DLC-1 基因在MCF-7 人乳腺癌细胞系中低表达的机制探究

目的:探究DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制。方法:应用甲基化特异性PCR(MSP)检测人乳腺癌细胞MCF-7的DLC-1基因甲基化状态,不同浓度的5-氮杂-2’-脱氧胞嘧啶(5-Aza-CdR)处理人乳腺癌细胞MCF-7,RT-PCR及Real-time PCR定量检测用药前后细胞中DLC-1基因mRNA表达水平变化。结果:DLC-1基因启动子区CpG岛呈甲基化状态,经过5-Aza-CdR处理后,DLC-1基因启动子区呈去甲基化状态,并且其mRNA恢复表达。结论:抑癌基因DLC-1 CpG岛甲基化是导致该基因低表达的原因之一,5-Aza-CdR能逆转DLC-1基因甲基化状态。     

DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制探究

目的：探究DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制。方法：应用甲基化特异性PCR（MSP）检测人乳腺癌细胞MCF-7的DLC-1基因甲基化状态,不同浓度的5-氮杂-2＇-脱氧胞嘧啶（5-Aza-CdR）处理人乳腺癌细胞MCF-7,RT-PCR及Real-time PCR定量检测用药前后细胞中DLC-1基因mRNA表达水平变化。结果：DLC-1基因启动子区CpG岛呈甲基化状态,经过5-Aza-CdR处理后,DLC-1基因启动子区呈去甲基化状态,并且其mRNA恢复表达。结论：抑癌基因DLC-1 CpG岛甲基化是导致该基因低表达的原因之一,5-Aza-CdR能逆转DLC-1基因甲基化状态。     

乙酰哈巴苷通过Wnt信号通路诱导结肠癌HCT116细胞凋亡

本文探讨乙酰哈巴苷诱导结肠癌HCT116细胞凋亡机制。采用流式细胞术检测乙酰哈巴苷对HCT116细胞周期及凋亡的影响,发现细胞周期被阻滞在G1期,0.75 mmol/L乙酰哈巴苷处理48 h后,细胞凋亡明显增加。转录组测序显示乙酰哈巴苷引起HCT116细胞1 921个mRNA上调和2 208个mRNA下调,可显著影响Wnt信号通路。RT-qPCR和Western blot验证实验表明,乙酰哈巴苷处理后,HCT116细胞中β-catenin、CyclinD1、Survivin、Bcl-2和c-Myc蛋白显著降低(P<0.05),凋亡相关蛋白Bax和cleaved-caspase3表达增加(P<0.05)。上述结果表明,乙酰哈巴苷主要通过Wnt信号通路抑制结肠癌细胞增殖并诱导细胞凋亡。     

DLC-1基因表达对结肠癌细胞侵袭转移能力的影响

目的:研究DLC-1基因对结肠癌细胞侵袭迁移能力的影响.方法:将DLC-1 shRNA(短发夹状RNA,short hairpin RNA)序列克隆到质粒pGCsi-U6/Neo载体,采用脂质体介导的转染方法将构建的DLC-1 shRNA表达质粒转入结肠癌细胞系LoVo细胞.采用RT-PCR技术和Western Blot技术分别检测LoVo细胞中DLC-1mRNA和蛋白表达水平的变化.Transwell小室人工重组基底膜侵袭转移实验观察LoVo细胞侵袭迁移能力的改变.结果:结肠癌细胞系LoVo细胞表达DLC-1分子.所构建质粒表达载体能有效地干扰LoVo细胞DLC-1 mRNA和蛋白质表达水平;Transwell小室人工重组基底膜侵袭转移实验结果显示,转染后LoVo细胞侵袭转移能力明显增强(p＜0.05).结论:结肠癌细胞系LoVo细胞表达DLC-1基因,应用RNAi技术可特异性降低其表达.DLC-1的表达水平与结肠癌细胞侵袭转移相关.     

曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

芍药苷对结肠癌SW480细胞增殖、侵袭、迁移的影响

目的观察芍药苷对人结肠癌SW480细胞株增殖、侵袭、迁移的影响,探究其干预机制。方法含10%胎牛血清的DMEM/F12培养基常规培养人结肠癌SW480细胞株,CCK-8以及EdU-488法检测芍药苷对SW480细胞增殖的影响,Transwell小室检测芍药苷对SW480细胞侵袭、迁移的影响,Westernblot法检测beclin1、Bcl-2蛋白的表达。结果不同浓度芍药苷分别处理24h、48h、72h的结肠癌SW480细胞增殖活性受到显著抑制:相比较对照组,160μg/ml芍药苷处理48h后,SW480细胞内黄绿色荧光减弱,细胞增殖率显著下降,为(58.91±4.99)%;SW480细胞的侵袭细胞数、迁移细胞数显著下降:侵袭抑制率为26.50%,迁移抑制率为24.67%;beclin1蛋白表达高于对照组,Bcl-2蛋白表达低于对照组,beclin1与Bcl-2蛋白表达呈负相关。结论芍药苷能够抑制结肠癌SW480细胞增殖、侵袭和迁移,其机制可能通过抑制Bcl-2蛋白表达,上调beclin1蛋白的表达。     

蛋白酶体抑制剂硼替佐米抑制PI3K/Akt通路致结肠癌SW480细胞凋亡

目的:研究蛋白酶体抑制剂硼替佐米对结肠癌SW480细胞凋亡作用,并进一步探讨其作用机制.方法:硼替佐米1-500nmol/L处理结肠癌SW480细胞24-48小时,MTT法检测细胞存活率、药物IC50值.流式细胞术检测细胞凋亡率.Western blot技术检测caspase-3,p-Akt和PTEN蛋白表达水平变化.结果:硼替佐米以时间-剂量依赖方式抑制结肠癌SW480细胞增殖,48小时IC50值:87.36 nmol/L.细胞凋亡实验显示药物作用24小时细胞开始出现凋亡,48小时凋亡明显.硼替佐米作用24小时后细胞周期明显阻滞在G0/G1期.Westemblot实验显示,80 nmol/L硼替佐米处理结肠癌SW480细胞后PTEN蛋白表达水平随时间明显增加,而p-Akt蛋白随时间表达下降.结论:硼替佐米可以抑制结肠癌SW480细胞增殖.其机制可能与抑制PTEN蛋白降解,抑制p-Akt途径有关.为结肠癌治疗药物的发展和更新提供了新的候选分子.     

蟾毒灵可抑制人红白血病细胞增殖并下调肾母细胞瘤1基因表达

已有研究证实蟾毒灵具有抑制肿瘤细胞增殖及诱导细胞凋亡的作用,在白血病治疗中疗效显著,然而其机制尚未阐明。本研究试图探讨蟾毒灵对人红系白血病(HEL)细胞增殖,肾母细胞瘤基因1 (Wilms'tumor 1 gene, WT1)甲基化的影响及其可能的作用机制。本研究采用不同浓度的蟾毒灵处理HEL细胞,观察细胞形态、增殖情况和细胞周期,采用RT-PCR、Western blotting和免疫细胞化学法检测WT1的mRNA和蛋白表达水平,并用甲基化特异性分析WT1的DNA甲基化和DNA甲基转移酶3a (DNMT3a)的蛋白表达水平。研究结果表明,蟾毒灵对HEL细胞的增殖抑制作用呈剂量依赖性,抑制率为23.13%～84.62%。在蟾毒灵处理的HEL细胞中观察到典型的凋亡形态特征;细胞周期增殖指数由75.45降至49.67;WT1 mRNA及其蛋白表达水平随着蟾毒灵剂量的增加而逐渐降低,同时WT1基因的甲基化状态由未甲基化状态变为部分或完全甲基化状态。而蟾毒灵处理后DNMT3a蛋白的表达水平逐渐增加,呈剂量依赖性。我们的研究初步说明蟾毒灵不仅能显著抑制HEL细胞增殖,阻滞G0/G1期细胞周期,还能诱导细胞凋亡,下调WT1的表达水平。     

