N6-methyladenosine demethylase FTO related to hyperandrogenism in PCOS via AKT pathway.

BACKGROUND: Polycystic ovary syndrome (PCOS) was known as the common endocrine disease in women, featured as hyperandrogenism, ovulation disorders, etc. Fat mass and obesity-associated protein (FTO), a m6A demethylase, is abnormal in the occurrence of ovarian diseases. However, the mechanism of FTO in the pathogenesis of PCOS is still unclear. METHODS: The level of FTO in clinical samples, PCOS rat with hyperandrogenism and granulosa cells (GCs) lines effected by DHT were investigated by ELISA, qRT-PCR, WB, and IHC, while m6A RNA methylation level was studied by m6A Colorimetric and androgen level was tested through ELISA. Changes in steroid hormone synthetase and androgen receptor (AR)/prostate-specific antigen (PSA) levels in vitro were visualized by WB after transient transfection silenced FTO. The effect of DHT combined with FTO inhibitor meclofenamic acid (MA) on FTO, AR/PSA, and AKT phosphorylation were also demonstrated by WB. The co-localization of FTO and AR in KGN cells was analyzed by confocal microscopy, and the physiological interaction between FTO and AR was studied by Co-IP assay. The effect of FTO-specific inhibitor MA, AKT phosphorylation inhibitor LY294002, and the combined them on GCs proliferation and cell cycle were evaluated by drug combination index, EDU assay, and flow cytometry analysis. RESULTS: FTO expression was upregulated in follicular fluid and GCs in PCOS patients clinically. The high FTO expression in patients was negative with the level of m6A, but positive with the level of androgen. The upregulation of FTO was accompanied with a decrease in the level of m6A in PCOS rat with hyperandrogenism. Dihydrotestosterone (DHT) promoted the FTO expression and inhibited m6A content as a dose-dependent way in vitro. In contrast, suppression of FTO with siRNA attenuated the expression of steroid hormone synthetase such as CYP11A1, CYP17A1, HSD11B1, HSD3B2 except CYP19A1 synthetase, ultimately inducing the decrease of androgen level. Suppression of FTO also decreased the biological activity of androgen through downregulation AR/PSA. MA treatment as the specific FTO antagonist decreased cell survival in time- and dose-dependent way in GCs lines. Correspondingly, MA treatment decreased the expression of FTO, AR/PSA expression, and AKT phosphorylation in the presence of DHT stimulation. Additionally, we also speculate there is a potential relation between FTO and AR according to FTO was co-localized and interacted with AR in KGN cells. Compared with AKT phosphorylation inhibitor LY294002 or MA alone, LY294002 combined with MA synergistically inhibited cell survival and increased G2/M phase arrest in GC line. CONCLUSIONS: We first evaluated the correlation of FTO and m6A in PCOS clinically, and further explored the mechanism between FTO and hyperandrogenism in PCOS animal and cell models. These findings contributed the potential therapy by targeting the FTO for hyperandrogenism in PCOS.

Down-regulation of the FTO gene in follicular fluid of infertile women with ovarian endometriosis.

OBJECTIVE: To evaluate FTO concentrations in follicular fluid (FF) of women with ovarian endometriosis (OE) and controls women without OE undergoing in vitro fertilization-embryo transfer (IVF-ET). METHODS: FTO concentrations in FF were measured in 74 patients (37 in the control group and 37 in the OE group) by ELISA. We measured the expression of FTO in GCs of 40 patients (19 in the control group and 21 in the OE group) by RT-qPCR. The level of m6A in GCs was measured in 20 patients (10 in the control group and 10 in the OE group) by colorimetry. RESULTS: Compared with the control group, FTO concentrations in FF (6.92 ± 0.44 vs. 5.67 ± 0.40 ng/ml) (p <.05) and FTO mRNA level in GCs of OE group were decreased significantly (p <.05), and the level of m6A was increased (0.21 ± 0.01 vs. 0.17 ± 0.03 ng) (p >.05). CONCLUSIONS: The FTO concentrations in FF of infertility women with OE are decreased, which may be related to the impaired oocyte quality in endometriosis patients.

Mechanism of Procyanidin B2 in the Treatment of Chronic Myeloid Leukemia Based on Integrating Network Pharmacology and Molecular Docking.

