miR-140-5 p通过调控Wnt1通路对非小细胞肺癌迁移与侵袭能力的影响

目的:探究miR-140-5p通过Wnt1通路对非小细胞肺癌(NSCLC)细胞迁移、侵袭的影响,为临床对NSCLC的治疗提供理论依据.方法:临床收集人NSCLC组织及癌旁组织20组,RT-PCR检测miR-140-5p与Wnt1基因的表达水平,Spearman相关分析法比较miR-140-5p与Wnt1基因表达水平的相...     

miR-147a靶向ATF2抑制非小细胞肺癌细胞侵袭和迁移北大核心CSCD

目的:探究miR-147a靶向激活转录因子2(ATF2)对非小细胞肺癌迁移、侵袭和上皮-间质转化的影响。方法:RT-qPCR检测非小细胞肺癌组织和细胞miR-147a和ATF2 mRNA表达,Western blot检测ATF2蛋白表达。采用miR-NC、pc-NC、miR-147a mimic、pc-ATF2质粒分别或联合转染H1703细胞,双荧光素酶报告验证其靶向关系,Transwell检测细胞侵袭,划痕实验检测细胞迁移,Western blot检测上皮钙黏蛋白(E-cadherin)、间质钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和ATF2蛋白表达。裸鼠分为4组:control组、miR-147a mimic组、pc-ATF2组、miR-147a+pc-ATF2组。裸鼠左腋下皮下注射转染后的非小细胞肺癌H1703细胞,第30天颈椎脱位法处死,完整取出皮下肿瘤,免疫组化检测N-cadherin阳性表达,Western blot检测E-cadherin、N-cadherin和Vimentin蛋白表达。结果:与正常肺组织和细胞相比,非小细胞肺癌组织和细胞miR-147a表达显著下调,ATF2 mRNA和蛋白表达显著上调(P<0.01)。miR-147a靶向下调ATF2表达。与对照组相比,miR-147a mimic组H1703细胞ATF2表达显著下调,侵袭数显著减少,划痕闭合率显著降低,E-cadherin表达显著上调,N-cadherin和Vimentin表达显著下调(P<0.01),共转染pc-ATF2逆转miR-147a mimic对H1703细胞的作用。与对照组相比,miR-147a mimic组移植瘤中N-cadherin阳性细胞百分比降低,E-cadherin表达显著上调,N-cadherin和Vimentin表达显著下调(P<0.01),共转染pc-ATF2逆转miR-147a mimic对H1703移植瘤的作用。结论:miR-147a靶向ATF2可抑制非小细胞肺癌迁移、侵袭和上皮-间质转化。     

鞘氨醇激酶1通过ERK1/2信号通路促进非小细胞肺癌细胞的侵袭和迁移

目的:探讨鞘氨醇激酶1(SphK1)对非小细胞肺癌(NSCLC)细胞迁移和侵袭的影响及其作用机制。方法:选取31例外科手术切除并经常规组织学检查确诊为NSCLC的肿瘤组织标本和配对癌旁肺组织标本,应用免疫组织化学染色和RT-qPCR检测SphK1的表达。将pcDNA3. 1-SphK1载体(SphK1组)、空白pcDNA3. 1载体对照(NC组)、SphK1 siRNA(siSphK1组)和对照siRNA(siNC组)分别转染人肺腺癌A549细胞,Western blot法检测SphK1、E-cadherin、fibronectin和p-ERK1/2的表达;利用Transwell实验评估过表达SphK1和抑制ERK1/2对A549细胞迁移和侵袭的影响。结果:SphK1在NSCLC组织中高表达,并与肿瘤分期相关。SphK1过表达可显著促进A549细胞的迁移和侵袭,提高p-ERK1/2和fibronectin蛋白水平,减少E-cadherin蛋白表达(P<0. 05),而干扰SphK1则呈现相反的结果。ERK1/2抑制剂U0126可显著抑制SphK1过表达诱导的p-ERK1/2和fi...     

