MiR-21在大肠癌中的表达及其对SW480细胞增殖侵袭的影响

目的检测microRNA-21(miR-21)在大肠癌患者中的表达情况,并探讨miR-21对大肠癌SW480细胞增殖和侵袭的影响。方法收集大肠癌患者标本32例,同时收取癌组织及相应的癌旁组织。通过荧光定量PCR(qRT-PCR)方法检测各组织中miR-21的表达水平。以脂质体为载体将miR-21抑制剂转染至SW480细胞,分别用MTT方法与Transwell实验检测转染后SW480细胞的增殖侵袭能力变化,利用qRT-PCR和免疫印迹实验检测第10号染色体同源缺失的磷酸酶及张力蛋白同源的基因(PTEN)mRNA和蛋白水平的改变。结果 miR-21在32例患者癌组织及正常黏膜组织中的表达量分别为(1.57±0.79)与(0.56±0.32),差异有统计学意义(P0.01)。下调miR-21的表达能够显著抑制SW480细胞的增殖和侵袭能力,PTEN mRNA和蛋白的表达水平均显著升高(P0.01)。结论 microRNA-21在大肠癌中高表达,下调microRNA-21表达抑制大肠癌SW480细胞增殖和侵袭可能与上调PTEN表达相关。     

敲低丝酶抑制蛋白E2(serpin E2)抑制SW480结肠癌细胞增殖、侵袭和迁移

目的探讨丝酶抑制蛋白E2(serpin E2)对结肠癌细胞增殖、侵袭和迁移的影响。方法免疫组织化学染色对30名结肠癌患者癌组织和癌旁组织中serpin E2的表达量进行差异性检测,通过转染serpin小干扰RNA(si-serpin)至SW480细胞中,实时定量PCR检测SW480细胞中serpin E2 mRNA水平,Western blot法检测serpin E2蛋白水平,CCK-8法和平板克隆形成实验检测SW480细胞的增殖,TranswellTM实验分别检测SW480细胞的侵袭与迁移能力。结果结肠癌组织中serpin E2表达明显增加,敲低SW480细胞serpin E2水平后,SW480细胞的增殖、侵袭、迁移能力均显著降低。结论下调SW480细胞中serpin E2水平抑制SW480细胞的增殖、侵袭和迁移。     

上调miR-433表达抑制结直肠癌细胞系增殖

目的研究miR-433在结直肠癌中的表达,并初步探讨其功能。方法选择不同分期结直肠癌癌组织(CRC)、癌旁组织与正常结直肠组织,用实时定量PCR检测miR-433表达并比较;体外培养结直肠癌细胞系HT-29、HCT-116和SW480以及正常结直肠细胞系NCM460,用实时定量PCR检测miR-433表达。随后将miR-433模拟物转染SW480细胞,CCK-8法检测细胞增殖;划痕实验检测细胞迁移;Transwell小室法检测细胞侵袭;流式细胞测量术检测细胞周期。结果与癌旁组织相比,miR-433在结直肠癌组织中表达显著降低(P0.05)。在结肠癌患者血清中miR-433水平也显著低于健康人群(P0.05)。此外,从TNM分期Ⅰ期～Ⅳ期,结直肠癌组织内miR-433表达逐渐降低(P0.05)。与NCM460相比,HT-29、HCT-116和SW480中,miR-433表达下调(P0.05)。SW480转染miR-433模拟物后,与对照miRNA组相比,在SW480细胞增殖水平明显降低(P0.05),G_2和M期细胞比例增多。上调miR-433后,细胞侵袭和迁移明显减弱(P0.05)。结论 miR-433在结直肠癌中表达降低。在SW480细胞中上调其表达后,可抑制细胞系增殖、侵袭与迁移。     

