ERCC1 siRNA对鼻咽癌细胞HNE-1/DDP顺铂耐药的影响及机制

目的:研究切除修复交叉互补基因1(excision repair cross complement 1,ERCC1)siRNA对鼻咽癌细胞HNE-1/顺铂(cisplatin,DDP)耐药的影响及机制。方法:以qRT-PCR和Western印迹法测定人鼻咽癌细胞HNE-1/DDP和HNE-1细胞中ERCC1表达水平。人鼻咽癌细胞HNE-1/DDP转染ERCC1siRNA重组慢病毒载体和阴性对照载体,以qRT-PCR和Western印迹法测定干扰效果。用DDP处理转染ERCC1 siRNA的HNE-1/DDP细胞,MTT检测细胞增殖变化,克隆形成实验检测细胞克隆形成能力,流式细胞术检测细胞凋亡变化,Western印迹法检测细胞中激活型caspase-3(C-caspase-3)、激活型caspase-9(C-caspase-9)蛋白水平,同时用Western印迹法检测细胞质和线粒体中细胞色素C(cy tochromeC)蛋白水平。结果:HNE-1/DDP细胞中ERCC1表达水平高于HNE-1细胞。ERCC1siRNA可明显下调HNE-1/DDP细胞中ERCC1的表达和转录。DDP和ERCC1siRNA可以降低HNE-1/DDP细胞增殖能力和克隆形成能力,提高细胞凋亡率,促进细胞中C-caspase-3,C-caspase-9蛋白表达,提高细胞质中cytochrome C蛋白水平,降低线粒体中cytochrome C蛋白水平。DDP处理转染ERCC1 siRNA的HNE-1/DDP细胞,细胞的增殖和克隆能力下降更多,细胞凋亡率升高更多,细胞中C-caspase-3,C-caspase-9蛋白水平更高,细胞质中cytochrome C蛋白水平也更高,线粒体中cytochromeC蛋白水平下降更多。结论:ERCC1siRNA能够逆转鼻咽癌细胞HNE-1/DDP对DDP耐药,作用机制可能与激活线粒体凋亡途径有关。     

259例结直肠癌患者ERCC1基因多态性分析

目的 分析结直肠癌患者切除修复交叉互补基因1(ERCC1)的基因多态性,以期能为结直肠癌患者的个体化治疗提供指导。方法 采用PCR测序法分析259例结直肠癌患者ERCC1的基因多态性。结果 男性患者ERCC1的基因型主要为C/C(占55.71%),其次是C/T(占37.86%),T/T基因型仅占6.43%;女性患者的基因型也主要是C/C(占59.66%),其次是C/T(占36.97%),T/T基因型仅占3.36%。25~44岁患者中,基因型主要为C/C(占51.72%),其次是C/T(占41.38%),T/T基因型仅占6.90%;>44~59患者中,基因型主要为C/C(占57.95%),其次是C/T(占40.91%),T/T基因型仅占1.14%;>59岁患者中,基因型主要为C/C(占58.45%),其次是C/T(占34.51%),T/T基因型仅占7.04%。结论 结直肠癌患者ERCC1的基因型主要为C/C。     

晚期非小细胞肺癌中切除修复交叉互补基因1的表达及临床意义

目的：探讨非小细胞肺癌组织中切除修复交叉互补基因1（ERCC1）表达水平与顺铂化疗耐药及预后的相关性。方法：应用免疫组化链霉素抗生物素蛋白-过氧化物酶连接（SP）法检测104例非小细胞肺癌组织中ERCC1的表达并分析其与顺铂化疗耐药及1年生存率的关系。结果：非小细胞肺癌癌组织ERCC1表达水平与非小细胞肺癌患者性别及组织学类型无关,而与非小细胞肺癌患者以顺铂为基础联合化疗的耐药性有关。非小细胞肺癌化疗无效组ERCC1的阳性表达率为69.2%,明显高于化疗有效组12.8%,差异有统计学意义（P〈0.05）;ERCC1高表达者有较短的中位总生存期（11.0个月）和无进展生存（8.0个月）,而ERCC1低表达患者有较长的中位总生存期（16.0个月）和无进展生存（13.0个月）。患者性别、年龄和组织类型与中位总生存期及无进展生存无相关性。结论：ERCC1在非小细胞肺癌组织中的表达水平与顺铂化疗耐药有关,ERCC1的高表达往往提示预后不佳。     