OBJECTIVE: To study the pharmacological mechanism of procyanidin B2 (PCB2) on chronic myeloid leukemia (CML) by integrating network pharmacological methods systematically. METHODS: Firstly, the potential target genes of PCB2 were predicted by the pharmacological database and analysis platform (TCMSP and Pharmmapper). Meanwhile, the relevant target genes of CML were collected from GeneCards and DisGene. Pooled data were collected to screen for common target genes. Furthermore, the above intersection genes were imported into the String website to construct a protein-protein interaction (PPI) network, and the Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were further analyzed. Besides, molecular docking was performed to verify the possible binding conformation between PCB2 and candidate targets. Finally, MTT and RT-PCR experiments of K562 cells were performed to verify the above results of network pharmacology. RESULTS: A total of 229 PCB2 target genes were retrieved, among which 186 target genes had interaction with CML. The pharmacological effects of PCB2 on CML were related to some important oncogenes and signaling pathways. The top ten core targets predicted by Network Analysis were as follows: AKT1, EGFR, ESR1, CASP3, SRC, VEGFA, HIF1A, ERBB2, MTOR, and IGF1. Molecular docking studies confirmed that hydrogen bonding was the main interaction force of PCB2 binding targets. According to the molecular docking score, the following three target proteins were most likely to bind to PCB2: VEGFA (-5.5 kcal/mol), SRC (-5.1 kcal/mol), and EGFR (-4.6 kcal/mol). After treatment of PCB2 for 24h, mRNA expression levels of VEGFA and HIF1A decreased significantly in K562 cells. CONCLUSION: Through integrating network pharmacology combined with molecular docking, the study revealed the potential mechanism of PCB2 anti-chronic myeloid leukemia.

Recombinant human granulocyte colony stimulating factor (rhG-CSF) participates in the progression of implantation via the hsa_circ_0001550-miRNA-mRNA interaction network.

Inadequate endometrial receptivity is a key factor affecting the successful implantation of embryos. Recombinant human granulocyte colony stimulating factor (rhG-CSF) can increase endometrial thickness and improve the outcomes of assisted reproductive technologies (ARTs). In this preliminary study, the function and possible molecular mechanisms of recombinant human granulocyte colony stimulating factor (rhG-CSF) which affects endometrial receptivity and implantation in human Embryonic Stem Cells (hESCs) were investigated. The cell viability of endometrial stromal cells treated with rhG-CSF 0.5 ng/ml for 24 h was significantly increased. Moreover, the expression of hsa_circ_0001550 was downregulated in endometrial stromal cells treated with rhG-CSF. Furthermore, the hsa_circ_0001550-miRNA-mRNA network was constructed and the downstream target genes (including 4 miRNAs and 117 mRNAs) of hsa_circ_0001550 were mainly involved in the cAMP and calcium signalling pathways, which play important roles in regulating endometrial receptivity and embryo implantation. We conclude that rhG-CSF participates in the regulation of embryo implantation by regulating the hsa_circ_0001550-miRNA-mRNA interaction network.

Associations between a new day 4 embryo grading system and implantation rates in frozen embryo transfer (FET) cycles.

The purpose of this study is to present a new day 4 (D4) embryo grading system for the assessment of embryos in frozen-thawed embryo transfer (FET) cycles.A new grading system (grades A-E) was modified from the 2011 ESHRE Istanbul Consensus for D4 embryos in FET cycles. In total, we retrospectively analyzed 5640 embryos with known implantation data after D4 transfer in FET cycles by using this proposed grading model.The transferred embryos exhibited a similar declining trend in implantation rates from the top grade A to the lowest grade E. The implantation rates of grade B and E embryos in the in vitro fertilization group were significantly higher than that in the intracytoplasmic sperm injection group (grade B: 41.82%, 35.23%, χ = 5.85, P < .05 and grade E: 18.53%, 14.81, χ = 76.86, P < .01, respectively). The receiver operating characteristic analysis showed that our proposed model predicted the implantation outcomes of all embryos (area under the ROC curve = 0.65; 95% CI, 0.63-0.66; P < .01).This study demonstrated that the new grading system provided by us turned out to be a useful tool in assisting embryo selection via embryo morphological changes, and D4 embryo transfer provided a simple and applicable method for a daily routine in FET cycles.

Induction of apoptotic DNA fragmentation mediated by mitochondrial pathway with caspase-3-dependent BID cleavage in human gastric cancer cells by a new nitroxyl spin-labeled derivative of podophyllotoxin.

