YAP/TAZ对发育和肿瘤的调控及其功能的研究进展

Hippo通路是一种在进化中形成的保守的蛋白激酶级联通路,它与发育中器官的大小和肿瘤的形成有关。Hippo通路的中枢是从肿瘤抑制子Hippo到原癌蛋白YAP/TAZ的激酶级联反应。YAP/TAZ是Hippo通路下游的主要的效应分子,它们广泛表达于多种组织器官中。在哺乳动物细胞中,Hippo通路激酶级联反应通过对YAP/TAZ磷酸化作用,促使其从细胞核转入细胞质中,从而抑制了YAP/TAZ的功能作用。TEAD家族转录子被鉴定为YAP/TAZ发挥生物学功能的重要调节因子。YAP/TAZ的失调引起的相关的基因的表达改变,将会影响细胞的增殖,分化,以及凋亡,从而会影响器官的大小以及肿瘤的形成。本文综述Hippo通路的最新进展,重点关注的是该通路中的YAP/TAZ调控的缺失对发育缺陷和肿瘤的影响。这将为我们研究再生医学,组织工程技术,肿瘤的干预防治提供新的思路与策略。     

Hippo/YAP和Wnt/β-catenin通路的对话

Hippo/YAP通路和Wnt/β-catenin通路是在细胞的生长分化、组织器官形成以及成体干细胞的维持等方面都起着重要作用的两条信号通路。在哺乳动物细胞中,Wnt/β-catenin通路通过一系列胞质蛋白的相互作用,使β-catenin蛋白在胞质内累积,进而入核传递生长刺激信号。Hippo/YAP通路通过激酶级联反应磷酸化YAP/TAZ,使其滞留在细胞质中,抑制了YAP/TAZ的转录活性,从而限制细胞的生长增殖,诱导细胞凋亡。这两条通路的异常调控往往会导致肿瘤的发生。近年来越来越多的研究证实,Hippo/YAP和Wnt/β-catenin在很多方面相互影响,共同参与组织生长和胚胎发育的调控。研究这两个通路在肿瘤发生过程中的转导和调控以及它们相互作用的机制,有助于为肿瘤的防治提供新的思路与策略。文章对这两条通路的协同作用及其分子机制进行了综述。     

吉祥草活性成分RCE-4抑制宫颈癌Ca Ski细胞增殖、侵袭和迁移的活性及机制研究

本文探讨吉祥草活性单体RCE-4对宫颈癌Ca Ski细胞增殖、侵袭和迁移的影响及机制。采用MTT法和克隆形成实验检测RCE-4对Ca Ski细胞增殖的影响,划痕实验及Transwell小室实验研究RCE-4对Ca Ski细胞的迁移和侵袭的影响,间接免疫荧光实验和Western blot法检测RCE-4对β-catenin和YAP/TAZ蛋白入核转运的影响,采用Western blot法检测RCE-4对Wnt/β-catenin、Hippo/YAP信号路通及下游靶蛋白表达的影响。结果显示,RCE-4可以显著抑制Ca Ski细胞的增殖、迁移和侵袭;RCE-4处理后,β-catenin和YAP/TAZ蛋白入核减弱,GSK3β、p-β-catenin、p-YAP、p-LATS1的蛋白表达量增加,PAK4、p-GSK3β、β-catenin、MST1、LATS1、NUAK2、YAP/TAZ、YAP的蛋白表达量减少,下游靶蛋白MMPs、c-Myc、SOX2、CYR61、VCAM-1、ICAM-1的表达被抑制,提示RCE-4体外可能通过对Wnt/β-catenin和Hippo/YAP信号通路的调控发挥其抗Ca Ski细胞侵袭和迁移的作用。     

shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 *

目的 该研究旨在探讨磷脂酰肌醇特异性磷脂酶C epsilon(phospholipase C epsilon, PLCε)对前列腺癌细胞丝氨酸/甘氨酸代谢及细胞增殖的影响。方法 慢病毒及质粒转染LNCAP、PC3细胞,q-PCR、Western blot分别检测LNCAP、PC3细胞中 PLCε、Yes相关蛋白(yes associated protein,YAP)、丝氨酸/甘氨酸生成酶[包括磷酸丝氨酸转氨酶1(phosphoserine aminotransferase1,PSAT1)、磷酸丝氨酸磷酸酶(phosphoserine phosphatase,PSPH)、丝氨酸羟甲基转移酶2(serine hydroxymethyltransferase2,SHMT2)及增殖相关基因细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)]的表达情况;克隆形成实验及MTT实验检测细胞的克隆形成率及增殖活性。结果 (1)感染LV-shPLCε可显著下调前列腺癌细胞LNCAP、PC3中的PLCε、YAP、PSAT1、PSPH、SHMT2及增殖相关基因的mRNA及蛋白质水平,同时抑制细胞的克隆形成能力和增殖活性;(2)在shPLCε组细胞中加入过表达YAP质粒后,能明显逆转YAP、PSAT1、PSPH、SHMT2及增殖相关基因的下调,但加入干扰YAP质粒后结果相反。结论 shPLCε可通过下调YAP的表达抑制前列腺癌细胞的丝氨酸/甘氨酸生成,从而抑制细胞的增殖。     

Angiomotin在肿瘤形成及Hippo信号通路中调控研究进展

Angiomotin(AMOT)是一种血管抑制素结合蛋白,AMOT在血管内皮细胞的迁移、紧密连接和管状形成等方面起着重要调控作用。AMOT及其同源家族蛋白AMOTL1和AMOTL2可能与Hippo信号通路的下游效应分子YAP相互作用来参与调控肿瘤细胞的生长。在乳腺癌、前列腺癌等癌症中,AMOT能够增加YAP进入细胞核的水平从而促进癌细胞的增殖和迁移;但在胶质母细胞瘤、肺癌等癌细胞中,AMOT将YAP滞留在细胞质或紧密连接处,从而抑制YAP的活性。另外,AMOT也可以促进Hippo信号通路中核心激酶LATS来发挥抑制肿瘤细胞增殖的作用。AMOT在肿瘤细胞生长中发挥的不同作用还需要更深入的研究,现对AMOT在癌症中的调控作用及在Hippo信号通路中的调控机制等方面的研究进展进行综述。     

ERK1/2在食管鳞状细胞癌中促进细胞增殖、抑制凋亡及其调控机制的研究

探讨ERK1/2在食管鳞状细胞癌(ESCC)中对肿瘤细胞增殖、凋亡的调控及其机制。平板克隆、细胞凋亡和细胞周期实验结果发现ERK1/2 MAPK通路抑制可减弱Eca109细胞克隆形成和增殖,促进细胞凋亡,减慢细胞周期;进一步发现ERK1/2 MAPK通路抑制可以反转由mi R-21过表达诱导的Eca109细胞增殖、凋亡和周期的变化;q RT-PCR和Western-blot免疫印迹结果发现ERK1/2 MAPK信号通路抑制可以下调内源性mi R-21表达和反转外源性mi R-21诱导的ERK1/2 MAPK信号通路活化。实验结果提示ERK1/2 MAPK通路抑制可能通过下调Eca109细胞中mi R-21表达阻碍Eca109细胞增殖、促进细胞凋亡和减慢细胞周期,最终导致ESCC细胞生长抑制。     

基于CRISPR/Cas9技术敲除SAMHD1肝癌细胞系的构建及对增殖的影响

运用CRISPR/Cas9基因编辑技术,建立SAMHD1基因敲除的Huh7细胞系,并检测敲除SAMHD1基因后对人肝癌细胞Huh7细胞增殖的影响.针对SAMHD1基因作用的功能区域,设计靶向SAMHD1的小向导sgRNA(Small Guide RNA).构建lentiCRISPRv2-SAMHDl-gRNA重组质粒转化后测序.筛选稳定敲除SAMHD1的稳定细胞系并测序鉴定.采用克隆形成实验分析基因SAMHD1敲除后肝癌细胞Huh7细胞的增殖能力.测序结果显示lentiCRISPRv2-SAMHDl-gRNA载体构建成功.Western blot结果和测序结果表明成功构建敲除SAMHD1的Huh7稳定细胞系.克隆形成实验结果表明,相对于Huh7-WT细胞,Huh7-KO SA-MHD1的细胞增殖能力增强(P＜0.01).成功构建基于CRISPR/Cas9技术敲除SAMHD1基因的Huh7细胞系,SAMHD1的表达缺失促进肝癌细胞的增殖.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G_1期阻滞

