自分泌IL-6经Ras/MEK/ERK、PI3K/Akt通路促进卵巢癌细胞黏附和侵袭功能的研究

目的探讨内源性IL-6表达改变对卵巢癌细胞黏附和侵袭功能的影响及相关信号转导通路。方法利用以往构建的内源性过表达IL-6的人卵巢癌A2780细胞系和内源性抑制IL-6表达的人卵巢癌SKOV-3细胞,分别采用细胞体外黏附实验、Transwell小室体外侵袭实验、免疫印迹技术等观察IL-6对卵巢癌细胞黏附、侵袭能力的影响,并对其可能的信号通路进行研究。结果与对照组相比,内源性过表达IL-6可促进卵巢癌细胞的体外黏附和侵袭功能;抑制IL-6表达则可抑制卵巢癌细胞的上述功能。过表达或抑制表达IL-6可增加或减少卵巢癌细胞磷酸化ERK、Akt的表达水平,应用ERK或Akt特异性信号阻断剂可显著抑制高表达IL-6的卵巢癌细胞黏附和侵袭作用,提示可能与其活化Ras/MEK/ERK、PI3K/Akt通路有关。结论卵巢癌细胞产生的IL-6可经Ras/MEK/ERK和PI3K/Akt通路增强自身的黏附和侵袭能力,调节IL-6表达及相关信号转导通路可能是未来临床控制卵巢癌进展的一种良好策略。     

IL-6经MEK/ERK途径促进ERα-Ser118磷酸化影响卵巢癌细胞他莫昔芬耐药

目的研究IL-6信号通路对ERα的Ser118位点磷酸化影响、对ERα转录活性的影响及IL-6诱导卵巢癌TAM耐药的机制。方法脂质体转染法获得内源性过表达IL-6的人卵巢癌A2780细胞株和内源性抑制IL-6表达的人卵巢癌CAOV3细胞株,Western blot法检测内源性和外源性IL-6对ERK和ERα的Ser118位点磷酸化影响,MTT法检测IL-6的MEK/ERK信号通路对A2780细胞TAM耐药性的影响,荧光素酶活性检测IL-6的MEK/ERK信号通路对ERα转录活性的影响。结果过表达IL-6能上调A2780细胞中ERα-Ser118的磷酸化水平,而抑制IL-6表达下调CAOV3细胞中ERα-Ser118的磷酸化水平;外源性IL-6上调A2780细胞中ERK的磷酸化水平,而MEK1/2特异性抑制剂PD98059则可以逆转IL-6介导的ERK及ERα-Ser118的磷酸化;PD98059可明显降低IL-6导致的A2890细胞对TAM的耐药性;IL-6明显促进ERα转录活性,而PD98059可逆转这一作用。结论 IL-6经MEK/ERK通路磷酸化ERα的Ser118位点促进ERα转录活性而激活ER途径,诱导OVCA细胞对TAM的耐药性。     

阻断Sonic Hedgehog信号对不同人肝癌细胞生长的影响

 目的: 研究阻断Sonic Hedgehog (Shh)信号对不同人肝癌细胞生长的影响,探讨阻断Shh信号抑制肝癌细胞生长的机制。方法: RT-PCR法检测Shh信号分子在3株人肝癌细胞(BEL-7402、Huh7和HepG2)中的表达,并检测Shh阻断抗体作用后BEL-7402细胞Shh信号效应分子表达变化;MTT法检测人肝癌细胞增殖活性;流式细胞术检测人肝癌细胞凋亡;Western blot 检测凋亡相关蛋白表达。结果: Shh信号分子在3株人肝癌细胞中均有表达,Shh阻断抗体可以下调Shh信号效应分子patched (Ptch)、Gli1和Gli2的表达;Shh阻断抗体可以抑制3株肝癌细胞生长,增加G₀/G₁期细胞,并诱导细胞凋亡;Shh阻断抗体作用后,BEL-7402细胞pro-caspase-3、pro-caspase-8和pro-caspase-9蛋白表达水平下降,cleaved caspase-3、cleaved caspase-8和cleaved caspase-9蛋白表达水平升高。结论: 阻断Shh信号可抑制Shh高表达的人肝癌细胞生长,阻滞细胞周期于G₀/G₁期,并诱导肝癌细胞凋亡。     

