FSH induces EMT in ovarian cancer via ALKBH5-regulated Snail m6A demethylation.

Background: The therapeutic benefits of targeting follicle-stimulating hormone (FSH) receptor in treatment of ovarian cancer are significant, whereas the role of FSH in ovarian cancer progresses and the underlying mechanism remains to be developed. Methods: Tissue microarray of human ovarian cancer, tumor xenograft mouse model, and in vitro cell culture were used to investigate the role of FSH in ovarian carcinogenesis. siRNA, lentivirus and inhibitors were used to trigger the inactivation of genes, and plasmids were used to increase transcription of genes. Specifically, pathological characteristic was assessed by histology and immunohistochemistry (IHC), while signaling pathway was studied using western blot, quantitative RT-PCR, and immunofluorescence. Results: Histology and IHC of human normal ovarian and tumor tissue confirmed the association between FSH and Snail in ovarian cancer metastasis. Moreover, in epithelial ovarian cancer cells and xenograft mice, FSH was showed to promote epithelial mesenchymal transition (EMT) progress and metastasis of ovarian cancer via prolonging the half-life of Snail mRNA in a N6-methyladenine methylation (m6A) dependent manner, which was mechanistically through the CREB/ALKBH5 signaling pathway. Conclusions: These findings indicated that FSH induces EMT progression and ovarian cancer metastasis via CREB/ALKBH5/Snail pathway. Thus, this study provided new insight into the therapeutic strategy of ovarian cancer patients with high level of FSH.

Mutation of aspartic acid 262 on estrogen receptor abrogates estradiol signaling pathway.

OBJECTIVE: ERα (estrogen receptor alpha) exerts nuclear genomic actions and membrane-initiated non-genomic effects. The mutation of aspartic acid into alanine in vitro revealed the critical role of aspartic acid 258 (corresponding to mouse amino acid site 262) of ERα for non-nuclear function. Our previous in vitro study revealed that this mutation blocked estrogen's non-genomic effects on vascular endothelial H2S release. Here, we studied the in vivo role of the aspartic acid 262 of ERα in the reproductive system and in the vascular tissue. APPROACH AND RESULTS: We generated a mouse model harboring a point mutation of the murine counterpart of this aspartic acid into alanine (ERαD262A). Our results showed that the ERαD262A females are fertile with standard hormonal serum levels, but the uterine development and responded with estrogen and follicular development are disrupted. In line with our previous study, we found that the rapid dilation of the aorta was abrogated in ERαD262A mice. In contrast to the previously reported R264-ERα mice, the classical estrogen genomic effector SP1/NOS3/AP1 and the nongenomic effectors p-eNOs, p-AKT, and p-ERK were disturbed in the ERαD262A aorta. Besides, the serum H2S concentration was decreased in ERαD262A mice. Together, ERαD262A mice showed compromised both genomic and non-genomic actions in response to E2. CONCLUSIONS: These data showed that aspartic acid 262 of ERα are important for both genomic and non-genomic effects of E2. Our data provide a theoretical basis for further selecting an effective non-genomic mouse model and provide a new direction for developing estrogen non-genomic effect inhibitors.

Association between greenness and dyslipidemia in patients with coronary heart disease: A proteomic approach.

BACKGROUND: Residential surrounding greenness may be protective of dyslipidemia are often theorized but remain poorly quantified. In particular, the underlying biological mechanisms of blood lipid changes with green spaces remain unclear. METHODS: Our observational epidemiology study included a residentially stable sample of 1035 coronary heart disease patients, and proteomics study included 16 participants. Normalized Difference Vegetation Index (NDVI) was used to evaluate residential greenness exposures. Proteomics technology was used to identify plasma greenness-related proteome disturbance, and the pathway analysis was employed to evaluate the potential biological mechanisms of greenness decreasing dyslipidemia risk. RESULT: Higher residential surrounding greenness in the 500-m area was associated with lower risks of dyslipidemia (odds ratio (OR) = 0.871, 95% confidence interval (CI): 0.763, 0.994 for per one-quartile NDVI increase). Lymphocytes mediated 18.7% of the association between greenness and dyslipidemia. Greenness related proteins (including PLXDC1, IGFBP2 and LY6D) may regulate the biological functions of lipid metabolism and transport-related proteins (including ADIPOQ and CES1) through a series of biological processes. CONCLUSION: People in greener surroundings have a lower risk of dyslipidemia, which may be due to their lower inflammation, stronger lipid transporter activity, and normal cholesterol metabolism.

17ß-estradiol promotes angiogenesis through non-genomic activation of Smad1 signaling in endometriosis.