卡铂(carboplatin,CBP)是一种抗肿瘤活性较强的化疗药物,通过诱导细胞周期阻滞抑制肿瘤细胞生长,但其诱导细胞周期阻滞的报告不甚一致.本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响.MTS结果显示,卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长,联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强.采用Giemsa染色法观察到,卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化.流式细胞术检测细胞周期发现,随卡铂浓度的增高,S期阻滞作用增强;抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用,增加G1期阻滞作用,而对G2/M期细胞影响不明显.Western印迹结果显示,随卡铂浓度的增高,p-ERK1/2、Cdc2(Y15)和p-Cdc2(T161)的表达逐渐升高,Cyclin E1和Cyclin B1的表达逐渐降低;抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用,上调Cdc2(Y15)的表达受阻,抑制Cyclin B1的下调作用,促进Cyclin E1的下调作用.本研究结果提示,卡铂通过抑制ERK1/2激活,诱导人卵巢癌HO-8910细胞S和G1期阻滞,抑制卵巢癌细胞生长.     

白花地胆草单体EM-12诱导2774-C10细胞G_1/S期阻滞及细胞凋亡的分子机制研究

目的:研究白花地胆草(Elephantopus mollis H.B.K.)的乙醇提取物EM-12抗肿瘤作用分子机制。方法:MTT检测EM-12对卵巢癌细胞活性影响;克隆形成抑制实验检测EM-12对卵巢癌细胞增殖能力的影响;GO功能分析(gene ontology analysis)分析EM-12对卵巢癌2774-C10细胞的影响;PI单染检测细胞周期;Annexin V-FITC/PI双染法检测细胞凋亡;Western blotting检测细胞凋亡、周期相关蛋白。结果:MTT实验结果表明,EM-12可以显著抑制卵巢癌细胞的活性;RNASeq及GO功能分析表明EM-12诱导2774-C10发生G1/S期阻滞;克隆形成抑制实验结果表明,EM-12可以显著抑制卵巢癌细胞的增殖;流式细胞术结果表明,随着药物浓度的增加,凋亡率逐渐增加,并且G_1期细胞比例逐渐增加,S期细胞比例减少,发生G_1/S期阻滞。Western blotting结果表明,随着药物浓度的增加cyclin E_2、cyclin D_1、CDK2、CDK6蛋白水平下降,并伴随caspase-8、caspase-3活化及多聚ADP核糖聚合酶PARP酶切失活。结论:EM-12诱导卵巢癌细胞发生G_1/S期阻滞,且通过死亡受体通路诱导细胞凋亡。     

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.     

沉默脂肪细胞增强子结合蛋白2通过PI3K/Akt途径抑制肝癌细胞增殖和迁移

脂肪细胞增强子结合蛋白2(AEBP2)作为多梳抑制复合物2(PRC2)的组成蛋白质,参与多种肿瘤细胞的增殖和迁移,然而其在肝癌中的作用尚不清楚。本研究基于UALCAN和Kaplan-Meier Plotter数据库分析发现,AEBP2在肝癌组织中高表达,并且与患者的不良预后呈正相关。实时荧光定量PCR和蛋白质印迹结果证实,AEBP2在肝癌细胞中的表达高于正常肝细胞。在HepG2和Huh-7细胞中转染AEBP2 siRNA,平板克隆、CCK-8、流式细胞术、划痕愈合和Transwell结果显示,沉默AEBP2可以抑制肝癌细胞增殖、迁移和侵袭,并促进细胞凋亡(P＜0.05)。免疫荧光检测和蛋白质印迹结果显示,沉默AEBP2能够抑制肝癌细胞上皮-间质转化(EMT)(P＜0.05)。生物信息学分析结果表明,AEBP2参与调控PI3K/Akt信号通路。蛋白质印迹结果证实,沉默AEBP2能下调PI3K、p-AKT (S473)、mTOR、MMP-2和MMP-9的蛋白质表达水平(P＜0.05)。此外,沉默AEBP2对HepG2细胞迁移和侵袭的影响可被PI3K/Akt通路激动剂胰岛素样生长因子1(IGF-1)部分逆转(P＜0.01)。综上所述,AEBP2可能通过调节PI3K/Akt途径促进肝癌细胞增殖和迁移。本研究为AEBP2在肝癌中的作用提供理论依据。     