DCST1-AS1在非小细胞肺癌中的表达及其生物学作用

目的 检测DCST1-AS1在非小细胞肺癌中的表达,并探讨其对癌细胞恶性生物学行为的影响。方法 收集65例非小细胞肺癌患者的癌组织及其癌旁组织,体外培养正常人支气管上皮细胞16HBE和非小细胞肺癌细胞系（A549、H1299、H1650和HCC827）。RT-qPCR法检测组织和细胞中DCST1-AS1、miR-29b表达水平,并分析DCST1-AS1表达与非小细胞肺癌患者临床特征的相关性。将A549细胞分为对照组、si-NC组、si-DCST1-AS1组、si-DCST1-AS1+anti-NC组和si-DCST1-AS1+anti-miR-29b组。采用CCK-8法、克隆形成实验检测细胞增殖;Transwell检测细胞侵袭及迁移;Western blot检测E-cadherin、Vimentin、N-cadherin表达。结果 非小细胞肺癌组织和细胞中DCST1-AS1的表达均升高（P<0.05）,miR-29b的表达均降低（P<0.05）;DCST1-AS1的表达与非小细胞肺癌患者TNM分期、组织分化程度、淋巴结转移及病理类型有关（P<0.05）。与对照组或si...     

miR-205调控YES1表达对人膀胱癌细胞增殖的影响

目的探讨miR-205对膀胱癌T24细胞增殖的影响。方法合成miR-205的模拟物并转染膀胱癌T24细胞,荧光显微镜下观察转染效率,实时荧光定量PCR法检测转染48 h后,T24细胞中miR-205的表达情况,MTT法检测转染24、48和72 h后,T24细胞的增殖情况,实时荧光定量PCR法和western blot检测转染48 h后膀胱癌细胞内源性YES1 mRNA及蛋白的表达变化。结果 miR-205的模拟物转染至T24细胞的效率为80%,转染miR-205模拟物48 h后,T24细胞内miR-205的表达水平显著升高(P0.05),T24细胞的增殖率显著低于阴性对照组(P0.05),且呈时间依赖性,YES1 mRNA及蛋白的表达水平明显低于阴性对照组(P0.05)。结论 miR-205抑制膀胱癌T24细胞增殖可能与靶向YES1基因相关。     

miR-183及MAPK1蛋白在非小细胞肺癌中的表达及其意义

目的探讨miR-183及丝裂原激活的蛋白激酶1（MAPKl）mRNA与蛋白在非小细胞肺癌（NSCLC）组织中的表达及意义。方法实时定量PCR方法检测NSCLC组织miR-183及MAPKlmRNA的表达水平,Westernblot方法检测NSCLC组织MAPKl蛋白表达;统计分析miR-183与MAPKl蛋白的相关性。结果miR-183在非小细胞肺癌组织中的表达量较癌旁组织明显降低（P〈0．01）,MAPKlmRNA与蛋白在癌组织中较癌旁组织表达明显增高（P〈0．05）;统计分析显示,miR-183表达与MAPKl表达呈显著地负相关关系。结论miR-183在NSCLC组织中低表达,与MAPKl表达呈显著负相关关系。     

长链非编码RNA PVT1调控miR-551通过Wnt信号通路对卵巢癌迁移和侵袭的影响

目的:探讨长链非编码RNA PVT1在卵巢癌组织中的表达情况及其在卵巢癌细胞迁移和侵袭过程中的作用及机制。方法:q PCR检测卵巢癌和正常卵巢组织及不同卵巢癌细胞中PVT1的表达情况;Transwell侵袭实验和细胞划痕实验分别检测沉默PVT1后卵巢癌细胞侵袭和迁移能力的变化;双萤光素酶报告基因检测PVT1与微小RNA(miR)-551的相互作用;Transwell侵袭实验和细胞划痕实验分别检测沉默PVT1后miR-551-inhibitor对卵巢癌细胞侵袭和迁移能力的影响;Western blot法检测沉默PVT1后Wnt信号通路相关蛋白的表达情况。裸鼠皮下成瘤实验检测沉默PVT1对卵巢癌成瘤重量及体积的影响。结果:与正常卵巢组织相比,卵巢瘤组织中PVT1表达明显增高(P0.05);卵巢癌细胞株ES-2中PVT1表达水平最高(P0.05);沉默PVT1可以抑制卵巢癌细胞侵袭和迁移能力;PVT1能与miR-551的位点特异性结合;沉默PVT1后,miR-551-inhibitor可以促进卵巢癌细胞侵袭和迁移能力;沉默PVT1后Wnt信号通路蛋白的表达相应下调;与阴性对照组相比,PVT1-siRNA组荷瘤小鼠肿瘤体积和重量都明显减小(P0.05)。结论:PVT1在卵巢癌发生发展过程中起重要作用,它可以靶向调节miR-551,通过Wnt信号通路调控卵巢癌细胞的侵袭和迁移能力。     

