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Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P 4 ) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N 6 -methyladenosine (m 6 A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P 4 signaling. Conditional deletion of methyltransferase-like 3 ( Mettl3 ), encoding the m 6 A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter ( Pgr-Cre ) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m 6 A modification. A luciferase assay revealed that the m 6 A modification in the 5′ untranslated region (5′-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P 4 signaling during embryo implantation via m 6 A-mediated translation control of Pgr mRNA.

Evidence suggests that intracellular angiotensin II type 1 receptor (AT1) contributes to peritoneal fibrosis (PF) under high glucose (HG)-based dialysates. It is generally believed that AT2 antagonisticly affects AT1 function. The aim of this study was to explore whether AT2 activation is beneficial for attenuating human peritoneal mesothelial cell (HPMC) injury due to HG. We treated a HPMC line with HG to induce extracellular matrix (ECM) formation. AT2 was increased and blocked using CGP42112A and AT2 siRNA. Lipid deposition was detected, signaling molecules associated with lectin-like oxidized lipoprotein receptor-1 (LOX-1) and ECM proteins were evaluated by real-time PCR and western blot. The results showed that HG led to AT2 inhibition in HPMCs, inhibition of AT2 further aggravated the expression of ECM proteins, including α-smooth muscle actin, fibroblast specific protein-1 and collagen I, while AT2 decreased the expression of ECM proteins, even during HG stimulation. Interestingly, there was a parallel change in lipid accumulation and ECM formation when AT2 was increased or depressed. Moreover, AT2-mediated decreased ECM production was associated with reduced lipid accumulation in HPMCs and depended on the downregulation of LOX-1. Further analysis showed that HG increased oxidized low-density lipoprotein (ox-LDL) deposition in HPMCs concomitant with an enhanced expression of ECM components, whereas blocking LOX-1 reversed ox-LDL deposition even in the presence of HG. This effect was also accompanied by the remission of ECM accumulation. Our results suggested that AT2 prevented ECM formation in HG-stimulated HPMCs by ameliorating lipid

Polycystic ovary syndrome (PCOS) causes anovulatory infertility in women of reproductive age, but etiopathogenesis of PCOS remains undetermined. Taurine up-regulated 1 (TUG1), an evolutionarily con...

This study aimed to investigate the role of connexin 43 (CX43) in thoracic ossification of ligamentum flavum (TOLF) based on the p38 mitogen-activated protein kinase (p38MAPK)-runt-related transcription factor 2 (RUNX2) pathway. Immunohistochemistry was used to detect CX43 expression in TOLF and non-TOLF patients, fibroblasts of TOLF were isolated and induced osteogenic differentiation, and CX43 expression was detected by western blot analysis (WB). In addition, si-CX43 was used to intervene CX43, and SB203580 was used to inhibit the p38MAPK. The expressions of bone differentiation marker protein were detected by WB, and the ossification ability was analyzed by alizarin red staining. The interaction between RUNX2 and CX43 was identified by dual-luciferase reporter assay. Results found that CX43 was highly expressed during TOLF, and si-CX43 could inhibit the expression of alkaline phosphatase (ALP) and osteopontin (OPN), as well as inhibit TOLF and the p38MAPK-RUNX2 pathway. In addition, SB203580 showed a synergistic effect with si-CX43 to further inhibit TOLF and the expression of RUNX2. The dual-luciferase reporter assay confirmed that RUNX2 could bind to the CX43 promoter. In conclusion, CX43 promotes TOLF, which may be mediated by p38MAPK-RUNX2, and RUNX2 binds to the CX43 promoter to form a positive feedback regulatory loop during TOLF.

