospadias.

Background: Puberty is among the most important stages in human development. Timely activation of hypothalamic gonadotropin-releasing hormone (GnRH) neurons underlies the initiation of pubertal development. The fat mass and obesity-associated (FTO), which is expressed in the hypothalamus, may regulate the m6A methylation of its target genes, influencing GnRH expression in the hypothalamus during puberty onset. This study aimed to explore the mechanism by which FTO regulates the function of its target neurotrophins within the hypothalamus and to clarify the molecular pathway underlying precocious puberty. Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to assess m6A methylation in the hypothalamus of female rats. Serum brain-derived neurotrophic factor (BDNF) levels were measured in girls with central precocious puberty (CPP) and matched controls. BDNF was applied to GnRH neurons in vitro . The functions of FTO were assessed through overexpression and knockdown in animal models, with downstream signaling evaluated via BDNF/PI3K/Akt pathway analysis. Intracerebroventricular (ICV) injections of a BDNF-overexpressing lentiviral vector or a negative control (NC) were used to investigate the role of central BDNF in regulating puberty onset and reproductive function. Results: Hypothalamic GnRH and FTO expression progressively increased whereas m6A methylation decreased during puberty. MeRIP-seq revealed significantly reduced m6A methylation of Bdnf mRNA in the arcuate nucleus (ARC) of female rats during early puberty. In the ARC, BDNF was expressed adjacent to GnRH-positive fibers and terminals. In addition, serum BDNF levels were higher in girls with CPP girls than in the control group. In vitro , BDNF treatment stimulated GnRH expression in GT1–7 cells. FTO positively regulated BDNF expression in an m6A methylation-dependent manner. FTO overexpression activated BDNF/PI3K/Akt signaling in the ARC and accelerated puberty onset. Conversely, FTO knockdown delayed vaginal opening and suppressed BDNF/PI3K/Akt signaling. ICV delivery of a BDNF-overexpressing lentivirus increased hypothalamic BDNF and GnRH expression, leading to a distinct endocrine profile. Conclusion: These findings suggest that FTO regulates the m6A demethylation of BDNF and promotes GnRH expression through the BDNF/PI3K/Akt signaling pathway. This study improves our understanding of the function of BDNF, which is critical for the development of therapeutic strategies for preventing and treating of precocious puberty.

N⁶-methyladenosine (m⁶A) plays a crucial role in the development and functional homeostasis of the central nervous system. The fat mass and obesity-associated (FTO) gene, which is highly expressed in the hypothalamus, is closely related to female pubertal development. In this study, we found that m⁶A methylation decreased in the hypothalamus gradually with puberty and decreased in female rats with precocious puberty. FTO expression was increased at the same time. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that the m⁶A methylation of PLCβ₃, a key enzyme of the Ca²⁺ signalling pathway, was decreased significantly in the hypothalamus in precocious rats. Upregulating FTO increased PLCβ3 expression and activated the Ca²⁺ signalling pathway, which promoted GnRH expression. Dual-luciferase reporter and MeRIP-qPCR assays confirmed that FTO regulated m⁶A demethylation of PLCβ₃ and promoted PLCβ₃ expression. Upon overexpressing FTO in the hypothalamic arcuate nucleus (ARC) in female rats, we observed advanced puberty onset. Meanwhile, PLCβ₃ and GnRH expression in the hypothalamus increased significantly, and the Ca²⁺ signalling pathway was activated. Our study demonstrates that FTO enhances GnRH expression, which promotes puberty onset, by regulating m⁶A demethylation of PLCβ3 and activating the Ca²⁺ signalling pathway.

