骨肉瘤患者血清中miR-34a、miR-449a表达及与新辅助化疗耐药的关系

目的 分析骨肉瘤患者血清中miR-34a、miR-449a表达水平及与新辅助化疗耐药的关系.方法 随机选择骨肉瘤83例,将其纳入研究组.另选取同期体检健康受试者83例,将其纳入对照组.检测血清miR-34a、miR-449a表达水平.问卷调查骨肉瘤患者临床病理特征,给予新辅助化疗,统计耐药情况.结果 研究组患者血清mi...     

miR-135a在调节小细胞肺癌多药耐药中的作用

目的:探讨miR-135a在小细胞肺癌(small cell lung cancer,SCLC)多药耐药中的作用。方法:收集2008年1月至2013年12月本院肿瘤科及呼吸内科就诊的48例SCLC患者外周血液标本,将其分为化疗敏感组(28例)和耐药组(20例),采用QRT-PCR法检测患者血液标本、SCLC敏感细胞株H69及耐药细胞株H69AR中miR-135a的表达,分析其与临床预后的关系,并应用miR-135a模拟物和抑制剂分别上、下调细胞株中miR-135a的表达,采用CCK-8法检测细胞对各种化疗药物(ADM、DDP和VP-16)敏感性的影响。结果:与化疗敏感组相比,miR-135a在化疗耐药患者血液标本中的表达明显增高[(2.174±3.981)vs(-1.963±3.750),P<0.001];在临床患者血液标本中,miR-135a的表达与患者的性别、年龄无关(P>0.05),与疾病的分期、化疗敏感性及患者的生存时间相关(均P<0.01)。miR-135a在H69AR细胞中的表达明显高于H69细胞[(7.841±0.392)vs(1.047±0.081),P<0.01];通过miR-135a抑制剂下调H69AR中miR-135a的表达能够显著增加细胞对化疗药物的敏感性,H69AR细胞对DDP、ADM及VP-16的IC50值明显下降[(247.09±11.55)vs(76.64±10.00)、(150.83±8.03)vs(40.72±5.06)、(436.63±61.19)vs(163.35±20.00);H69细胞转染miR-135a mimics后对化疗药物DDP、ADM和VP-16的IC50值明显增加[(18.58±1.37)vs(159.27±3.29)、(24.37±2.63)vs(129.19±2.64),(48.55±2.59)vs(359.87±2.92)μg/ml,P<0.01]。结论:miR-135a参与调节SCLC的多药耐药,下调miR-135a基因的表达可能增加细胞对化疗药物的敏感性。     

miR-21促进肺癌EGFR-TKI耐药细胞株上皮—间质细胞转化的研究

[目的]研究miR-21是否可以调控上皮—间质转化（EMT）而参与肺癌获得性耐药。[方法]使用HCC827细胞（EGFR基因19外显子缺少的肺腺癌细胞株）,在此细胞的基础上培养吉非替尼耐药细胞株HCC827/GR。检测耐药细胞株中miR-21的表达,同时观察耐药细胞的形态变化,及在耐药细胞株中抑制miR-21表达后检测耐药细胞株的侵袭能力及EMT相关蛋白E-cadherin和Vimentin的表达。[结果]与敏感细胞株HCC827相比,HCC827/GR细胞株对吉非替尼的耐药倍数约为100倍;同时在耐药细胞株中我们发现miR-21较敏感株表达增高约5.3倍,同时细胞形态发生了明显变化。通过检测EMT相关蛋白,我们发现耐药细胞株中间质相关蛋白Vimentin明显高表达,而上皮相关蛋白E-cadherin明显低表达,当miR-21被抑制后,耐药细胞株侵袭能力下降,同时Vimentin表达量下降,而E-cadherin表达量增高。[结论]miR-21可能通过促进EMT参与肺癌EGFR-TKI的获得性耐药。     

