趋化因子CXCL5对宫颈癌HeLa细胞恶性表型的影响及机制

目的研究趋化因子CXCL5对宫颈癌HeLa细胞恶性表型的影响及其机制。方法通过基因转染构建过表达趋化因子CXCL5的宫颈癌HeLa细胞株,研究过表达CXCL5对宫颈癌HeLa细胞恶性行为和肿瘤相关基因表达的影响。结果 CCK-8、集落形成和划痕实验结果显示,过表达CXCL5可明显促进HeLa细胞的增殖和迁移能力;Western Blot实验结果表明,与空载体转染细胞株相比较,CXCL5过表达HeLa细胞株的ERK和p-ERK蛋白表达水平显著提高。结论趋化因子CXCL5通过ERK信号通路经自分泌途径促进宫颈癌细胞的增殖与迁移。     

LINC00665通过激活WNT-CTNNB1/β-catenin信号通路促进HeLa细胞增殖、迁移、侵袭和上皮-间质转化（英文）

越来越多的证据表明,长链非编码RNA(long non-coding RNA, lncRNA)在癌症进展中起关键作用。但是,人们对lncRNA 00665 (LINC00665)在大多数癌症中的作用了解甚少。本研究旨在揭示LINC00665在宫颈癌细胞中的功能。用小发夹RNA (short hairpin RNA, shRNA)敲减HeLa细胞中LINC00665的表达以研究宫颈癌细胞在体外和体内的转移和增殖表型。将LINC00665敲减组和对照组HeLa细胞进行转录组测序,筛选差异表达基因(differentially expressed genes, DEGs)。对DEGs进行Metascape数据库功能分析和基因集富集分析(gene set enrichment analysis, GSEA)。用Western blot和免疫荧光染色法检测WNT-CTNNB1/β-catenin通路和上皮-间质转化(epithelial-mesenchymal transition, EMT)相关标记物的表达水平。结果显示,沉默LINC00665降低HeLa细胞活力,上调E-cadherin蛋白表达水平,下调N-cadherin、Vimentin和CTNNB1蛋白表达水平,抑制HeLa细胞的迁移和侵袭。生物信息学分析结果显示LINC00665可能通过激活WNT-CTNNB1/β-catenin信号转导通路促进EMT。以上结果提示,LINC00665通过WNT-CTNNB1/β-catenin信号传导途径调控HeLa细胞增殖、迁移、侵袭和EMT。LINC00665可能作为一个潜在的宫颈癌药物治疗靶点。     

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

丝裂原活化蛋白激酶4通过激活AKT促进宫颈癌细胞增殖

丝裂原活化蛋白激酶(MAPK)家族在基因表达调控和细胞功能活动中发挥了关键作用,并且在肿瘤的发生发展中也起着重要作用。最近发现,MAPK家族的一个新成员MAPK4在肺癌的进展中起着重要作用,然而MAPK4在宫颈癌中的作用尚未见报道。本研究首先通过GSCA在线软件在12个组织类型共65种肿瘤细胞系中分析了MAPK4的mRNA水平。根据分析的结果,选取了宫颈癌细胞中MAPK4表达最高的HeLa细胞和表达最低的SiHa细胞作为研究模型进行下一步的研究。在HeLa细胞中,干扰MAPK4的表达后,通过CCK8试验检测了宫颈癌细胞的增殖能力,发现HeLa细胞的增殖能力显著下降。在SiHa细胞中,转染MAPK4过表达质粒后检测细胞的增殖能力,发现MAPK4过表达后,SiHa细胞的增殖能力显著上升。最后,本研究探索了MAPK4诱导宫颈癌细胞增殖的分子机制,发现在宫颈癌细胞中MAPK4促进了蛋白激酶B(AKT)的活性,这表明MAPK4可能通过激活AKT促进了宫颈癌细胞的增殖。上述研究结果表明,MAPK4能作为判断宫颈癌增殖潜能的标志物以及宫颈癌治疗的潜在分子靶点。本研究为宫颈癌的发病机制提供了新的见解,对开发新的靶向药物治疗宫颈癌具有一定的价值。     

