RNAi沉默Snail增加结肠癌对5氟尿嘧啶敏感性研究

目的:研究Snail的抑制是否能增加耐药结肠癌细胞对5-FU的敏感性,评估其可能的信号转导通路。方法:使用5-氟尿嘧啶耐药HCT116细胞(HCT116/5-FU),评估细胞形态及分子的变化。通过靶向人Snail基因小干扰RNA(si RNA)抑制Snail的表达。Annexin V/PI染色用于评估5-FU诱导的细胞凋亡。Western blot检测caspase以及可能的丝裂原活化蛋白激酶(MAPK)和线粒体途径。结果:HCT116细胞对5-Fu耐药性的获得诱导了与EMT一致的形态学变化。RNA干扰沉默Snail逆转HCT116/5-FU细胞EMT并增加了5-FU耐药HCT116细胞对5-FU的敏感性。可能的机制涉及JNK与线粒体途径的激活。结论:EMT样表型的改变与HCT116细胞对5-FU耐药相关;si RNA介导的Snail下调可能是一个潜在的克服5-FU化疗耐药的治疗方法。     

MUC1对人结肠癌细胞HCT116生物学行为的影响

目的:观察MUC1对人结肠癌细胞HCT116增殖、侵袭及化疗敏感性的影响。方法:采用MUC1表达阴性的结肠癌细胞株HCT116,通过慢病毒转染、嘌呤霉素筛选、半定量RT-PCR和Western blot鉴定构建稳定表达MUC1的HCT116细胞株;实验分空病毒组和MUC1病毒组;CCK实验和软琼脂克隆形成实验检测两组细胞的增殖能力,Transwell侵袭实验检测两组细胞的侵袭能力;MTT法和流式细胞仪检测两组细胞对奥沙利铂的敏感性,酶底物法检测Caspase-3活性。结果:获得稳定表达MUC1的HCT116细胞株;两组细胞贴壁生长无差异;与空病毒组相比,MUC1病毒组细胞软克隆形成数增加,穿过小室的细胞数增加(P0.05);MUC1病毒组细胞对奥沙利铂的敏感性降低,MUC1病毒组Caspase-3活性水平低于空病毒组(P0.05)。结论:MUC1与结肠癌的非锚定依赖生长、侵袭和化疗敏感性有关。     

miR-155-TP53INP1调节轴在结直肠癌化疗药物敏感性中的作用机制

摘要 目的：初步揭示miR-155通过靶向调节TP53INP1表达水平影响结直肠癌细胞对5-FU化疗敏感性。方法：将人结肠直肠癌细胞系HCT116进行培养,提取细胞总RNA后,采用miR-155逆转录特异性引物构建反转录体系进行PCR扩增,通过qRT-PCR检测miR-155在5-FU耐药细胞HCT116/FU及敏感细胞株HCT116中的表达情况;取对数生长期细胞,分别转染miR-155mimics、miR-155抑制剂、miR-155阴性对照后,采用CCK-8法检测miR-155对细胞5-FU药物敏感性的影响,双荧光素酶报告基因系统验证miR-155与TP53INP1的靶基因关系,Western blot检测miR-155对 TP53INP1表达的影响。结果：miR-155在HCT116 /Fu细胞中的表达量是HCT116细胞的7.25倍;在相同5-FU浓度时,HCT116+阴性对照的细胞生长抑制率均高于HCT116+mimics、半数抑制浓度显著低于HCT116+mimics,差异均具有统计学意义(P＜0.05);TP53INP1是miR-155的靶基因,能显著降低野生型TP53INP1 3''-UTR的荧光素酶活性;转染miR-155 mimics后,TP53INP1的相对表达量显著下降,转染miR-155抑制剂后,TP53INP1的相对表达量显著升高,差异均具有统计学意义(P＜0.05)。结论：miR-155水平升高使HCT116细胞对5-FU的敏感性降低,miR-155可能通过靶向调节TP53INP1的表达水平,从而影响结直肠癌细胞对5-FU的敏感性。     

Skp2RNA干扰对人结肠癌HCT116细胞p27表达及对5-氟尿嘧啶敏感性影响的研究

目的:细胞S期激酶相关蛋白2(S-phase kinase associated protein,Skp2)作为泛素化降解过程中的一种F-box蛋白,在泛素化降解中起着特异性的底物识别和关键性的速率限制作用.依赖Skp2的泛素化降解途径参与p27、p21等众多细胞周期调控因子的降解,进而通过影响G1-S检测点而调控细胞周期.本研究探讨靶向干扰人结肠癌HCT116细胞中Skp2基因对细胞p27表达的影响及细胞对5-氟尿嘧啶(5-FU)敏感性的变化.方法:将已合成好的特异性的Skp2RNA干扰载体转染人结肠癌HCT116细胞,G418稳定筛选后,通过RT-PCR和Western印迹法分别检测细胞内p27mRNA和蛋白的表达情况,MTT法检测细胞对5-Fu敏感性的变化.结果:在转染干扰载体后,HCT116细胞内的p27mRNA表达无明显变化,蛋白水平却明显上升.MTT显示HCT116细胞对5-FU敏感性较其他组显著提高,IC50值明显下降(P＜0.05).结论:Skp2基因的RNA干扰能够上调结肠癌细胞内源性p27蛋白的表达,并提高5-FU对癌细胞的杀伤作用,为大肠癌的基因治疗提供了新的思路.     