CD44 acts through RhoA to regulate YAP signaling

The Hippo pathway plays an important role in both physical and pathogenesis processes. As crucial downstream effectors of Hippo pathway, YAP is inhibited by Lats1/2 through phosphorylation. However, upstream signals that regulate the Hippo pathway have been still poorly understood. Here, we found that knockdown of CD44 reduced YAP expression and nuclear localization, but nearly had no effect on its upstream effectors, Mst1 and Lats1. Downregulated CD44 expression also significantly decreased the expression of YAP downstream effectors CTGF, Cyr61 and EDN1 at mRNA level. Our next study showed that knockdown of CD44 inhibited RhoA expression, which was consistent with RhoA knockdown mediated YAP downregulation. Furthermore, we demonstrated that over expression of the constitutively active RhoA (RhoA-V14) could block the YAP expression decrease mediated by CD44 knockdown. Moreover, downregulation of CD44 significantly promoted cell apoptosis and inhibited cell proliferation, cell cycle progression and migration, which were consistent with the effects of RNAi-mediated YAP knockdown in both A549 and HepG2 cells. Overall, data are presented showing that CD44 could act through RhoA signaling to regulate YAP expression and this study also provide new insights into the regulatory mechanisms of the Hippo–YAP pathway.     

MicroRNA-21 promotes the proliferation and inhibits apoptosis in Eca109 via activating ERK1/2/MAPK pathway

The aim of this study was to investigate how miR-21 promotes proliferation and inhibits apoptosis in esophageal squamous cell carcinoma (ESCC). MTT, wound healing assay and cell cycle showed that proliferation and migration of ESCC cell line Eca109 cells were increased in miR-21 mimics group, and decreased in anti-miR-21 Oligonucleotide (AMO) group after transfection into Eca109 cells with miR-21 mimics, AMO and scramble sequence, respectively. Cell apoptosis assay indicated that cell apoptosis can be obviously inhibited by overexpression of miR-21 and promoted by downregulation of miR-21. Meanwhile, western-blot results showed that p-ERK1/2 expression was elevated in miR-21 mimics group, whereas decreased in AMO group. Furthermore, the ERK1/2, a key component of MAPK signaling pathway, was knocked down, and overexpressed successfully using shRNA-ERK1/2 and overexpressing plasmids containing full length cDNA of ERK1/2, respectively. It was observed that shRNA-ERK1/2 can significantly decreased the level of miR-21 expression, while overexpression of ERK1/2 can up-regulate expression of miR-21. As further confirmation, Eca109 cells were treated with gradient concentration of U0126, a kind of MEK inhibitor, and expression of miR-21 was subsequently examined. It was found that U0126 can significantly decreased endogenous expression of miR-21. In parallel, U0126 decreased cell proliferation, migration and increased the apoptosis in Eca109 cells, with the expression of miR-21 being reduced significantly in U0126 group as compared with control groups. Our findings indicated that miR-21 promoted the proliferation, migration and inhibited apoptosis of Eca109 cells through activating ERK1/2/MAPK pathway, and that targeting miR-21 could be a promising therapeutic strategy in ESCC.     

