PI3K/Akt信号通路与卵巢早衰相关性的研究进展

近年研究发现,PI3K/Akt信号通路参与卵巢早衰(POF)疾病发生。磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(Akt)的过度激活可使原始卵泡过早发育及卵泡过快凋亡,进而发展成为POF。本文就关于PI3K/Akt信号通路与POF关系的研究进展做一综述。     

过表达MicroRNA-195抑制子宫内膜癌细胞系Ishikawa的增殖、迁移与侵袭能力

目的:观察MicroRNA-195对子宫内膜癌细胞增殖、迁移与侵袭能力的影响。方法:取对数生长期人子宫内膜癌细胞系Ishikawa,分别转染MicroRNA-195模拟物(MicroRNA-195过表达组)、MicroRNA-195抑制物(MicroRNA-195低表达组)及阴性对照质粒MicroRNA-NC(空白质粒组)。转染48h后,qRT-PCR法检测细胞中MicroRNA-195表达,CCK-8法检测细胞增殖,Transwell小室实验检测细胞迁移与侵袭能力,Western blot法检测Akt、p-Akt蛋白表达。将转染MicroRNA-195模拟物、MicroRNA-195抑制物及阴性对照质粒MicroRNA-NC的人子宫内膜癌细胞系Ishikawa分别注射至裸鼠皮下,每天检测肿瘤体积,实验周期为3周。结果:MicroRNA-195过表达可抑制人子宫内膜癌细胞系Ishikawa的增殖、迁移与侵袭能力(P<0.05),提高p-Akt蛋白表达(P<0.05);MicroRNA-195低表达可促进人子宫内膜癌细胞Ishikawa的增殖、迁移与侵袭能力(P<0.05),降低p-Akt蛋白表达(P<0.05)。MicroRNA-195过表达可抑制裸鼠体内人子宫内膜癌细胞Ishikawa的增殖(P<0.05),MicroRNA-195低表达可促进裸鼠体内人子宫内膜癌细胞Ishikawa的增殖(P<0.05)。结论:MicroRNA-195可抑制子宫内膜癌细胞Ishikawa的增殖、迁移与侵袭能力,其作用机制可能与激活PI3K/Akt信号通路有关。     

MiR-506通过PI3K/AKT信号通路调控人绒毛膜癌细胞增殖、迁移和侵袭

目的 探讨MiR-506是否通过PI3K/AKT通路对人绒毛膜癌细胞JEG-3的增殖、迁移、侵袭和凋亡发挥调控作用。方法 采用qRT-PCR法测定绒毛膜滋养层细胞HTR8/Svneo和绒毛膜癌细胞JEG-3中MiR-506的表达水平,将JEG-3细胞分组后转染MiR-506模拟物或抑制物后进行培养,采用CCK-8法测定细胞增殖情况,采用流式细胞术测定各组细胞凋亡情况,采用Western blot法测定PI3K/AKT通路相关蛋白表达情况。结果JEG-3细胞中MiR-506表达水平较HTR8/Svneo细胞降低;MiR-506模拟物组JEG-3细胞中MiR-506表达水平升高,MiR-506抑制物组MiR-506表达水平降低;MiR-506模拟物组24 h、48 h、72 h时吸光度增加,而MiR-506抑制物组吸光度显著降低;MiR-506模拟物组细胞凋亡数量升高（P <0.05）,而MiR-506抑制物组细胞凋亡数量降低（P <0.05）;MiR-506模拟物组PI3K和AKT蛋白表达水平降低（P <0.05）,MiR-506抑制物组PI3K和AKT蛋白表达水平增加...     

