Hsa-miR-125a-3p和Hsa-miR-125a-5p对非小细胞肺癌侵袭转移的作用研究
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摘要
MicroRNA(miRNA)是近年来备受关注的一类普遍存在于动、植物中的非编码小RNA,大约20-24个核苷酸,来自于长的转录子前体pri-miRNA和pre-miRNA。MiRNA与其靶mRNA分子的3’端非编码区(3’-UTR)不完全互补结合,在多细胞生物体中,转录后调控基因的表达,影响着几乎所有的信号通路,参与多种生理病理过程,在肿瘤的发生和发展中也发挥了重要的调节作用。目前研究最为清楚的Ras相关单体GTP酶家族为Rho, Racl和CDC42,它们参与细胞内多样的调节作用,包括细胞骨架的重构,细胞的迁移和侵袭。Rho相关的丝／苏蛋白激酶(Rho-associated serine/threonine protein kinase,Rock)是迄今为止功能研究最为清楚的Rho下游靶效应分子之。活化的GTP·Rho可以和Rock相互作用而激活Rock。通过与Rho相互作用,Rock在肿瘤细胞的转移过程中参与调控细胞迁移和侵袭。
肿瘤的侵袭和转移是世界上癌症患者死亡最常见的原因。最近,越来越多的研究报道表明,miRNA与肿瘤细胞的侵袭和转移有关,其调控肿瘤细胞侵袭和转移的作用机制成为miRNA研究领域的热点之一。在miRNA与Rho/Rock-1传导通路的关系中,Kong等人在鼠正常乳腺上皮细胞中发现,miR-155在TGF-β诱导的上皮间质转化以及RhoA引起的细胞迁移侵袭过程中具有重要作用。TWISTl能够诱导miR-1Ob, miR-1Ob通过直接转录后抑制HOXD 10的表达来间接的增加RhoC的水平,从而影响了肿瘤细胞的迁移和侵袭。研究Rho和miRNAs之间的关系加强了miRNA在肿瘤形成中的重要性,并且为癌症转移的患者提供了新的治疗策略。
miR-125a是一种功能以及机制尚未研究清楚的miRNAs之一。Yanainhara等人发现miR-125a定位在19q13.41,并用miRNA芯片分析了104对肺癌组织以及相应的癌旁肺组织,发现与癌旁肺组织相比较,miR-125a在肺癌组织中下调表达。至目前为止,已发现的成熟体miR-125a家族有两个成员,即hsa-miR-125a-3p和hsa-miR-125a-5p。本研究目的旨在探讨非小细胞肺癌(non-small cell lung cancer, NSCLC)组织中hsa-miR-125a-3p和hsa-miR-125a-5p的表达及其临床意义,分析hsa-miR-125a-3p和hsa-miR-125a-5p与NSCLC细胞系转移潜能的关系。并在细胞水平上,通过向肺癌细胞中转染正义和反义的2’-O-甲基寡核苷酸的方法,探讨增加hsa-miR-125a-3p和hsa-miR-125a-5p的表达以及下调hsa-miR-125a-3p和hsa-miR-125a-5p的表达对Rho/Rockl途径,以及肺癌细胞迁移和侵袭能力的影响,为hsa-miR-125a-3p和hsa-miR-125a-5p可能是Rho/Rockl信号通路非常重要的调节因子提供依据。
材料与方法
1、NSCLC组织标本
52例配对的非小细胞肺癌和癌旁肺组织(远离原发灶)标本均来自中国医科大学病理教研室。患者术前均未接受过任何化学或放射治疗。福尔马林固定石蜡包埋的肿瘤组织切片常规进行HE染色,分别由两名病理诊断医师,根绝WHO肺及肺膜肿瘤分类标准(2004)和TNM分期系统(1997),确定肿瘤的组织学分型以及临床病理分期。52例NSCLC包括：男性33例,女性29例；年龄40～78岁(平均58岁±9岁)；鳞癌22例,腺癌30例；病理分级,高分化8例,中分化28例,低分化16例；病理分期,Ⅰ期18例,Ⅱ期15例,Ⅲ期19例；淋巴结转移20例,无淋巴结转移32例。52例配对组织在液氮中快速冰冻,以后用于提取mRNA。常规病理检查手术切除的肺门、纵膈以及肺内淋巴结,用于确定肺癌的淋巴结转移情况。患者的临床病理参数包括性别,年龄,组织学类型,病理分级,病理分期,淋巴结转移,摘自患者的病历资料。
2、细胞培养
冻存于液氮的A549细胞经复苏后,用DMEM培养液(含有10%新生牛血清,100μ／ml的青霉素(?)100μg/ml的链霉素),在37℃,5%CO2饱和湿度条件下培养,经3次传代稳定后,进行转染。
3、细胞转染
2’-O-甲基寡核苷酸(2'-O-Me-sense-3p:5'-ACA GGU GAG GUU CUU GGG AGCC-3'2'-O-Me-antisense-3p:5'-GGC UCC CAA GAA CC U CAC CUGU-3', 2'-O-Me-scramble-3p:5'-GGU CGG UGC UCG AUG CAG GUAA-3', 2'-O-Me-sense-5p:5'-UCC CUG AGA CCC UUU AAC CUG UGA-3', 2'-O-Me-antisense-5p:5'-UCA CAG GUU AAA GGG UCU CAG GGA-3', 2'-O-Me-scramble-5p:5'-GGA CG G CGA UCA GAU AAG AGU UCU-3'.)用结合DNA技术合成(GeneChem, China)。用LipofetamineTM 2000 (Invitrogen, USA)将20gM 2’-O-甲基寡核苷酸转染入6孔板中A549和SPC细胞中为RNA提取和transwell备用。untreated为未加任何处理因素的细胞,scramble为转染乱码序列2’-O-甲基寡核苷酸的细胞,sense为转染正义序列2’-O-甲基寡核苷酸的细胞,antisense为转染反义序列2’-O-甲基寡核苷酸的细胞。其中对照组细胞为(untreated和scramble)。
4、Real-time PCR
根据操作说明应用TaqMan MicroRNA试剂检测成熟体miRNA (Applied Biosystems, USA)。所有的RT产物,在ABI Prism 7900HT序列检测系统中运行。用特异的RT引物和TaqMan探针定量检测hsa-miR-125a-3p (PN:4395310)和hsa-miR-125a-5p (PN:4395309), RNU6B (PN:4373381)和U18(PN:4380904)为内参。组织样本用2次独立样本经过2次独立实验,细胞样本用3次独立样本经过3次独立实验后得到的数据用公式RQ=2-△△Ct的方法进行分析。
5、RT-PCR
TRIzol试剂提取总RNA,紫外分光光度计测定RNA纯度并定量,在1%甲醛变性凝胶电泳验证RNA完整性。RT-PCR采用两步法试剂盒(宝生物公司)按照厂家推荐步骤进行,扩增体系为20μl,以持家基因β-actin为内参照,对样品模板用量标准化。引物参照文献及使用Primer Designer5.0软件设计。RT条件按照厂家推荐,PCR条件：95℃lmin,随后进行35个循环的94℃30s,55℃30s,72℃45s,最后72℃充分延伸1Omin; p-actin PCR条件：94℃5min,随后94℃40s,50℃40s,72℃40s共35个循环,最后72℃充分延伸5min。扩增产物进行1.5%琼脂糖凝胶电泳,UVP图像处理系统采集图像,ImageJ灰度分析软件进行条带灰度分析。产物经1.75%琼脂糖凝胶电泳,凝胶成像系统摄像,分析各条带强度。
6、Western blot检测
提取各组织蛋白,根据蛋白的分子量制备不同浓度的SDS—聚丙烯酰胺凝胶,每孔上样量50μg,常规电泳、转膜、封闭,一抗及相应二抗37℃孵育1h,DAB显色,β-actin作为内对照。UVP图像处理系统采集图像,ImageJ灰度分析软件进行条带灰度分析。以平均吸光度值INT和信号面积S的乘积代表信号强度。分别计算不同指标和P-actin的INT×S,用二者的比值显示不同指标的表达水平。
7、Transwell细胞迁移和侵袭实验
在具有8μm小孔聚碳酸酯滤膜的培养小室上铺用预冷无血清培养基稀释的Matrigel基质胶,接种100μl无血清培养基稀释的A549细胞或NCI-H460细胞(5×105个／mL),下室加入600μl含10%血清的DMEM培养液或1640培养液,每组设6复孔。37℃、5%CO2条件下培养24h后,取出培养小室,用2.5%戊二醛固定15min,并以0.5%Triton X-100处理3min,苏木精中染核15min。将培养小室倒置,在光学显微镜(Leica,德国)下观察、照相,并计数每高倍视野滤膜底面的平均细胞数。细胞迁移实验在无Matrigel基质胶包被的Transwell小室中进行,方法同细胞侵袭实验。
8、统计学分析
SPSS13.0用于所有的统计学分析。Real-time PCR结果数据即hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌及相应的癌旁肺组织中的表达水平经过log2转换。配对T检验用于分析两种成熟体miRNA-125a在非小细胞肺癌和癌旁肺组织中的显著差异。Chi-square检验(Chi-square test)和双侧fisher精确检验(Two-sided fisher's exact test)用于分析hsa-miR-125a-3p和hsa-miR-125a-5p的表达与临床病理因素的关系。Mann-whitney检验(Mann-whitney test)用于病理分级,病理分期等级资料的分析。Spearman相关分析(Spearmancorrelation analysis)用于分析hsa-miR-125a-3p和hsa-miR-125a-5p与病理分期和淋巴结转移的相关性。其它实验采用t检验。用mean±SD, p<0.05为有统计学意义。
