Insulin receptor tyrosine kinase activity and substrate 1 (IRS-1) expression in human myometrium and leiomyoma

BACKGROUND: Uterine leiomyomas are the commonest tumors of the genital tract. Growth factors seem to be implicated in the development of leiomyoma. OBJECTIVE: To determine the insulin receptor (IR) tyrosine kinase activity--phosphorylation of exogenous substrate poly(Glu 4: Tyr 1)--and insulin receptor substrate 1 expression in normal myometrium and leiomyoma. STUDY DESIGN: The study group consisted of 12 women with leiomyoma undergoing routine hysterectomy. Samples of leiomyoma and adjacent normal myometrium were obtained at the time of operation. Plasma membrane fractions were prepared and samples were incubated with and without insulin and incubated with exogenous substrate poly(Glu 4: Tyr 1). IRS-1 expression was studied in the whole lysate via Western blotting using specific antibodies. Data were analyzed using Student's t-test. RESULTS: The phosphorylation of the exogenous substrate poly(Glu 4: Tyr 1) in myometrium (1.566+/-0.177) and in leiomyoma (1.98+/-0.612) were similar (P=0.774). The IRS-1 levels in myometrium (0.190+/-0.022) and in leiomyoma (0.226+/-0.022) were not different (P=0.184). CONCLUSIONS: There was no difference in IR tyrosine kinase activity (phosphorylation of exogenous substrate) and IRS-1 expression between normal myometrium and leiomyomata. Other steps in the insulin signaling cascade require further study to investigate the role of insulin receptor in leiomyomata.     

Prevalence of the insulin receptor substrate-1(IRS-1) Gly972Arg and the insulin receptor substrate-2(IRS-2) Gly1057Asp polymorphisms in PCOS patients and non-diabetic healthy women

Objective  

The aim of our study was to investigate the frequency and compare the prevalence of IRS-1Gly972Arg and IRS-2 Gly1057Asp polymorphisms in PCOS patients and non-diabetic healthy women.     

瘦素、胰岛素及IGF-2影响滋养层细胞中PKB和P70S6K蛋白表达的机制

目的:探讨瘦素、胰岛素及IGF-2对滋养层细胞中PKB和P70S6K蛋白表达的影响是否与PI3K/PKB/mTOR信号途径存在联系。方法:应用Western blot检测瘦素、胰岛素及IGF-2作用后滋养层细胞中PKB和P70S6K蛋白的表达,以及分别加入PI3K的特异性抑制剂LY294002和mTOR的特异性抑制剂雷帕霉素后对PKB和P70S6K蛋白表达的影响。结果:瘦素、胰岛素及IGF-2刺激组滋养层细胞中PKB和P70S6K蛋白表达与对照组相比明显增加(P0.05)。瘦素、胰岛素及IGF-2刺激组分别加入LY294002和雷帕霉素,PKB和P70S6K蛋白表达均明显降低(P0.05)。结论:瘦素、胰岛素及IGF-2可能通过PI3K/PKB/mTOR信号通路促进滋养层细胞中PKB和P70S6K蛋白表达。     

Enhancement of the efficiency of oocyte vitrification through regulation of histone deacetylase 6 expression

Objective

Successful oocyte vitrification (OV) is critical for cryopreservation of the oocytes from female patients with infertility, polycystic ovaries, and gynecologic cancers. Recent evidence suggests that relatively low levels of histone acetylation are critical for maintenance of the maturation capacity of cryopreserved oocytes. However, previous studies have only demonstrated a key role of histone deacetylases (HDAC) 1 and 2 in the cryopreservation of oocytes.

Methods

In this study, we investigated the role of HDAC6 in these settings. We found that mouse oocytes with low HDAC6 levels decreased survival rate, cleavage rate, and blastocyst rate after OV. Bioinformatics analyses were used to predict HDAC6-targeting microRNAs (miRNAs), while the functional binding of miRNAs to HDAC6 mRNA was evaluated by a dual luciferase reporter assay.

Results

Among all HDAC6-targeting miRNAs, we detected expression of miR-558, miR-527, and miR-762 in mouse oocytes. Specifically, we found that only miR-762 significantly inhibited protein translation of HDAC6 via binding to the 3′-UTR of the HDAC6 mRNA. Transfection of oocytes with HDAC6 or antisense of miR-762 significantly increased the survival rate, the cleavage rate, and blastocyst rate after OV.

Conclusion

As a result, our data suggest that induction of HDAC6 levels by miR-762 suppression may improve the current protocol for OV.

     

Gonadotropin-releasing hormone (GnRH)-I regulation of interleukin (IL)-1b and IL-1 receptor antagonist expression in cultured human endometrial stromal cells

Aim:  Human endometrium is an active site of cytokine production and action. Among these cytokines, the interleukin-1 (IL-1) system seems to be relevant to the embryonic implantation process. We have previously reported the production of GnRH-I by human blastocyst, as well as the presence of GnRH-I receptor in human endometrium. This suggests a close interaction between the immune and endocrine systems through these cytokine mediators in embryonic implantation.
Methods:  To test the relevance of this interaction during embryonic implantation, we investigated GnRH-I regulation of IL-1b and IL-1ra mRNA and protein expression in human endometrial stromal cells using quantitative competitive polymerase chain reaction and ELISA.
Results:  IL-1b mRNA and protein expression in cultured human endometrial stromal cells was significantly enhanced by GnRH-agonist in comparison to control groups. IL-1ra mRNA and protein was significantly decreased by GnRH-agonist in comparison to control groups. In contrast, the GnRH-antagonist ablated the regulatory effects of GnRH agonist in 1b and IL-1ra mRNA and protein levels in a dose-dependent manner.
Conclusions:  In conclusion, these results suggest a possible close interaction between the immune and endocrine systems in human embryonic implantation through the classical neuropeptide hormone GnRH and its receptor.     