MicroRNA-34a调控KLF4转录因子

摘 要：［目的］ 研究microRNA-34a（miR-34a）与其靶基因KLF4转录因子在结直肠癌发生化疗耐药中的作用。［方法］ 利用miRDB和microRNA公共数据库筛选出miR-34a的直接作用靶点KLF4 3′-UTR区。运用real-time PCR方法检测人5-氟尿嘧啶（5-Fu）耐药和敏感的结直肠癌组织及癌旁组织中miR-34a与KLF4的表达水平;建立5-Fu抵抗性结直肠癌细胞系,运用real-time PCR方法检测耐药细胞中KLF4的表达;上调5-Fu耐药结直肠癌细胞中miR-34a的表达,明确在结直肠癌5-Fu耐药细胞系中miR-34a对KLF4的调控作用。［结果］ 在40例5-Fu耐药的结直肠癌组织中miR-34a呈低表达,KLF4呈高表达,且miR-34a在癌组织及癌旁组织中的表达与KLF4呈显著负相关（r=-0.678,P<0.001）;在5-Fu耐药结直肠癌细胞系HCT-8/5-Fu和SW-480/5-Fu中KLF4呈高表达,且上调耐药细胞中miR-34a可抑制耐药细胞中KLF4的表达。［结论］ KLF4转录因子在5-Fu化疗耐药的结直肠癌组织中呈低表达,并与miR-34a呈负相关,上调miR-34a的表达可直接抑制KLF4的表达,提示miR-34a负向调控KLF4具有逆转结直肠癌细胞化疗耐药的可能。     

LncRNA SOX2OT海绵吸附miR-34a促进SOX2表达增强大肠癌多药耐药

目的验证长链非编码RNA SOX2OT(Long noncoding RNA SOX2OT,SOX2OT)增强大肠癌HCT-116细胞多药耐药(Multidrug resistance,MDR),并从SOX2OT/miR-34a/SOX2信号通路探讨其可能的有效机制。方法双荧光素酶报告基因验证SOX2OT与miR-34a、miR-34a与SOX2的靶向结合;HCT-116细胞瞬时转染SOX2OT质粒、miR-34a mimic,RT-qPCR验证转染效率;RT-qPCR检测SOX2OT、miR-34a和SOX2表达水平并进行相关性分析;CCK-8检测细胞增殖,评估SOX2OT、miR-34a对HCT-116细胞5-氟尿嘧啶(5-FU)、长春新碱(VCR)、顺铂(CDDP)、紫杉醇(TAXOL)化疗敏感性的影响。结果 SOX2OT与miR-34a、miR-34a与SOX2之间存在靶向关系;SOX2OT抑制miR-34a(P<0.05),上调SOX2(P<0.01);miR-34下调SOX2OT(P<0.01)和SOX2(P<0.01);转染SOX2OT质粒后HCT...     

miR-34a通过靶基因Notch1调控子宫内膜癌细胞的增殖和凋亡

目的:探讨miR-34a通过靶基因Notch1调控子宫内膜癌细胞的增殖和凋亡。方法:通过荧光定量PCR检测miR-34a在子宫内膜癌和癌旁组织中的表达,并通过双荧光素酶报告基因检测miR-34a的靶基因,子宫内膜癌Ishikawa细胞转染miR-34a mimics及对照miR-NC,并转染Notch表达载体,通过MTT和流式细胞术分别检测miR-34a和Notch1对子宫内膜癌Ishikawa细胞增殖和凋亡的影响,并通过Western blot检测miR-34a对Notch1蛋白表达的影响。结果:miR-34a在人子宫内膜癌组织中表达显著下调（P<0.05）,双荧光素酶报告基因实验证实miR-34a能与Notch1 3' UTR结合,miR-34a mimics能够显著抑制细胞活力（P<0.05）,促进细胞凋亡（P<0.05）,并且miR-34a mimics显著抑制Notch1蛋白表达（P<0.05）。结论:miR-34a能够通过调控靶基因Notch1的表达,抑制子宫内膜癌细胞的增殖,促进细胞凋亡。     