microRNA-130b在结肠癌中的表达及生物学意义研究

目的:研究微小RNA 130b(microRNA-130b,miR-130b)在结肠癌(Colonic carcinoma,CRC)中的表达情况及生物学意义。方法:收集2013年1月至2015年3月于我院普通外科行手术切除的CRC组织及对应癌旁组织共60例,运用qRT-PCR技术检测miR-130b在CRC组织及癌旁组织中的表达水平,统计分析miR-130b表达水平与患者临床病理资料间的相关性;采用人工合成的miR-130b模拟物转染人结肠癌SW480细胞,分别采用CCK-8分析、Brd U检测转染后细胞增殖变化,流式细胞仪及Caspase3/7活性分析检测转染后细胞凋亡变化。结果:miR-130b在CRC组织中表达水平显著高于对应癌旁组织(P0.05);miR-130b高表达与肿瘤体积增大(≥5 cm,P0.05)及高TNM分期(Ⅲ+Ⅳ期,P0.05)显著相关;在人结肠癌SW480细胞中过表达miR-130b可显著抑制细胞增殖并促进细胞凋亡(P0.05)。结论:miR-130b在结肠癌组织中表达下调并与肿瘤恶性临床病理特征有关,miR-130b可能通过抑制结肠癌细胞增殖、促进凋亡来抑制结肠癌的生长及发展。     

基因沉默PRDX1 表达对人结直肠癌SW480 细胞侵袭迁移的影响

目的:探讨RNA 干扰过氧化还原酶1(Peroxiredoxin 1,PRDX1)表达对人结直肠癌SW480 细胞侵袭转移能力的影响。方法:筛选RNA 干扰PRDX1 的慢病毒质粒,与阴性对照慢病毒质粒分组转染结直肠癌SW480 细胞,转染后的SW480 细胞可分为PRDX1 基因沉默组(si-PRDX1)和阴性对照组(Vector)。实时荧光定量PCR(qRT-PCR)和免疫印迹法(Western blot)分别检测两组细胞中PRDX1 mRNA 和蛋白表达;采用Transwell 侵袭和迁移实验检测基因沉默PRDX1 表达对结直肠癌细胞侵袭及迁移能力的影响;通过Western blot 检测两组细胞中基质金属蛋白酶(MMP)家族部分蛋白表达水平。结果:基因沉默PRDX1 表达可有效抑制结直肠癌SW480 细胞中PRDX1 mRNA 和蛋白水平的表达,与阴性对照组相比(Vector),差异均具有统计学意义(P<0.01),说明基因沉默PRDX1 的SW480 细胞系构建成功;Transwell 侵袭和迁移实验显示si-PRDX1组细胞的侵袭及迁移能力较对照组均明显降低(P<0.01);Western blot 结果显示,与Vector 组相比,si-PRDX1 组细胞中组织基质金属蛋白酶抑制剂2(TIMP-2)的表达明显增加,而MMP-2 及MMP-9 的表达显著下降,且差异均具有统计学意义(P<0.05)。结论:基因沉默人结直肠癌SW480 细胞的PRDX1 表达可有效抑制细胞的侵袭、迁移及转移能力,其机制可能会通过调控TIMP-2、MMP-2 及MMP-9 的表达介导。     

过表达miR-10b促进肺癌细胞系A549增殖

目的探讨非小细胞肺癌(NSCLC)患者肺癌及癌旁组织中miR-10b(miR-10b)表达水平及miR-10b是否通过调控锌指转录蛋白基因(KLF4)对肺癌细胞系A549恶性化的影响。方法 40例NSCLC患者病理切片,原位杂交检测肺癌及癌旁组织中miR-10b的表达量;对肺癌细胞系A549转染miR-10b mimics后,CCK-8法检测肺癌细胞增殖;real-time PCR及Western blot检测KLF4 mRNA及蛋白水平;软琼脂克隆形成实验检测过表达miR-10b对A549细胞的肿瘤恶性化程度的影响。结果肺癌细胞A549及肺癌组织中miR-10b的表达量分别高于正常肺上皮细胞16HBE及癌旁组织;过表达miR-10b模拟物的A549细胞中,KLF4蛋白水平显著下降(P0.05);过表达的miR-10b可显著增加A549细胞的增殖速度及在软琼脂内的成瘤性。结论 miR-10b在不同类型细胞及组织中具有分布差异性、可能是通过抑制KLF4的表达促进肺癌细胞增殖及恶性化。     