抑制VEGF基因表达对乳腺癌细胞细胞周期的影响

目的：研究针对VEGF基因的siRNA（small interferenceRNA）对乳腺癌MCF-7细胞细胞周期的影响。方法：依据Promega公司在网上提供的设计软件,设计针对VEGF基因的siRNA,合成DNA模板,体外转录合成siRNA。脂质体转染法将合成的siRNA转染入MCF-7细胞,以未转染细胞以及错义序列siRNAscr转染细胞为对照。用细胞计数法检测siRNA对MCF-7细胞增殖的影响：流式细胞法检测细胞周期变化,RT—PCR法比较转染前后p21、CyclinDl表达水平的变化,Westemblot检测转染前后磷酸化ERK的表达。结果：细胞计数法结果显示,转染24h后siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。siRNA转染后能有效地抑制MCF-7细胞的增殖,阻滞细胞周期于G0／G1期,S期细胞明显减少,G0／G1期细胞比例逐渐增多;p21mRNA表达显著上调,抑制CyclinD1mRNA及磷酸化ERK蛋白的表达。结论：体外转录合成的siRNA可能通过上调细胞周期蚤白激酶抑制剂p21的表达,下调CyclinDl及磷酸化ERK的表达,将细胞周期阻滞于G0／G1期,从而显著抑制MCF-7细胞的增殖。     

塞来昔布诱导HCT-116结肠癌细胞G2/M阻滞

目的:研究选择性COX-2抑制剂塞来昔布诱导结肠癌细胞株HCT-116细胞周期阻滞的作用及其可能的机制。方法:应用流式细胞仪检测塞来昔布对HCT-116细胞周期的影响,定量PCR检测细胞周期素cyclinB1及COX-2 mRNA表达水平,Western-Blot检测细胞周期素cyclinB1的蛋白水平。结果:塞来昔布诱导HCT-116细胞G2/M阻滞的作用呈剂量依赖性,塞来昔布在mRNA及蛋白水平下调HCT-116细胞的cyclinB1。结论:塞来昔布能在体外抑制HCT-116细胞的增殖,诱导G2/M的阻滞,其作用与下调细胞周期素cyclinB1有关。     

Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells

12(S)-Lipoxygenase (LOX) and its product 12(S)-hydroxyeicosatetraenic (HETE) acid have been implicated in angiogenesis and tumour invasion in several tumour types while their role in colorectal cancer progression has not yet been studied. We have analysed 12(S)-LOX expression in colorectal tumours and found gene expression up-regulated in colorectal cancer specimens for which the pathology report described involvement of inflammation. Using cell line models exposed to 12(S)-HETE or over-expressing 12(S)-LOX malignant cell growth as well as tumour cell migration was found to be stimulated. Specifically, Caco2 and SW480 cells over-expressing 12(S)-LOX formed fewer colonies from sparse cultures, but migrated better in filter-migration assays. SW480 LOX cells also had higher anchorage-independent growth capacity and a higher tendency to metastasise in vivo. Knock-down or inhibition of 12(S)-LOX inhibited cell migration and anchorage-independent growth in both 12(S)-LOX transfectants and SW620 cells that express high endogenous levels of 12(S)-LOX. On the cell surface E-cadherin and integrin-β1 expression were down-regulated in a 12(S)-LOX-dependent manner disturbing cell-cell interactions. The results demonstrate that 12(S)-LOX expression in inflammatory areas of colorectal tumours has the capacity to induce an invasive phenotype in colorectal cancer cells and could be targeted for therapy.     

Effect of RSK4 on biological characteristics of colorectal cancer

Background

This study aimed to investigate the expression of P90 Ribosomal Protein S6 kinase 4 (RSK4) in colorectal cancer cells and its biological function.

Methods

We selected early SW480 and HCT116 colorectal cancer cell lines, using Lipofectamine? 2000 transfection reagent carrying RSK4 gene transfected into cells to establish the colorectal cancer cell lines with high expression of RSK4. RT-PCR and western blot (WB) analysis confirmed RSK4 expression in SW480 and HCT116 cancer cell lines. We used methylthiazoltetrazolium (MTT) assay and flow cytometry to detect the proliferation of colorectal cancer cells. After transfection of RSK4, the effect of RSK4 on the RNA levels associated with epithelial–mesenchymal transition (EMT) of colorectal cancer cells was analyzed by real-time fluorescence quantitative PCR and the expression of EMT-related protein was detected by WB analysis.