PURPOSE: 4-[4''-(2'', 2'', 6'', 6''-tetramethyl-l''-piperidinyloxy) amino]-4'-demethyl-epipodophyllotoxin (GP7) is a new semi-synthesized nitroxyl spin-labeled derivative of podophyllotoxin with anti-leukemic and anti-osteosarcoma effects. The purpose of the present study is to investigate the anti-gastric cancer (GC) effects of GP7 and the possible involvement of caspase pathway in GP7-induced apoptotic DNA fragmentation in human GC cells. MATERIALS AND METHODS: Effects of GP7 on the proliferation of human GC cell lines MKN28, AGS, BGC-823 and HGC-27 in different degrees of differentiation and normal human gastric epithelial cell line GES-1 were studied by MTT assay and compared with the effects of etoposide. Effects of GP7 on cell viability and heat shock protein 90 expression of BGC-823 and HGC-27 cells were analyzed by trypan blue exclusion test and western blotting, respectively. Effects of GP7 on apoptotic DNA fragmentation and caspase pathway of BGC-823 and HGC-27 cells were detected by agarose gel electrophoresis, colorimetric assay and western blotting. Caspase-3 inhibitor was used to manipulate the activity of caspase-3. RESULTS: GP7 inhibited concentration- and time-dependently the proliferation of human GC cells, and the inhibitory effect of GP7 on the proliferation of BGC-823 or HGC-27 cells was 1.15- or 1.21-fold higher than that of etoposide. GP7 downregulated heat shock protein 90, improved the anti-GC effects of adriamycin, cisplatin, 5-fluorouracil and their combinations, induced apoptotic DNA fragmentation, activations of caspase-9 and -3 but not -8, cytochrome-c release and BID cleavage in BGC-823 and HGC-27 cells. Caspase-3 inhibitor abrogated GP7-induced BID cleavage, decreased cytochrome-c release, caspase-9 and -3 activities and apoptotic DNA fragmentation but increased cell viability in BGC-823 and HGC-27 cells. CONCLUSION: Our findings indicate that GP7 is a promising anti-GC derivative of podophyllotoxin, and GP7-induced apoptosis in human GC cells may be mediated by mitochondrial pathway with caspase-3-dependent BID cleavage.

[5'-flanking regulatory sequence methylation of the Boule gene in the testis tissue of infertile men with Sertoli cell-only syndrome].

OBJECTIVE: To investigate the 5'-flanking regulatory sequence methylation status of the Boule gene in the testis tissue of infertile men with Sertoli cell-only syndrome (SCOS). METHODS: We collected biopsy samples of the testis tissue from 12 men with obstructive azoospermia (the control group) and 15 cases of SCOS, all without varicocele, cryptorchidism, or infectious disease. We extracted genomic DNA from the testis tissue of the SCOS patients, analyzed the characteristics of the 5'-flanking regulatory sequence of the Boule gene using the bioinformatics method, and detected the methylation status of the Boule gene by sodium bisulfite sequencing. RESULTS: A CpG island was observed in the 5'-flanking regulation region of the Boule gene. The methylation level of the Boule gene was remarkably higher in the SCOS group than in the obstructive azoospermia controls (61.4% vs 21.7%, P<0.01), with significant differences in the methylation levels of 14 CpG sites, namely, －58 bp, －50 bp, －48 bp, －38 bp, －28 bp, －24 bp, －20 bp, －15 bp, －1 bp, +5 bp, +8 bp, +15 bp, +29 bp, and +58 bp. CONCLUSIONS: The methylation level of the Boule gene is significantly higher in the SCOS patients than in the obstructive azoospermia males, which suggests that the changes in Boule methylation may be associated with spermatogenic dysfunction.

Reversal of mdr1-mediated multidrug resistance in human leukemia cells by a new spin-labeled derivative of podophyllotoxin.

GP7 (4-[4"-(2", 2", 6", 6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin) is a promising anticancer drug of the podophyllotoxin class. However, little is known about its anti-multidrug resistance effects. In the present study, we investigated the effects of GP7 on P-glycoprotein (P-gp) overexpression multidrug-resistant human leukemia K562/ADM cells with the comparison of VP-16 and K562 cells. GP7 inhibited the proliferation of K562/ADM cells in a concentration- or time-dependent manner, and the inhibitory effect of GP7 on K562/ADM cells was 1.50-fold higher than that of VP-16. GP7 caused G2/M phase accumulation but VP-16 caused S phase accumulation in K562/ADM and K562 cells. GP7 could induce apoptosis of both K562/ADM and K562 cell lines, but there was no significant difference between GP7- and VP-16-induced apoptotic ratios. GP7 could also induce typical apoptotic morphological changes and internucleosomal DNA fragmentation of K562/ADM and K562 cells, but DNA fragmentation induced by GP7 in K562/ADM cells was weaker than that in K562 cells. When treated with GP7 or VP-16 for 48 h, 128-256 microM GP7 induced more DNA fragmentation than VP-16 did, but 32-64 microM GP7 induced less DNA fragmentation than VP-16 did. GP7 could down-regulate the expression of P-gp in K562/ADM cells but VP-16 could not. Our findings suggest that GP7 may reverse multidrug resistance in human leukemia K562/ADM cells via down-regulation of P-gp expression.

GP7 induces internucleosomal DNA fragmentation independent of caspase activation and DNA fragmentation factor in NB4 cells.

DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process.