miR-34b抑制非小细胞肺癌A549的侵袭和迁移

目的：本研究旨在探索miR-34b对非小细胞肺癌A549侵袭和迁移的影响及HIF1α在其中所起的作用。方法：A549细胞转染miR-34b mimic和pc-HIF1α过表达载体以过表达miR-34b和缺氧诱导因子-1α (HIF1α)。A549细胞分为4组：A549(空对照)组,miR-34b mimic组,pc-HIF1α组和miR-34b mimic+pc-HIF1α组。实时定量PCR(qRT-PCR)检测miR-34b表达和HIF1α的mRNA水平。荧光素酶分析验证miR-34b和HIF1α的靶向关系。蛋白印迹检测HIF1α、基质金属蛋白酶2 (MMP-2),Snail和血管内皮细胞生长因子(VEGF)的蛋白水平。Transwell分析细胞侵袭。划痕实验检测细胞迁移。结果：转染miR-34b mimic可提高A549细胞miR-34b的表达水平,降低HIF1α的mRNA水平(P<0.001)。miR-34b可直接靶向HIF1α。miR-34b 过表达可抑制 pc-HIF1α诱导的HIF1α蛋白水平升高(P<0.01)。与对照组相比,miR-34b 过表达可显著降低细胞侵袭数目和愈合率(P<0.01)。此外,miR-34b 过表达可显著抑制 HIF1α过表达导致的细胞侵袭和愈合率增加(P<0.01)。miR-34b mimic组MMP-2、Snail和VEGF相对蛋白水平低于对照组(P<0.01)。pc-HIF1α组MMP-2、Snail和VEGF相对蛋白水平高于对照组(P<0.01)。与pc-HIF1α组相比,miR-34b mimic+pc-HIF1α组MMP-2、Snail和VEGF相对蛋白水平下降(P<0.01)。结论：miR-34b-5p过表达通过靶向HIF1α抑制非小细胞肺癌A549的侵袭和迁移。     

miR-183及Akt1蛋白在非小细胞肺癌中的表达及其意义

目的探讨mi R-183及Akt1蛋白在非小细胞肺癌(NSCLC)组织中的表达,以及抑制或过表达mi R-183对Akt1蛋白表达的影响。方法实时定量PCR方法检测NSCLC组织mi R-183m RNA及Akt1m RNA的表达水平,Western blot方法检测NSCLC组织Akt1蛋白表达;将mi R-183 inhibitors及mi R-183 mimics转染至A549细胞,实时定量PCR方法及Western blot方法分别检测转染后Akt1 m RNA及蛋白的表达变化。结果 mi R-183m RNA在非小细胞肺癌组织中的表达量较癌旁组织明显降低(0.01),Akt1 m RNA及蛋白在癌组织中较癌旁组织表达明显增高(0.05);转染mi R-183 inhibitors的细胞内Akt1蛋白与m RNA水平与对照组相比显著升高(0.05),转染mi R-183 mimics的细胞内Akt1蛋白与m RNA水平对照组相比显著降低(0.05)。结论 mi R-183在NSCLC组织中低表达,并能够调节Akt1的表达。     