miRNA-10b靶向调控Twist促进骨肉瘤细胞增殖与侵袭的研究

目的探讨microRNA-10b对骨肉瘤细胞MG-63增殖与侵袭能力的影响及潜在作用机制。方法应用实时荧光定量PCR检测人骨肉瘤组织和癌旁正常骨组织中miR-10b的表达差异。将miR-10b mimic和Twist特异性的siRNA (Twist siRNA)分别通过脂质体转染法转染入骨肉瘤细胞系MG-63,应用RT-PCR检测细胞中miR-10b和Twist mRNA的表达,采用蛋白质印迹法(Western blot)检测Twist及内参β-actin蛋白表达水平,采用MTT [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]法检测miR-10b mimic及Twist siRNA对人骨肉瘤细胞系MG-63增殖能力的影响;采用Transwell侵袭实验检测miR-10b mimic及Twist siRNA对人骨肉瘤细胞系MG-63侵袭能力的影响。结果与正常癌旁骨组织相比,miR-10b在骨肉瘤组织中呈现明显高表达,差异具有极显著性统计学意义(P0.01); miR-10b mimic可使Twist mRNA和蛋白表达水平升高,差异具有统计学意义(P0.05),同时可显著增强MG-63细胞的增殖与侵袭能力(P0.05),而转染miR-10b mimic和Twist siRNA的MG-63细胞的增殖与侵袭能力较仅转染miR-10b mimic的MG-63细胞显著减弱(P0.05)。结论人骨肉瘤细胞系MG-63中miR-10b的表达上调,可能通过靶向激活Twist信号通路促进骨肉瘤细胞的异常增殖与远处侵袭。     

lncRNA RAB11B-AS1对结直肠癌SW480细胞增殖、侵袭、迁移及凋亡的影响

目的探讨lncRNA RAB11B-AS1对结直肠癌SW480细胞增殖、侵袭、迁移及凋亡的影响及可能机制。方法 qRT-PCR方法检测34例结直肠癌组织及相应的癌旁组织、人正常结肠黏膜上皮细胞NCM460与人结直肠癌细胞株SW480中lncRNA RAB11B-AS1的表达水平。构建稳定过表达RAB11B-AS1的SW480细胞系,MTT方法检测SW480细胞的增殖能力变化;流式细胞术检测SW480细胞凋亡率;Transwell检测SW480细胞侵袭能力变化,划痕实验检测SW480细胞迁移能力变化;Western blot检测SW480细胞中细胞周期蛋白1(cyclin D1)和CDK6的表达水平。结果 RAB11B-AS1在结直肠癌组织中的表达显著低于癌旁组织(P0.01);在SW480中的表达显著低于NCM460细胞(P0.01)。RAB11B-AS1过表达后,SW480细胞的增殖、侵袭与迁移能力显著降低,凋亡率显著升高,cyclin D1与CDK6的蛋白表达降低(P0.01)。结论 lncRNA RAB11B-AS1在结直肠癌组织中表达降低,过表达RAB11B-AS1能够抑制SW480细胞的增殖、迁移与侵袭,并促进凋亡。     