吉非替尼获得性耐药非小细胞肺癌患者miR-21、miR-155表达水平及临床意义

目的探讨吉非替尼获得性耐药非小细胞肺癌患者miR-21、miR-155表达水平及临床意义。方法选取接受吉非替尼治疗出现耐药的80例晚期NSCLC患者作为研究组,另选取60例吉非替尼治疗效果良好、6个月以上病情无进展NSCLC患者为对照组。采用RT-PCR检测患者组织及血清中miR-21、miR-155表达水平;采用免疫组织化学法检测肺癌组织中EGFR的表达;采用Kaplan-Meier生存曲线比较患者3年内生存情况。结果研究组血清、组织中miR-21水平及血清miR-155水平明显高于对照组(P<0.001),但组织中miR-155水平与对照组相比无统计学差异(P>0.05);EGFR低表达患者血清、组织中miR-21及血清miR-155水平明显高于EGFR高表达患者(P<0.05),而组织中miR-155水平无统计学差异(P>0.05)。生存曲线结果显示血清中miR-21、miR-155高表达患者生存率明显低于低表达患者(χ^(2)=14.179,P<0.001;χ^(2)=18.338,P<0.001);组织中miR-21高表达患者生存率明显低于低表达患者(χ^(2)=12.195,P<0.001),但miR-155高表达患者生存率较低表达患者无统计学差异(χ^(2)=1.162,P>0.05)。结论吉非替尼获得性耐药NSCLC患者血清及组织中miR-21、miR-155水平与患者组织中EGFR水平及预后相关,可能在吉非替尼获得性耐药中发挥作用。     

miR-34b和miR-320a的表达与前列腺癌组织临床病理特征及预后的相关性

目的 探讨前列腺癌组织中miR-34b、miR-320a的表达与其临床病理特征及预后的相关性.方法 选择前列腺癌患者80例,另选40例良性前列腺增生患者作为对照组.采用定量聚合酶链式反应(RT-PCR)检测两组患者组织中miR-34b、miR-320a的表达量,分析前列腺癌组织中miR-320a、miR-34b与各临床...     

非小细胞肺癌中MET抑制剂的耐药机制及应对策略研究进展

MET基因是非小细胞肺癌的重要肿瘤驱动基因,针对MET 14外显子跳跃突变的靶向药物为患者带来新的治疗希望。虽然以tepotinib和沃利替尼等为代表的MET抑制剂显示出良好的抗肿瘤效果,但MET抑制剂的耐药不可避免。通过对HGF/MET信号通路的研究,不仅有助于探索MET抑制剂的耐药机制,有利于找到抑制和逆转耐药的方法,而且能够扩大新药研发的领域。初步研究显示HGF/MET信号通路抑制剂与其他药物的联合应用可能具有更大的临床应用潜力。本文就MET基因异常的特点,MET抑制剂的耐药机制和应对耐药策略进行综述,并提出MET抑制剂未来的发展方向和面临的挑战。     

结直肠癌中医辨证分型与miR-29、miR-34a表达的相关性分析

目的:探究结直肠癌患者血清miR-29、miR-34a表达水平及其与中医辨证分型的相关性。方法:选取2014年06月至2015年06月本院收治的127例结直肠癌患者作为研究对象（结直肠癌组）,并根据中医辨证分型标准分为湿热内蕴型21例、气滞血瘀型40例、脾肾阳虚型29例、气血两虚型18例、肝肾阴虚型19例;另选取同期在本院体检的127例健康者作为健康对照组。采用实时荧光定量PCR（qRT-PCR）技术检测血清miR-29、miR-34a表达水平,并进行比较;采用Pearson法分析血清miR-29与miR-34a表达水平的相关性;采用Logistic回归模型分析影响结直肠癌患者不良预后发生的危险因素。结果:结直肠癌组患者血清miR-29、miR-34a表达水平均明显低于健康对照组（P＜0.05）。低分化、浸润程度T3-T4、TNM分期Ⅲ-Ⅳ期、淋巴结转移、远处转移患者血清miR-29、miR-34a表达水平明显低于高中分化、浸润程度T1-T2、TNM分期Ⅰ-Ⅱ期、无淋巴结转移、无远处转移患者（P＜0.05）。结直肠癌患者血清miR-29与miR-34a表达水平呈正相关（r=0.529,P=0.000）。低分化、浸润程度T3-T4、TNM分期Ⅲ-Ⅳ期、淋巴结转移、低miR-29水平、低miR-34a水平是影响结直肠癌患者不良预后发生的危险因素（P＜0.05）。脾肾阳虚型、气滞血瘀型、湿热内蕴型预后不良患者比例依次明显增加（P＜0.05）。肝肾阴虚型、气血两虚型、脾肾阳虚型、气滞血瘀型、湿热内蕴型结直肠癌患者血清miR-34a表达水平依次显著降低（P＜0.05）,miR-29表达水平差异无统计学意义（P＞0.05）。结论:结直肠癌患者血清中miR-29、miR-34a低表达,与肿瘤分期升高、分化程度降低、浸润程度增加、淋巴结转移、远处转移、不良预后等密切相关,且miR-34a对结直肠癌不同中医辨证分型有一定的分型参考意义。     