双歧杆菌脂磷壁酸对结肠癌细胞抑制增殖和促凋亡的研究

目的 探讨双歧杆菌脂磷壁酸(lipoteichoic acid,LTA)抑制人类结肠癌细胞株HCT116细胞增殖和促进凋亡情况.方法 培养结肠癌细胞株HCT116和提取双歧杆菌LTA;实验分为4组,即LTA低剂量组(20 μg/mL)、中剂量(60 μg/mL)、高剂量(100μg/mL)和HCT116对照组(Control);用四甲基偶氮唑蓝法(MTT)检测LTA对结肠癌细胞增殖的抑制率,流式细胞仪检测LTA对结肠癌细胞周期分布变化和凋亡率,免疫组化分析bcl-2、Cytochrome C和NF-kBp65含量变化,RT-PCR检测TLR2mRNA和TLR4m RNA的表达.结果 MTT法测得LTA各组对结肠癌细胞HCT116均具有较好的抑制(P＜0.01);流式细胞仪检得G0/G1期细胞显著增多(P＜0.01),而G2和S期细胞减少(P＜0.05),细胞凋亡率增加(P＜0.05);免疫组化分析IOD/Area值,bcl-2和NF-kBp65表达显著下调,Cytochrome C显著上升(P＜0.05);RT-PCR测得TLR2 mRNA和TLR4 mRNA的表达均上升(P＜0.05).结论 LTA具有明显的抑制结肠癌细胞增殖和促进凋亡作用,为研制高效、无毒的新型抗结肠癌药物奠定一定的基础.     

核受体PXR对人结肠癌细胞增殖和化疗敏感性的影响

目的:探讨核受体PXR在结肠癌细胞增殖和化疗敏感性中的作用.方法:分别用RT-PCR和westernblot方法检测PXR在人结肠癌细胞株LS174T、LOVO、HT29、HCT116中的表达情况.通过质粒稳定转染方法建立PXR敲低的细胞株.用MTT方法分析利福平活化PXR或稳定转染敲低PXR后,细胞增殖和化疗敏感性的改变.结果:在结肠癌细胞株LS174T、LOVO、HT29、HCT116中,LS174T细胞的PXR表达水平最高.利福平处理后,LS174T细胞中PXR表达增强.利福平活化PXR或稳定转染敲低PXR后,相应地促进或抑制细胞增殖,降低或提高细胞对化疗药物的敏感性.结论:PXR能促进结肠癌细胞增殖,提高细胞对化疗药物的敏感性,可能在结肠癌多药耐药机制中具有重要作用.     

PXR基因外显子3甲基化与肠癌细胞对5氟尿嘧啶的耐药性相关

孕烷X受体（pregnane X receptor, PXR）可通过调节细胞色素P450同工酶3A4 -CYP3A4的表达而影响肿瘤细胞对化疗的敏感性,而其表达水平则会受到自身基因 甲基化的影响.本文研究了结肠癌组织中pxr基因甲基化的分布情况及其对pxr, cyp3a4表达的影响,并在多种结肠癌细胞系中分析了pxr基因甲基化是否与5-氟尿嘧 啶 (5-FU)耐药性相关.收集结肠癌病灶区、癌旁区及正常结肠组织样本,分别提取基因组DNA及RNA.PCR限制性酶切分析检测pxr基因外显子3甲基化;real-time PCR检测pxr及cyp3a4基因的表达.鉴定LOVO、LS180、LS174T、HT29、HCT116等5种结 肠癌细胞中pxr外显子3甲基化与pxr, cyp3a4表达的相关性并分别筛选出PXR高/低表达的细胞株进行5-FU耐药性分析.结果显示,结肠癌病灶组织中pxr外显子3甲基化频率显著增加,伴有pxr,cyp3a4表达的增强.在结肠组织及结肠癌细胞系中,pxr与cyp3a4的表达均密切相关,且均与pxr甲基化程度相关.PXR高表达细胞株LS180对5-FU的耐药性显著升高,以siRNA分别下调pxr及cyp3a4的表达,均可增加LS180对5 -FU的敏感性.结果提示,pxr基因外显子3区甲基化与PXR及CYP3A4的高表达密切相关,并与结肠癌细胞对5-FU的抗药性相关.     