IncRNA RAB11B-AS1对结直肠癌SW480细胞增殖、侵袭、迁移及凋亡的影响

目的 探讨IncRNA RAB11B-AS1对结直肠癌SW480细胞增殖、侵袭、迁移及凋亡的影响及可能机制.方法 qRT-PCR方法检测34例结直肠癌组织及相应的癌旁组织、人正常结肠黏膜上皮细胞NCM460与人结直肠癌细胞株SW480中IncRNA RAB11B-AS1的表达水平.构建稳定过表达RAB11B-AS1的SW480细胞系,MTT方法检测SW480细胞的增殖能力变化;流式细胞术检测SW480细胞凋亡率;Transwell检测SW480细胞侵袭能力变化,划痕实验检测SW480细胞迁移能力变化;Western blot检测SW480细胞中细胞周期蛋白1(cyclin D1)和CDK6的表达水平.结果 RAB11B-AS1在结直肠癌组织中的表达显著低于癌旁组织(P＜O.Ol);在SW480中的表达显著低于NCM460细胞(P＜0.01).RAB11B-AS1过表达后,SW480细胞的增殖、侵袭与迁移能力显著降低,凋亡率显著升高,cyclinDl与CDK6的蛋白表达降低(P＜O.Ol).结论 IncRNA RAB11B-AS1在结直肠癌组织中表达降低,过表达RAB11B-AS1能够抑制SW480细胞的增殖、迁移与侵袭,并促进凋亡.     

MicroRNA-10b对人胃癌SGC-7901细胞增殖、迁移和侵袭能力的影响

目的探讨Micro RNA-10b(miR-10b)对人胃癌SGC-7901细胞增殖、迁移和侵袭的影响。方法应用Real-time PCR方法检测21例胃癌组织和癌旁正常组织中miR-10b的表达水平,应用Lipofectamine?LTX试剂将化学合成的miR-10b mimics和miR-10b inhibitor转染至胃癌SGC-7901细胞,采用MTT方法检测SGC-7901细胞增殖能力的变化,Transwell小室法检测SGC-7901细胞迁移和侵袭能力的变化;应用Western Blot方法检测P21蛋白表达水平。结果与正常组织相比,miR-10b在胃癌组织中的表达水平显著增高。与对照组相比,miR-10b表达沉默能够显著抑制人胃癌SGC-7901细胞的增殖、迁移和侵袭能力,显著增加P21的蛋白表达水平。miR-10b过表达的作用与之相反。结论 MiR-10b沉默抑制胃癌SGC-7901细胞的增殖、迁移和侵袭可能与上调P21蛋白表达相关。     

脑胶质瘤患者血清miR-21、miR-221、miR-222和miR-10b的相对表达量及临床意义

目的 探讨血清中miR-21、miR-221、miR-222和miR-10b对脑胶质瘤恶性程度的鉴别价值.方法 采用Real-timePCR技术检测34例低级别脑胶质瘤患者和50例高级别脑胶质瘤患者miR-21、miR-221、miR-222和miR-10b的相对表达量,使用受试者工作特征曲线(receiver operating characteristic curve,ROC)评价其诊断价值.结果 与低级别脑胶质瘤组相比,高级别脑胶质瘤组血清miR-10b、miR-21和miR-221的相对表达量均显著升高,且差异具有统计学意义,miR-222相对表达量的差异无统计学意义.ROC曲线评价血清miR-10b、miR-21和miR-221对于脑胶质瘤恶性程度的诊断价值,miR-10b的曲线下面积(area under curve,AUC)值最大,为0.722(0.694,0.751),对于低级别脑胶质瘤的诊断敏感性和特异性分别为79.41％和78.00％.使用二元Logistic回归分析评价血清miR-10b、miR-21和miR-221联合检测对于脑胶质瘤恶性程度的诊断价值,其曲线下面积为0.915(0.855,0.977),对于低级别脑胶质瘤的诊断敏感性和特异性分别为85.29％和88.00％.与miR-10b、miR-21和miR-221单独检测的诊断价值相比,其曲线下面积均有显著提高(P=0.026、P=0.012、P=0.008).结论 miR-10b、miR-21和miR-221联合诊断可以为临床脑胶质瘤患者的鉴别诊断提供一种潜在的辅助诊断方法.     