S1P/S1PR对人卵巢癌SKOV3细胞的促血管生成作用的研究

目的：探究鞘氨醇-1-磷酸/鞘氨醇-1-磷酸受体（S1P/S1PR）对人卵巢癌SKOV3细胞促血管生成作用的影响和机制。方法：管腔形成实验探究S1P对人卵巢癌SKOV3细胞的促血管生成作用的影响;实时定量聚合酶链式反应（qRT-PCR）检测S1P处理后的人卵巢癌SKOV3细胞中血管生成因子白细胞介素8（IL-8）、IL-6、血管内皮生长因子（VEGF）的变化情况;设计合成小干扰RNA（siRNA）干扰序列基因沉默SKOV3细胞中S1PR1、S1PR2和S1PR3的表达,蛋白质印迹（Western-blot）和qRT-PCR检测SKOV3细胞中S1PR的下调结果;qRT-PCR检测S1PR对SKOV3细胞IL-8、IL-6、VEGF表达的影响。结果：管腔形成实验结果显示,S1P处理后的SKOV3细胞培养上清液重悬人脐静脉内皮细胞融合细胞（EA.hy926）,其管腔形成的数量多于对照组（t=3.667,P=0.021）,表明S1P促进了人卵巢癌细胞SKOV3的促血管形成能力。S1P处理后的SKOV3细胞中血管生成因子IL-8、IL-6、VEGF mRNA的表达水平较对照组升高,差异有统计学意义（均P＜0.05）。S1PR1和S1PR3基因沉默可显著降低SKOV3细胞中IL-8、IL-6、VEGF的mRNA表达水平,差异有统计学意义（均P＜0.05）,而S1PR2基因沉默后IL-8、IL-6、VEGF的mRNA变化不明显。结论：S1P通过S1PR1/3上调人卵巢癌SKOV3细胞中IL-8、IL-6、VEGF的表达,从而增强了SKOV3细胞的促血管生成作用,S1P/S1PR通路有望成为抑制卵巢癌生长的治疗新靶点。     

Study on the expression of angiotensin II (ANG II) receptor subtype 1 (AT1R) in the placenta of pregnancy-induced hypertension

OBJECTIVE: To study the expression of angiotensin II (ANG II) receptor subtype 1 (AT(1)R) in the human placenta with pregnancy-induced hypertension (PIH). METHODS: Immunohistochemistry was used to detect the expression of AT(1)R in placental tissues of 30 patients with PIH and 10 patients with normal pregnancies (control group). The PIH tissues were further divided into 3 groups: mild PIH group, moderate PIH group and severe PIH group. Each group consisted of 10 patients. A high-resolution pathological image analysis system (HPIAS-1000) was used to determine the quantity of AT(1)R expression. RESULTS: The integral optical density and area of staining in the syncytiotrophoblast (STB) layer and villous endothelium of the placenta were significantly increased in PIH patients, in the moderate and severe PIH groups, as compared with the control group (P < 0.05), indicating that the expression of AT(1)R was highly increased in PIH. However, there was no significant difference between normal pregnancy and the mild PIH group (P > 0.05). Furthermore, statistically significant differences in AT(1)R expression were observed between mild, moderate and severe PIH groups (P < 0.05). CONCLUSION: The expression of AT(1)R is statistically significantly increased in the STB layer and villous endothelium of human placenta with PIH. Expression increases with the severity of the disease. Increased expression may be involved in the pathogenesis of PIH.     

米非司酮对子宫肌瘤胰岛素样生长因子-1的影响

目的观察米非司酮对子宫肌瘤组织中胰岛素样生长因子-1(IGF-1)表达及比不同治疗效果间肌瘤组织IGF—1表达的差异,探讨疗效的个体差异原因。方法子宫肌瘤患者手术前每天服用米非司酮10mg,共3个月,于治疗前后B超测定最大肌瘤三维径线,判定肌瘤体积变化,另采用免疫组化法(ABC法)对米非司酮治疗组和对照组不同月经期的子宫肌瘤组织中IGF-1的表达进行分析,以观察不同组间IGF—1水平的差别。结果①对照组肌瘤IGF-1的染色在增生期和分泌期无明显差异,均呈强阳性;②治疗组肌瘤IGF-1的染色呈不同程度减弱或阴性,与对照组差异显著(P<0.05);③治疗组中肌瘤缩小明显者IGF-1的染色弱于肌瘤缩小不明显者,并有显著差异(P<0.05)。结论①米非司酮可能通过抑制IGF-1的合成使肌瘤细胞生长受抑,肌瘤体积缩小。②不同治疗效果与IGF-1表达的受抑程度有关。     

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration

IntroductionFailure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function.MethodsA transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)₂D₃. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression.ResultsEVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)₂D₃ for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)₂D₃ (p < 0.05).DiscussionThis study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα_12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.