MicroRNA-34a overcomes HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells partly by targeting MET

In non-small-cell lung cancer (NSCLC) that harbours an activating epidermal growth factor receptor (EGFR) mutation, over-expression of hepatocyte growth factor (HGF) is an important mechanism involved in the acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) by restoring activity of the PI3K/Akt pathway via phosphorylation of MET. In our study, we found that the forced expression of miR-34a inhibited cell growth and induced apoptosis partly by targeting MET in HGF-induced gefitinib-resistant HCC827 and PC-9 cells. Furthermore, dramatic tumour regression was observed in the miR-34a plus gefitinib group in HGF-induced gefitinib resistant mouse xenograft models. This study demonstrates for the first time that miR-34a rescues HGF-induced gefitinib resistance in EGFR mutant NSCLC cells.     

miR-497 and miR-34a retard lung cancer growth by co-inhibiting cyclin E1 (CCNE1)

Cyclin E1, encoded by the CCNE1 gene, promotes G1/S transition, chromosome instability, and oncogenesis. Here, we show that miR-497 and miR-34a target the 3′-UTR of CCNE1. miR-497 and miR-34a are downregulated in cancer cells and their ectopic expression inhibited cell proliferation and colony formation in vitro, and inhibited tumor growth in a xenograft model. The effect of simultaneous overexpression of miR-497 and miR-34a on the inhibition of cell proliferation, colony formation, and tumor growth, and the downregulation of cyclin E1 was stronger than the effect of each miRNA alone. The synergistic actions of miR-497 and miR-34a partly correlated with cyclin E1 levels. When cells stably expressing CCNE1 were transfected with the Hi-miR-497/34a plasmid, there was no effect on colony formation, compared with that of cells transfected with either Hi-miR497 or Hi-miR34a. These results indicate cyclin E1 is downregulated by both miR-497 and miR-34a, which synergistically retard the growth of human lung cancer cells.     

miR-449a Suppresses Tumor Growth,Migration, and Invasion in Non-Small Cell Lung Cancer by Targeting a HMGB1-Mediated NF-kB Signaling Pathway

MicroRNAs (miRNAs) have been reported to be involved in many human cancers and tumor progression. The dysregulation of miR-449a is found in many types of malignancies and is associated with tumor growth, migration, and invasion. However, its expression and function in non-small cell lung cancer (NSCLC) still remains unclear. In our study, miR-449a was found to be downregulated in both NSCLC tissues and cell lines, and low miR-449a expression was obviously associated with tumor differentiation, TMN stage, and poor overall survival (OS). Moreover, we demonstrated that miR-449a could inhibit tumor proliferation, migration, and invasion in NSCLC. We also confirmed that HMGB1 was a direct target gene of miR-449a in NSCLC with dual-luciferase reporter assay, and upregulation of HMGB1 could reverse the miR-449a-induced suppression of growth, migration, and invasion in NSCLC cells. Last, we found that miR-449a suppressed tumor initiation and development through the NF- B signaling pathway. These results indicate that miR-449a functions as a tumor suppressor in NSCLC by targeting the HMGB1-mediated NF- B signaling pathway in NSCLC.     