miR-193b在宫颈癌中的表达与功能及其对化疗敏感性的影响

目的:探讨微小RNA-193b(miR-193b)在不同宫颈组织中的表达以及其对顺铂作用于宫颈癌细胞敏感性的影响,并初步探讨其作用机制。方法:实时荧光定量PCR(qPCR)检测不同宫颈组织中miR-193b的表达水平。采用脂质体将miR-193b-inhibitor转染至HeLa细胞,Transwell法、CCK-8法和免疫蛋白印迹法分别检测细胞迁移数、细胞对顺铂的敏感性以及细胞中PTEN、Akt、p-Akt和P-糖蛋白(P-gp)的蛋白水平。结果:qPCR结果显示miR-193b在正常宫颈组织及宫颈糜烂组织中呈低表达,在宫颈上皮内瘤变和宫颈癌组织中的表达明显上升;转染miR-193b-inhibitor后,HeLa细胞迁移数明显减少(P0.05),顺铂对HeLa细胞活力的抑制作用进一步增强(P0.05),细胞中PTEN的蛋白表达明显上调(P0.05),Akt的蛋白表达无明显变化,p-Akt和P-gp的蛋白水平明显下调(P0.05)。结论:miR-193b在宫颈癌组织中呈高表达,沉默miR-193b可增加HeLa细胞对顺铂的敏感性,作用机制可能与PTEN-PI3K/Akt通路有关。     

miR-130b在胶质瘤对替莫唑胺耐药中的作用

目的:探讨微小RNA-130b(microRNA-130b,miR-130b)在胶质瘤细胞中的表达及其调控体外胶质瘤细胞对替莫唑胺(temozolomide,TMZ)耐药中的作用。方法:利用RT-qPCR法检测miR-130b在胶质瘤细胞株U251、SHG-44和U87中的表达水平;计算TMZ对不同胶质瘤细胞株(U251、SHG-44和U87)的半数抑制浓度(IC_(50));通过不同浓度梯度的TMZ作用于体外U251细胞,从而获得相对稳定的对TMZ耐药的U251(U251/TMZ resistance,U251/TR)细胞,计算TMZ对U251/TR细胞的IC_(50)及耐药指数(resistance factor,RF);使用miR-130b模拟物(miR-130b mimics)和miR-130b抑制物(miR-130b inhibitor)瞬时转染体外胶质瘤细胞;CCK-8法检测体外胶质瘤细胞的活力;流式细胞术检测胶质瘤细胞凋亡;通过生物信息学工具分析miR-130b可能的靶基因,萤光素酶报告基因实验测定萤光素酶活性;电泳迁移率变动分析检测核因子κB(nuclear factor-κB,NF-κB)的活性;Western blot法测定肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、Bcl-2、X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)和survivin蛋白在胶质瘤细胞中的表达水平。结果:TMZ对不同胶质瘤细胞株(U251、SHG-44和U87)的IC_(50)分别为54.8、94.8和149.6μmol/L;TMZ对U251/TR细胞的IC_(50)为(446.5±61.3)μmol/L,其RF为8.1;转染miR-130b mimics可明显增强TMZ对U251/TR细胞的生长抑制和凋亡诱导作用;转染miR-130b inhibitor可显著抑制TMZ对U251/TR细胞的生长抑制和凋亡诱导作用;萤光素酶报告基因实验验证TNF-α是miR-130b的直接作用靶点;与mimics NC组相比,转染miR-130b mimics后U251/TR细胞中的NF-κB活性及TNF-α、Bcl-2、XIAP和survivin蛋白表达均明显下调;与inhibitor NC组相比,转染miR-130b inhibitor后U251细胞中的NF-κB活性及TNF-α、Bcl-2、XIAP和survivin蛋白表达均显著上调(P0.05);NF-κB抑制剂Bay 11-7082可增强TMZ对U251/TR细胞凋亡的诱导作用。结论:miR-130b在耐药型胶质瘤细胞中表达下调,并通过靶向调控TNF-α/NF-κB通路增强TMZ对体外胶质瘤细胞的作用。     

miR-193b Regulates Mcl-1 in Melanoma

MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737-resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3' untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression.     