实验结果
1、Hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌组织中表达下调与非小细胞肺癌淋巴结转移和病理分期有关。
(1) hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌组织中的表达：real-time PCR检测52例非小细胞肺癌及相应的癌旁肺组织中hsa-miR-125a-3p和hsa-miR-125a-5p相对表达水平的结果显示,与癌旁肺组织相比较,52例非小细胞肺癌组织中,hsa-miR-125a-3p和hsa-miR-125a-5p表达下降(p＜0.05)。hsa-miR-125a-3p表达的平均水平降低约4.5倍(p<0.001), hsa-miR-125a-5p表达的平均水平下降近6倍(p＜0.001)。
(2) hsa-miR-125a-3p和hsa-miR-125a-5p与临床病理因素的关系：根据hsa-miR-125a-3p和hsa-miR-125a-5p在52例非小细胞肺癌中表达的平均水平分别分成2组,即hsa-miR-125a-3p低表达(低于平均值1.2352,log2转换数据)和高表达组(高于平均值1.2352,log2转换数据),hsa-miR-125a-5p低表达(低于平均值1.8594,log2转换数据)和高表达组(高于平均值1.8594,log2转换数据)。统计学分析结果显示,hsa-miR-125a-3p表达与年龄(p=0.031)、病理分期(p=0.012)和淋巴结转移(p=0.034)有关,与性别、组织分型以及病理分级无关；hsa-miR-125a-5p与病理分期(p=0.002)和淋巴结转移(p=0.042)有关,与年龄、性别、组织分型以及病理分级无关。sepearman相关检验结果显示,hsa-miR-125a-3p表达与病理分期(r=-0.352,p=0.011)和淋巴结转移(r=-0.326,p=0.018)呈负相关,hsa-miR-125a-5p表达与病理分期(r=0.439,p=0.001)和淋巴结转移(r=0.300,p=0.031)呈正相关。
2、hsa-miR-125a-3p和hsa-miR-125a-5p通过Rho/Rockl信号通路调控肺癌细胞的迁移和侵袭。
(1)hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌细胞系中的表达：real-time PCR检测了4种肺癌细胞系LH7,A549,SPC-A-1和NCI-H460中hsa-miR-125a-3p和hsa-miR-125a-5p的表达情况,人正常支气管上皮细胞(Human bronchiolar epithelium cell line, HBE)作为正常对照细胞系,U18作为内对照。结果显示,hsa-miR-125a-3p和hsa-miR-125a-5p在4种肺癌细胞系中的表达均低于HBE (p<0.01)。hsa-miR-125a-3p相对表达水平在NCI-H460细胞中表达最低,在A549细胞中呈中度表达(p<0.001), hsa-miR-125a-5p相对表达水平在A549细胞中表达最低,在SPC-A-1细胞中呈中度表达(p＜0.001)。
(2)2’-O-甲基寡核苷酸对细胞内hsa-miR-125a-3p和hsa-miR-125a-5p的影响：Real-time PCR的结果显示,转染正义2’-O-甲基寡核苷酸后,细胞内hsa-miR-125a-3p和hsa-miR-125a-5p的含量明显增加(p＜0.001)；转染反义2’-O-甲基寡核苷酸后,细胞内hsa-miR-125a-3p和hsa-miR-125a-5p明显降低了(p＜0.001)。
(3) hsa-miR-125a-3p对肺癌细胞迁移和侵袭的影响：hsa-miR-125a-3p的迁移实验显示,A549细胞的穿膜细胞数在未处理组细胞中是34.40±2.47个,与未处理组细胞相比较,转染乱序组细胞的穿膜细胞数没有明显改变(34.40±2.38,p=1.000),转染sense-3p组细胞的穿膜细胞数明显降低(25.33±3.15,p＜0.001),转染antisen-3p组细胞的穿膜细胞数明显增加(48.80±2.65, p< 0.001)。hsa-miR-125a-3p的侵袭实验显示,A549细胞侵过基质胶的穿膜细胞数在未处理组细胞中是24.33±2.06。与未处理组细胞相比较,转染乱序组细胞侵过基质胶的穿膜细胞数没有明显改变(24.00±2.54,p=0.696),转染sense-3p组细胞侵过基质胶的穿膜细胞数明显降低(11.13±1.60,p＜0.001),转染antisen-3p组细胞侵过基质胶的穿膜细胞数明显增加(34.80±2.88,p＜0.001)。
(4) hsa-miR-125a-5p对肺癌细胞迁移和侵袭的影响：hsa-miR-125a-5p的迁移实验显示,SPC-A-1细胞的穿膜细胞数在未处理组细胞中是24.20±1.78个,与未处理组细胞相比较,转染乱序组细胞的穿膜细胞数没有明显改变(28.80±2.70,p=0.445),转染sense-5p组细胞的穿膜细胞数明显增加(45.27±2.71,p＜0.001),转染antisen-5p组细胞的穿膜细胞数明显降低(16.93±3.24, p<0.001)。hsa-miR-125a-5p的侵袭实验显示,SPC-A-1细胞侵过基质胶的穿膜细胞数在未处理组细胞中是24.20±1.78。与未处理组细胞相比较,转染乱序组细胞侵过基质胶的穿膜细胞数没有明显改变(23.80±2.00,p=0.568),转染sense-5p组细胞侵过基质胶的穿膜细胞数明显增加(35.80±1.74,p＜0.001),转染antisen-5p组细胞侵过基质胶的穿膜细胞数明显降低(17.47±2.07,p＜0.001)。
(5) hsa-miR-125a-3p靶基因的预测和确认：用microrna.org和Target ScanHuman 5.1预测靶基因的网络数据库,对在细胞迁移和侵袭中起作用的潜在靶基因进行了预测。在候选基因调查中,我们发现RhoA基因的3’UTR包含2个高保守区域,分别由miRanda和TargetScan算法计算得到的,这两个高保守区域可能为hsa-miR-125a-3p与靶基因的作用连接位点。
(6) Hsa-miR-125a-3p通过靶基因RhoA调控Rho/Rockl信号途径：Western blotting和RT-PCR分析显示RhoA蛋白在转染sense-3p细胞中明显减少(p=0.001),在转染antisense-3p细胞中明显增多(p＜0.001),而RhoA mRNA(p=0.482)变化并不是很明显。如预期的那样,Rockl蛋白和mRNA在转染sense-3p细胞中明显减少(p＜0.05),在转染antisense-3p细胞中明显增多(p＜0.05)。
为了进一步说明RhoA是hsa-miR-125a-3p的潜在靶基因,我们转染了包含RhoA基因3’UTR全长的荧光素酶报告基因。三次结果显示,增加细胞内hsa-miR-125a-3p含量能够降低荧光素酶活性(p＜0.001)。这些结果表明hsa-miR-125a-3p可直接通过抑制靶基因RhoA而抑制细胞迁移和侵袭。
在A549细胞中用Rho抑制剂CT04(最终浓度2.0μg／ml)阻断Rho,与未处理组相比较,迁移和侵袭的细胞数量在用CT04处理后明显减少(p＜0.001)。与阻断Rho未转染组相比较,转染sense-3p组或转染antisense-3p组细胞数目并没有明显的减少或增加(p＞0.05)。结果表明hsa-miR-125a-3p可能通过调控Rho/Rockl途径而影响细胞的迁移和侵袭。
(7) Hsa-miR-125a-5p通过RhoC调控Rho/Rockl信号途径：Western blotting和RT-PCR分析显示RhoA蛋白(p=0.001)在sense-5p和antisense-5p转染细胞中都没有明显变化,RhoA mRNA (p=0.482)变化亦不是很明显。然而,RhoC mRNA和蛋白在转染sense-5p细胞中明显增加(p＜0.05),在转染antisense-5p细胞中明显减少(p＜0.05)。如预期的那样,Rockl蛋白和mRNA在转染sense-5p中明显增加(p＜0.01),在转染antisense-5p细胞中明显减少(p＜0.01)。
在SPC-A-1细胞中用Rho抑制剂CT04(最终浓度2.0μg/ml)阻断Rho,与未处理组相比较,迁移和侵袭的细胞数量在用CT04处理后明显减少(p＜0.001)。与阻断Rho未转染组相比较,转染sense-5p组或转染antisense-5p组细胞数目也并没有明显的增加或减少(p＞0.05)。结果表明hsa-miR-125a-5p可能通过调控Rho/Rockl途径而影响细胞的迁移和侵袭。
结论
1、hsa-miR-125a-3p与病理分期和淋巴结转移呈负相关,而hsa-miR-125a-5p与病理分期和淋巴结转移呈正相关。
