HDAC6-dependent deacetylation of NGF dictates its ubiquitination and maintains primordial follicle dormancy.

Rationale: Primordial follicles are limited in number and cannot be regenerated, dormant primordial follicles cannot be reversed once they enter a growth state. Therefore, the length of the female reproductive lifespan depends on the orderly progression and selective activation of primordial follicles, the mechanism of which remains unclear. Methods: We used human ovarian cortical biopsy specimens, granulosa cells from diminished ovarian reserve (DOR) patients, Hdac6-overexpressing transgenic mouse model, and RNA sequencing to analyze the crucial roles of histone deacetylase 6 (HDAC6) in fertility preservation and primordial follicle activation. Results: In the present study, we found that HDAC6 was highly expressed in most dormant primordial follicles. The HDAC6 expression was reduced accompanying reproductive senescence in human and mouse ovaries. Overexpression of Hdac6 delayed the rate of primordial follicle activation, thereby prolonging the mouse reproductive lifespan. Short-term inhibition of HDAC6 promoted primordial follicle activation and follicular development in humans and mice. Mechanism studies revealed that HDAC6 directly interacted with NGF, reducing acetylation modification of NGF and thereby accelerating its ubiquitination degradation. Consequently, the reduced NGF protein level maintained the dormancy of primordial follicles. Conclusions: The physiological significance of the high expression of HDAC6 in most primordial follicles is to reduce NGF expression and prevent primordial follicle activation to maintain female fertility. Reduced HDAC6 expression increases NGF expression in primordial follicles, activating their development and contributing to reproduction. Our study provides a clinical reference value for fertility preservation.

MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation.

The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.

The UDPase ENTPD5 regulates ER stress-associated renal injury by mediating protein N-glycosylation.

Impaired protein N-glycosylation leads to the endoplasmic reticulum (ER) stress, which triggers adaptive survival or maladaptive apoptosis in renal tubules in diabetic kidney disease (DKD). Therapeutic strategies targeting ER stress are promising for the treatment of DKD. Here, we report a previously unappreciated role played by ENTPD5 in alleviating renal injury by mediating ER stress. We found that ENTPD5 was highly expressed in normal renal tubules; however, ENTPD5 was dynamically expressed in the kidney and closely related to pathological DKD progression in both human patients and mouse models. Overexpression of ENTPD5 relieved ER stress in renal tubular cells, leading to compensatory cell proliferation that resulted in hypertrophy, while ENTPD5 knockdown aggravated ER stress to induce cell apoptosis, leading to renal tubular atrophy and interstitial fibrosis. Mechanistically, ENTPD5-regulated N-glycosylation of proteins in the ER to promote cell proliferation in the early stage of DKD, and continuous hyperglycemia activated the hexosamine biosynthesis pathway (HBP) to increase the level of UDP-GlcNAc, which driving a feedback mechanism that inhibited transcription factor SP1 activity to downregulate ENTPD5 expression in the late stage of DKD. This study was the first to demonstrate that ENTPD5 regulated renal tubule cell numbers through adaptive proliferation or apoptosis in the kidney by modulating the protein N-glycosylation rate in the ER, suggesting that ENTPD5 drives cell fate in response to metabolic stress and is a potential therapeutic target for renal diseases.

JUND/linc00976 promotes cholangiocarcinoma progression and metastasis, inhibits ferroptosis by regulating the miR-3202/GPX4 axis.