冬凌草甲素对人食管鳞癌细胞系KYSE-150和KYSE-450增殖及迁移的影响

目的 探讨冬凌草甲素（ORI）对食管鳞癌细胞系KYSE-150和KYSE-450增殖、凋亡、周期及迁移的作用。 方法 MTT法检测ORI对食管癌细胞增殖的影响;集落形成实验检测ORI对集落形成的影响;流式细胞术检测ORI对食管癌细胞凋亡和周期的影响;Transwell迁移实验检测ORI对食管癌细胞迁移的作用;Western blotting检测ORI对抗凋亡蛋白Bcl-2、细胞周期抑制蛋白p21Cip1/Waf1及上皮-间质转化（EMT）标志蛋白表达水平的影响。 结果 ORI对KYSE-150和KYSE-450细胞的增殖、迁移和集落形成有显著的抑制作用（P<0.05）,且抑制作用呈一定的时间、剂量依赖性;流式结果显示,随着ORI浓度的增加,细胞的凋亡率明显增加（P<0.05）,G₂/M期细胞比例显著增加（P<0.05）,G₀/G₁期细胞比例明显下降（P<0.05）;ORI处理食管癌细胞48 h,Bcl-2、间质细胞标志蛋白,波形蛋白（vimentin）、β-连环蛋白（β-catenin）表达下调,p21Cip1/Waf1、上皮细胞标志蛋白,E-钙黏蛋白（E-cadherin）表达上调。 结论 冬凌草甲素可能通过诱导细胞凋亡,阻滞细胞在G₂/M期抑制食管癌细胞的增殖,并通过抑制EMT转化从而抑制食管癌细胞的迁移。     

HOTAIR 促进卵巢癌恶性生物学行为

目的:研究沉默HOTAIR后对上皮性卵巢癌恶性生物学行为的影响。方法:实时荧光定量聚合酶链反应检测HOTAIR在卵巢癌细胞株SKOV3、OVCAR3和A2780的表达情况。沉默HOTAIR后,划痕实验检测卵巢癌细胞转移能力,侵袭实验检测卵巢癌细胞侵袭能力,胸腺嘧啶核苷类似物检测卵巢癌细胞增殖能力。结果:HOTAIR在SKOV3和OVCAR3细胞中表达高于A2780细胞;沉默HOTAIR后,卵巢癌SKOV3细胞转移、侵袭和增殖能力降低;结论:沉默HOTAIR可以抑制卵巢癌SKOV3细胞恶性生物学行为。     

多发性骨髓瘤中MAPK信号通路对BLyS表达的调控研究

目的 研究多发性骨髓瘤(MM)细胞中丝裂原活化蛋白激酶(MAPK)信号通路的表达及活化情况,探讨MAPK信号通路对MM细胞B淋巴细胞刺激因子(BLyS)表达变化的影响及对MM细胞增殖与存活的影响,并初步探讨MAPK信号通路在IFN-γ(MM重要的促生长因子)上调MM细胞BLyS表达过程中的作用.方法 应用Western blot方法检测MM细胞中蛋白ERK、p-ERK、JNK、p-JNK、p38及p-p38的表达情况;应用RT-PCR及Western blot检测MAPK信号通路对BLyS表达的影响;应用WST-1法检测靶向JNK的MAPK信号通路抑制剂SP600125对MM细胞增殖与存活的影响.结果 MM细胞株中,除了ERK、JNK及p38的表达外,还有活化蛋白p-JNK的表达;靶向JNK的MAPK信号通路抑制剂SP600125可下调MM细胞BLyS的表达,其激动剂茴香霉素(anisomycin)可上调BLyS的表达;IFN-γ可上调MM细胞BLyS的表达,SP600125可部分抵消IFN-γ对BLyS的上调作用;SP600125可抑制MM细胞的增殖与存活.结论 MM细胞中有JNK/SAPK信号通路的活化;JNK/SAPK信号通路的活化程度与BLyS的表达高低呈正相关;JNK/SAPK信号通路在IFN-γ上调MM细胞BLyS表达过程中发挥重要作用.     