Background: NONO-TFE3 translocation renal cell carcinoma (tRCC), one of the RCCs that are associated with Xp11.2 translocation/TFE3 gene fusion (Xp11.2 tRCCs), involves an X chromosome inversion between NONO and TFE3 with the characteristics of endonuclear aggregation of NONO-TFE3 fusion protein. The oncogenic mechanisms of NONO-TFE3 fusion have not yet been fully elucidated. Objective: This study aimed at investigating the mechanism of NONO-TFE3 fusion regulating HIF1A as well as the role of HIF-1α in the progression of NONO-TFE3 tRCC under hypoxia. Methods: Immunohistochemistry and Western Blotting assays were performed to profile HIF-1α expression in renal clear cell carcinoma (ccRCC) or in Xp11.2 tRCC. Chromatin immunoprecipitation (ChIP), a luciferase reporter assay, and real-time quantitative PCR (RT-qPCR) were used to evaluate the regulation of HIF1A expression by NONO-TFE3 fusion. Then, the flow cytometry analysis, tube formation assays, and cell migration assays were used as well as glucose or lactic acid levels were measured to establish the impact of HIF-1α on the progression of NONO-TFE3 tRCC. Besides, the effect of HIF-1α inhibitor (PX-478) on UOK109 cells was analyzed. Results: We found that HIF1A was the target gene of NONO-TFE3 fusion. In UOK109 cells, which were isolated from NONO-TFE3 tRCC samples, NONO-TFE3 fusion promoted aerobic glycolysis and angiogenesis by up-regulating the expression of HIF-1α under hypoxia. Furthermore, the inhibition of HIF-1α mediated by PX-478 suppressed the development of NONO-TFE3 tRCC under hypoxia. Conclusion: HIF-1α is a potential target for therapy of NONO-TFE3 tRCC under hypoxia.

Xp11.2 translocation renal cell carcinoma (tRCC) is mainly caused by translocation of the TFE3 gene located on chromosome Xp11.2 and is characterized by overexpression of the TFE3 fusion gene. Patients are diagnosed with tRCC usually before 45 years of age with poor prognosis. We investigated this disease using two tRCC cell lines, UOK109 and UOK120, in this study. The purpose of this study was to investigate the pathogenic mechanism of TFE3 fusions in tRCC based on its subcellular localization, nuclear translocation and transcriptional activity. The expression of TFE3 fusions and other related genes were analyzed by quantitative reverse transcription PCR (qRT-PCR) and Western blot. The subcellular localization of TFE3 was determined using immunofluorescence. The transcriptional activity of TFE3 fusions was measured using a luciferase reporter assay and ChIP analysis. In some experiments, TFE3 fusions were depleted by RNAi or gene knockdown. The TFE3 fusion segments were cloned into a plasmid expression system for expression in cells. Our results demonstrated that TFE3 fusions were overexpressed in tRCC with a strong nuclear retention irrespective of treatment with an mTORC1 inhibitor or not. TFE3 fusions lost its co-localization with lysosomal proteins and decreased its interaction with the chaperone 14–3-3 proteins in UOK109 and UOK120 cells. However, the fusion segments of TFE3 could not translocate to the nucleus and inhibition of Gsk3β could increase the cytoplasmic retention of TFE3 fusions. Both the luciferase reporter assay and ChIP analysis demonstrated that TFE3 fusions could bind to the promoters of the target genes as a wild-type TFE3 protein. Knockdown of TFE3 results in decreased expression of those genes responsible for lysosomal biogenesis and other target genes. The ChIP-seq data further verified that, in addition to lysosomal genes, TFE3 fusions could regulate genes involved in cellular responses to hypoxic stress and transcription. Our results indicated that the overexpressed TFE3 fusions were capable of escaping from the control by the mTOR signaling pathway and were accumulated in the nucleus in UOK109 and UOK120 cells. The nuclear retention of TFE3 fusions promoted the expression of lysosomal genes and other target genes, facilitating cancer cell resistance against an extreme environment.

Humans are inevitably exposed to ubiquitous phthalate esters (PAEs). In utero exposure to di-n-butyl phthalate (DBP) induces abnormal development of the testis and reproductive tract in male offspring, which correspond closely with the human condition of testicular dysgenesis syndrome (TDS)-like syndrome. However, the underlying mechanisms have not been elucidated in detail. In this study, pregnant rats were orally exposed to either corn oil (controls) or DBP at three different doses by gavage during Gestational Days 12.5–21.5. Pathological examinations were performed for toxicity evaluation. Proliferation and apoptosis related proteins (ras related dexamethasone induced 1 (Rasd1), mitogen-activated protein kinase kinases1/2 (MEK1/2), Bcl-2, and Bax) were measured for mechanisms exploration. The results showed that different doses of DBP caused male developmental and reproductive toxicity in rats, including the decrease of anogenital distance (AGD), the histological damage of testis, and apoptosis of seminiferous tubule cells. Our data suggested that DBP played chronic and continuous toxic roles on male reproductive system by disrupting expression of Rasd1 and MEK1/2 as well as Bcl-2/Bax ratio. Further research is warranted.