掌叶半夏有效提取物对宫颈癌细胞株增殖的抑制作用

目的既往的临床研究证实中药掌叶半夏可有效治疗宫颈癌。在此基础上,课题组观察了中药掌叶半夏有效提取物(Pinellia Extract,PE)对宫颈癌细胞株CaSki和HeLa细胞增殖的作用,先前采用MTT法检测细胞活性以及倒置相差显微镜观察细胞形态的变化,初步显示了PE对CaSki和HeLa细胞的增殖有抑制作用;本文将对PE的增殖抑制作用进行进一步的研究。方法采用扫描电镜和透射电镜观察PE作用后CaSki和HeLa细胞表面及内部超微结构的变化,In-cell Western blot检测不同浓度PE作用15min后CaSki和HeLa细胞磷酸化ERK的表达。结果0.15mg/mlPE作用24h后CaSki和HeLa细胞表面结构及内部结构均发生了显著的变化,提示细胞增殖受到抑制;不同浓度PE作用15min后CaSki和HeLa细胞磷酸化ERK的表达随PE浓度的增加逐渐下降。结论PE对CaSki和HeLa宫颈癌细胞株的增殖有明显抑制作用,表现在细胞表面及内部超微结构的变化;增殖抑制作用之一是阻断ERK的磷酸化。实验结果为进一步研究PE的抗宫颈癌作用机制以及新药的研制开发奠定了一定的理论依据和实验基础。     

姜黄素通过MAPK信号通路诱导人肝癌SMMC-7721细胞凋亡

本文阐述了姜黄素(Curcumin)对体外培养的人肝癌SMMC-7721细胞增殖和凋亡的影响,并探讨了其诱导凋亡的信号转导机制。采用MTT法和细胞计数法检测不同浓度姜黄素对人肝癌细胞株SMMC-7721增殖的影响,利用流式细胞术检测姜黄素对人肝癌SMMC-7721细胞凋亡的影响,通过RT-PCR及Western blot检测姜黄素对人肝癌SMMC-7721细胞中凋亡相关蛋白Caspase-3、Survivin、Bcl-2和Bax表达的影响,最后通过检测MAPK的磷酸化水平分析姜黄素诱导SMMC-7721细胞凋亡的信号转导机制,通过MAPK抑制剂实验进一步证实诱导凋亡的分子机制。研究结果显示,姜黄素呈时间和剂量依赖性抑制人肝癌SMMC-7721细胞的增殖,其中40μmol/L姜黄素可明显诱导SMMC-7721细胞的凋亡,并呈时间依赖性上调促凋亡蛋白Caspase-3和Bax的表达、下调抗凋亡蛋白Survivin和Bcl-2的表达,姜黄素对凋亡相关蛋白表达的调节及诱导凋亡可以通过激活JNK、抑制ERK和p38 MAPK信号通路实现。表明姜黄素可诱导人肝癌SMMC-7721细胞凋亡,其机制与姜黄素激活JNK、抑制ERK和p38 MAPK信号通路从而上调Caspase-3和Bax的表达,下调Survivin和Bcl-2的表达有关。     

STAT3基因沉默降低小鼠乳腺癌细胞的体外迁移能力

信号转导子与转录活化子3(STAT3)是一个具有信号转导和转录调控双重功能的转录因子,有文献报道STAT3在乳腺癌中的表达显著升高,并能促进乳腺癌的转移。为了深入探索STAT3在肿瘤发生发展中的作用和影响乳腺癌转移的分子机制,采用RNA干扰技术在小鼠乳腺癌细胞株4T1中沉默STAT3的表达。MTT实验结果显示STAT3沉默对4T1细胞的增殖能力没有影响;细胞迁移实验结果表明STAT3表达被沉默后4T1细胞的迁移能力明显被抑制;定量PCR结果显示,STAT3基因沉默后4T1细胞中VEGF和IL-6的mRNA水平下降,E-cadherin表达上升,mosin表达下降;信号通路检测显示STAT3基因表达沉默后MAPK的活化明显降低。研究表明STAT3在小鼠乳腺癌细胞的迁移过程中发挥重要作用,为以STAT3基因为靶向的治疗提供了一定的实验依据。     