miR-135b-3p调控XAF1表达影响甲状腺癌细胞的迁移、侵袭和放射增敏性

目的探讨miR-135b-3p对甲状腺癌细胞迁移、侵袭和放射敏感性的影响以及其可能的调控机制。方法培养正常甲状腺细胞Nthy-ori 3-1、甲状腺癌细胞K1和TPC-1,RT-qPCR检测细胞中miR-135b-3p表达,Western blot检测细胞中X染色体连锁凋亡抑制蛋白相关因子1(XAF1)表达。转染anti-miR-135b-3p至TPC-1细胞抑制miR-135b-3p表达,Transwell、Western blot及克隆形成实验分别检测抑制miR-135b-3p表达对TPC-1细胞迁移和侵袭、E-cadherin和MMP-2蛋白表达及对TPC-1细胞放射敏感性的影响。双荧光素酶报告基因实验验证miR-135b-3p与XAF1之间的靶向调控关系。结果与Nthy-ori 3-1细胞相比,K1和TPC-1细胞中miR-135b-3p表达水平显著升高(P<0.05),XAF1蛋白表达水平显著降低(P<0.05)。抑制miR-135b-3p表达可抑制TPC-1细胞的迁移和侵袭能力(P<0.05),促进TPC-1细胞E-cadherin蛋白表达(P<0.05),抑制MMP-2蛋白表达(P<0.05),增强TPC-1细胞的放射敏感性(P<0.05)。miR-135b-3p靶向负调控XAF1表达。抑制XAF1表达可降低抑制miR-135b-3p表达对TPC-1细胞迁移、侵袭及放射敏感性的影响。结论miR-135b-3p通过下调XAF1表达促进甲状腺癌细胞的迁移和侵袭能力,并降低其放射增敏性,是甲状腺癌的潜在治疗靶点。     

LncRNA HEIH通过miR-98-5p调节肺癌细胞增殖和凋亡

目的探讨lncRNA HEIH对肺癌细胞增殖和凋亡的影响及其作用机制。方法培养人正常肺上皮细胞BEAS-2B和肺癌细胞系A549、A427、H1299和TKB-1,RT-qPCR检测细胞中HEIH表达水平;分别转染si-HEIH和miR-98-5p mimics至A549细胞,沉默A549细胞中HEIH表达或过表达miR-98-5p;MTT法检测细胞增殖;流式细胞仪检测细胞凋亡;Western blot检测CCND1、caspase-3、SHH、GLI-1、PTCH和SUFU蛋白表达。双荧光素酶报告基因实验验证HEIH与miR-98-5p之间的关系。结果与正常肺上皮细胞BEAS-2B相比,肺癌细胞系A549、A427、H1299和TKB-1中HEIH表达水平显著升高(P<0.05).其中A549细胞中的HEIH表达最高。因此,后续实验选择A549细胞为研究对象。沉默HEIH表达或过表达miR-98-5p均可降低A549细胞培养12、48和72 h后吸光度值(A值)(P<0.05)(MTT法);升高凋亡率(P<0.05);抑制CCND1蛋白表达(P<0.05),促进caspase-3蛋白表达(P<0.05)。并且过表达miR-98-5p还抑制了A549细胞中SHH和GLI-1的mRNA和蛋白表达(P<0.05),促进了PTCH和SUFU的mRNA和蛋白表达水平(P<0.05)。过表达HEIH逆转了过表达miR-98-5p对A549细胞增殖、凋亡以及SHH、GLI-1、PTCH和SUFU的mRNA和蛋白表达的影响。结论沉默HEIH表达可能通过靶向miR-98-5p经Hedgehog信号通路抑制肺癌细胞的增殖,并促进其凋亡。     

MiR-152 regulates metastases of non-small cell lung cancer cells by targeting neuropilin-1

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been suggested to play critical roles in tumorigenesis. Recently, miR-152 was reported to be dysregulated in some human cancers. However, the function and mechanism of miR-152 in non-small cell lung cancer (NSCLC) is still unclear. In the present study, our findings showed that the expression of miR-152 was significantly down-regulated and neuropilin-1 was up-regulated in the NSCLC specimens. Moreover, the levels of miR-152 and neuropilin-1 were inversely correlated. Bioinformatics analyses and luciferase reporter assay showed that miR-152 targeted the 3’-UTR of neuropilin-1 mRNA to inhibit its translation. Furthermore, overexpression of miR-152 inhibited neuropilin-1 mediated cell invasiveness, while down-regulated expression of miR-152 increased neuropilin-1 mediated cell invasiveness in NSCLC cells. Together, these findings indicated that miR-152 suppression in NSCLC cells might promote neuropilin-1 mediated cancer metastasis and suggested a new therapeutic application of miR-152 in the treatment of NSCLC.     

miR-425 regulates cell proliferation,migration and apoptosis by targeting AMPH-1 in non-small-cell lung cancer