Results

After transfection of RSK4 overexpression, the MTT assay detected that RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro; flow cytometry detected that S-phase cells decreased significantly, and G0/1 cells increased significantly (P?<?0.05). The invasion ability of SW480 and HCT116 cells transfected with RSK4 was markedly lower than that in the control group, and the difference was statistically significant (P?<?0.05). Fluorescent quantitative PCR and WB analysis showed that the expression of EMT-associated molecular E-cadherin was remarkably increased and the expression of Snail was significantly decreased (P?<?0.01).

Conclusion

RSK4 gene in colorectal cancer cell lines with low expression of RSK4 after transfection can inhibit the growth and invasion of tumor cells. RSK4 gene may inhibit EMT and inhibit metastasis of colorectal cancer cells, may be a potential tumor suppressor gene and inhibit tumor distant metastasis, and may provide the biological basis for new therapeutic targets.

     

Proteomics identification of ITGB3 as a key regulator in reactive oxygen species-induced migration and invasion of colorectal cancer cells

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females worldwide. Unfortunately 40-50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of <10%. Reactive oxygen species (ROS) have been proposed to play a crucial role in tumor metastasis. We now show that higher levels of ROS accumulation are found in a colorectal cancer-derived metastatic cell line (SW620) compared with a cell line (SW480) derived from the primary lesion from the same patient. In addition, ROS accumulation can affect both the migratory and invasive capacity of SW480 and SW620 cells. To explore the molecular mechanism underlying ROS-induced migration and invasion in CRC, we have compared protein expression patterns between SW480 and SW620 cells using a two-dimensional electrophoresis-based proteomics strategy. A total of 63 altered proteins were identified from tandem MS analysis. Cluster analysis revealed dysregulated expression of multiple redox regulative or ROS responsive proteins, implicating their functional roles in colorectal cancer metastasis. Molecular and pathological validation demonstrated that altered expression of PGAM1, GRB2, DJ-1, ITGB3, SOD-1, and STMN1 was closely correlated with the metastatic potential of CRC. Functional studies showed that ROS markedly up-regulated expression of ITGB3, which in turn promoted an aggressive phenotype in SW480 cells, with concomitant up-regulated expression of STMN1. In contrast, knockdown of ITGB3 expression could mitigate the migratory and invasive potential of SW620 or H(2)O(2)-treated SW480 cells, accompanied by down-regulated expression of STMN1. The function of ITGB3 was dependent on the surface expression of integrin αvβ3 heterodimer. Furthermore, STMN1 expression and the PI3K-Akt-mTOR pathway were found to be involved in ROS-induced and ITGB3-mediated migration and invasion of colorectal cancer cells. Taken together, these studies suggest that ITGB3 plays an important role in ROS-induced migration and invasion in CRC.     

DLC-1与肝癌关系研究

DLC-1基因是一种肿瘤抑制基因,位于人类染色体8p21．3-22。它是RhoA特异性GTP酶的激动蛋白,与调控细胞增殖和粘附的信号传导通路关系密切,在人类多种肿瘤中呈低表达或表达缺失。研究发现DLC-1基因在原发性肝癌(HCC)及肝癌细胞系中表达缺失,提示该基因在原发性肝癌中抑制了肝癌细胞的增殖。DLC-1表达的恢复引起了caspase-3介导的细胞凋亡,抑制肝癌细胞的生长和癌细胞的浸润,从而在肝细胞癌的转移、侵袭及肿瘤细胞的生物特性方面发挥作用。因其与肝癌发生,转移乃至复发关系密切,使其在肝癌早期发现,早期预测肝癌的转移复发及肝癌的预后方面发挥重要角色。     