2、hsa-miR-125a-3p能够通过Rho A/Rockl信号途径抑制肺癌细胞的迁移和侵袭。
3、hsa-miR-125a-5p能够通过RhoC/Rockl信号途径促进肺癌细胞的迁移和侵袭。
MicroRNAs (miRNAs) are short (21-24-nt) non-coding RNAs generated from longed transcripts, termed pri-miRNAs and pre-miRNAs. They act as post-transcriptional regulators of gene expression and induce translational repression. Recently, miRNAs have been discovered to play important roles in tumourigenesis. By targeting 3'untranslated regions (3'UTRs) of cognate mRNAs, miRNAs are involved in diverse processes, including cell differentiation, proliferation, apoptosis and metastasis. Understanding the specific functions of individual miRNAs may contribute to our knowledge of the molecular properties of cancers and may provide new biomarkers for cancer diagnosis and therapy.
The Rho family of small GTPases, of which the best studied members are Rho, Racl and CDC42, are involved in regulation of a variety of cellular processes includ-ing reorganization of the actin cytoskeleton, cell motility and invasive phenotypes. Rho-associated serine-threonine protein kinase (Rock), one of the best characterized downstream effectors of Rho, is activated when it selectively binds to the active GTP-bound form of Rho. As with Rho, Rock has been implicated in altering cell migration and invasion during tumor cell metastasis.
Tumor invasion and metastasis are the critical steps in determining the death of cancer patients. Recently, miRNA have been discovered to have a role in invasion and metastasis. Emerging studies have indicated that miRNAs participate in the signaling of the Rho pathway. Among the tested miRNAs, miR-155 may play an important role in TGF-β-induced epithelial-mesenchymal transition (EMT) and cell migration and invasion by targeting RhoA. During tumor progression, TWIST 1 induces miR-10b, which in turn indirectly increases RhoC levels by direct down-regulation of its transcriptional repressor HOXD10. Studies on the association of Rho with miRNAs could highlight the importance of miRNAs in tumorigenesis and lead to the development of novel therapeutic strategies for patients with metastases.
MiR-125a is one of the many miRNAs that remain to be fully characterized. Using miRNA microarray analysis, Yanainhara and colleagues found that miR-125a, specifically the hsa-miR-125a-5p mature miRNA, is located at 19q13.41 and that its expression is downregulated in NSCLC. Recently, a new member of the mature miR-125a family has been identified and named hsa-miR-125a-3p. Unfortunately, the expression and function of hsa-miR-125a-3p are currently unknown. In the present study, we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues, and explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells. In add-ition, the effects of hsa-miR-125a-3p/5p on the invasive and migratory capabilities of lung cancer cells were studied by investigating its effects on Rho/Rock pathway. These results provide basis that hsa-miR-125a-3p and hsa-miR-125a-5p maybe very important regulators in Rho/Rock pathway.
Materials and Methods
Samples preparation:We analyzed 52 pairs of non-small cell lung cancer specimens and corresponding normal lung tissues (LAC) collected at the time of surgery and prior to chemotherapy. Specimens were obtained from patients at the First Affiliated Hospital of China Medical University from 1 January 2006 to 1 December 2007 with informed consent. For the majority of samples, clinical and biological information was available. The study has been approved by the Hospitals'Ethical Review Committee.
Cell culture: The HBE (Human Bronchiolar Epithelium) cell line and the human lung cancer cell lines A549 and SPC-A-1 (adenocarcinoma), LH7 and NCI-H460 (large cell cancinoma) were propagated in RPMI1640 (Gibco). The A549 (adenoca-rcinoma) cell line was propagated in Dulbecco's Modifed Eagle Medium (Gibco). In both cases, the medium was supplemented with 10% fetal bovine serum (FBS),100 U/ml penicillin, and 100 U/ml streptomycin. Cells were cultured at 37℃in 5% CO2 until they reached a confluency of 75%.