MiR-506通过PI3K/AKT信号通路调控人绒毛膜癌细胞增殖、迁移和侵袭#br#

Objective?To investigate the regulation of miR-506 on proliferation, migration, invasion and apoptosis of choriocarcinoma JEG-3 cells via PI3K/AKT signal pathway. Methods?qRT-PCR was used to determine miR-506 in JEG-3 cells and normal HTR8/Svneo cells. JEG-3 cells were cultured after transfection and divided into different groups. CCK 8 method was used to determine cell proliferation. Flow cytometry was used to determine cell apoptosis. Western-blot were used to determine and protein related to PI3K/AKT signal pathway. Results?The expression of miR-506 in JEG-3 cells was increased compared with that in HTR8/Svneo cells; miR-506 in miR-506 mimics group increased, while MiR-506 in miR-506 inhibitor group decreased. The absorbance value in miR-506 mimic group at 24 h, 48 h and 72 h decreased , while absorbance value in miR-506 inhibitor group at 24 h, 48 h and 72 h increased. The apoptosis rate in miR-506 group increased, while the apoptosis rate in miR-506 inhibitor group decreased. The mRNA and protein levels of PI3K and AKT in miR-506 mimic group decreased, while the mRNA and protein levels of PI3K and AKT in miR-506 inhibitor group increased. Conclusion?MiR-506 is a tumor suppressor gene of choriocarcinoma, which may regulate the proliferation, migration, invasion and apoptosis of choriocarcinoma of choriocarcinoma cells via the PI3K/AKT signal pathway.     

芬太尼通过PI3 K/Akt信号通路调控宫颈癌细胞增殖迁移

目的:探究芬太尼对宫颈癌细胞增殖迁移的作用及分子机制.方法:将宫颈癌细胞系C33 A分为对照组和芬太尼组(使用不同浓度芬太尼处理),分别采用CCK-8、Transwell法、流式细胞术检测芬太尼对C33 A细胞增殖、迁移及凋亡的影响.Western blot法检测活化Caspase 3、PARP1含量,MMP-2、MM...     

PI3K/Akt/mTOR信号通路靶向治疗在子宫内膜癌中的研究进展

子宫内膜癌是女性高发的恶性肿瘤之一,在生殖系统中常见的恶性肿瘤位居第三,近年其发病率呈上升趋势。磷磷脂酰肌醇-3-激酶/蛋白激酶B/雷帕霉素靶蛋白(PI3K/Akt/m TOR)信号通路是子宫内膜癌中发生最频繁的信号转导通路之一。有研究证实,24%~39%的子宫内膜癌患者存在PI3K/Akt/m TOR信号通路的异常激活。众多研究表明,其通路中分子的改变不同程度的参与并影响了多种生物功能,PI3K/Akt/m TOR信号通路的抑制剂在子宫内膜癌治疗中也起到了重要作用,并引发了其机制研究的热潮。临床前期实体肿瘤和临床各期试验研究均在证明不同种类PI3K/Akt/m TOR信号通路抑制剂的作用。本文将对子宫内膜癌中PI3K/Akt/m TOR信号通路整体阐述,进而对该通路各个组件相关的抑制剂做总结,从而对于药物靶向治疗或联合其他方案治疗子宫内膜癌提供广阔的治疗思路。     

过表达miR-198通过靶向调控Wnt2 B表达抑制滋养细胞HTR-8/SVneo的增殖、转移与侵袭

目的:研究过表达miR-198对人滋养细胞株HTR-8/SVneo增殖、转移和侵袭能力的影响,并探讨其可能作用机制.方法:qRT-PCR检测子痫前期(PE)患者及正常产妇胎盘组织中miR-198表达水平.培养人滋养细胞株HTR-8/SVneo,采用脂质体法将miR-198 mimics及其阴性对照mimics NC转染...     

色素上皮衍生因子通过抑制PI3K/Akt通路降低宫颈癌HeLa细胞活性和侵袭相关蛋白表达

目的：探讨色素上皮衍生因子（PEDF）通过抑制磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号通路,从而下调人宫颈癌HeLa细胞的活性和侵袭相关蛋白c-Met表达的相关机制。方法：选取本院病理已确诊的40例宫颈癌样本及对应的40例癌旁正常组织样本,通过免疫组织化学法检测PEDF、PI3K和p-Akt蛋白的表达。在细胞水平分为正常宫颈上皮细胞HCerEpiC组、HeLa组、HeLa+PEDF组、HeLa+PEDF+PI3K/Akt激活剂组和HeLa+PI3K/Akt抑制剂组。通过蛋白质印迹（Western blotting）检测各组细胞内PEDF、PI3K、p-Akt、Akt及c-Met蛋白的表达水平,CCK-8法检测各组细胞的活性。结果：宫颈癌样本和HeLa细胞中的PI3K/Akt通路显著激活,且PEDF表达显著下降（P＜0.01）。PEDF可以显著抑制HeLa细胞中PI3K/Akt激活和细胞活性以及侵袭相关蛋白水平的增加（P＜0.01）。Akt激活剂可以逆转PEDF对HeLa细胞活性和侵袭蛋白表达的影响,而激活PI3K无法改变PEDF的作用。单纯的药物抑制PI3K/Akt通路不足以模拟PEDF对HeLa细胞的作用。结论：PEDF通过PI3K/Akt双重抑制作用降低HeLa细胞活性和侵袭相关蛋白表达,这为临床上PEDF治疗宫颈癌提供了新的思路和理论依据。     