CaSR在oxLDL诱导的A7r5细胞增殖及迁移中的作用

目的:探讨钙敏感受体(calcium-sensing receptor,Ca SR)在氧化型低密度脂蛋白(oxidized low-density lipoprotein,ox LDL)诱导的大鼠胸主动脉平滑肌细胞(A7r5细胞)增殖及迁移中的作用及信号机制。方法:Brd U掺入法检测细胞增殖;伤口愈合实验及Transwell迁移分析检测细胞迁移情况;Western blot方法检测Ca SR、PCNA、ERK MAPK通路及PI3K/AKT通路的蛋白表达。结果:(1)小剂量(10 mg/L)ox LDL作用A7r5细胞24 h促进细胞的增殖和迁移;(2)ox LDL增加A7r5细胞的Ca SR表达;(3)Ca SR拮抗剂NPS2390抑制了ox LDL的作用,而激动剂Gd Cl_3进一步增强了ox LDL的作用;(4)ox LDL可促进p-AKT、pERK蛋白表达;(5)PI3K/AKT通路抑制剂LY294002、ERK MAPK通路抑制剂PD98059能够抑制ox LDL诱导的细胞增殖和迁移效应;(6)NPS2390抑制了ox LDL诱导的p-AKT和p-ERK蛋白表达,而Gd Cl_3作用相反。结论:(1)ox LDL诱导A7r5细胞增殖及迁移效应;(2)Ca SR参与ox LDL诱导的A7r5细胞增殖及迁移作用;(3)Ca SR通过活化PI3K/AKT通路及ERK MAPK信号通路参与ox LDL诱导的A7r5细胞增殖及迁移作用。     

TRIM19通过调控p53信号促进卵巢癌进展的作用及机制研究

目的:探讨TRIM19在卵巢癌进展中的作用及其分子机制。方法:qPCR检测人卵巢表皮细胞IOSE80、卵巢癌细胞SKOV-3、A2780、OVCAR3中TRIM19表达水平;以siRNA敲减SKOV-3细胞中TRIM19基因表达;以CCK-8方法、Transwell实验、流式细胞术分别检测细胞增殖能力、迁移行为及凋亡率;以RT-qPCR技术检测信号通路分子表达。结果:在卵巢癌细胞SKOV-3、A2780、OVCAR3中,TRIM19表达量较正常表皮细胞IOSE80中明显增强。当TRIM19基因在卵巢癌SKOV-3细胞中被成功敲减后,细胞增殖能力降低74.2%,侵袭能力降低70.0%,凋亡率增加约30倍。在分子水平,敲减TRIM19后,p53、p21、CyclinD、CDK2、Bax基因表达显著增加;同时敲减TRIM19与p53后,p53、p21、CyclinD、CDK2、Bax基因表达略有提高。结论:沉默TRIM19基因抑制卵巢癌细胞增殖、侵袭,诱导凋亡,并且激活p53依赖的凋亡信号,TRIM19促进卵巢癌进程。TRIM19具有成为卵巢癌治疗的新靶点潜力。     

内源性IL-8诱导卵巢癌细胞对顺铂与紫杉醇产生耐药的机制研究

目的探讨内源性IL-8诱导卵巢癌细胞对顺铂和紫杉醇产生耐药的机制及相关信号转导通路。方法在原有工作基础上,以2种人卵巢癌细胞系A2780(不分泌IL-8,对顺铂、紫杉醇敏感)和SKOV-3(高分泌IL-8,对顺铂、紫杉醇耐药)为研究模型,分别将正义(sense,ss)IL-8基因或反义(antisense,as)IL-8基因稳定转染至A2780细胞或SKOV3细胞,应用MTT法、Caspase-3活性测定、RT-PCR及Western blot技术等观察内源性IL-8是否影响卵巢癌细胞对顺铂和紫杉醇的敏感性,并对其作用的机制和可能的信号传导通路进行研究。结果 1)内源性过表达IL-8可诱导A2780细胞对顺铂和紫杉醇产生耐药,而抑制IL-8表达可恢复SKOV3细胞对顺铂和紫杉醇的敏感性,IL-8诱导的卵巢癌细胞化疗耐药是通过降低Caspase-3活性来实现的;2)内源性过表达IL-8可上调A2780细胞的耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达,而抑制IL-8表达可使上述基因的表达明显降低;3)Wortmannin(PI3K抑制剂)和PD98059(MEK1/2抑制剂)能分别阻断IL-8诱导下卵巢癌细胞的Akt和ERK活化及化疗耐药作用。结论 IL-8诱导的卵巢癌细胞化疗耐药可能与其上调耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达以及活化Raf/MEK/ERK和PI3K/Akt信号通路相关,提示调节IL-8表达或其相关信号通路可能是治疗耐药性卵巢癌的一种良好策略。     