In the present study, we focused on characterizing the proteome in granulosa cells in patients with biochemical premature ovarian insufficiency (bPOI) in order to identify differential proteins and investigate the fundamental mechanisms of POI. A total of 2688 proteins were identified based on the data-independent acquisition method, and 70 differentially expressed proteins were significant. Bioinformatic analyses, including gene expression pattern analysis, gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and Search Tool for the Retrieval of Interacting Genes/Proteins analysis, revealed discrete modules and the underlying molecular mechanisms in bPOI. Importantly, we observed that Ras-related C3 botulinum toxin substrate 1 (RAC1) was downregulated in the granulosa cells of bPOI. Low expression of RAC1 may affect the development process of POI by affecting the proliferation, apoptosis, and hormone synthesis of granulosa cells. Downregulation of RAC1 expression in the KGN and COV434 cells inhibited cell proliferation, blocked cells in the G1/G0 phase, and promoted apoptosis. Western blot results showed that β-catenin and cyclin D1 in the KGN and COV434 cells transfected with RAC1-siRNA were downregulated, while P21 and Bax were upregulated. Knocking down RAC1 in the KGN cells or adding the RAC1 enzyme inhibitor to the human luteinized granulosa cells (hLGC) inhibited the synthesis of E

Polycystic ovary syndrome (PCOS) causes anovulatory infertility in women of reproductive age, but etiopathogenesis of PCOS remains undetermined. Taurine up-regulated 1 (TUG1), an evolutionarily conserved long non-coding RNA, performs various biological functions; however, the role of TUG1 in PCOS remains unclear. Herein, TUG1 expression was assayed in granulosa cells (GCs) of 100 patients with PCOS and 100 control participants. Receiver operating characteristic (ROC) curve analysis was conducted to determine the diagnostic value of TUG1 in PCOS. TUG1 expression was also silenced in KGN cells to explore the role of TUG1 in cellular proliferation, apoptosis, cell-cycle progression, autophagy, and steroidogenesis. We found that TUG1 levels were dramatically increased in the PCOS group compared with those of the control group; this increased expression was related to a rising antral follicle count (R = 0.209, P

Aim: To identify the expression profiles and potential functions of circular RNAs (circRNAs) in granulosa cells (GCs) from women with biochemical premature ovarian insufficiency (bPOI). Patients & methods: CircRNAs microarray analysis was performed to GCs from 8 patients with bPOI and 8 control women, followed by qRT-PCR in 15 paired samples. CircRNA–miRNA networks and the prediction of their enriched signaling pathways were conducted by bioinformatics analysis. Results: A total of 133 upregulated and 424 downregulated circRNAs was identified in women with bPOI. We constructed circRNA–miRNA networks and found that the most predominantly enriched signaling pathways were the FoxO signaling pathway and cellular senescence. Conclusion: CircRNAs are differentially expressed in bPOI, which might contribute to the pathogenesis of bPOI.

Chronic inflammation plays an important role in the progression of vascular calcification (VC). This study was designed to explore the effects and underlying mechanisms of inflammation on VC in the radial arteries of patients with end-stage renal disease (ESRD) with arteriovenostomy. Forty-eight ESRD patients were divided into control (n = 25) and inflammation groups (n = 23) according to plasma C-reactive protein (CRP) level. Surgically removed tissues from the radial arteries of patients receiving arteriovenostomy were used in this study. Alizarin Red S staining was used to examine calcium deposition. The expression of inflammation markers, bone structure-associated proteins and mammalian target of rapamycin complex1 (mTORC1) pathway-related proteins was assessed by immunohistochemical staining. The expression of tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) was increased in the radial arteries of the inflammation group. Additionally, Alizarin Red S staining revealed a marked increase in calcium deposition in the inflammation group compared to controls. Further analysis by immunohistochemical staining demonstrated that the deposition was correlated with the increased expression of bone-associated proteins such as bone morphogenetic proteins-2 (BMP-2) and osteocalcin and collagen I, which suggested that inflammation induces osteogenic differentiation in vascular tissues and that osteogenic cells are the main cellular components involved in VC. Interestingly, there was a parallel increase in the expression of phosphorylated mTOR (p-mTOR) and pribosomal protein S6 kinase 1 (p-S6K1) in the inflammation group. Furthermore, mTORC1 pathway-related proteins were significantly associated with the enhanced expression of bone formation biomarkers. Inflammation contributed to VC in the radial arteries of ESRD patients via the induction of osteogenic differentiation in vessel walls, which could be regulated by the activation of the mTORC1 pathway.