Long noncoding RNAs (lncRNAs) are a novel class of noncoding RNAs that have emerged as critical regulators and biomarkers in various cancers. Nevertheless, the expression profile and mechanistic function of lncRNAs in cholangiocarcinoma (CCA) remain unclear. Herein, we examined the expression levels of linc00976 in clinical specimens and cell lines using reverse transcription-quantitative PCR. In total, 50 patients with CCA were enrolled to analyze the correlation between linc00976 expression and clinical characteristics of CCA. Loss- and gain-of-function experiments were performed to investigate the biological effects of linc00976 on proliferation, ferroptosis, migration, and invasion of CCA cells in vitro and in vivo. In situ hybridization, RNA immunoprecipitation, bioinformatic databases, RNA pull-down assay, a dual-luciferase reporter assay, mRNA sequencing, chromatin immunoprecipitation-PCR, and rescue experiments were performed to elucidate the underlying mechanisms of linc00976-induced competitive endogenous RNA regulatory networks. We characterized a novel and abundant lncRNA, linc00976, that functions as a pro-oncogenic regulator of CCA progression. Compared with normal controls, linc00976 was dramatically upregulated in CCA tissue samples and cell lines. Patients with CCA exhibiting high linc00976 expression had a highly advanced clinical stage, substantial lymph node metastasis, and poor overall survival. Knockdown of linc00976 significantly repressed proliferation and metastasis and promoted ferroptosis of CCA cells both in vitro and in vivo, whereas linc00976 overexpression exerted the opposite effect. Mechanistically, linc00976 competitively interacted with miR-3202 to upregulate GPX4 expression, thus contributing to the malignant biological behavior of CCA cells. Moreover, we demonstrated that JUND specifically interacts with the linc00976 promoter and activates linc00976 transcription. Accordingly, JUND promotes linc00976 transcription, and linc00976 plays a crucial role in accelerating CCA tumorigenesis and metastasis and inhibiting ferroptosis by modulating the miR-3202/GPX4 axis. These findings suggest that targeting linc00976 may afford a promising therapeutic strategy for patients with CCA.

Loss of MEN1 leads to renal fibrosis and decreases HGF-Adamts5 pathway activity via an epigenetic mechanism.

BACKGROUND: Renal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear. METHODS: Kidney histology was examined on paraffin sections stained with hematoxylin-eosin staining. Masson's trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real-time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA-sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP-seq) was carried out for identification of menin- and H3K4me3-enriched regions within the whole genome in the mouse kidney tissue. ChIP-qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh-HGF on renal fibrosis. RESULTS: The expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α-SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial-to-mesenchymal transition (EMT) induced by TGF-ß treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO-induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency. CONCLUSIONS: These findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

MEN1 deficiency leads to neuroendocrine differentiation of lung cancer and disrupts the DNA damage response.

The MEN1 gene, a tumor suppressor gene that encodes the protein menin, is mutated at high frequencies in neuroendocrine (NE) tumors; however, the biological importance of this gene in NE-type lung cancer in vivo remains unclear. Here, we established an ATII-specific KrasG12D/+/Men1-/- driven genetically engineered mouse model and show that deficiency of menin results in the accumulation of DNA damage and antagonizes oncogenic Kras-induced senescence and the epithelial-to-mesenchymal transition during lung tumorigenesis. The loss of menin expression in certain human primary lung cancers correlates with elevated NE profiles and reduced overall survival.

Secreted recombinant human IL-24 protein inhibits the proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro and in vivo.

Interleukin-24 (IL-24) displays cancer-specific apoptosis-inducing properties in a broad spectrum of human tumors without harmful effects on normal cells. The human IL-24 protein is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and a potent antiangiogenic molecule. However, the function of secreted recombinant human IL-24 (srhIL-24) protein in esophageal squamous cell carcinoma (ESCC) cells has not been studied. In the present study, we prepared a stable site-specific-integrated cell line, Flp-InTMCHO/IL-24 (FCHO/IL-24), which secreted rhIL-24 at a higher level than three random-integrated cell lines. In vitro, we identified that the purified srhIL-24 inhibited proliferation and induced the apoptosis of ESCC Eca-109 cells and activated STAT3, which was related with the IL-20 receptors. In vivo, the tumorigenicity of Eca-109 cells was significantly inhibited by s.c. injection of FCHO/IL-24 cells. Decreased tumor microvessel density and an increased number of TUNEL-positive tumor cells were associated with tumor growth inhibition, indicating the presence of antiangiogenic activity and induction of apoptotic activity. In summary, the present study demonstrated that srhIL-24 induced growth inhibition and apoptosis in ESCC Eca-109 cells in vitro and in vivo, which may be mediated by the receptor pathway.

A novel human interleukin-24 peptide created by computer-guided design contributes to suppression of proliferation in esophageal squamous cell carcinoma Eca-109 cells.