17ß-estradiol (E2) plays a key role in endometriosis through regulation of angiogenesis. Smad1 has been reported to be up-regulated in patients with endometriosis. However, the role of Smad1 in E2-mediated angiogenesis during the development of endometriosis remains to be determined. This study aimed to explore the role of Smad1 in E2-mediated angiogenesis during endometriosis and its underlying mechanisms. Immunofluorescence staining and Western blotting were performed to examine the expression of p-Smad1 in ectopic and control endometrium. Western blotting was used to examine activation of Smad1 signaling in NMECs, EMECs and HUVECs. Tube formation assay was performed to examine the effect of E2 on angiogenesis. Cell proliferation and migration was determined using in real-time by xCELLigence RTCA DP instrument. We found that the expression of p-Smad1 was significantly up-regulated in ectopic endometrium and ectopic intima microvascular endothelial cells. E2 non-genomically stimulated phosphorylation of Smad1 in HUVECs. c-Src and p44/42 MAPK(ERK1/2) signaling pathways are required for E2's induction on Smad1 phosphorylation. Moreover, caveolae is involved in E2-induced Smad1 phosphorylation in vascular endothelial cells. E2 promoted tube formation of vascular endothelial cells through c-Src/ERK1/2/Smad1 signaling pathway. Knockdown of Smad1 expression attenuated E2-induced proliferation and migration of HUVECs. In conclusion, E2 promotes proliferation, migration and tube formation of HUVECs through c-Src/ERK1/2/Smad1 signaling pathway. Our data shed new lights on the mechanisms through which E2 contributes to endometriosis, and may provide novel strategies to treat endometriosis.

Association Between Circular RNAs and Intracranial Aneurysm Rupture Under the Synergistic Effect of Individual Environmental Factors.

Introduction: To study the association between specific circular RNAs and rupture of intracranial aneurysm. To explore its clinical diagnostic significance and synergistic effects with individual environmental influencing factors. Methods: Three hundred and forty seven cases and controls were included in this study. Multivariate analysis was used to explore the main individual environmental factors. Intracranial aneurysm rupture related circular RNAs screened based on sequencing was verified in peripheral blood by PCR. ROC curve, logistic regression model and fork analysis were used to study the association, diagnostic values, and synergistic effects of circular RNA with intracranial aneurysms and individual environmental factors. Results: Smoking, hair dyeing, sitting time ≥6 h/day, single animal oil intake and hypertension are the main risk factors for intracranial aneurysm rupture; People with higher education, sleeping time ≥7 h/day, tea drinking, diabetes, higher levels of (hemoglobin, low density lipoprotein, serum calcium, and apolipoprotein-A1) have a low risk of intracranial aneurysm rupture. Hsa_circ_0008433 and hsa_circ_0001946 are closely related to intracranial aneurysm rupture and have certain clinical diagnostic significance (AUC = 0.726; 95% CI: 0.668~0.784). Hsa_circ_0008433 (OR = 0.497, 95% CI: 0.338~0.731), hsa_circ_0001946 (OR = 0.682, 95% CI: 0.509~0.914) were independent epigenetic factors affecting intracranial aneurysm rupture, and have a multiplicative interaction with age (OR = 3.052, 95% CI: 1.006~9.258). Conclusions: Low expressions of hsa_circ_0008433 and hsa_circ_0001946 are risk factors for intracranial aneurysms rupture and have good clinical diagnostic value. There was a multiplicative interaction between epigenetic score and age. The older and the higher the epigenetic score was, the more likely to have intracranial aneurysm rupture.

lncRNA ZFAS1 Is Involved in the Proliferation, Invasion and Metastasis of Prostate Cancer Cells Through Competitively Binding to miR-135a-5p.

BACKGROUND: Prostate cancer (PCa) is a common malignant tumor in men. lncRNA ZFAS1 plays a carcinogenic role in many types of cancer; however, its potential role in PCa remains unclear. The current study aimed to determine the expression and function of ZFAS1 in PC. METHODS: The ZFAS1 expression in PC tissues and cells was determined by quantitative polymerase chain reaction (qPCR). SiZFAS1, miR-135a-5p mimic and miR-135a-5p inhibitor were transfected into PCa cells. The direct target of ZFAS1 was predicted by Starbase and verified by dual-luciferase reporter. Cell viability, proliferation, apoptosis, migration and invasion of the PCa cells were determined by cell counting kit-8, clone formation assay, flow cytometer, scratch and Transwell assay, respectively. The expression levels of related proteins and mRNAs were determined by Western blotting and qPCR. RESULTS: ZFAS1 expression was up-regulated in PCa cells and tissues. ZFAS1 could competitively bind to miR-135a-5p in PCa cells, and down-regulation of ZFAS1 inhibited cell viability, proliferation, migration, invasion of PCa cells and the occurrence of epithelial-mesenchymal transformation (EMT) and promoted apoptosis of PCa cells and increased the miR-135a-5p expression. Moreover, the function of miR-135a-5p mimic in PCa cells was consistent with ZFAS1 knockdown, while the function of miR-135a-5p inhibitor was opposite to that of miR-135a-5p mimic in PCa cells. The results showed that knocking down ZFAS1 could attenuate the effects of miR-135a-5p inhibitor on cell proliferation, invasion and EMT of PCa cells. CONCLUSION: Knocking down ZFAS1 could inhibit the proliferation, invasion and metastasis of PCa cells through regulating miR-135a-5p expression.

17ß-Estradiol nongenomically induces vascular endothelial H2S release by promoting phosphorylation of cystathionine γ-lyase.