NOP14 regulates the growth,migration, and invasion of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. Recently, nucleolar complex protein 14 (NOP14) has been discovered to play a critical role in cancer development and progression, but the mechanisms of action of NOP14 in colorectal cancer remain to be elucidated. In this study, we used collected colorectal cancer tissues and cultured colorectal cancer cell lines (SW480, HT29, HCT116, DLD1, Lovo), and measured the mRNA and protein expression levels of NOP14 in colorectal cancer cells using qPCR and Western blotting. GFP-NOP14 was constructed and siRNA fragments against NOP14 were synthesized to investigate the importance of NOP14 for the development of colorectal cells. Transwell migration assays were used to measure cell invasion and migration, CCK-8 kits were used to measure cell activity, and flow cytometry was applied to the observation of apoptosis. We found that both the mRNA and protein levels of NOP14 were significantly upregulated in CRC tissues and cell lines. Overexpression of GFP-NOP14 markedly promoted the growth, migration, and invasion of the CRC cells HT19 and SW480, while genetic knockdown of NOP14 inhibited these behaviors. Overexpression of NOP14 promoted the expression of NRIP1 and phosphorylated inactivation of GSK-3β, leading to the upregulation of β-catenin. Genetic knockdown of NOP14 had the opposite effect on NRIP1/GSK-3/β-catenin signals. NOP14 therefore appears to be overexpressed in clinical samples and cell lines of colorectal cancer, and promotes the proliferation, growth, and metastasis of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.Key words: Colorectal cancer, NOP14, proliferation, migration, invasion     

AEG-1对大肠癌进展的影响

目的研究AEG-1在大肠癌组织和细胞中的表达,探讨AEG-1通过调控上皮间质转化和耐药参与大肠癌的进展。方法采用qRT-PCR检测AEG-1在大肠癌组织和细胞中的表达,统计AEG-1对大肠癌患者生存率的影响,分析其在不同癌症分期患者中的表达差异,并分析AEG-1表达量与大肠癌诊断敏感性的关系。采用体外实验将si-NC、pc-DNA-NC、si-AEG-1、pc-DNA-AEG-1转染到大肠癌SW116和LOVO细胞中,然后通过qRT-PCR检测转染效率以及AEG-1在两细胞系中的表达情况。采用CCK-8和克隆形成实验检测AEG-1对大肠癌细胞增殖的影响;采用流式细胞术检测转染后上皮间质转化和耐药情况的变化;采用Western blotting检测转染后上皮间质转化和耐药相关蛋白N-cadherin、E-cadherin、MRP的变化情况。结果 77例大肠癌患者组织中AEG-1表达水平明显高于对照组。浸润T3+T4期的患者中AEG-1的表达水平高于浸润T1+T2期患者。Ⅲ+Ⅳ期患者中AEG-1的表达水平高于Ⅰ+Ⅱ期。AEG-1高表达组患者OS时间明显低于低表达组。AEG-1表达量与大肠癌诊断敏感性之间呈显著正相关。在LOVO细胞系中,降低AEG-1表达后其细胞活力、侵袭力明显降低,同时间质细胞标志蛋白N-cadherin、上皮细胞标志蛋白E-cadherin、多药耐药相关蛋白MRP表达量降低。在SW116细胞系中,过表达AEG-1后上皮间质转化、耐药相关蛋白表达量显著升高。结论 AEG-1在大肠癌组织和细胞中的表达量明显上升,AEG-1通过调控上皮间质转化和耐药参与大肠癌的发生发展,为大肠癌的治疗提供了新的理论依据和新的靶点。     

A functional variant of IC53 correlates with the late onset of colorectal cancer

The IC53 gene was reported to be upregulated in the colon adenocarcinoma cell line SW480. Here, we show that the expression level of IC53 is positively correlated with the grade and depth of invasion in adenocarcinoma of the colon. Injection of IC53 stably transfected HCT-116 cells into athymic nude mice promoted tumor growth. Furthermore, overexpression of IC53 increased cell invasive growth, which could be dramatically prevented by knocking down IC53 with siRNA. The effects of IC53 on cell-invasive growth were mediated by upregulation of integrins, activation of phosphatidylinositol 3-kinase and phosphorylation of Akt. A single-nucleotide polymorphism rs2737 in the IC53 gene created a potential microRNA379 target site, and microRNA379 expression inhibited IC53 translation. Among 222 patients with colorectal cancer, the C/C rs2737 genotype was associated with late onset of colorectal cancer (median age 63.0 versus 55.3 years, P = 0.003). The frequency of the C/C rs2737 genotype was much lower in patients who developed colorectal cancer below the age of 45 years than in individuals over age 45 years (10.8% versus 26.6%, P = 0.039). These data indicated that IC53 is a positive mediator for colon cancer progression, and IC53-rs2737 may serve as protection from the onset of colorectal cancer.