Transfection:Depletion of hsa-miR-125a-3p/5p in the A549 and SPC-A-1 cell lines was achieved through transfection with antisense 2'-O-methyl oligonucleotides directed against hsa-miR-125a-3p/5p. Cells were transfected using LipofetamineTM 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, complexes con-taining the oligonucleotides were prepared according to the recommended protocol and added directly to cells at a final oligonucleo tide concentration of 0.4 nmol/ml. Oligonucleotides composed entirely of 2'-O-methyl bases were chemically synthe-sized by Integrated DNA Technologies (GeneChem) and were comprised of the following sequences:2'-O-Me-sense-3p:5'-ACA GGU GAG GUU CUU GGG AGCC-3'2'-O-Me-antisense-3p:5'-GGC UCC CAA GAA CC U CAC CUGU-3', 2'-O-Me-scramble-3p:5'-GGU CGG UGC UCG AUG CAG GUAA-3',2'-O-Me-sense-5p:5'-UCC CUG AGA CCC UUU AAC CUG UGA-3',2'-O-Me-antisense-5p:5'-UCA CAG GUU AAA GGG UCU CAG GGA-3',2'-O-Me-scramble-5p: 5'-GGA CG G CGA UCA GAU AAG AGU UCU-3'. Cells were divided into four groups:an untreated group incubated only in the normal media (untreated), a group transfected with the scrambled 2'-O-methyl oligonucleotide (scramble); a group transfected with the sense 2'-O-methyl oligonucleotide (sense) and a group transfected with the antisense 2'-O-methyl oligonucleotide (antisense). The untreated and scramble groups served as negative controls.
Quantitative real-time polymerase chain reaction (qRT-PCR):Expression of mature miRNAs was assayed using the TaqMan MicroRNA Assay in accordance with the manufacturer's instructions (Applied Biosystems). All reactions, including no-template controls and RT-minus controls, were run in an ABI Prism 7900HT Sequence detection system (Applied Biosystems). Specific RT primers and TaqMan probes were used to quantify the expression of hsa-miR-125a-3p (PN:4395310) and hsa-miR-125a-5p (PN:4395309). Samples were normalized to RNU6B (PN:4373381) or U18 (PN:4380904) as indicated. For quantification of tissue samples, RT-PCR analysis was performed in two independent experiments, each using two independent samples. For quantification of cell samples, RT-PCR analysis was performed in three independent experiments, each using three independent samples. MiRNA expression data is presented as fold difference relative to either RNU6B or U18 based on the following equation:RQ=2-△△Ct.
Semi-quantitative RT-PCR: Total RNA was prepared using Trizol (Invitrogen, USA). cDNA was generated by reverse transcription of 500ng total RNA using PrimeScript RT reagent Kit (TaKaRa, Japan). Primer sequences of RhoA were 5'-TAT GTG CCC ACA GTG TTT GA-3'(forward) and 5'-CAT TCC GAA GAT CCT CCT TCT TATT-3'(reverse); Rockl primer sequences were 5'-GCA ACT ATG ATG TGC CTG AA-3'(forward) and 5'-CAC CAT TTC GCC CTA ACC-3'(reverse), and that ofβ-actin were 5'-AAA TCG TGC GTG ACA TTA A-3'(forward) and 5'-CTC GTC ATA CTC CTG CTTG-3'(reverse). The lengths of RhoA, Rockl andβ-actin PCR products were 270 bp,489 bp and 513 bp respectively. PCR was carried out by an initial denaturation step at 94℃for 5min, followed by 35 cycles of denaturation at 94℃for 40s, annealing at 56℃(RhoA) or 57.2℃(Rock1) for 40s, and elongation at 72℃for 40s. Cycling was completed by a final elongation step at 72℃for 5min. The PCR forβ-actin was performed in the same manner as above, except that 30 cycles of annealing at 50℃was used. The EC3 Imaging system (UVP Inc.) was used to catch up the specific bands, and the optical density of each band was measured using the Image J software.
Western blot: Cells were washed twice with ice-cold phosphate buffered saline (PBS) and lysed in M-PER reagent (Pierce Biotechnology) containing 1 mM PMSF and phosphatase inhibitors for 1h at 4℃. The supernatants were centrifuged at 12 OOO×g for 30 min at 4℃. The supernatant containing total protein was harvested. Aliquots containing 80μg of proteins were separated on 12% (RhoA) or 8% (Rockl) or 10%(β-actin) SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk, respectively incubated with mouse anti-RhoA, mouse anti-Rockl and rabbit anti-β-actin (1:200, Santa Cruz Biotechnology, USA) at 4℃overnight, and followed by each corresponding second antibody (1:4000, Chemicon, USA) at room temperature for 2 h. Immunoreactive straps were identified using Super ECL reagent (Pierce Biotechnology) according to the manufacturer's protocol. Specific bands for RhoA, Rockl andβ-actin were identified by pre-stained protein molecular weight marker (SM0441, MBI Fermentas). The EC3 Imaging System (UVP Inc.) was used to catch up the specific bands, and the optical density of each band was measured using the Image J software.
Cell migration and invasion assays:For the migration assay,5×104 cells were trypsinised, washed, resuspended in serum-free RPMI1640 or DMEM, and placed in the top portion of the chamber. The lower portion of the chamber contained 10% FBS as a chemo-attractant. The chambers were incubated at 37℃,5% CO2 for 12 h, and then cells on the membrane were washed with PBS, and fixed in 100% methanol, stained with Haematoxylin, photographed, and counted. For the invasion assay, pre-cooled serum-free RPMI1640 or DMEM was mixed with Matrigel (1:7 dilution, BD Biosciences, USA). The upper compartments were filled with 100μl of the mixture, and the Matrigel was allowed to solidify at room temperature for 4h. Other procedures followed the migration protocol, except that the chambers were incubated for 24h. Five random fields for each chamber were counted. Chambers were conducted in duplicate in three separate experiments.
Statistical analysis:The statistical package SPSS 13.0 was used for all analyses. For real-time PCR data, the statistical analysis of hsa-miR-125a-3p expression level between NSCLC tissues and corresponding LAC tissues was log2 transformed. Paired-Samples T Test was used to analyze the significant differences of hsa-miR-125a-3p expression between NSCLCs and LACs. All values were expressed as mean±SD. The Chi-square test and Two-sided fisher's exact test were used to determine the relationship between hsa-miR-125a-3p expression and clinicopathological factors. Mann-whitney test was used for pathological grade and pathological stage ranked data analysis, and Spearman correlation analysis was determined the correlation between hsa-miR-125a-3p expression and clinical stage and lymph node metastasis status. Other results were analyzed using Independent-Samples T Test. Results were considered statistically significant at the values of p<0.05.
Results
1、Hsa-miR-125a-3p and hsa-miR-125a-5p are downregulated in non-small cell lung cancer and have inverse effects on invasion and migration of lung cancer cells.
(1) Downregulation of hsa-miR-125a-3p/-5p expression in NSCLC tissues:The results, the relative expression of hsa-miR-125a-3p and hsa-miR-125a-5p in 52 NSCLCs and paired LACs by real-time PCR, showed that the expressions of hsa-miR-125a-3p and hsa-miR-125a-5p decreased in NSCLCs in comparison to matched LACs (p<0.05). The mean expression level of hsa-miR-125a-3p in NSCLCs was decreased about 4.5 folds (p<0.001) and hsa-miR-125a-5p was reduced about 6 folds (p<0.001) in NSCLCs of that in LACs.