人miR-34a真核表达载体的构建及其表达

目的:构建人miR-34a的真核表达载体,并使其在骨肉瘤SOSP-9607细胞中表达.方法:从人骨肉瘤SOSP-9607细胞基因组DNA中用PCR法扩增出miR-34a的前体序列,并引入酶切位点和保护碱基,双酶切后定向插入真核表达载体pcD-NA3.1(+)中,构建miR-34a真核表达载体pcD-NA-miR34a,然后进行菌落PCR、酶切和测序鉴定.同时用构建好的载体pcDNA-miR34a转染骨肉瘤细胞SOSP-9607细胞,筛选miR-34a稳定表达细胞系,用Northern blot及real time RT-RCR法鉴定pcD-NA-miR34a可于真核细胞中过表达miR-34a的生物学特性.结果:成功构建了人miR-34a真核表达载体pcDNA-miR34a.pcDNA-miR34a可于骨肉瘤SOSp-9607细胞中上调miR-34a的表达,获得了miR-34a高表达骨肉瘤细胞系.结论:miR-34a的真核表达载体可在骨肉瘤细胞SOSP-9607细胞中有效表达,为进一步研究miR-34a在骨肉瘤中的功能及基因调控机制奠定了实验基础.     

MiR-34b和miR-155在非小细胞肺癌中的表达及临床意义

目的:检测肺癌组织及癌旁正常组织中miR-34b和miR-155的表达水平及其与肺癌临床病理特征之间的关系。方法:收集50例非小细胞肺癌患者的肺癌组织和癌旁正常肺组织标本,运用 Real-time PCR检测 miR-34b和miR-155在肺癌组织和癌旁正常肺组织中的表达情况,分析其表达与肺癌临床病理特征之间的关系。结果:Real-time PCR 检测显示在50例非小细胞肺癌组织中 miR-34b的表达显著低于癌旁正常肺组织(P=0.019),而miR-155的表达显著高于癌旁正常肺组织(P=0.000)。MiR-34b和miR-155的表达与淋巴结转移情况显著相关（P均＜0.05,miR-34b呈负相关,miR-155呈正相关）;与性别、年龄、病理类型及肿瘤分化程度无明显相关;miR-34b的表达与临床病理分期无显著相关,而miR-155表达与临床病理分期显著相关。结论:MiR-34b和miR-155的异常表达与非小细胞肺癌的发生发展相关,其可能成为肺癌的筛查、诊断及治疗的肿瘤标志物。     

人乳腺癌组织中miR-34a的表达及其在乳腺癌细胞系中的功能研究

目的 探讨miR-34a在人乳腺癌组织和细胞中的表达情况及对乳腺癌细胞系MDA-MB-231增殖、迁移侵袭和凋亡等生物学行为的影响,为研究乳腺癌组织中miR-34a的作用及深入了解乳腺癌发生发展的分子机制奠定理论基础。方法 通过实时荧光定量PCR（qRT-PCR）法检测miR-34a在20例人乳腺癌组织和癌旁正常组织中表达量的差异并比较其在人乳腺癌细胞系MDA-MB-231、MCF-7和正常乳腺上皮细胞MCF-10A中的表达差异;体外利用脂质体转染技术,转染miR-34a的模拟物（miR-34a mimic）和标记FAM（绿色荧光）的阴性对照(negative control ,miR-NC)进入MDA-MB-231细胞,研究miR-34a对细胞增殖活性、迁移和侵袭能力以及凋亡和周期分布的影响。结果 miR-34a在乳腺癌组织中的表达量较正常癌旁组织下调(P＜0.01);在MDA-MB-231、MCF-7和MCF-10A中的表达呈依次增高的趋势(P＜0.01);转染miR-34a mimic与转染miR-NC的MDA-MB-231相比,其增殖活力、迁移和侵袭能力均下降(P＜0.01),凋亡增加(P＜0.01),细胞周期被阻滞在G1/G0期（P＜0.01）。结论 miR-34a在乳腺癌组织和细胞系MDA-MB-231及MCF-7中的表达较正常组织和MCF-10A中都明显下调;miR-34a能够抑制肿瘤细胞MDA-MB-231的增殖、侵袭迁移,增加细胞凋亡率,使细胞周期阻滞在G0/G1;miR-34a可能起到抑癌作用,其表达水平与乳腺癌的发生发展密切相关。     

MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real‐time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in‐situ hybridization. Total and phospho‐MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High‐level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR‐TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho‐MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho‐MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA‐mediated knockdown of EGFR abolished phospho‐MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho‐MET expression in 2 cell lines with amplified MET gene was not down‐regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. © 2008 Wiley‐Liss, Inc.