Augmented miR-10b expression associated with depressed expression of its target gene KLF4 involved in gastric carcinoma

Previous studies have revealed several targets of miR-10b, such as syndecan-1, HOXD10, TBX5, and E-cadherin. In this study, we aimed to assess whether Krüppel-like factor 4 (KLF4) is a target gene of miR-10b in gastric cancer (GC). Targeting of KLF4 by miR-10b was confirmed by dual-luciferase reporter assays. The expression levels of miR-10b and KLF4 mRNA in 5 different gastric cancer cell lines and 65 pairs of gastric cancer tissues were detected by Real-time PCR. In addition, KLF4 protein in gastric cancer cell lines and 30 GC tissues was measured by western blotting and immunochemistry, respectively. KLF4 is a direct target gene of miR-10b in GC, and its expression is reduced by miR-10b at both mRNA and protein levels. In addition, the expression level of miR-10b was tendentiously upregulated in GC tissues while the expression levels of KLF4 mRNA and protein were decreased in gastric cancer tissues compared with normal adjacent tissue. There was a dramatically inverse correlation between the expression levels of miR-10b and KLF4 mRNA in GC (r = -0.339, P = 0.006). These findings indicate that miR-10b was upregulated in GC and may have a key role in GC pathogenesis and development through the downregulation of its target gene KLF4.     

The potential value of miR-1 and miR-374b as biomarkers for colorectal cancer

The mortality of colorectal cancer (CRC) is growing due to the unsatisfactory specificity and sensitivity of the existing screening methods. Previous studies have focused on the role of miRNAs as CRC biomarkers. However, few studies have examined the miRNA profiles at each stage. The objective of this study was to identify miRNAs that distinguish CRC patients from normal people to prevent the misdiagnosis of patients with certain stages of CRC. We performed miRNA profiling of 1547 human miRNAs by qRT-PCR in CRC patients with stage II and stage III disease. The statistical analyses showed that there were 96 miRNAs that were significantly dysregulated in CRC relative to normal tissues (P<0.05). There were 28 dysregulated miRNAs associated with separate or combined stages II and III disease. There were 25 downregulated miRNAs, including the following: miR-1, -145, -145*, -137, -363, -143, -4770, -490-5p, -9, -144*, -99a, -99b, -23b, -143*, -100, -768-3p, -24-1*, -125a-5p, -30e*, -574-3p, -126, let-7b, miR-1979, -374b, and -140-3p. We found an upregulation of miR-203, 182, and 96. Our results demonstrated that the expression of miR-1 and miR-374b was significantly decreased in each stage and may function as a biomarker of CRC. Furthermore, 20 miRNAs were dysregulated both in stage II disease without lymph node or distant metastasis and in stage II-III tumors but not in stage III tumors. Only miR-4794 was involved exclusively with stage II tumors, and there were 19 miRNAs that were dysregulated only in stage III disease with lymph node metastasis and in stage II-III disease. There were only 6 miRNAs that were uniquely dysregulated in stage III. Our results indicate that miRNA expression may be valuable in the clinic. However, large prospective studies are required to confirm the role of miRNAs. This study provides a new model for analyzing novel CRC biomarkers by considering more clinical factors.     

HLF细胞高氧暴露后miR-15b及miR-16上调并抑制VEGF表达

目的探讨高氧暴露下人胚肺成纤维细胞(HLF)中miR-15b及miR-16对VEGF蛋白的调控作用。方法用转染技术分别上调及下调HLF细胞miR-15b和miR-16水平;用Western blot技术检测细胞中VEGF蛋白;同时,用体外细胞培养方法,观察高氧暴露下HLF细胞miR-15b、miR-16及VEGF蛋白水平的影响。结果上调miR-15b和miR-16可使HLF细胞中VEGF蛋白降低,反之可使其升高;高氧暴露可诱导HLF细胞miR-15b和miR-16上调,而VEGF表达则下降。结论高氧暴露可诱导HLF细胞中miR-15b及miR-16上调并抑制VEGF的表达,可能参与支气管肺发育不良(BPD)的发生过程。