A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响

为了观察A-Raf的突变体A-Raf-S214C对MAPK(mitogen-activated protein kinase,丝裂原活化蛋白激酶)信号通路的影响,并探讨其诱发肺癌和朗格汉斯增生症的可能分子机制。通过定点突变构建pc DNA5-FRT-TO-A-Raf-S214C突变质粒,并利用该质粒构建稳定表达A-Raf-S214C突变体的HEK293细胞系,之后通过检测下游ERK磷酸化程度,确定该突变体对MAPK信号通路的影响。结果显示,通过实验成功构建了稳定表达A-Raf-S214C突变体的细胞系,在不同浓度的多西环素诱导下,其表达量有所不同。与野生型相比,A-Raf-S214C突变体可明显增强MAPK信号通路中下游信号分子ERK的磷酸化。结果表明A-Raf-S214C突变体是A-Raf激酶的激活突变体,可以导致MAPK信号通路下游信号分子ERK磷酸化明显增强。     

OPN对卵巢癌Hey细胞增殖、迁移和侵袭能力的影响

目的:探讨骨桥蛋白(Osteopontin,OPN)对卵巢癌Hey细胞增殖、迁移和侵袭能力的影响及其可能涉及的分子机制。方法:选择卵巢癌细胞株Hey、HO8910、A2780和正常卵巢上皮细胞IOSE80,采用Western blot检测OPN蛋白的表达情况。对OPN表达量相对较高的Hey细胞株的OPN基因敲减或过表达,运用CCK-8、平板克隆实验、细胞划痕、Transwell侵袭实验等方法研究OPN对细胞增殖、迁移及侵袭能力的影响,采用Western blot检测Wnt/β-catenin通路相关蛋白β-catenin、CyclinD1、c-myc的表达情况。结果:OPN在卵巢癌细胞株Hey、HO8910、A2780内均有表达,且表达量均高于正常卵巢上皮细胞IOSE80。si RNA-OPN转染卵巢癌Hey细胞沉默OPN表达,CCK-8和平板克隆实验显示沉默OPN能够降低Hey细胞的增殖能力,划痕实验和Transwell侵袭实验显示下调OPN可降低Hey细胞的迁移和侵袭能力,Wnt/β-catenin通路相关蛋白β-catenin、CyclinD1、c-myc表达下降。ex-OPN转染Hey细胞使OPN表达升高,与对照组相比,OPN过表达能够增强Hey细胞的增殖、迁移和侵袭能力,并且Wnt/β-catenin通路相关蛋白β-catenin、CyclinD1、c-myc表达升高。结论:OPN能够促进卵巢癌细胞的增殖、迁移及侵袭能力,该作用可能通过促进Wnt/β-catenin信号通路实现。     

The expression of the phosphotyrosine phosphatase DEP-1/PTPeta dictates the responsivity of glioma cells to somatostatin inhibition of cell proliferation

Here we characterize the intracellular effectors of the antiproliferative activity of somatostatin in glioma cell lines and post-surgical specimens. The responsiveness to somatostatin correlated with the expression of the phosphotyrosine phosphatase DEP-1/PTPeta, identified in C6 and U87MG cells, in which somatostatin inhibited cell growth. The expression of a dominant negative mutant of DEP-1/PTPeta in C6 cells abolished somatostatin effects, confirming the involvement of this phosphotyrosine phosphatase in such effects. Somatostatin treatment increased the activity of DEP-1/PTPeta and inhibited ERK1/2 activation. Conversely, basic fibroblast growth factor-dependent MEK phosphorylation was not affected, suggesting a direct effect on ERK1/2. In vitro experiments showed that PTPeta was able to interact and dephosphorylate ERK1/2 activated by basic fibroblast growth factor. Furthermore, by transfecting PTPeta in the somatostatin-unresponsive, DEP-1/PTPeta-deficient U373MG cells, the somatostatin-dependent control of cell proliferation was recovered. Finally we evaluated the requirement for DEP-1/PTPeta in somatostatin inhibition of cell proliferation in post-surgical specimens derived from different grade human gliomas. Although all of the glioma analyzed expressed somatostatin receptor mRNA, DEP-1/PTPeta expression was limited to 8 of 22 of the tumors. Culturing seven gliomas, a correlation between the expression of DEP-1/PTPeta and the somatostatin antiproliferative effects was identified. In conclusion we propose that the expression and activation of DEP-1/PTPeta is required for somatostatin inhibition of glioma proliferation.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