(2) Correlation between hsa-miR-125a-3p/5p expression and clinicopathological variables of NSCLC:To determine the effects of hsa-miR-125a-3p/5p expression on tumor initiation and progression, lung cancer patients were divided into two groups based on the mean level of hsa-miR-125a-3p and hsa-miR-125a-5p expression in 52 NSCLCs. The two groups were defined as follows:hsa-miR-125a-3p low expression and high expression (1.2352 of the log2 value) and hsa-miR-125a-5p low expression and high expression (1.8594 of the log2 value). The results indicated that statistically significant associations between hsa-miR-125a-3p and hsa-miR-125a-5p expression and pathological stage were observed (p=0.012 and p=0.002, respectively). Changes in expression of hsa-miR-125a-3p and hsa-miR-125a-5p were also statistically significantly associated with lymph node metastasis in lung cancer (p=0.034 and p=0.042, respectively).No correlation was observed between hsa-miR-125a-3p/5p expression and gender, histology type, or pathological grade. However, the hsa-miR-125a-3p expression level was correlated with age (p=0.031), in contrast to that of hsa-miR-125a-5p. Spearman correlation test results showed a negative correlation between hsa-miR-125a-3p expression and pathological stage (r=-0.352, p=0.011), as well as lymph node metastasis (r=-0.326, p=0.018). However the correlations between hsa-miR-125a-5p expression and pathological stage (r=0.439, p=0.001) and lymph node metastasis (r=0.300, p=0.031) were positive.
2、Hsa-miR-125a-3p and hsa-miR-125a-5p regulate migration and invasion in lung cancer cell lines by Rho/Rockl pathway.
(1) Downregulation of hsa-miR-125a-3p/5p expression in NSCLC cell lines:In order to identify suitable cell lines for further studies, we examined the relative expression levels of hsa-miR-125a-3p/5p in four lung cancer cell lines (LH7, A549, SPC-A-1, and NCI-H460). Expression of hsa-miR-125a-3p/5p in the lung cancer cell lines was normalized to that of a control human bronchiolar epithelium (HBE) cell line. U18 was used as an internal standard for real-time PCR. We found that the expression levels of hsa-miR-125a-3p and hsa-miR-125a-5p were lower in each of the four lung cancer cell lines than in the HBE cell line (p<0.01). The mean level of hsa-miR-125a-3p expression was moderately decreased in A549 cells (p<0.001), and the mean level of hsa-miR-125a-5p expression was moderately decreased in SPC-A-1 cells (p<0.001).
(2) Effects of 2'-O-methyl oligonucleotides on the expressions of hsa-miR-125a-3p and hsa-miR-125a-5p:Real-time PCR results ensured that transfection with sense 2'-O-methyl oligonucleotides increased hsa-miR-125a-3p (p<0.001) and hsa-miR-125a-5p expressions (p<0.001), and antisense 2'-O-methyl oligonucleotides decreased hsa-miR-125a-3p (p<0.001) and hsa-miR-125a-5p expressions after 24 hours (p<0.001). Expression in transfected cells was normalized to that of untreated cells, and U18 expression was used as an internal standard.
(3) Effects of hsa-miR-125a-3p on migratory and invasive capabilities of A549 cells:For analysis of hsa-miR-125a-3p, the number of A549 cells in the untreated group that migrated through a microporous membrane was 34.40±2.47. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (34.40±2.38, p=1.000). However, the number of migrating cells was significantly decreased when cells were transfected with the sense-3p oligonucleotide (25.33±3.15, p<0.001), and the number of migrating cells was significantly increased when cells were transfected with the antisense-3p oligonucleotide(48.80±2.65, p<0.001). In the hsa-miR-125a-3p invasion analysis, the number of A549 cells in the untreated group that invaded through the Matrigel was 24.33±2.06. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (24.00±2.54, p=0.696). The number of invading cells decreased significantly when cells were transfected with the sense-3p oligonucleotide (11.13±1.60, p<0.001), and the number of invading cells was significantly increased when cells were transfected with the antisense-3p oligonucleotide (34.80±2.88, p<0.001).
(4) Effects of hsa-miR-125a-5p on migratory and invasive capabilities of SPC-A-1:For analysis of hsa-miR-125a-5p, the number of A549 cells in the untreated group that migrated through a microporous membrane was 24.20±1.78. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (28.80±2.70, p=0.445). However, the number of migrating cells was significantly increased when cells were transfected with the sense-3p oligonucleotide (45.27±2.71, p<0.001), and the number of migrating cells was significantly decreased when cells were transfected with the antisense-3p oligonucleotide (16.93±3.24, p<0.001). In the hsa-miR-125a-5p invasion analysis, the number of A549 cells in the untreated group that invaded through the Matrigel was 24.20±1.78. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (23.80±2.00, p=0.568). The number of invading cells increased significantly when cells were transfected with the sense-3p oligonucleotide (35.80±1.74, p<0.001), and the number of invading cells was significantly decreased when cells were transfected with the antisense-3p oligonucleotide (17.47±2.07, p<0.001).
(5) Predicted and confirmed hsa-miR-125a-3p target:We proceeded to identify potential targets known to play a role in cell mobility and invasion by using microrna.org and TargetScanHuman 5.1 webservers. Among the candidates surveyed, we found that the 3'UTR of the RhoA gene contains two highly conserved regions that may serve as a binding site for hsa-miR-125a-3p as determined by the miRanda and TargetScan algorithm.
(6) Hsa-miR-125a-3p inhibited migration and invasion by targeting RhoA: Western blotting and RT-PCR analyses revealed that RhoA protein (p=0.001) but not RhoA mRNA (p=0.482) was considerably decreased in sense-3p transfected cells. As expected, Rockl protein (p=0.001) and mRNA (p=0.003) were also reduced clearly. Transfection with 20μM of antisense-3p resulted in an increase in RhoA protein measured by Western blotting (p<0.001), however, RhoA mRNA remained unchanged (p=0.374). As expected, Rockl mRNA and protein were also increased obviously (p<0.001). These results indicated that hsa-miR-125a-3p could post-transcriptionally regulate RhoA mRNA and repressed its translation and contribute to mediate Rockl.
To further demonstrate that RhoA is a potential target of hsa-miR-125a-3p, we generated luciferase reporters that contained the full length of the RhoA gene 3'UTR. Results from triplicate experiments showed that reporter activity was reduced by the ectopic expression of hsa-miR-125a-3p (p<0.001). Taken together, these results indicated that hsa-miR-125a-3p inhibited cell migration and invasion in SPC-A-1 cells by targeting RhoA.
To further validate whether inhibitor of hsa-miR-125a-3p regulate migration and invasion via the Rho pathway, we blocked Rho with Rho inhibitor CT04 at a final concentration of 2.0μg/ml according to the previous studies of our laboratory in A549 cell line. The number of migratory cells and invasive cells were decreased clearly after treatment with CT04 when compared with the untreated group (p<0.001). The number of migratory cells and invasive cells were not significantly decreased in cells transfected with sense-3p (p>0.05), and also not increased obviously in cells transefected with antisense-3p (p>0.05), when compared with CT04 group. These results suggest that hsa-miR-125a-3p regulate migration and invasion in Rho-dependent manner.