BackgroundMicroRNAs (miRNAs) have been proved to act as vital roles on non-small-cell lung cancer (NSCLC), and miR-425 has been proven to serve an important function in several tumors. However, the functional role of miR-425 on NSCLC is still unclear.MethodsThe mRNA and protein expression of miR-425 and AMPH-1 were determined by qRT-PCR and western blot analysis, respectively. NSCLC cells (SK-MES-1 and A549) proliferation and migration were measured by CCK-8 and transwell assay, respectively. Cell apoptosis was assessed by flow cytometry and western blotting, In addition, luciferase reporter assay was carried out to confirm the direct targeting of AMPH-1 by miR-425. Xenograft experiments were performed to observe the tumorigenesis of miR-425 in vivo.ResultsThe results showed that miR-425 was overexpressed and AMPH-1 expression was downregulated in SK-MES-1 and A549 cells. Silencing miR-425 inhibited proliferation, migration and promoted apoptosis of NSCLC cells. Moreover, we proved that miR-425 could target AMPH-1. The expression of AMPH-1was upregulated in A549 with miR-425 inhibitor. Moreover, miR-425 knockdown were less tumorigenic than the control in vivo.ConclusionsTaken together, miR-425 could promote the proliferation, invasion and suppress apoptosis by targeting AMPH-1 in NSCLC cells. miR-425/AMPH-1 axis may represent a potential therapeutic strategy or novel prognostic biomarkers to NSCLC.     

LncRNA NCK1-AS1 promotes non-small cell lung cancer progression via regulating miR-512-5p/p21 axis

ObjectiveThis study aimed at probing into the effect of lncRNA NCK1-AS1 on proliferation, migration and invasion of non-small cell lung cancer (NSCLC) cells and its regulatory function on miR-512-5p/p21 molecular axis.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expressions of NCK1-AS1 and miR-512-5p in NSCLC tissues and cell lines. The alterations of cell proliferation, migration, invasion and cell cycle were examined by cell counting kit-8 (CCK-8) assay, BrdU experiment, Transwell experiment and flow cytometry, respectively. The dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the binding relationships between miR-512-5p and NCK1-AS1, and miR-512-5p the 3'UTR of p21 mRNA. Western blot was used to determine the effects of NCK1-AS1 and miR-512-5p on p21 protein expression.ResultsNCK1-AS1 expression was up-regulated in NSCLC tissues and cells, and its high expression was correlated with shorter overall survival time and faster progression of patients. Overexpression of NCK1-AS1 promoted NSCLC cell proliferation, migration and invasion, and accelerated the cell cycle, whereas NCK1-AS1 siRNA inhibited these malignant biological behaviors, and arrested cell cycle. NCK1-AS1 could bind to miR-512-5p, p21 was verified as a target gene of miR-512-5p, and NCK1-AS1 could up-regulate the expression of p21 in NSCLC cells via repressing miR-512-5p expression.ConclusionNCK1-AS1 promotes NSCLC progression by regulating miR-512-5p/p21 molecular axis.     

下调TCF3抑制非小细胞肺癌细胞的增殖和迁移能力

目的:探索下调T细胞因子3(TCF3)抑制非小细胞肺癌细胞增殖和迁移的分子机制。方法:运用Lipofectamine 2000转染法将siTCF3和阴性对照siRNA(NCsiRNA)转染非小细胞肺癌A549和H1299细胞;运用real-time PCR和Western blot分别测定TCF3的mRNA和蛋白水平;运用萤光素酶报告基因实验测定TCF3的转录活性;MTT、克隆形成实验、Transwell实验和Annexin V-FITC/PI染色联合流式细胞术分别测定细胞的活力、克隆形成能力、转移能力及细胞凋亡率;Western blot检测Wnt、c-Myc、基质金属蛋白酶(MMP)-9、MMP-13、金属蛋白酶组织抑制物(TIMP)-1的蛋白表达水平。结果:与NCsiRNA转染组的细胞比较,siTCF3显著抑制A549细胞和H1299细胞中TCF3的mRNA和蛋白水平(P0.01)。TCF3转录活性和c-Myc蛋白表达水平明显低于NCsiRNA细胞(P0.05)。MTT实验结果显示,培养24 h、48 h、72 h和96 h的A549-siTCF3和H1299-siTCF3细胞活力均显著低于NCsiRNA细胞(P0.05)。与NCsiRNA细胞相比,siTCF3显著抑制A549细胞和H1299细胞的克隆形成能力(P0.01)。Transwell实验结果显示A549-siTCF3和H1299-siTCF3细胞迁移数显著低于A549-NCsiRNA和H1299-NCsiRNA组细胞(P0.05)。流式细胞术分析结果显示A549-siTCF3细胞和H1299-siTCF3细胞的凋亡率显著高于A549-NCsiRNA和H1299-NCsiRNA细胞(P0.01)。Western blot实验结果显示,下调TCF3表达能抑制Wnt蛋白的表达,MMP-9和MMP-13的蛋白表达明显降低,TIMP-1的蛋白表达增高。结论:siTCF3显著抑制A549细胞和H1299细胞的增殖和迁移能力,并诱导细胞凋亡,其分子机制可能通过下调Wnt通路活性以及调控MMP家族关键成员的表达而实现。     