Human Pinx1 protein, associated with shelterin proteins, is widely revealed as a haploinsufficient tumor suppressor. Growing evidence has manifested the deregulation of PinX1 in distinct cancers. Nonetheless, the loss status of PinX1 and its diagnostic, prognostic and clinicopathological significance in Basal-like breast cancer are still unclear. In the present study, the PinX1 expression levels of breast cancer tissues were investigated by qRT-PCR and immunoblotting assays. Then immunohistochemistry (IHC) was performed to detect PinX1 expression on a tissue microarray. The optimal threshold for PinX1 positivity was determined by receiver operating characteristic (ROC) curve analysis. To clarify the probable role of PinX1 in BLBC, the PinX1 knockout and stably over-expressed MDA-MB-231 cell lines were constructed by the CRISPR-Cas9 system and gene transfection. The association of PinX1 expression with cell proliferation, migration and apoptosis of MDA-MB-231 cells were observed by CCK-8 assay, wound healing assay, Transwell assay, flow cytometric analysis and immunoblotting of the cleaved caspase-3 protein level. Our results showed that both PinX1 mRNA and protein expression were downregulated in breast cancer tissues (P

Peritoneal fibrosis (PF) is characterized by progressive accumulation of extracellular matrix (ECM) components in the peritoneum under high glucose conditions. Rapamycin has previously been shown to inhibit ECM accumulation of peritoneal mesothelial cells (PMCs) and prevent PF. Here we explored the undefined mechanisms by which rapamycin inhibits ECM accumulation of PMCs. We used high-glucose peritoneal dialysis solution (PDS) in a mouse peritoneal dialysis model to induce in vivo PF and in human PMCs in vitro to stimulate ECM accumulation. The mice that received chronic PDS infusions showed typical features of PF, including markedly increased peritoneal thickness, excessive matrix deposition, increased peritoneal permeability, and higher expressions of α-smooth muscle actin and collagen I. Rapamycin significantly ameliorated these pathological changes. There was a parallel decrease in lipid accumulation in the peritoneum of rapamycin-treated mice. Rapamycin significantly inhibited high-glucose PDS-induced ECM accumulation and reduced the lipid droplet in human PMCs in the presence of PDS. The effects of rapamycin on intracellular lipid metabolism correlated with a series of steps in lipid homeostasis; namely, a decrease in low density lipoprotein receptor-mediated lipid influx, which was mediated through the downregulation of sterol regulatory element-binding protein-2 (SREBP-2) and SREBP cleavage-activating protein (SCAP), and an increase in adenosine triphosphate-binding cassette transporter A1-mediated lipid efflux, which was mediated through the upregulation of the liver X receptor α and peroxisome proliferator-activated receptor α. We conclude that rapamycin shows a clear protective effect on high-glucose PDS-induced PF by improving the disruption of intracellular lipid homeostasis.