p53 can inhibit cell proliferation through caspase-mediated cleavage of ERK2/MAPK

Stimulation of the Ras/MAPK cascade can either activate p53 and promote replicative senescence and apoptosis, or degrade p53 and promote cell survival. Here we show that p53 can directly counteract the Ras/MAPK signaling by inactivating ERK2/MAPK. This inactivation is due to a caspase cleavage of the ERK2 protein and contributes to p53-mediated growth arrest. We found that in Ras-transformed cells, growth arrest induced by p53, but not p21(Waf1), is associated with a strong reduction in ERK2 activity, phosphorylation, and protein half-life, and with the appearance of caspase activity. Likewise, DNA damage-induced cell cycle arrest correlates with p53-dependent ERK2 downregulation and caspase activation. Furthermore, caspase inhibitors or expression of a caspase-resistant ERK2 mutant interfere with ERK2 cleavage and restore proliferation in the presence of p53 activation, indicating that caspase-mediated ERK2 degradation contributes to p53-induced growth arrest. These findings strongly point to ERK2 as a novel p53 target in growth suppression.     

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.     

TBP-like Protein（TLP）通过G2／M期阻滞抑制HeLa细胞的增殖

TBP—likeprotein（TLP）是真核细胞中一种常见的转录因子,在调节生长发育方面起着重要的作用。该实验构建重组质粒pEGFP—N1．TLP,研究TLP对人宫颈癌细胞HeLa增殖的影响。利用流式细胞仪检测质粒的转染效率,通过激光共聚焦显微镜观察外源TLP蛋白的亚细胞定位。经过MTT检测、RNAi—TLP诱导的基因沉默7LHoechst33258染色研究TLP对HeLa细胞的增殖抑制作用。流式细胞术、Westernblot和RT-PCR实验结果表明,TLP将HeLa细胞周期阻滞于G2／M期,并抑制周期相关基因CDKl和CyclinBl的转录和翻译。研究表明,外源TLP在HeLa细胞的细胞核中表达,通过降低细胞周期相关基因CDKl和CDKl的表达水平,将HeLa细胞的细胞周期阻滞于G2／M期,从而抑制细胞的增殖。     

DNA损伤诱导的长链非编码RNA-UCA1促进HeLa细胞增殖

尿路上皮癌抗原1 (UCA1)是一种长链非编码RNA,在多种肿瘤内高表达.然而,其在宫颈癌细胞和组织中的表达报告颇不一致,且功能尚未确定.本文探索UCA1在宫颈癌HeLa细胞中的生物学功能.实时定量PCR（qRT-PCR）结果显示,UCA1、p21和p53 mRNA在阿霉素（doxorubicin,DOX）或γ射线照射的HeLa细胞中表达上调|相反,敲减p53表达则可抑制DOX诱导的UCA1上调.表明DNA损伤诱导的UCA1可能与p53有关.转染结合CCK8检测HeLa细胞增殖活力结果显示,与对照比较,过表达UCA1促进HeLa细胞增殖,干扰UCA1表达则减缓细胞增殖.此外,流式细胞术结果显示,过表达UCA1导致阿霉素诱导的凋亡率下降;siRNA抑制UCA1表达后引起细胞G2/M期比例上升,S期下降,且阿霉素诱导的细胞凋亡率上升.上述结果说明,DNA损伤诱导的UCA1可促进HeLa细胞增殖,减少细胞凋亡.然而,是否DNA损伤诱导的UCA1上调依赖p53尚需进一步实验证明.     

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.