Based on the three-dimensional modeling structure of human interleukin-24 (hIL-24) and its most likely active position predicted by solvent accessibility and apparent electrostatic properties, a novel hIL-24 peptide M1 was created by computer-guided molecular design. The cytotoxicity and cell selectivity of M1 were examined in three human carcinoma cell lines and one normal human embryo lung fibroblast cell line (HEL). MTT assay showed that M1 induced growth arrest in two IL-20 receptor complex-positive cancer cell lines (the esophageal squamous cell carcinoma cell line Eca-109 and the melanoma cell line A375), and antibodies against IL-24 or IL-20 receptor complexes significantly neutralized the inhibitory activity. Moreover, M1 had almost no cytotoxicity on the lung cancer A549 cell line, which lacks a full complement of the IL-20 receptor complexes, or on HEL cells that express the IL-20 receptor complexes. These findings demonstrate that M1 could act as an excellent candidate for the induction of growth arrest on receptor complex-positive cancer cells. In summary, the M1 peptide may represent a novel anticancer agent for esophageal squamous cell carcinoma therapy due to its cancer cell selectivity and its relatively low cytotoxicity to normal cells.

[Induction effects of pentachlorophenol on vitellogenin and p53 in Chinese rare minnow (Gobiocypris rarus)].

Taking the Chinese rare minnow (Gobiocypris rarus) as the test animal, the studies were designed to investigate induction effects of pentachlorophenol (PCP) on vitellogenin (VTG) protein, VTG gene and tumor suppressor p53 gene in the liver of Gobiocypris rarus. The endocrine disrupting of PCP was evaluated by detecting VTG, and sensitive biomarkers of PCP were screened at both protein and mRNA levels. Gobiocypris rarus were exposed to PCP at 1.5, 15, 40, 80, 120, 150, 160 microg x L(-1) respectively, while setting blank, solvent control and 17alpha-ethynylestradiol (EE2) as positive control. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), VTG protein expression differences were detected in the liver of Gobiocypris rarus after exposure to PCP. Cloning the VTG and p53 gene new fragments of Gobiocypris rarus based on conserved regions, mRNA expression levels of VTG and p53 gene in the liver of Gobiocypris rarus were determined by quantitative real-time PCR assay after PCP treatment. The results showed that 40, 80, 120, 160 microg x L(-1) PCP induced the liver of male and female Gobiocypris rarus to produce VTG protein, and had a significant concentration effect. VTG and p53 mRNA levels significantly increased in the liver of Gobiocypris rarus after exposure to 1.5, 15, 150 microg x L(-1) PCP, and had remarkable concentration and time effects. The studies suggested that PCP had estrogenic effects, and VTG protein, VTG and p53 gene in the liver of Gobiocypris rarus could be used as candidate sensitive biomarkers for detecting PCP.

[Effects of pentachlorophenol on DNA damage and cytotoxicity of HeLa cells].

Using human cervical carcinoma HeLa cells, the cell viability was determined by MTT assay after pentachlorophenol (PCP) treatment, the cytotoxicity of PCP was evaluated by detecting lactate dehydrogenase (LDH) leakage rate and total superoxide dismutase (SOD) activity in cell culture medium; DNA damage was detected by comet assay. The results indicated that HeLa cells proliferation was inhibited by PCP and the median inhibitory concentration (IC50) was 66.59 micromol x L(-1); PCP did not induce DNA damage in the concentration range from 6.25 micromol x L(-1) to 50 micromol L(-1); LDH leakage rate increased gradually with the increasing of exposure time when HeLa cells were treated by PCP in the concentration range from 12.5 micromol x L(-1) to 200 micromol x L(-1); SOD activity decreased gradually as the increasing of exposure time when HeLa cells were treated by PCP at lower concentration of 12.25 micromol x L(-1), 17.5 micromol x L(-1), 25 micromol x L(-1) respectively, LDH leakage rate increased significantly at 25 micromol x L(-1) and activity of SOD decreased markedly at 12.25 micromol x L(-1) in HeLa cells following PCP-treatment respectively. Results suggested that SOD and LDH might be regarded as candidate sensitive biomarkers for evaluating toxicity of PCP at low concentration on human and wildlife.