(7) Hsa-miR-125a-5p enhanced migration and invasion by targeting RhoA: Western blotting and RT-PCR analyses revealed that RhoA protein (p=0.001) and mRNA (p=0.263) remained unchanged (p=0.146). Interestingly, RhoC protein and mRNA were considerably increased in sense-3p transfected cells (p<0.001). As expected, Rockl protein (p<0.001) and mRNA (p<0.001) were also increased clearly. Transfection with 20μM of antisense-3p resulted in a decrease in RhoC protein measured by Western blotting (p<0.001), however, RhoA mRNA (p=0.087) and protein (p=0.147) remained unchanged. As expected, Rockl mRNA and protein were also decreased obviously (p<0.001).
To further validate whether inhibitor of hsa-miR-125a-5p induce migration and invasion via the Rho pathway, we blocked Rho with Rho inhibitor CT04 in SPC-A-1 cell line. The number of migratory cells and invasive cells were decreased clearly after treatment with CT04 when compared with the untreated group (p<0.001). The number of migratory cells and invasive cells were not significantly decreased in cells transfected with antisense-5p (p>0.05), and also not increased obviously in cells transefected with sense-5p (p>0.05), when compared with CT04 group. These results suggest that hsa-miR-125a-5p regulate migration and invasion in Rho-dependent manner.
Conclusions
1、Hsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC.
2、Hsa-miR-125a-3p inhibited migration and invasion by RhoA/Rockl pathway.
3、Hsa-miR-125a-5p enhanced migration and invasion by RhoC/Rockl pathway.
肿瘤的侵袭和转移是世界上癌症患者死亡最常见的原因。最近,越来越多的研究报道表明,miRNA与肿瘤细胞的侵袭和转移有关,其调控肿瘤细胞侵袭和转移的作用机制成为miRNA研究领域的热点之一。在miRNA与Rho/Rock-1传导通路的关系中,Kong等人在鼠正常乳腺上皮细胞中发现,miR-155在TGF-β诱导的上皮间质转化以及RhoA引起的细胞迁移侵袭过程中具有重要作用。TWISTl能够诱导miR-1Ob, miR-1Ob通过直接转录后抑制HOXD 10的表达来间接的增加RhoC的水平,从而影响了肿瘤细胞的迁移和侵袭。研究Rho和miRNAs之间的关系加强了miRNA在肿瘤形成中的重要性,并且为癌症转移的患者提供了新的治疗策略。
miR-125a是一种功能以及机制尚未研究清楚的miRNAs之一。Yanainhara等人发现miR-125a定位在19q13.41,并用miRNA芯片分析了104对肺癌组织以及相应的癌旁肺组织,发现与癌旁肺组织相比较,miR-125a在肺癌组织中下调表达。至目前为止,已发现的成熟体miR-125a家族有两个成员,即hsa-miR-125a-3p和hsa-miR-125a-5p。本研究目的旨在探讨非小细胞肺癌(non-small cell lung cancer, NSCLC)组织中hsa-miR-125a-3p和hsa-miR-125a-5p的表达及其临床意义,分析hsa-miR-125a-3p和hsa-miR-125a-5p与NSCLC细胞系转移潜能的关系。并在细胞水平上,通过向肺癌细胞中转染正义和反义的2’-O-甲基寡核苷酸的方法,探讨增加hsa-miR-125a-3p和hsa-miR-125a-5p的表达以及下调hsa-miR-125a-3p和hsa-miR-125a-5p的表达对Rho/Rockl途径,以及肺癌细胞迁移和侵袭能力的影响,为hsa-miR-125a-3p和hsa-miR-125a-5p可能是Rho/Rockl信号通路非常重要的调节因子提供依据。
材料与方法
1、NSCLC组织标本
52例配对的非小细胞肺癌和癌旁肺组织(远离原发灶)标本均来自中国医科大学病理教研室。患者术前均未接受过任何化学或放射治疗。福尔马林固定石蜡包埋的肿瘤组织切片常规进行HE染色,分别由两名病理诊断医师,根绝WHO肺及肺膜肿瘤分类标准(2004)和TNM分期系统(1997),确定肿瘤的组织学分型以及临床病理分期。52例NSCLC包括：男性33例,女性29例；年龄40～78岁(平均58岁±9岁)；鳞癌22例,腺癌30例；病理分级,高分化8例,中分化28例,低分化16例；病理分期,Ⅰ期18例,Ⅱ期15例,Ⅲ期19例；淋巴结转移20例,无淋巴结转移32例。52例配对组织在液氮中快速冰冻,以后用于提取mRNA。常规病理检查手术切除的肺门、纵膈以及肺内淋巴结,用于确定肺癌的淋巴结转移情况。患者的临床病理参数包括性别,年龄,组织学类型,病理分级,病理分期,淋巴结转移,摘自患者的病历资料。
2、细胞培养
冻存于液氮的A549细胞经复苏后,用DMEM培养液(含有10%新生牛血清,100μ／ml的青霉素(?)100μg/ml的链霉素),在37℃,5%CO2饱和湿度条件下培养,经3次传代稳定后,进行转染。
3、细胞转染
2’-O-甲基寡核苷酸(2'-O-Me-sense-3p:5'-ACA GGU GAG GUU CUU GGG AGCC-3'2'-O-Me-antisense-3p:5'-GGC UCC CAA GAA CC U CAC CUGU-3', 2'-O-Me-scramble-3p:5'-GGU CGG UGC UCG AUG CAG GUAA-3', 2'-O-Me-sense-5p:5'-UCC CUG AGA CCC UUU AAC CUG UGA-3', 2'-O-Me-antisense-5p:5'-UCA CAG GUU AAA GGG UCU CAG GGA-3', 2'-O-Me-scramble-5p:5'-GGA CG G CGA UCA GAU AAG AGU UCU-3'.)用结合DNA技术合成(GeneChem, China)。用LipofetamineTM 2000 (Invitrogen, USA)将20gM 2’-O-甲基寡核苷酸转染入6孔板中A549和SPC细胞中为RNA提取和transwell备用。untreated为未加任何处理因素的细胞,scramble为转染乱码序列2’-O-甲基寡核苷酸的细胞,sense为转染正义序列2’-O-甲基寡核苷酸的细胞,antisense为转染反义序列2’-O-甲基寡核苷酸的细胞。其中对照组细胞为(untreated和scramble)。
4、Real-time PCR
根据操作说明应用TaqMan MicroRNA试剂检测成熟体miRNA (Applied Biosystems, USA)。所有的RT产物,在ABI Prism 7900HT序列检测系统中运行。用特异的RT引物和TaqMan探针定量检测hsa-miR-125a-3p (PN:4395310)和hsa-miR-125a-5p (PN:4395309), RNU6B (PN:4373381)和U18(PN:4380904)为内参。组织样本用2次独立样本经过2次独立实验,细胞样本用3次独立样本经过3次独立实验后得到的数据用公式RQ=2-△△Ct的方法进行分析。
5、RT-PCR
TRIzol试剂提取总RNA,紫外分光光度计测定RNA纯度并定量,在1%甲醛变性凝胶电泳验证RNA完整性。RT-PCR采用两步法试剂盒(宝生物公司)按照厂家推荐步骤进行,扩增体系为20μl,以持家基因β-actin为内参照,对样品模板用量标准化。引物参照文献及使用Primer Designer5.0软件设计。RT条件按照厂家推荐,PCR条件：95℃lmin,随后进行35个循环的94℃30s,55℃30s,72℃45s,最后72℃充分延伸1Omin; p-actin PCR条件：94℃5min,随后94℃40s,50℃40s,72℃40s共35个循环,最后72℃充分延伸5min。扩增产物进行1.5%琼脂糖凝胶电泳,UVP图像处理系统采集图像,ImageJ灰度分析软件进行条带灰度分析。产物经1.75%琼脂糖凝胶电泳,凝胶成像系统摄像,分析各条带强度。