RNA干扰MBP-1基因对胃癌细胞SGC-7901增殖影响

目的：探讨 MBP-1（c-myc promter binding protein 1,MBP-1）基因表达沉默对胃癌细胞株 SGC-7901细胞增殖影响。方法实验分3组：空白对照组（未转染胃癌细胞）、阴性对照组（转染错义序列）和干扰组（转染 MBP-1 shRNA）。设计2条针对MBP-1基因的小干扰 RNA 片段及1条阴性对照 siRNA,并构建入 pSIREN-retroQ 质粒。将构建的重组 pSIREN-retroQ 质粒通过 Lipofectamine 2000脂质体转染胃癌 SGC-7901细胞,嘌呤霉素筛选稳转株细胞。Real time PCR 和 Western blot 分别检测MBP-1表达。MTT 法对 MBP-1干扰后 SGC-7901细胞增殖进行检测。结果通过 PCR 扩增阳性克隆及测序,说明已成功构建MBP-1干扰及对照重组 pSIREN-retroQ 质粒。通过 Lipofectamine 2000脂质体将重组质粒转染胃癌 SGC-7901细胞,并通过嘌呤霉素筛选2周,说明已成功构建 MBP-1干扰及对照 SGC-7901稳转株细胞。Real time PCR 检测,干扰组 MBP-1 mRNA 相对表达量与空白对照组相比显著下调（P ＜0.05）。Western blot 检测 MBP-1蛋白表达,干扰组 MBP-1表达量与空白对照组相比也都显著下调。MTT 法检测结果表明,MBP-1干扰组细胞在48、72、96和120 h 增殖能力比空白对照组都有显著的升高（P ＜0.05）。结论下调 MBP-1基因表达能明显促进胃癌细胞 SGC-7901的增殖,从而为胃癌基因治疗提供了新靶点。     

高能X射线对肺腺癌A549细胞ERCC1表达的影响

目的检测高能X射线对肺腺癌A549细胞与顺铂耐药相关的基因——切除修复交叉互补基因1（ERCC1）表达的影响。方法对A549细胞进行X射线照射,总剂量10 Gy/（5 d.5f）。利用RT-PCR检测照射前及照射开始后第1~4周细胞的ERCC1基因的表达;免疫组织化学SABC法检测照射前后细胞ERCC1蛋白的表达。结果照射后第1~3周A549细胞ERCC1 mRNA及蛋白的表达水平比照射前显著升高,差异有显著性（F=152.00、26.63,q=6.03~28.31,P〈0.01）。结论肺腺癌A549细胞照射后可能出现顺铂耐药。     

RNA干扰敲除Smac基因的表达促进人肺癌细胞的生长及增强顺铂耐药性

目的 探讨RNA干扰敲除Smac基因的表达对肺癌细胞的生长及顺铂(cDDP)耐药性的影响.方法 通过转染含有以人Smac基因为靶基因的慢病毒载体系统,即含有小分子干扰RNA序列的pGC-FU载体,分别在A549和95D细胞中实施Smac基因敲除.通过转染含有Smac全长编码序列的pGC-FU载体来实现Smac的高表达.细胞生长、细胞周期及凋亡采用四甲基偶氮唑盐(MTT)法、克隆形成实验及流式细胞仪测定.药物耐药用10 μg/ml顺铂检测.结果 Smac下调表达促进A549和95D细胞中肺癌细胞的生长及增强顺铂耐药性;Smac高表达抑制A549细胞生长并且增强其对顺铂的敏感性.结论 Smac抑制肺癌细胞生长并且增强其对顺铂的敏感性.     

特异性RNA干扰阻抑骨髓基质细胞SDF-1表达对Jurkat细胞黏附及药物敏感性的影响

目的观察特异性RNA干扰(RNA i)阻抑急性白血病骨髓基质细胞衍生因子-1(SDF-1)表达对共培养的急性白血病细胞系Jurkat细胞黏附及药物敏感性的影响。方法脂质体介导SDF-1特异性RNA i质粒转染培养的急性白血病患者骨髓基质细胞,G418筛选阳性混合克隆,ELISA法检测SDF-1表达;Jurkat细胞与之共培养,通过细胞计数计算黏附率,MTT法检测对阿霉素的药物敏感性。以未转染急性白血病骨髓基质细胞及正常骨髓基质细胞作为对照。结果转染阳性克隆组、未转染急性白血病组及正常对照组骨髓基质细胞培养上清SDF-1水平分别为每周(1920±205)pg/105细胞、(12 370±1355)pg/105细胞和(6620±770)pg/105细胞;与之共培养的Jurkat细胞黏附率分别为(28.8±2.6)%,(57.4±3.8)%和(45.2±4.0)%,阿霉素50%抑制浓度分别为585,6162,1758 nmol/L。结论RNA i阻抑SDF-1表达的骨髓基质细胞使Jurkat细胞黏附减少,对阿霉素的敏感性增加。     