TFF3激活PI3K/Akt通路参与宫颈腺癌的发病机制

目的:探讨TFF3通过激活PI3K/Akt信号通路调控宫颈腺癌发生发展的机制。方法:TFF3细胞因子和PI3K/Akt信号通路抑制剂(LY294002)或两者共同处理Hela细胞,CCK-8法检测Hela细胞增殖能力,Transwell小室观察细胞迁移能力,流式细胞学方法检测细胞凋亡,Western blot检测PI3K/Akt信号通路关键蛋白Akt及其激活状态蛋白p-Akt的表达。结果:与对照组相比,TFF3处理后Hela细胞的增殖和迁移能力明显提高,细胞凋亡减弱,差异有统计学意义(P0.05);LY294002处理后Hela细胞的增殖及迁移能力明显降低,细胞凋亡增多,差异有统计学意义(P0.05);TFF3+LY294002处理后Hela细胞的增殖、迁移能力及细胞凋亡与TFF3和LY294002处理组比较,差异均有统计学意义(P0.05)。Western blot结果表明,TFF3能激活p-Akt基因表达。结论:TFF3能通过提高p-Akt蛋白表达激活PI3K/Akt信号通路,从而参与调节宫颈腺癌细胞的恶性行为。     

抵抗素促进子宫内膜癌细胞生长作用机制的探讨

目的:研究抵抗素对子宫内膜癌细胞增殖的影响及其对子宫内膜癌细胞PI3K/Akt信号传导通路的作用。方法:CCK-8法检测不同浓度抵抗素(0ng/ml、10ng/ml、50ng/ml、100ng/ml、200ng/ml)对Ishikawa和KLE两种子宫内膜癌细胞系增殖的影响。Western blot法检测不同浓度抵抗素作用下PI3K/Akt信号通路的变化及PI3K/Akt信号通路抑制剂对抵抗素活化PI3K/Akt信号通路作用的影响。结果:抵抗素能促进两种子宫内膜癌细胞系Ishikawa和KLE的生长,并呈时间和浓度依赖性。抵抗素可促进两种子宫内膜癌细胞系中PI3K/Akt信号通路的激活。PI3K/Akt信号通路抑制剂LY29004可减弱抵抗素诱导的Akt磷酸化水平。结论:抵抗素能促进子宫内膜癌细胞增殖,这种作用可通过激活PI3K/Akt信号通路来实现。     

CDX1 restricts the invasion of HTR-8/SVneo trophoblast cells by inhibiting MMP-9 expression

Introduction

Pathogenesis of early-onset preeclampsia (PE) is generally recognized by impaired trophoblast invasion of the myometrial arteries, which results in placental insufficiency. Recently, we reported that CDX1 is hypermethylated in the human preeclampsia placenta. However, whether CDX1 participates in trophoblast invasion has not been clearly elucidated.

Methods

We investigated the function of CDX1 in the extravillous trophoblast cell line HTR-8/SVneo using stable transfection of CDX1. Using a CDX1 stable transfected cell line, we determined the cell invasion using a QCM ECMatrix 24-well kit. The cell viability was detected using an MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Quantitative RT-PCR and western blotting analyses were performed to examine the changes in the expression of downstream target genes and proteins. To disrupt PI3K/AKT signaling, we used the PI3K inhibitor perifosine.

Results

Cell invasion assays demonstrated that CDX1 restricts trophoblast cell invasiveness. In contrast, quantification of cumulative cell numbers revealed that CDX1 did not affect cell proliferation. Western blotting analysis and quantitative real time PCR demonstrated that MMP-9 expression was reduced, whereas TIMP-1 expression was increased in CDX1-overexpressed cells. However, overexpression of CDX1 did not affect PI3K/AKT signaling in HTR-8/SVneo trophoblast cells. In contrast, CDX1 was regulated by the PI3K/AKT signaling pathway.