6、Western blot检测
提取各组织蛋白,根据蛋白的分子量制备不同浓度的SDS—聚丙烯酰胺凝胶,每孔上样量50μg,常规电泳、转膜、封闭,一抗及相应二抗37℃孵育1h,DAB显色,β-actin作为内对照。UVP图像处理系统采集图像,ImageJ灰度分析软件进行条带灰度分析。以平均吸光度值INT和信号面积S的乘积代表信号强度。分别计算不同指标和P-actin的INT×S,用二者的比值显示不同指标的表达水平。
7、Transwell细胞迁移和侵袭实验
在具有8μm小孔聚碳酸酯滤膜的培养小室上铺用预冷无血清培养基稀释的Matrigel基质胶,接种100μl无血清培养基稀释的A549细胞或NCI-H460细胞(5×105个／mL),下室加入600μl含10%血清的DMEM培养液或1640培养液,每组设6复孔。37℃、5%CO2条件下培养24h后,取出培养小室,用2.5%戊二醛固定15min,并以0.5%Triton X-100处理3min,苏木精中染核15min。将培养小室倒置,在光学显微镜(Leica,德国)下观察、照相,并计数每高倍视野滤膜底面的平均细胞数。细胞迁移实验在无Matrigel基质胶包被的Transwell小室中进行,方法同细胞侵袭实验。
8、统计学分析
SPSS13.0用于所有的统计学分析。Real-time PCR结果数据即hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌及相应的癌旁肺组织中的表达水平经过log2转换。配对T检验用于分析两种成熟体miRNA-125a在非小细胞肺癌和癌旁肺组织中的显著差异。Chi-square检验(Chi-square test)和双侧fisher精确检验(Two-sided fisher's exact test)用于分析hsa-miR-125a-3p和hsa-miR-125a-5p的表达与临床病理因素的关系。Mann-whitney检验(Mann-whitney test)用于病理分级,病理分期等级资料的分析。Spearman相关分析(Spearmancorrelation analysis)用于分析hsa-miR-125a-3p和hsa-miR-125a-5p与病理分期和淋巴结转移的相关性。其它实验采用t检验。用mean±SD, p<0.05为有统计学意义。
实验结果
1、Hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌组织中表达下调与非小细胞肺癌淋巴结转移和病理分期有关。
(1) hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌组织中的表达：real-time PCR检测52例非小细胞肺癌及相应的癌旁肺组织中hsa-miR-125a-3p和hsa-miR-125a-5p相对表达水平的结果显示,与癌旁肺组织相比较,52例非小细胞肺癌组织中,hsa-miR-125a-3p和hsa-miR-125a-5p表达下降(p＜0.05)。hsa-miR-125a-3p表达的平均水平降低约4.5倍(p<0.001), hsa-miR-125a-5p表达的平均水平下降近6倍(p＜0.001)。
(2) hsa-miR-125a-3p和hsa-miR-125a-5p与临床病理因素的关系：根据hsa-miR-125a-3p和hsa-miR-125a-5p在52例非小细胞肺癌中表达的平均水平分别分成2组,即hsa-miR-125a-3p低表达(低于平均值1.2352,log2转换数据)和高表达组(高于平均值1.2352,log2转换数据),hsa-miR-125a-5p低表达(低于平均值1.8594,log2转换数据)和高表达组(高于平均值1.8594,log2转换数据)。统计学分析结果显示,hsa-miR-125a-3p表达与年龄(p=0.031)、病理分期(p=0.012)和淋巴结转移(p=0.034)有关,与性别、组织分型以及病理分级无关；hsa-miR-125a-5p与病理分期(p=0.002)和淋巴结转移(p=0.042)有关,与年龄、性别、组织分型以及病理分级无关。sepearman相关检验结果显示,hsa-miR-125a-3p表达与病理分期(r=-0.352,p=0.011)和淋巴结转移(r=-0.326,p=0.018)呈负相关,hsa-miR-125a-5p表达与病理分期(r=0.439,p=0.001)和淋巴结转移(r=0.300,p=0.031)呈正相关。
2、hsa-miR-125a-3p和hsa-miR-125a-5p通过Rho/Rockl信号通路调控肺癌细胞的迁移和侵袭。
(1)hsa-miR-125a-3p和hsa-miR-125a-5p在非小细胞肺癌细胞系中的表达：real-time PCR检测了4种肺癌细胞系LH7,A549,SPC-A-1和NCI-H460中hsa-miR-125a-3p和hsa-miR-125a-5p的表达情况,人正常支气管上皮细胞(Human bronchiolar epithelium cell line, HBE)作为正常对照细胞系,U18作为内对照。结果显示,hsa-miR-125a-3p和hsa-miR-125a-5p在4种肺癌细胞系中的表达均低于HBE (p<0.01)。hsa-miR-125a-3p相对表达水平在NCI-H460细胞中表达最低,在A549细胞中呈中度表达(p<0.001), hsa-miR-125a-5p相对表达水平在A549细胞中表达最低,在SPC-A-1细胞中呈中度表达(p＜0.001)。
(2)2’-O-甲基寡核苷酸对细胞内hsa-miR-125a-3p和hsa-miR-125a-5p的影响：Real-time PCR的结果显示,转染正义2’-O-甲基寡核苷酸后,细胞内hsa-miR-125a-3p和hsa-miR-125a-5p的含量明显增加(p＜0.001)；转染反义2’-O-甲基寡核苷酸后,细胞内hsa-miR-125a-3p和hsa-miR-125a-5p明显降低了(p＜0.001)。
(3) hsa-miR-125a-3p对肺癌细胞迁移和侵袭的影响：hsa-miR-125a-3p的迁移实验显示,A549细胞的穿膜细胞数在未处理组细胞中是34.40±2.47个,与未处理组细胞相比较,转染乱序组细胞的穿膜细胞数没有明显改变(34.40±2.38,p=1.000),转染sense-3p组细胞的穿膜细胞数明显降低(25.33±3.15,p＜0.001),转染antisen-3p组细胞的穿膜细胞数明显增加(48.80±2.65, p< 0.001)。hsa-miR-125a-3p的侵袭实验显示,A549细胞侵过基质胶的穿膜细胞数在未处理组细胞中是24.33±2.06。与未处理组细胞相比较,转染乱序组细胞侵过基质胶的穿膜细胞数没有明显改变(24.00±2.54,p=0.696),转染sense-3p组细胞侵过基质胶的穿膜细胞数明显降低(11.13±1.60,p＜0.001),转染antisen-3p组细胞侵过基质胶的穿膜细胞数明显增加(34.80±2.88,p＜0.001)。
(4) hsa-miR-125a-5p对肺癌细胞迁移和侵袭的影响：hsa-miR-125a-5p的迁移实验显示,SPC-A-1细胞的穿膜细胞数在未处理组细胞中是24.20±1.78个,与未处理组细胞相比较,转染乱序组细胞的穿膜细胞数没有明显改变(28.80±2.70,p=0.445),转染sense-5p组细胞的穿膜细胞数明显增加(45.27±2.71,p＜0.001),转染antisen-5p组细胞的穿膜细胞数明显降低(16.93±3.24, p<0.001)。hsa-miR-125a-5p的侵袭实验显示,SPC-A-1细胞侵过基质胶的穿膜细胞数在未处理组细胞中是24.20±1.78。与未处理组细胞相比较,转染乱序组细胞侵过基质胶的穿膜细胞数没有明显改变(23.80±2.00,p=0.568),转染sense-5p组细胞侵过基质胶的穿膜细胞数明显增加(35.80±1.74,p＜0.001),转染antisen-5p组细胞侵过基质胶的穿膜细胞数明显降低(17.47±2.07,p＜0.001)。
(5) hsa-miR-125a-3p靶基因的预测和确认：用microrna.org和Target ScanHuman 5.1预测靶基因的网络数据库,对在细胞迁移和侵袭中起作用的潜在靶基因进行了预测。在候选基因调查中,我们发现RhoA基因的3’UTR包含2个高保守区域,分别由miRanda和TargetScan算法计算得到的,这两个高保守区域可能为hsa-miR-125a-3p与靶基因的作用连接位点。
(6) Hsa-miR-125a-3p通过靶基因RhoA调控Rho/Rockl信号途径：Western blotting和RT-PCR分析显示RhoA蛋白在转染sense-3p细胞中明显减少(p=0.001),在转染antisense-3p细胞中明显增多(p＜0.001),而RhoA mRNA(p=0.482)变化并不是很明显。如预期的那样,Rockl蛋白和mRNA在转染sense-3p细胞中明显减少(p＜0.05),在转染antisense-3p细胞中明显增多(p＜0.05)。
为了进一步说明RhoA是hsa-miR-125a-3p的潜在靶基因,我们转染了包含RhoA基因3’UTR全长的荧光素酶报告基因。三次结果显示,增加细胞内hsa-miR-125a-3p含量能够降低荧光素酶活性(p＜0.001)。这些结果表明hsa-miR-125a-3p可直接通过抑制靶基因RhoA而抑制细胞迁移和侵袭。
