ZNF695, A Potential Prognostic Biomarker, Correlates with Immune Infiltrates in Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma: Bioinformatic Analysis and Experimental Verification.

BACKGROUND: The role of Zinc Finger Protein 695 (ZNF695) is unclear in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). OBJECTIVE: The objective of this study was to conduct a comprehensive analysis and experimental validation of ZNF695 in CESC. METHODS: The study investigated the expression of ZNF695 in both pan-cancer and CESC, utilizing data from The Cancer Genome Atlas (TCGA) database to assess its diagnostic value. The present study investigated the association between ZNF695 expression levels and clinical characteristics, as well as prognosis, in patients with CESC. The study explored potential regulatory networks involving ZNF695, including its association with immune infiltration, immune score, stemness index based on mRNA expression (mRNAsi), and drug sensitivity in CESC. We explored the expression of ZNF695 in CESC single cells. ZNF695 expression was validated using GSE29570. RESULTS: ZNF695 was found to be aberrantly expressed in pan-cancer and CESC. There was a significant correlation observed between an elevated level of ZNF695 expression in patients with CESC and histological grade (p = 0.017). Furthermore, a strong association was found between high ZNF695 expression in CESC patients and poorer overall survival (OS) (HR: 1.87; 95% CI: 1.17-3.00; p = 0.009), Progression-free Survival (PFS) (HR: 1.86; 95% CI: 1.16-2.98; p = 0.010), and Disease-specific Survival (DSS) (HR: 1.98; 95% CI: 1.15-3.42; p = 0.014). The expression of ZNF695 in CESC patients (p = 0.006) was identified as an independent prognostic determinant. ZNF695 was associated with steroid hormone biosynthesis, oxidative phosphorylation, and so on. ZNF695 expression correlated with immune infiltration, immune score, and mRNAsi in CESC. ZNF695 expression significantly and negatively correlated with AICA ribonucleotide, BIX02189, QL-XI-92, STF-62247, and SNX-2112 in CESC. ZNF695 gene was upregulated in CESC tissues and cell lines. ZNF695 was significantly upregulated in the CESC cell lines. CONCLUSION: ZNF695 may be a potential prognostic biomarker and immunotherapeutic target for CESC patients.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

NOTCH Pathway Genes in Ovarian Cancer: Clinical Significance and Associations with Immune Cell Infiltration.

BACKGROUND: Activation of the NOTCH signaling pathway is associated with tumorigenesis. The aim of this study was to investigate NOTCH pathway gene functions and regulatory mechanisms in ovarian cancer (OC). METHODS: We conducted a bioinformatics analysis of publicly available datasets in order to identify potential NOTCH-related mechanisms, associated genes, biological pathways, and their relation to immune function. RESULTS: Significant differential expression of the NOTCH pathway genes DLL1, DLL3, DLL4, HES1, HEY1, JAG1, NOTCH2, NOTCH3, and NOTCH4 was observed between OC samples and normal controls. Low expression of DLL4 and of NOTCH4 in OC patients was associated with International Federation of Gynecology and Obstetrics (FIGO) stage (p <0.001 and p = 0.036, respectively), while high expression of NOTCH3 was associated with race (p = 0.039) and age (p = 0.044). JAG2 and NOTCH1 expression were significantly associated with progression-free interval (PFI) (p = 0.011 and p = 0.039, respectively). DLL1 (Hazard Ratio (HR): 2.096; 95% CI: 1.522-2.886, p < 0.001) and NOTCH1 (HR: 0.711; 95% CI: 0.514-0.983, p = 0.039) expression were independently associated with PFI in multivariate analysis. DLL1, DLL3, JAG1, JAG2, NOTCH3 and NOTCH4 expression could significantly differentiate OC from non-cancer samples. Genes associated with the NOTCH pathway were mainly enriched in five signaling pathways: the NOTCH signaling pathway, breast cancer, endocrine resistance, Th1 and Th2 cell differentiation, and oxidative phosphorylation. The expression of NOTCH pathway genes was significantly associated with immune cell infiltration. CONCLUSIONS: NOTCH pathway genes appear to play an important role in the progression of OC by regulating immune cells, endocrine resistance, Th1 and Th2 cell differentiation, and oxidative phosphorylation. JAG2 and NOTCH1 are potential biomarkers and therapeutic targets for the treatment of OC.

Canonical Wnt/ß-catenin activity and differential epigenetic marks direct sexually dimorphic regulation of Irx3 and Irx5 in developing mouse gonads.

Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/ß-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/ß-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote ß-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and ß-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection.

Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.

CREB Signaling Is Involved in Rett Syndrome Pathogenesis.

Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene. To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation (MECP2T158M/T158M ), hESC line expressing no MECP2 (MECP2-KO), congenic pair of wild-type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function. At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated from MECP2T158M/T158M , MECP2-KO, and V247fs-MT stem cell lines. Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the female Mecp2 heterozygous mice rescued several behavioral defects. Together, our study establishes a robust in vitro platform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT.SIGNIFICANCE STATEMENT Our study establishes a robust human stem cell-based platform for consistent quantitative evaluation of genotype-dependent Rett syndrome (RTT) phenotypes at the cellular level. By providing the first evidence that enhancing cAMP response element binding protein signaling can alleviate RTT phenotypes both in vitro and in vivo, we reveal a previously unappreciated role of cAMP response element binding protein signaling in RTT pathogenesis, and identify a potential therapeutic target for RTT.