Conclusions

Altogether, we found that in trophoblast cells, CDX1 reduced invasion independently of the PI3K/AKT signaling pathway. Furthermore, CDX1 functions in concert with PI3K/AKT signaling to regulate trophoblast invasion. We concluded that CDX1 restricts the invasion of HTR-8/SVneo trophoblast cells by inhibiting MMP-9 expression independently of the PI3K/AKT pathway.     

CCL2通过抑制白细胞介素-24表达增强人绒毛膜滋养细胞株的细胞活力

目的探讨白细胞介素-24(interleukin-24,IL-24)对人滋养细胞活力的调节作用。方法免疫组织化学法检测IL-24及其受体(IL-20R1、IL-20R2和IL-22R1)在正常早孕期绒毛组织的表达;In-cell Western法分析重组胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)和趋化因子CCL2对人绒毛膜滋养细胞株HTR-8/SVneo细胞中IL-24表达的影响;MTT法分析重组IL-24和CCL2对HTR-8/SVneo细胞活力的影响。结果 IL-24及其受体均在正常早孕期绒毛组织滋养细胞中强阳性表达。与对照组相比,重组CCL2体外刺激后HTR-8/SVneo细胞IL-24表达水平显著下降(P0.001)。重组IL-24处理后,HTR-8/SVneo细胞活力显著降低(P0.05或P0.001);相反,IL-24中和性抗体明显促进其细胞活力(P0.05或P0.01),重组CCL2(100 ng/mL)可体外增强HTR-8/SVneo细胞活力(P0.01),但这一作用可被重组IL-24所抑制。结论 CCL2通过抑制IL-24分泌增强人滋养细胞株HTR-8/SVneo细胞的体外活力,从而可能利于囊胚植入和胎盘发育。     

MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1

MiR-155 is known to participate in various cellular processes by targeting gene expression. We previously revealed a link between miR-155 and perturbation of trophoblast invasion and differentiation. This study aimed to investigate the target molecule(s) of miR-155 on the influence on the proliferation and migration of trophoblast cells. Bioinformatics analysis showed that, at the 3' untranslated region (UTR) of cyclin D1, six bases are complementary to the seed region of miR-155. Luciferase assays and cyclin D1 3'UTR transfection assays validated that cyclin D1 3'UTR was the target of miR-155 in HTR-8/SVneo cells. Overexpression of miR-155 in HTR-8/SVneo cells reduced the level of cyclin D1 protein, decreased cell proliferation and invasion, and increased cell number at the G1 stage. Furthermore, the increased expression of miR-155 also regulated the protein levels of kinase inhibitory protein p27 and phosphorylated cytoskeletal protein filamin A. In conclusion, we found that cyclin D1 may be a target of miR-155 in HTR-8/SVneo cells, and demonstrated a negative regulatory role of miR-155 involved in cyclin D1/p27 pathway in proliferation and migration of the cells.     

miR-219a suppresses human trophoblast cell invasion and proliferation by targeting vascular endothelial growth factor receptor 2 (VEGFR2)

ObjectiveVascular endothelial growth factor (VEGF) plays a critical role in regulating trophoblast cell invasion and proliferation, involved in a variety of pregnancy complications, such as spontaneous abortion and pre-eclampsia. Numerous studies have revealed that microRNAs (miRNAs) are participated in a series of molecular processes that regulate cell function, such as cell invasion, proliferation, and apoptosis. Vascular endothelial growth factor receptor 2 (VEGFR2), a receptor of VEGF, has been shown to be involved in trophoblast function. However, the relation between miRNA and VEGFR2 and their role in trophoblast function remain to be elucidated.MethodsThe effect of miR-219a on the trophoblast function has been explored using luciferase reporter, transwell, qRT-PCR, western blot, bromodeoxyuridine (BrdU), ELISA, immunofluorescent staining, and tube formation assays.ResultsIn the current study, we observed that through targeted inhibition of VEGFR2 expression by miR-219a, the function of VEGFR2 as well as the downstream PI3K/AKT/NF-κB signaling pathway were suppressed, leading to suppression of trophoblastic proliferation and invasion. Moreover, upregulation of VEGFR2 restored the miR-219a–inhibited cell proliferation, invasion, and tube formation.ConclusionsThese results revealed that miR-219a played crucial roles in negatively regulating trophoblastic proliferation and invasion by suppression of the PI3K/AKT/NF-κB signaling pathway by targeting VEGFR2, therefore serving as a potential treatment method for the complications of pregnancy caused by trophoblastic dysregulation.     