Snail在卵巢癌细胞侵袭转移潜能方面研究

目的研究Snail与卵巢癌侵袭转移问的关系,并探讨其分子机制。方法分别构建正义全长Snail真核表达载体和小分子干扰RNA,分别转染卵巢癌细胞系A2780、C13＊。采用Western印迹方法分析相关蛋白表达,Transwell试验检测细胞侵袭转移能力。结果①成功构建Snail正义全长真核表达载体,并合成Snail小分子RNA干扰片段;②在转染PEGFPCI／Snail的卵巢癌细胞株A2780、C13＊中E-eadhefin表达明显下调,Snail和Vimentin则表达上调;而在转染Snail／SiRNA的卵巢癌细胞株A2780、C13‘中E—cadherin表达明显上调,Snail和Vimentin则表达下调;③Snail过表达可显著促进卵巢癌细胞株A2780、C13＊的侵袭转移潜能;封闭Snail表达水平可一定水平抑制卵巢癌细胞株A2780、C13＊的侵袭转移潜能。结论snail可通过促进卵巢癌上皮细胞间质化转变（epithelial—mesenchymaltransition,EMT）而提高其侵袭转移能力;封闭Snail表达可一定程度逆转卵巢癌上皮细胞间质化转变（EMT）而抑制其侵袭转移能力。     

FTO accelerates ovarian cancer cell growth by promoting proliferation,inhibiting apoptosis,and activating autophagy

ObjectiveFat mass and obesity-associated protein (FTO) is identified as a critical demethylase involved in various physiological processes. Despite efforts have been made to study the biological functions of FTO in certain cancers, the role of FTO in ovarian cancer is largely unknown. In this study, we sought to investigate the function of FTO on proliferation, apoptosis and autophagy of ovarian cancer cells.MethodsQuantitative real-time PCR was performed to detect FTO expression in ovarian tumor tissues and ovarian cancer cell lines OVCAR-3, SKOV-3, COC1, HO-8910 and A2780. SKOV-3 cells were constructed with FTO overexpression and A2780 cells were constructed with FTO knockdown. CCK-8 assay was used to examine cell viability and flow cytometry was used to detect cell apoptosis. Activity assay kits were applied to detect caspase-3 and caspase-9 levels. Western blot was performed to measure the expressions of FTO, PCNA, Bax, Bcl-2, LC3, ATG5, P62, p-AKT and AKT. Stable FTO-overexpression SKOV-3 cells or FTO-depletion A2780 cells were injected subcutaneously into male Balb/c-nu mice. Xenografted tumors were assayed by H&E staining. Immunohistochemistry was subjected to measure FTO and Ki67 expressions.ResultsFTO was up-regulated in ovarian tumor tissues compared with non-cancerous ovarian tissues. FTO overexpression markedly increased viability and autophagy function, but decreased apoptosis of ovarian cancer cells. In addition, FTO overexpression promoted AKT phosphorylation. In contrast, FTO silence showed the opposite effect.ConclusionFTO accelerated ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy.     

MiR-515-5p通过调控硫酸软骨素蛋白聚糖4表达对卵巢癌A2780细胞增殖和转移的影响及其机制

目的 探讨miR-515-5p对硫酸软骨素蛋白聚糖4(CSPG4)的靶向调控作用,以及对卵巢癌细胞系A2780细胞增殖和转移的影响.方法 通过生物信息学工具预测miR-515-5p的靶基因.Real-time PCR和Western blotting法检测65例卵巢癌组织和与其对应的癌旁组织中miR-515-5p和CS...     