1-硝基-2-酰基蒽醌-缬氨酸通过抑制ERK1/2激活和ERCC1表达诱导结肠癌细胞死亡

化学方法合成是新药研发的一种重要途径。结合抗肿瘤药物的作用机制以及蒽醌类衍生物的构效关系,设计合成了一类新的蒽醌类衍生物1-硝基-2-酰基蒽醌-缬氨酸(简称C3),发现其具有很好的抗肿瘤活性。为了确定蒽醌类衍生物C3对结肠癌HCT116和HT29细胞的作用及其分子机制,首先通过MTT比色法检测C3对结肠癌HCT116和HT29细胞活性的影响。结果显示,C3对这两种结肠癌细胞具有明显的抑制作用,呈时间和剂量依赖性。60μg/mL的C3处理HCT116和HT29细胞48 h,细胞活性分别是50.67%和59.77%,达到了半抑制浓度;同时,其细胞形态和细胞核发生明显变化。进一步采用Western印迹和qRT-PCR技术,检测C3对DNA切除修复交叉互补1(excision repair cross-complementation group 1,ERCC1)转录水平和蛋白质水平表达及其稳定性的影响。结果表明,C3降低了ERCC1转录水平和蛋白质水平的表达,并且减弱了ERCC1转录水平和蛋白质水平的稳定性。最后,用U0126(MEK1/2抑制剂)和C3联合作用结肠癌HCT116和HT29细胞,通过Western印迹检测ERCC1蛋白质水平的表达。结果表明,C3通过降低p-ERK1/2的蛋白质水平的表达,从而抑制ERCC1的表达。上述结果证明,C3通过细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK1/2)信号通路,降低了ERCC1转录水平和蛋白质水平的稳定性,使ERCC1转录水平和蛋白质水平表达发生下调,进而抑制结肠癌HCT116和HT29细胞的活性。     

冬凌草甲素对人结肠癌细胞株HCT116生长的影响及其机制研究

目的:研究冬凌草甲素(ORI)对人结肠癌细胞株HCT116生长的影响及其可能机制。方法:以体外培养的HCT116细胞为研究对象,给予不同浓度(0、2.5、5、10、20μM)ORI处理HCT116细胞不同时间(0、24、48、72 h),通过MTT法检测其对HCT116细胞增殖的影响,DAPI染色观察其对细胞核的形态的影响,western blot检测细胞内β-catenin、c-myc蛋白表达的变化。结果:1ORI可显著抑制HCT116细胞的增殖,且此作用随着浓度和作用时间的增加或延长而增强(P0.05)。2ORI处理HCT116细胞24小时后,细胞核固缩的百分率随药物作用浓度的增加而增加。35、10、20μM ORI处理HCT116细胞24小时后,细胞内的β-catenin、c-myc蛋白水平均显著下调,且随着ORI浓度的增加逐渐减少。结论:ORI能以浓度和时间依赖性的方式抑制HCT116细胞的增殖,其机制可能与Wnt/β-catenin信号通路有关。     

分心木乙醇提取物对人结肠癌HCT116细胞增殖、凋亡和迁移的影响

为研究分心木对人结肠癌细胞HCT116增殖、凋亡和迁移的影响,该研究以75%乙醇作为溶剂提取分心木中的活性成分,利用MTT法检测分心木乙醇提取物对HCT116细胞增殖的影响;流式细胞术、AO/EB双染、TUNEL法和Western blot检测细胞凋亡;划痕愈合实验检测细胞迁移;体外建立肿瘤3D细胞模型(3D培养)检测分心木乙醇提取物对HCT116 3D肿瘤细胞球增殖的影响。结果显示,分心木乙醇提取物以剂量依赖的方式抑制HCT116细胞活性,促进细胞凋亡,同时促进凋亡因子Bax的表达,降低抗凋亡因子Bcl2的表达,并促进凋亡的关键执行蛋白PARP的裂解;划痕愈合实验和3D肿瘤细胞培养表明,分心木乙醇提取物抑制细胞迁移和3D肿瘤细胞的增殖。该研究表明,分心木乙醇提取物抑制肿瘤细胞的增殖和迁移,并通过促进cleaved PARP、Bax蛋白表达和抑制Bcl2蛋白的表达诱导肿瘤细胞凋亡。此外,分心木乙醇提取物抑制肿瘤3D细胞球的增殖,这提示结肠癌对分心木乙醇提取物的药物敏感性在体内体外没有显著差异。     

Lin28 Mediates Radiation Resistance of Breast Cancer Cells via Regulation of Caspase,H2A.X and Let-7 Signaling