Prostaglandin E2 inhibits proliferation and migration of HTR-8/SVneo cells, a human trophoblast-derived cell line

Normal placentation requires a highly coordinated control of proliferation, migration and invasiveness of extravillous trophoblast cells. Since prostaglandin E2 is a major prostanoid synthesized by intrauterine tissues and highly involved in pregnancy homeostasis, we examined the possibility that it modulates extravillous trophoblast cell functions. Here, we report the presence of mRNAs for prostaglandin E2 EP2 and EP4 receptor isoforms and of proteins in both first-trimester human chorionic villi and in the human trophoblast-derived HTR-8/SVneo cells. Moreover we found that: (i) this cell line releases prostaglandin E2 and the output is enhanced by interleukin-1beta; (ii) the prostanoid consistently inhibits serum- or epidermal growth factor-induced cell proliferation and also migration. An involvement of cAMP in the prostaglandin E2 antiproliferative action is suggested by the observation that the prostanoid greatly enhances cAMP level in HTR-8/SVneo cells and that forskolin inhibits cell proliferation; moreover the administration of prostaglandin E2 plus forskolin, a condition which evokes a synergistic enhancement of cAMP, induces a major impairment of cell growth. Provided that our data are applicable to the trophoblast tissue in vivo, we suggest that prostaglandin E2 exerts an important control on extravillous trophoblast cell functions, preventing an excessive proliferation and migration.     

Endocrine disruptors, polychlorinated biphenyls-induced gC1qR-dependent apoptosis in human trophoblast cell line HTR-8/SVneo

Although an association exists between exposure to polychlorinated biphenyls (PCBs) and spontaneous miscarriage, the mechanisms underlying this phenomenon remain unclear. In this study, PCBs content in plasma was detected by gas chromatography-mass spectrometry (GC-MS) and decidua tissues were examined for the expression of globular heads of C1q receptor (gC1qR) using Western blot in patients who underwent induced abortion and spontaneous abortion. Results showed increased PCBs content and gC1qR expression in patients who experienced spontaneous abortion. In vitro, Western blot analysis demonstrated significantly higher caspase 3 expression and apoptotic cell counts in green fluorescent protein (GFP)-gC1qR vector group. Additionally, gC1qR and caspase 3 showed decreased expression following PCBs plus gC1qR small interfering RNA (siRNA) treatment. The percentage of apoptotic cells increased in cells treated with PCBs alone or PCB plus negative siRNA. These data suggest that maternal exposure to PCBs is associated with adverse pregnancy outcomes and that upregulation of gC1qR is important for PCBs-mediated trophoblast cell apoptosis.     

HTR-8/SVneo cell line contains a mixed population of cells

IntroductionThe placenta, a transient organ in humans, is essential for pregnancy maintenance and fetal development. Trophoblast and stromal cells are the main cell types present in human placenta. Trophoblast cells are derivatives of the trophectoderm layer and fulfill the endocrine, exchange, invasion and implantation processes of the placenta, whereas stromal cells are of extraembryonic mesenchymal origin and are important for villous formation and maintenance. Different cell lines were developed to study trophoblast functions including BeWo, JEG-3 and JAR from chorioncarcinoma while HTR-8/SVneo was developed using first trimester extravillous trophoblast infected with simian virus 40 large T antigen (SV40). These cell lines are largely used to study trophoblast functions including cell fusion, migration and invasion. Therefore, the purity of each cell lines is crucial in order to be able to use them as a model recapitulating trophoblast cells.MethodsHTR-8/SVneo, BeWo, JEG-3 and JAR were analyzed for epithelial and mesenchymal markers using immunofluorescence, real time PCR and Western blot.ResultsOur results showed that HTR-8/SVneo cell line contains two populations of cells suggesting the presence of trophoblast and stromal/mesenchymal cells. While all cells in BeWo, JEG-3 and Jar are positive for the trophoblast/epithelial marker CK7, HTR-8/SVneo cells contained few clusters of CK7 positive cells. Interestingly, vimentin expression was detected in a subset of HTR-8/SVneo cells and was completely absent from all other tested placental cell lines.DiscussionOur results unveil the presence of a heterogeneous population of trophoblast and stromal cells within HTR-8/SVneo cell line. This mixed population of cells should be taken into consideration when using this cell line to study trophoblast functions.