MCT1 promotes the cisplatin-resistance by antagonizing Fas in epithelial ovarian cancer

This study was designed to investigate the role of MCT1 in the development of cisplatin-resistant ovarian cancer and its possible relationship with Fas. We found the expression of MCT1 was obviously increased both in cisplatin-resistant ovarian cancer tissue and A2780/CP cells compared with sensitive ovarian cancer tissue and cell lines A2780. And in A2780 cells treated with Cisplatin, the expression of MCT1 increased in a concentration-dependent manner, MCT1 knockdown attenuates cisplatin-induced cell viability. In A2780 and A2780/CP cells transfected with MCT1 siRNA, the activation of several downstream targets of Fas, including FasL and FAP-1 were largely prevented, whereas the expression of Caspase-3 was increased, accompanying with increased abundance of Fas. Coimmunoprecipitation and immunofluorescence showed that there is interaction between endogenous MCT1 with Fas in vivo and in vitro. In vivo, depletion of MCT1 by shRNA reverses cisplatin-resistance and the expression of Fas. This study showed that down regulation of MCT1 promote the sensibility to Cisplatin in ovarian cancer cell line. And this effect appeared to be mediated via antagonizing the effect of Fas.     

Ikaros的3种亚型对人卵巢癌SKOV3细胞增殖的影响

目的:探讨Ikaros的3种亚型对人卵巢癌SKOV3细胞增殖的影响。方法:利用逆转录病毒转染人卵巢癌SKOV3细胞,分别表达Ikaros的3种亚型(IK1、IK2和IK6);采用CCK-8法分析表达不同亚型后SKOV3细胞的增殖能力;流式细胞术检测细胞周期的改变;Western blot检测细胞周期相关蛋白的表达。结果:CCK-8结果显示IK1和IK2能明显抑制SKOV3细胞的增殖;细胞周期结果表明IK1和IK2能诱导SKOV3细胞发生G1期阻滞,IK6则对SKOV3细胞的增殖能力和细胞周期则无明显影响;Western blot检测结果显示,IK1和IK2明显降低cyclin D1和cyclin D2蛋白的表达水平,同时升高p21蛋白的表达水平。IK6则对SKOV3细胞的cyclin D1、cyclin D2及p21蛋白表达水平无明显改变。结论:IK1和IK2这2种亚型能明显抑制卵巢癌SKOV3细胞的增殖,其机制可能是由于IK1和IK2能降低细胞周期促进蛋白cyclin D1和cyclin D2的表达及增加细胞周期抑制蛋白p21的表达,从而诱导细胞周期发生G1期阻滞。而IK6亚型对SKOV3细胞增殖能力及细胞周期无明显影响。     

小干扰RNA沉默hPTTG1基因对卵巢癌细胞增殖和凋亡的影响

目的: 利用RNA干扰技术沉默人垂体瘤转化基因1( hPTTG1 )表达,观察卵巢癌细胞增殖能力和细胞凋亡的改变并探讨分子机制。方法: 化学合成靶定 hPTTG1 的小干扰RNA(siRNA)转染体外培养的A2780细胞,RT-PCR和Western blotting检测 hPTTG1 及c-myc表达水平;MTT法和 -TdR掺入实验检测 hPTTG1 对细胞增殖的影响;annexin V/PI染色流式细胞术和TUNEL法测定各组细胞凋亡情况。结果: hPTTG1 siRNA转染组 hPTTG1 mRNA和蛋白表达下降,抑制率分别为70.5%±3.9%和63.8%±4.5%;转染 hPTTG1 siRNA 24 h细胞吸光度值开始下降,48 h抑制效果最明显,抑制率为42.9%±5.2%;转染 hPTTG1 siRNA后细胞 -TdR放射性计数明显低于空白组和阴性对照组; hPTTG1 siRNA干扰组细胞存活率下降,细胞凋亡率和坏死率均增加;TUNEL染色分析结果可见 hPTTG1 siRNA干扰组凋亡指数明显高于空白组和阴性对照组; hPTTG1 干扰后c-myc mRNA和蛋白表达均下调。结论: hPTTG1 siRNA通过下调c-myc表达抑制A2780细胞增殖,诱导细胞凋亡, hPTTG1 可作为卵巢癌基因治疗的候选靶点。     