TDI-FP法检测肿瘤患者ERCC1 Asn118Asn基因多态性

目的建立一种可靠、敏感、全面的ERCC1 Asn118Asn基因多态性检测新方法,用于预测肿瘤患者对铂类的耐药性。方法以ERCC1基因第4个外显子Asn118Asn的一对引物行PCR扩增227例临床样本DNA,将荧光偏振检测技术与模板指导的末端延伸反应(TD I-FP)结合,应用探针杂交延伸反应对临床样本扩增产物进行ERCC1 Asn118Asn基因多态性检测,并以DNA序列测定验证检测结果。结果227例临床样本DNA中,119例(52.4%)ERCC1 Asn118Asn基因为C/C纯合子(AAC)基因型,88例(38.8%)为C/T杂合子基因型,20例(8.8%)为T/T纯合子(AAT)基因型。但DNA序列测定法检出杂合子基因型仅33例。结论本研究初步建立了特异性较好、操作简便、无需标记探针的临床标本ERCC1 Asn118Asn基因多态性检测新方法,有望用于肿瘤患者铂类耐药性的检测。     

ERCC1、BRCA1、RRM1表达与含铂类方案治疗晚期NSCLC的关系研究

目的研究探讨Ⅲ、Ⅳ期非小细胞肺癌(non-small cell lung cancer,NSCLC)患者癌组织内核苷酸切除修复交叉互补组1基因(ERCC1)、乳腺癌易感染基因1(BRCA1)、核苷酸还原酶M1(RRM1)表达与铂类标准化疗方案化疗疗效的关系。方法共入选Ⅲ、Ⅳ期NSCLC患者80例,所有患者都接受至少2周期的化疗,以GP、NP、TP方案为主。ERCC1、BRCA1及RRM1表达水平的检测采用免疫组化二步法。对患者化疗后的疗效与ERCC1、BRCA1、RRM1表达水平的关系进行评价。结果 80例患者中,ERCC1阳性患者与阴性患者有效率分别为28.8%、71.4%,BRCA1阳性患者与阴性患者化疗有效率分别为30.0%、57.5%,RRM1阳性患者与阴性患者化疗有效率分别为30.2%、70.4%,阳性患者与阴性患者有效率比较,差异均有统计学意义(P均<0.05)。ERCC1、BRCA1、RRM1均阴性表达者化疗有效率明显高于阳性表达者(66.1%vs31.8%,P均<0.05)。结论 ERCC1、BRCA1、RRM1表达不同可能是化疗方案中铂类药物敏感性差别的重要因素,可能为临床NSCLC患者个体化用药提供可参考的依据。     

切除修复交叉互补基因1在胃癌中的研究进展

胃癌是世界上最常见的恶性肿瘤之一，发病率和病死率居各种恶性肿瘤的第二位。由于早期胃癌无明显临床症状或症状不典型，多数患者确诊时已经是进展期胃癌（AGC），甚至已经转移，这些患者即使行根治性胃癌切除术，术后复发仍然是一个严峻的问题，术后辅助化疗成为AGC综合治疗的一个重要部分。     

ERCC1对晚期非小细胞肺癌一线含铂化疗疗效的预测价值

  目的  探讨肺癌组织ERCC1(excision repair cross-complementation group1)表达水平对晚期非小细胞肺癌含铂化疗疗效及生存期的预测价值。  方法  采用免疫组织化学方法对39例非小细胞肺癌患者的石蜡包埋肿瘤标本进行ERCC1检测, 分析其与化疗反应率及生存期间的关系。  结果  56.4%(22/39)的肿瘤组织呈ERCC1高表达。ERCC1表达与患者性别、年龄、病理类型、肿瘤分化程度、吸烟史及吸烟指数均无相关性; ERCC1低表达患者对含铂一线化疗的反应率优于ERCC1高表达者, P < 0.05。但ERCC1表达水平对患者总生存(overall survival, OS)和疾病进展(time to progression, TTP)时间没有明显影响。  结论  ERCC1在肺癌组织的表达水平可能成为预测晚期非小细胞肺癌患者一线含铂化疗方案疗效的因素之一。     