Resistance to radiation therapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to breast tumorigenesis; however, the relationship between Lin28 and radioresistance remains unknown. In this study, we investigated the association of Lin28 with radiation resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines. The results showed that the expression level of Lin28 was closely associated with resistance to radiation treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to radiation than MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which have low-level Lin28 expression. Transfection with Lin28 siRNA significantly led to an increase of sensitivity to radiation. By contrast, stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to radiation treatment. Stable expression of Lin28 also significantly inhibited radiation-induced apoptosis. Moreover, further studies have shown that caspases, H2A.X and Let-7 miRNA were the molecular targets of Lin28. Stable expression of Lin28 and treatment with radiation induced H2AX expression, while inhibited p21 and γ-H2A.X. Overexpression of Let-7 enhanced the sensitivities to radiation in breast cancer cells. Taken together, these results indicate that Lin28 might be one mechanism underlying radiation resistance, and Lin28 could be a potential target for overcoming radiation resistance in breast cancer.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

Lin28 mediates paclitaxel resistance by modulating p21, Rb and Let-7a miRNA in breast cancer cells

Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemoresistance remained unknown. In this study, we investigated the association of Lin28 with paclitaxel resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines and tumor tissues. We found that the expression level of Lin28 was closely associated with the resistance to paclitaxel treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to paclitaxel than the MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which had low-level expression of Lin28. Knocking down of Lin28 in Lin28 high expression T47D cells increased the sensitivity to paclitaxel treatment, while stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to paclitaxel treatment, resulting in a significant increase of IC50 values of paclitaxel. Transfection with Lin28 also significantly inhibited paclitaxel-induced apoptosis. We also found that Lin28 expression was dramatically increased in tumor tissues after neoadjuvant chemotherapy or in local relapse or metastatic breast cancer tissues. Moreover, further studies showed that p21, Rb and Let-7 miRNA were the molecular targets of Lin28. Overexpression of Lin28 in breast cancer cells considerably induced p21 and Rb expression and inhibited Let-7 miRNA levels. Our results indicate that Lin28 expression might be one mechanism underlying paclitaxel resistance in breast cancer, and Lin28 could be a potential target for overcoming paclitaxel resistance in breast cancer.     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

NDRG1对人肠癌细胞失巢凋亡的影响

目的:探讨NDRG1对体外培养的人肠癌细胞系失巣凋亡的影响。方法:采用慢病毒系统将NDRG1表达单元转入人肠癌细胞系SW620、HCT8中,建立相应的过表达稳定细胞系;通过siRNA的方法干扰HCT116和LOVO细胞系中NDRG1的表达,分别在非贴壁培养的情况下培养48小时,采用流式细胞术和TUNEL染色检测细胞的凋亡情况。结果:在贴壁培养条件下,NDRG1过表达并没有显著影响肠癌细胞的生长及增殖,而NDRG1特异性siRNA干扰HCT116细胞中NDRG1的表达后,其凋亡率无明显变化(P0.05)。在悬浮培养条件下,NDRG1过表达的肠癌细胞的失巢凋亡率显著低于正常对照组(P0.05),而用三种不同的siRNA干扰HCT116及LOVO细胞中NDRG1的表达后,其失巢凋亡率均显著高于正常对照组(P0.05)。结论:NDRG1在体外可抑制人肠癌细胞的失巢凋亡。     

Effect of DPC4 gene on invasion and metastasis of colorectal carcinoma cells

To investigate the effect of DPC4 gene on invasion and metastasis of colorectal carcinomacells,the expression of DPC4 was detected in sixty-three samples of colorectal tumors and seven cases ofcolorectal mucosa.The biological behavior of tumors expressing DPC4 was evaluated (including tumorstaging,differentiation degree and metastasis).pcDNA3.1-DPC4 plasmid was constructed and transferredinto HCT116 cells not expressing DPC4.The cell models (DPC4~ -HCT116) steadily expressing DPC4 wereobtained.Compared with HCT116 and pcDNA3.1-HCT116 cells,the doubling time of DPC4~ -HCT116 cellswas lengthened obviously (P<0.01),the apoptosis rate of DPC4~ -HCT 116 cells was significantly increased(P<0.01),the cloning efficiency,cell adherency,migration and invasion ability of DPC4~ -HCT116 cells weredropped obviously (P<0.01).The number of cancer nodules was decreased significantly in abdominal cavityand liver of the nude mice inoculated with DPC4~ -HCT116 cells.The activity of MMP-9 and MMP-2 wasdetected by gelatin zymography.In comparison with HCT116 and pcDNA3.1-HCT116 cells,the activity ofMMP-9 was decreased in DPC4~ -HCT116 cells.Therefore,the down-regulation of DPC4 expression may beassociated with the carcinogenesis of colorectal carcinoma.DPC4 may inhibit the proliferation of coloncancer cell by restraining growth and inducing apoptosis,and the invasion and metastasis of colorectalcarcinoma cells.MMP-9 may be one of the downstream target genes regulated by DPC4.