TROP-1/Ep-CAM and CD24 are potential candidates for ovarian cancer therapy

To clarify the possible roles of epithelial cell adhesion molecule (TROP-1/Ep-CAM) and CD24 molecule (CD24) in ovarian tumorigenesis, and explore the possible mechanism underlying this disease. Recombinant eukaryotic expression vectors pCIneo-TROP-1/Ep-CAM and pCIneo-CD24 were transfected into human normal ovarian surface epithelia cell line IOSE-80 respectively, with IOSE-80 cells transfected with the empty vector pCIneo as control. MRNA and protein expression of TROP-1/Ep-CAM and CD24 were detected by RT-PCR and Western blotting, respectively. Cell migration was assayed by trans-well inserts; cell proliferation and adhesion were analyzed by CCK-8 Cell Counting kit; cell cycle and cell apoptosis analysis were performed by flow cytometer. The expressions of TROP-1/Ep-CAM and CD24 were obviously up-regulated in TROP-1/Ep-CAM group and CD24 group compared to that in control group (P < 0.01). Cells of TROP-1/Ep-CAM group and CD24 group was significantly promoted migratory and proliferation abilities, but inhibited cell apoptosis and adhesive than that of control group (P < 0.05). Besides, the number of the cells in G1 and G2 stages was significantly lower in two disease groups than that in control group (P < 0.05). TROP-1/Ep-CAM and CD24 may play key roles in the progression of ovarian cancer through promoting migration, proliferation, inhibiting cell apoptosis and adhesion, and disturbing cell cycle. They may be used as specific therapeutic targets in the treatment of ovarian cancer. However, further experiments are still needed to confirm our results.     

Proteasome inhibition suppresses cisplatin-dependent ERCC-1 mRNA expression in human ovarian tumor cells

One of the major problems with ovarian cancer treatment is the clinical development of resistance to cisplatin. Considerable efforts have been directed toward the identification of biological and pharmacological agents that would reverse drug resistance to cisplatin. ALLnL is an inhibitor of the proteasome that can inhibit the ubiquitin-proteasome pathway, which plays an essential role in many processes in cell life. We have recently shown that ALLnL, at concentrations that do not appear harmful, has significantly enhanced DNA platination and decreased DNA repair of cisplatin-DNA adducts in human A2780/CP70 ovarian carcinoma cells. These activities of proteasome inhibition were also associated with substantially increased cisplatin toxicity in these cells. In this communication, we demonstrate that treatment of A2780/CP70 cells with ALLnL blocks cisplatin-induced ERCC-1 mRNA expression in a concentration- and time-dependent manner, as measured by Northern blot analysis. In addition, we showed that the cisplatin-dependent increase in steady-state levels of ERCC-1 mRNA was prevented by pretreatment with lactacystin, a potent and specific inhibitor of proteasome. These results suggest that the effect of proteasome inhibition on cisplatin cytotoxicity, DNA platination, and DNA repair of cisplatin adducts in ovarian cancer cells may be through down-regulating ERCC-1 expression.     

MiR-302a inhibits the tumorigenicity of ovarian cancer cells by suppression of SDC1

MicroRNA plays an important role in tumor proliferation and cell cycle. In this study, we suggested the level of miR-302a was increasing in the human ovarian cancer cells compared to the normal cells. We aimed to explore the role of miR-302a downregulation in human ovarian cancer cells. Functional studies demonstrate over expression of miR-302a could significant suppress ovarian cancer cells proliferation and promote the cell cycle progress. In vitro reporter assay suggested SDC1 is a direct target gene of miR-302a. Furthermore, the expressions of miR-302a in ovarian cancer cells were inversely corrected with that of SDC1. Upregulation of SDC1 could rescue the effect of over expressed miR-302a in the ovarian cancer cells. These findings provide evidence that miR-302a plays a key role in inhibition of the ovarian cancer cells proliferation, and enhancing the cells’ apoptosis through targeting SDC1, and strongly suggest that exogenous miR-302a may have therapeutic value in treating ovarian cancer.