Rab11a通过PI3K/AKT和Ras/MEK/ERK信号通路调节胰腺癌细胞凋亡和侵袭

目的:探究Rab11a在胰腺癌中的表达模式及其对肿瘤生长和转移的影响。方法:通过免疫组织化学法、RT-PCR和Western blot检测60例胰腺癌患者的癌组织和癌旁组织中Rab11a的表达。通过对人胰腺癌细胞系PANC1转染靶向Rab11a的小干扰RNA或过表达Rab11a的pcDNA3.1质粒考察Rab11a对细胞增殖、凋亡、迁移和侵袭的影响。通过Western blot检测PANC1细胞中PI3K、AKT、Ras、MEK、ERK1/2和GSK3β的磷酸化水平。结果:胰腺癌组织中Rab11a的表达水平均高于癌旁组织(P<0.05)。Rab11a的表达水平与TNM分期和淋巴结转移有关(P<0.05)。CCK-8测试和细胞集落形成实验显示,下调Rab11a抑制了PANC1细胞的增殖(P<0.05)。流式细胞术显示,下调Rab11a促进了PANC1细胞的凋亡(P<0.05)。细胞划痕实验显示,下调Rab11a抑制了PANC1细胞的迁移能力(P<0.05)。Matrigel Transwell实验显示,下调Rab11a抑制了PANC1细胞的侵袭能力(P<0.05)。然而,上调Rab11a则促进了PANC1细胞的增殖、迁移和侵袭,并抑制了细胞凋亡(P<0.05)。蛋白质印迹分析显示,下调Rab11a抑制了PANC1细胞中PI3K/AKT和Ras/MEK/ERK信号通路的活化(P<0.05)。此外,应用PI3K/AKT和Ras/MEK/ERK信号通路的选择性抑制剂处理PANC1细胞可阻断Rab11a对细胞增殖的促进作用(P<0.05)。结论:Rab11a的高表达是胰腺癌预后恶化的潜在生物标志物。靶向抑制Rab11a可通过抑制PI3K/AKT和Ras/MEK/ERK信号通路来降低胰腺癌的生长和转移能力。     

MST1/2调控先天免疫的功能和机制

Hippo信号通路通过一系列激酶级联反应,实现对细胞增殖、器官大小以及组织再生等方面的调控。其中,MST1/2是核心激酶Hippo蛋白在哺乳动物中的同源物,对于下游信号通路的激活至关重要。此外,MST1/2在细胞分化、形态和细胞骨架重排等方面也发挥重要作用。近期多项研究工作指出,MST1/2参与调控免疫T细胞的粘附、迁移、归巢和抑制性Treg细胞的成熟与功能,以及心肌细胞自噬等过程。有趣的是,这一功能是不依赖经典的Hippo信号通路的,被称为“非经典Hippo信号通路”。最新的研究结果揭示了MST1/2通过非经典Hippo信号通路调控先天免疫巨噬细胞对病原菌或病毒的免疫应答,包括巨噬细胞的吞噬、细胞因子(炎症因子、趋化因子、Ⅰ型干扰素等)和线粒体活性氧的产生,从而在机体抵抗细菌病毒感染、炎症相关癌症、动脉粥样硬化等疾病中发挥重要功能。本文对MST1/2调控先天免疫功能、相关分子机制和疾病进行了总结和讨论。     

Curcumin inhibits the growth of triple‐negative breast cancer cells by silencing EZH2 and restoring DLC1 expression

Enhancer of zeste homolog 2 (EZH2), an oncogene, is a commonly up‐regulated epigenetic factor in human cancer. Hepatocellular carcinoma deletion gene 1 (DLC1) is an antioncogene that is either expressed at low levels or not expressed in many malignant tumours. Curcumin is a promising anticancer drug that has antitumour effects in many tumours, but its mechanism of action is unclear. Our research demonstrated that EZH2 was up‐regulated in breast cancer (BC) tissues and cells, whereas DLC1 was down‐regulated, and the expression of EZH2 and DLC1 was negatively correlated in BC. By analysing the characteristics of clinical cases, we found that positive expression of EZH2 and negative expression of DLC1 may be predictors of poor prognosis in patients with triple‐negative breast cancer (TNBC). Moreover, knockdown of EZH2 expression restored the expression of DLC1 and inhibited the migration, invasion and proliferation, promoted the apoptosis, and blocked the cell cycle of MDA‐MB‐231 cells. Furthermore, we found that curcumin restored the expression of DLC1 by inhibiting EZH2; it also inhibited the migration, invasion and proliferation of MDA‐MB‐231 cells, promoted their apoptosis and blocked the cell cycle. Finally, xenograft tumour models were used to demonstrate that curcumin restored DLC1 expression by inhibiting EZH2 and also inhibited the growth and promoted the apoptosis of TNBC cells. In conclusion, our results suggest that curcumin can inhibit the migration, invasion and proliferation, promote the apoptosis, block the cycle of TNBC cells and restore the expression of DLC1 by inhibiting the expression of EZH2.