依据ERCC1及RRM1对晚期非小细胞肺癌实施个体化治疗的随机对照临床研究

目的对晚期非小细胞肺癌（NSCLC）患者根据切除修复交叉互补基因1（ERCC1）及核糖核苷酸还原酶M1（RRM1）的检测结果选择敏感药物,观察疗效差异,以期对个体化治疗起到指导作用。方法对经病理确诊的晚期NSCLC的肿瘤细胞进行ERCC1及RRM1基因的联合检测。入组患者按照1∶2随机分为2组,对照组使用吉西他滨联合顺铂化疗;基因型组根据ERCC1及RRM1的表达情况分为4组进行个体化治疗。观察近期疗效及远期疗效。结果本研究共纳入174例患者,总体有效率为44.2%,对照组有效率为37.5%,基因型组有效率为47.5%,结果无统计学差异。基因型组中ERCC1阴性组有效率（56.7%）高于ERCC1阳性组（37.9%）,结果有统计学差异。各组间中位无疾病进展生存期、中位生存期及1年生存率无统计学差异。结论 ERCC1低表达患者中使用个体化治疗可获得更高的有效率,而ERCC1高表达患者可能意味着对铂类耐药。     

Long non-coding RNA NEAT1 regulates ferroptosis sensitivity in non-small-cell lung cancer

ObjectivesFerroptosis is caused by iron-dependent lipid peroxide accumulation, the sensitivity of which might be regulated by acyl-CoA synthetase long chain family member 4 (ACSL4). Non-small-cell lung cancer (NSCLC) can resist oxidative stress and reduce the sensitivity of tumor cells to ferroptosis by changing the expression of some proteins. Mechanisms involving ferroptosis sensitivity in NSCLC are not fully understood.MethodsA dual-luciferase reporter assay was used to confirm a targeting relationship between long non-coding (lnc)RNA NEAT1 and ACSL4. Overexpression and silencing assays of NEAT1 function were used to determine its roles in cell death (by TUNEL staining) and lipid peroxidation (by malondialdehyde levels). Expression of ferroptosis-related proteins (SLCA11, GPX4, and TFR4) was evaluated by western blot in NSCLC cells treated or not with the ferroptosis inducer erastin.ResultsErastin-induced cell death was positively correlated with ACSL4 level. NEAT1 regulated levels of ACSL4 and proteins related to the ferroptosis and classical apoptosis pathways. Levels of ACSL4, SLC7A11, and GPX4 were decreased more by NEAT1 silencing plus erastin than by erastin alone.ConclusionNEAT1 regulates ferroptosis and ferroptosis sensitivity, with the latter depending on ACSL4, suggesting that targeting NEAT1 or ACSL4 may be a viable therapeutic approach to the treatment of NSCLC.     

microRNA regulation of cell viability and drug sensitivity in lung cancer

Introduction: microRNAs (miRNAs) are 19 – 23 nucleotide long RNAs found in multiple organisms that regulate gene expression and have been shown to play important roles in tumorigenesis. In the context of lung cancer, numerous studies have shown that tumor suppressor genes and oncogenes that play crucial roles in lung tumor development and progression are targets of miRNA regulation. Manipulation of miRNA levels that modulate lung cancer cell survival and drug sensitivity can therefore provide novel therapeutic targets and agents.

Areas covered: Here, the authors review the published in vitro, in vivo and preclinical studies on the functional role of miRNAs in modulating lung cancer cell viability and drug response, and discuss the limitations and promise of translating current findings into miRNA-based therapeutic and diagnostic strategies.

Expert opinion: Although many miRNAs have been identified as potent regulators of cell viability and drug sensitivity in lung cancer, most of them have not been characterized for potential clinical application. Further study is warranted to evaluate translation of the current findings to the clinic to improve the diagnosis and treatment of lung cancer. In addition, most studies have focused on non-small cell lung cancer (NSCLC). It is therefore important to raise interest in investigating miRNAs in small cell lung cancer (SCLC) as well as in comparative studies of miRNA expression and function in different histological subtypes of lung cancer.     