Estrogen exerts its cardiovascular protective role at least in part by regulating endothelial hydrogen sulfide (H2S) release, but the underlying mechanisms remain to be fully elucidated. Estrogen exerts genomic effects, i.e. those involving direct binding of the estrogen receptor (ER) to gene promoters in the nucleus, and nongenomic effects, mediated by interactions of the ER with other proteins. Here, using human umbilical vein endothelial cells (HUVECs), immunological detection, MS-based analyses, and cGMP and H2S assays, we show that 17ß-estradiol (E2) rapidly enhances endothelial H2S release in a nongenomic manner. We found that E2 induces phosphorylation of cystathionine γ-lyase (CSE), the key enzyme in vascular endothelial H2S generation. Mechanistically, E2 enhanced the interaction of membrane ERα with the Gα subunit Gαi-2/3, which then transactivated particulate guanylate cyclase-A (pGC-A) to produce cGMP, thereby activating protein kinase G type I (PKG-I). We also found that PKG-Iß, but not PKG-Iα, interacts with CSE, leading to its phosphorylation, and rapidly induces endothelial H2S release. Furthermore, we report that silencing of either CSE or pGC-A in mice attenuates E2-induced aorta vasodilation. These results provide detailed mechanistic insights into estrogen's nongenomic effects on vascular endothelial H2S release and advance our current understanding of the protective activities of estrogen in the cardiovascular system.

Elevated luteinizing hormone contributes to atherosclerosis formation by inhibiting nitric oxide synthesis via PI3K/Akt pathway.

BACKGROUND: The contentious effects of estrogen therapy on the risk of postmenopausal cardiovascular disease (CVD) indicate that this type of atherosclerosis is not solely induced by estrogen deficiency. Other sex hormones such as elevated luteinizing hormone (LH) may also affect CVD risk in this population. We therefore explored the relationship between LH and atherosclerosis in ovariectomized (OVX) female mice. METHODS: Aortic atherosclerotic lesions were assessed in OVX ApoE knock out (ApoE-/-) female mice administered with LH. Human umbilical vascular endothelial cells (HUVECs) were cultured as cell model. The influence of LH on NO release, phosphorylated endothelial nitric oxide synthase (eNOS) and Akt levels were evaluated. Immunoprecipitation and lentiviral particle transfection were applied to assess the role of Gαq on PI3K activity. RESULTS: LH increased the atherosclerotic lesion area and carotid artery intima-media thickness (IMT) in OVX ApoE-/- female mice. High levels of LH attenuated vasodilation induced by Ach and inhibited NO release from HUVECs. These effects were related to the findings that LH enhanced interaction between Gαq and p110α, which subsequently inhibited PI3K activity and suppressed the phosphorylation of Akt and eNOS. CONCLUSIONS: Elevated LH promotes atherosclerosis formation in OVX ApoE-/- female mice. This effect may be mediated by inhibiting endothelial NO synthesis via PI3K/Akt signaling pathway.

Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression.

Rationale: Postmenopausal atherosclerosis (AS) has for decades been attributed to estrogen deficiency. Although the follicular stimulating hormone (FSH) levels rise sharply in parallel, the direct effect of FSH on AS has never been investigated. In this study, we explored the possible role of FSH in the development of AS. Methods: This was a prospective cohort study of 48 healthy premenopausal and 15 postmenopausal women. ApoE knockout mice were used as atherosclerosis model and human umbilical vascular endothelial cells (HUVECs) were cultured as cell model. Serum hormones and vascular cell adhesion molecule-1 (VCAM-1) levels were measured. Real-time PCR, histology for atherosclerotic lesions, immunofluorescence, luciferase assay, transfection experiments, flow chamber adhesion assay and western blot were performed. Results: In ApoE knockout mice, administration of FSH increased the atherosclerotic lesions and serum VCAM-1 concentration. Importantly, in blood samples of postmenopausal women, we detected significantly higher levels of FSH and VCAM-1 compared with those from premenopausal women, and there was a positive correlation between these two molecules. In cultured HUVECs, FSH receptor (FSHR) mRNA and protein expression were detected and FSH enhanced VCAM-1 expression. This effect was mediated by the activation of nuclear factor κB (NF-κB), which was sequentially enhanced by the activation of PI3K/Akt/mTOR cascade. FSH first enhanced GαS activity resulting in elevated cAMP level and PKA activity, which relayed the signals from FSHR to the PI3K/Akt/mTOR cascade. Furthermore, FSHR was detected in endothelial caveolae fraction and interacted with caveolin-1 and GαS. The disruption of caveolae or the silencing of caveolin-1 blocked FSH effects on signaling activation and VCAM-1 expression, suggesting the existence of a functional signaling module in membrane caveolae. Finally, FSH increased human monocyte adhesion to HUVECs which was reversed by the VCAM-1 neutralizing antibody. Conclusion: FSHR was located in the membrane caveolae of HUVECs and FSH promoted VCAM-1 expression via FSHR/GαS /cAMP/PKA and PI3K/Akt/mTOR/NF-κB pathway. This may contribute to the deleterious role of FSH in the development of AS in postmenopausal women.