SEL1L expression in non-small cell lung cancer

SEL1L gene product plays a role in cell transformation and tumor progression in human breast, pancreas, esophageal, and prostate cancer. SEL1L expression was evaluated in a series of 76 surgically resected non-small cell lung carcinomas to investigate its clinical significance. SEL1L is scarcely detectable in normal lung, whereas in the initial stages of cell transformation, it becomes consistently expressed with evident staining in bronchial squamous metaplasia and in associated dysplastic changes. SEL1L immunoreactivity can be detected both in the cytoplasm and less commonly in the nuclei; the subcellular location correlates with tumor histotype, with cytoplasmic immunoreactivity being most prevalent in squamous cell carcinomas (P = .0005) and nuclear immunoreactivity being associated with adenocarcinomas (P = .02). Nuclear import and export signals are present in the SEL1L coding sequence, justifying the different subcellular location of the protein. SEL1L immunoreactivity was inversely correlated with tumor grade (P = .05); when considering only the adenocarcinomas, a stronger association was found (P = .006). SEL1L messenger RNA and protein evaluation in lung cancer cell lines confirmed the expression of the gene and the dual subcellular location of the protein in lung tumors. The data here reported suggest that, in non-small cell lung carcinoma, SEL1L may be an indicator of cell transformation, thus having important biologic and clinical implications.     

miR-106a promotes growth and metastasis of non-small cell lung cancer by targeting PTEN

MicroRNAs are a class of small non-coding RNAs that play essential roles in cancer development and progression. Recent studies suggested that abnormal expression of miRNAs occurs frequently in non-small cell lung cancer (NSCLC) tissues compared to adjacent normal tissues. In this study, we investigated the expression and the biological roles of miR-106a in non-small cell lung cancer. Our results showed that miR-106a was up-regulated in NSCLC tissues and cell lines. Inhibition of miR-106a in NSCLC cells substantially inhibited cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-106a, and over-expression of miR-106a suppressed PTEN by direct binding to its 3’-untranslated region (3’-UTR). Furthermore, the presence of miR-106a was inversely correlated with PTEN in NSCLC tissues. Overall, this study suggested that miR-106a inhibited the growth and metastasis of NSCLC cells by decreasing PTEN expression. These data provide novel insights with potential therapeutic applications for the treatment of NSCLC.     

miR-185 acts as a tumor suppressor by targeting AKT1 in non-small cell lung cancer cells

Increasing evidence has shown that microRNAs play critical roles in the initiation and progression of non-small cell lung cancer (NSCLC). miR-185 is deregulated in various cancers, whereas its functional mechanism in NSCLC is still unclear. Here, we confirmed that the expression of miR-185 was significantly down-regulated in NSCLC tissues and cell lines. miR-185 over-expression caused significant suppression of in vitro cell proliferation, migration and invasion, and in vivo tumor growth. We subsequently identified that AKT1 was a target gene of miR-185. Re-expression of AKT1 could partially rescue the inhibitory effects of miR-185 on the capacity of NSCLC cell proliferation and motility. Collectively, we conclude that miR-185 has a critical function by blocking AKT1 in NSCLC cells, and it may be a novel therapeutic agent for miRNA based NSCLC therapy.