RNA干扰Ku70基因对宫颈癌Hela细胞放射敏感性影响的实验研究

目的观察ku70基因短干扰RNA(siRNA)对宫颈癌Hela细胞放射敏感性的影响。方法采用集落形成技术测定不同剂量射线照射对Hela细胞存活率的影响,观察Hela细胞对射线照射的敏感性。采用时定量PCR技术(quantitiv real time PCR,QRT-PCR)检测Ku70 mRNA水平表达。采用Western Blot免疫印记技术检测Ku70蛋白表达。采用RNA干扰(RNAi)技术抑制Hela细胞中Ku70蛋白的表达,观察Ku70蛋白表达下调对Hela细胞放射敏感性的影响。采用集落形成技术测定4.0 Gy射线照射对Hela细胞存活率的影响。进而分析Hela细胞对射线的放射敏感性。结果宫颈癌Hela细胞对1.0-4.0 Gy射线照射不够敏感,有相当高的辐射抗性。量效实验证实,1.0-6.0 Gy射线照射后,HeLa细胞中Ku70 mRNA及Ku70蛋白表达均明显增高。时效实验证实,4.0 Gy射线照射后8 h-72 h,HeLa细胞中Ku70 mRNA及Ku70蛋白表达均明显增高。本实验成功的构建了Ku70基因RNA干扰载体pSilencer-4.1-Ku70,稳定转染HeLa细胞后,有效抑制Ku70蛋白的表达。稳定转染pSilencer-4.1-Ku70干扰质粒并抑制Ku70蛋白表达后,HeLa细胞对射线照射的放射敏感性显著增高(P0.001)。结论 Ku70 siRNA有效下调Ku70 mRNA水平,抑制Ku70蛋白的表达,可明显提高Hela细胞对辐射的敏感程度。本研究结果还提示,Ku70基因可作为衡量宫颈腺癌辐射敏感性指标,为临床合理筛选患者进行放疗、预测放疗疗效提供一条适用途径。     

Oncolytic-adenovirus-expressed RNA interference for cancer therapy

Importance of the field: RNA interference (RNAi) has generated considerable excitement for its potential cancer therapeutic applications. Because of the difficulties in delivering a large amount of siRNA to cancer cells and the short half-life of siRNA, it is important to choose an efficient delivery system for transduction of siRNA into target cells. Oncolytic adenovirus offers a better platform by virtue of its high transfection efficiency and selective replication in cancer cells.

Areas covered in this review: This review focuses on the synergism between oncolytic adenovirus and siRNA antitumor responses, and discusses recent progresses in oncolytic-adenovirus-expressed siRNA.

What the reader will gain: siRNA-expressing oncolytic adenovirus can generate a significantly enhanced antitumor effect through gene knockdown and viral oncolysis.

Take home message: Due to its potency and target specificity, using siRNA delivery by oncolytic adenovirus has generated much excitement and will open new avenues for treatment of human cancer.     

Effect of CLN3 silencing by RNA interference on the proliferation and apoptosis of human colorectal cancer cells

Apoptosis constitutes a system for the removal of aged, or damaged cells, which is regulated by the interplay of pro-apoptotic and antiapoptotic proteins. Previous study has shown that Juvenile Batten disease protein, CLN3, is antiapoptotic gene in NT2 neuronal precursor cells and a few types of cancers. However, in colorectal cancer, whether CLN3 also play its antiapoptotic role and the effect of targeted controlling CLN3 on the biological behavior of human colorectal cancer cell is unknown. We employed the sequence-specific siRNA silencing the CLN3 gene and investigated its effects on growth and apoptosis of colorectal cancer HCT116 cells, which has highest elevation of CLN3 expression among four colorectal cancer cell lines. After CLN3 specific siRNA transfection, mRNA and protein expression levels of CLN3 in HCT116 cells were noticeably decreased. Moreover, CLN3-siRNA inhibited the proliferation of colorectal cancer cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. Our current study demonstrated that CLN3 was expressed in colorectal cancer cells at a high frequency. Moreover, CLN3 down-regulation with RNA interference can inhibit proliferation, apoptosis, and cell cycle progression of colorectal cancer cells. Our study represented a potential new approach to understanding the role of CLN3 in cancer and provides a potential novel strategy colorectal cancer therapy.