The aim of this paper was to explore the effects and possible mechanisms in vitro of tea polyphenols (TP) delaying human glomerular mesangial cells (HGMCs) senescence induced by high glucose (HG). HGMCs were cultured in vitro and divided into the normal group (N, 5.5 mmol·L⁻¹ glucose), the mannitol group(MNT, 5.5 mmol·L⁻¹ glucose plus 24.5 mmol·L⁻¹ mannitol), the high dose of D-glucose group (HG, 30 mmol·L⁻¹ glucose), the low dose of TP group (L-TP, 30 mmol·L⁻¹ glucose plus 5 mg·L⁻¹ TP) and the high dose of TP group (H-TP, 30 mmol·L⁻¹ glucose plus 20 mg·L⁻¹ TP), which were cultured in 5% CO₂ at 37 °C, respectively. Firstly, the effects of TP on the cell morphology of HGMCs were observed after 72 h-intervention. Secondly, the cell cycle, the positive rate of senescence-associated-β-galactosidase (SA-β-gal) staining and the telomere length were detected, respectively. Finally, the protein expressions of p53, p21 and Rb in the p53-p21-Rb signaling pathway were investigated, respectively. And the expressions of p-STAT3 and miR-126 were examined severally. The results indicated that HG not only arrested the cell cycle in G₁ phase but also increased the positive rate of SA-β-gal staining, and shortened the telomere length. HG led to the protein over-expressions of p53, p21 and Rb and HGMCs senescence by activating the p53-p21-Rb signaling pathway. In addition, L-TP delayed HGMCs senescence by improving the cell cycle G₁ arrest, reducing SA-β-gal staining positive rate and lengthening the telomere length. L-TP reduced the protein over-expressions of p53, P21 and Rb induced by HG and inhibited the telomere-p53-p21-Rb signaling pathway. Moreover, the expression of p-STAT3 was increased and the expression of miR-126 was decreased in HGMCs induced by HG. L-TP reduced the expression of p-STAT3 and increased the expression of miR-126 in HGMCs. In conclusion, HG could induce HGMCs senescence by activating the telomere-p53-p21-Rb signaling pathway in vitro. L-TP could delay HGMCs senescence through regulating STAT3/miR-126 expressions and inhibiting the telomere-p53-p21-Rb signaling pathway activation. These findings could provide the effective interventions in clinic for preventing and treating renal cell senescence in diabetic kidney disease.

Background Bladder cancer (BCa) is one of the most common cancers in the urinary system among the world. Previous studies suggested that TMEM40 expression level was significantly associated with clinicopathological parameters including histological grade, clinical stage and pT status of bladder cancer. However, the molecular mechanism of TMEM40 in BCa remains poorly understood. Methods Real-time quantitative RT-PCR (qRT-PCR) and western blot (WB) were used to examine the expression levels of TMEM40 in BCa tissues, paired non-cancer tissues and cell lines. A series of experiments, including CCK-8, wound healing, flow cytometry, transwell and EdU assays were performed to assess the effects of TMEM40 on cell proliferation, cell cycle and apoptosis, migration and invasion. In addition, tumor growth was evaluated in vivo using a xenogenous subcutaneously implant model. All statistical analyses were executed by using the SPSS 20.0 software. All experimental data from three independent experiments were analyzed by Student’s t test and results were expressed as mean ± standard deviation. Results In this study, we identified the role of TMEM40 in the tumorigenesis of bladder cancer and found that it was upregulated in bladder cancer tissues and cell lines, compared with their normal counterparts. The results demonstrated that effective silence of TMEM40 expression suppressed cell proliferation, blocked G1-to-S cell cycle transition, and inhibited cell migration and invasion in human bladder 5637 and EJ cell lines. Consistently, in vivo data showed that TMEM40 silencing could dramatically decreased tumor growth. Further study revealed that TMEM40 knockdown resulted in accumulation of p53 and p21 protein and decrease of c-MYC and cyclin D1 protein. Conclusion These data suggest that TMEM40 represents a potential oncogene, which exert a crucial role in the proliferation and apoptosis via the p53 signaling pathway in BCa, thus probably serve as a novel candidate biomarker and a potential therapeutic target for patients with BCa.

Liver diseases are one of the leading causes of death in the world. In spite of tremendous advances in modern drug research, effective and safe hepatoprotective agents are still in urgent demand. Natural products are undoubtedly valuable sources for drug leads. A number of natural triterpenoids were reported to possess pronounced hepatoprotective effects, and triterpenoids have become one of the most important classes of natural products for hepatoprotective agents. However, the significance of natural triterpenoids has been underestimated in the hepatoprotective drug discovery, with only very limited triterpenoids being covered in the reviews of hepatoprotective natural products. In this paper, ca 350 natural triterpenoids with reported hepatoprotective effects in ca 120 references between 1975 and 2016 will be reviewed, and the structure-activity relationships of certain types of natural triterpenoids, if available, will be discussed. Patents are not included.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in reproductive-aged women; however, its etiology remains poorly understood. This study aimed to reveal the role of miR-29a in PCOS. MiR-29a levels were measured in the granulosa cells (GCs) of forty-seven PCOS patients and forty-seven controls. A receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of miR-29a in non-hyperandrogenism PCOS. MiR-29a was overexpressed in KGN and COV434 cells to examine its roles in proliferation, cell-cycle progression, and steroidogenesis. MiR-29a was significantly down-regulated in PCOS patients, and associated with an increased antral follicle count. The ROC curve showed a major separation between PCOS patients and controls. MiR-29a overexpression in KGN and COV434 cells inhibited cell proliferation, arrested cell-cycle progression, and decreased aromatase expression and estradiol production. These findings suggest that miR-29a is involved in GC proliferation and steroidogenesis, providing insights into PCOS pathogenesis.

The wavelet transform of pulse signal and noise have different properties, thus an improved wavelet transform modulus maximum algorithm was introduced to remove the noise in UWB system. Improvements include: a) a new adaptive threshold algorithm is used to process the wavelet transform modulus maxima on the largest scale level; b) getting modulus maxima on the first level through nonlinear least-square method. At last, Mallat alterative projection algorithm was adopted to reconstruct the signal. Simulation results show that the denoising effect of the novel method is far better than the traditional one, and it can locate the position of the narrowband pulse more accurately.

Next-generation sequencing was used to investigate 9 rare Chinese pedigrees with rare autosomal recessive neurologic Mendelian disorders. Five probands with ataxia-telangectasia and 1 proband with chorea-acanthocytosis were analyzed by targeted gene sequencing. Whole-exome sequencing was used to investigate 3 affected individuals with Joubert syndrome, nemaline myopathy, or spastic ataxia Charlevoix-Saguenay type. A list of known and novel candidate variants was identified for each causative gene. All variants were genetically verified by Sanger sequencing or quantitative polymerase chain reaction with the strategy of disease segregation in related pedigrees and healthy controls. The advantages of using next-generation sequencing to diagnose rare autosomal recessive neurologic Mendelian disorders characterized by genetic and phenotypic heterogeneity are demonstrated. A genetic diagnostic strategy combining the use of targeted gene sequencing and whole-exome sequencing with the aid of next-generation sequencing platforms has shown great promise for improving the diagnosis of neurologic Mendelian disorders.

Rho GTPases, including Rac1, Cdc42, play a critical role in the regulation of a variety of cellular processes such as cell morphology, cell migration, transcriptional activation and gene expression. We constructed several FRET-based single-molecule probes containing red fluorescent protein dsRed1, cyan fluorescent protein ECFP, the GTPase binding domain of the effector, Pak1 or N-WASP, and Rac1 or Cdc42. Rac1 and Cdc42 signaling pathways were activated in transfected cells by the inducer, insulin or bradykinin respectively. In vitro fluorescent spectroscopy assays showed that FRET phenomena were observed in transfected NIH3T3 and Hela cells. For all 3 signaling pathways in NIH3T3 cells, the values of FRET efficiency reached the highest after induction for 5 min, but the increasing extents of the values of FRET efficiency varied in 3 signaling pathways. The values of FRET efficiency decreased with the extention of the induction time, but differed significantly in the decreasing speed for the signaling pathways. Rac1 and Cdc42 activation assays indicated that Rac1 and Cdc42 were in the activated state (GTP-bound) in the induced cells. Their relative activated activities in the cells induced for different time were consistent with the values of FRET efficiency. The activated Rac1, Cdc42 signaling pathways led to the formation of lamelliopodia and filopodia in the transfected cells respectively. The results showed that these single-molecule probes could be used to directly monitor the spatial and temporal imaging of the induced activation of the signaling pathways in living cells. With these single-molecule probes, the GEF or GAP activities of putative regulatory proteins for Rac1 and Cdc42 were analyzed and judged, thus greatly simplifying the currently-used methods for identifying the regulatory proteins for Rho GTPases.