在A549细胞中用Rho抑制剂CT04(最终浓度2.0μg／ml)阻断Rho,与未处理组相比较,迁移和侵袭的细胞数量在用CT04处理后明显减少(p＜0.001)。与阻断Rho未转染组相比较,转染sense-3p组或转染antisense-3p组细胞数目并没有明显的减少或增加(p＞0.05)。结果表明hsa-miR-125a-3p可能通过调控Rho/Rockl途径而影响细胞的迁移和侵袭。
(7) Hsa-miR-125a-5p通过RhoC调控Rho/Rockl信号途径：Western blotting和RT-PCR分析显示RhoA蛋白(p=0.001)在sense-5p和antisense-5p转染细胞中都没有明显变化,RhoA mRNA (p=0.482)变化亦不是很明显。然而,RhoC mRNA和蛋白在转染sense-5p细胞中明显增加(p＜0.05),在转染antisense-5p细胞中明显减少(p＜0.05)。如预期的那样,Rockl蛋白和mRNA在转染sense-5p中明显增加(p＜0.01),在转染antisense-5p细胞中明显减少(p＜0.01)。
在SPC-A-1细胞中用Rho抑制剂CT04(最终浓度2.0μg/ml)阻断Rho,与未处理组相比较,迁移和侵袭的细胞数量在用CT04处理后明显减少(p＜0.001)。与阻断Rho未转染组相比较,转染sense-5p组或转染antisense-5p组细胞数目也并没有明显的增加或减少(p＞0.05)。结果表明hsa-miR-125a-5p可能通过调控Rho/Rockl途径而影响细胞的迁移和侵袭。
结论
1、hsa-miR-125a-3p与病理分期和淋巴结转移呈负相关,而hsa-miR-125a-5p与病理分期和淋巴结转移呈正相关。
2、hsa-miR-125a-3p能够通过Rho A/Rockl信号途径抑制肺癌细胞的迁移和侵袭。
3、hsa-miR-125a-5p能够通过RhoC/Rockl信号途径促进肺癌细胞的迁移和侵袭。
MicroRNAs (miRNAs) are short (21-24-nt) non-coding RNAs generated from longed transcripts, termed pri-miRNAs and pre-miRNAs. They act as post-transcriptional regulators of gene expression and induce translational repression. Recently, miRNAs have been discovered to play important roles in tumourigenesis. By targeting 3'untranslated regions (3'UTRs) of cognate mRNAs, miRNAs are involved in diverse processes, including cell differentiation, proliferation, apoptosis and metastasis. Understanding the specific functions of individual miRNAs may contribute to our knowledge of the molecular properties of cancers and may provide new biomarkers for cancer diagnosis and therapy.
The Rho family of small GTPases, of which the best studied members are Rho, Racl and CDC42, are involved in regulation of a variety of cellular processes includ-ing reorganization of the actin cytoskeleton, cell motility and invasive phenotypes. Rho-associated serine-threonine protein kinase (Rock), one of the best characterized downstream effectors of Rho, is activated when it selectively binds to the active GTP-bound form of Rho. As with Rho, Rock has been implicated in altering cell migration and invasion during tumor cell metastasis.
Tumor invasion and metastasis are the critical steps in determining the death of cancer patients. Recently, miRNA have been discovered to have a role in invasion and metastasis. Emerging studies have indicated that miRNAs participate in the signaling of the Rho pathway. Among the tested miRNAs, miR-155 may play an important role in TGF-β-induced epithelial-mesenchymal transition (EMT) and cell migration and invasion by targeting RhoA. During tumor progression, TWIST 1 induces miR-10b, which in turn indirectly increases RhoC levels by direct down-regulation of its transcriptional repressor HOXD10. Studies on the association of Rho with miRNAs could highlight the importance of miRNAs in tumorigenesis and lead to the development of novel therapeutic strategies for patients with metastases.
MiR-125a is one of the many miRNAs that remain to be fully characterized. Using miRNA microarray analysis, Yanainhara and colleagues found that miR-125a, specifically the hsa-miR-125a-5p mature miRNA, is located at 19q13.41 and that its expression is downregulated in NSCLC. Recently, a new member of the mature miR-125a family has been identified and named hsa-miR-125a-3p. Unfortunately, the expression and function of hsa-miR-125a-3p are currently unknown. In the present study, we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues, and explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells. In add-ition, the effects of hsa-miR-125a-3p/5p on the invasive and migratory capabilities of lung cancer cells were studied by investigating its effects on Rho/Rock pathway. These results provide basis that hsa-miR-125a-3p and hsa-miR-125a-5p maybe very important regulators in Rho/Rock pathway.
Materials and Methods
Samples preparation:We analyzed 52 pairs of non-small cell lung cancer specimens and corresponding normal lung tissues (LAC) collected at the time of surgery and prior to chemotherapy. Specimens were obtained from patients at the First Affiliated Hospital of China Medical University from 1 January 2006 to 1 December 2007 with informed consent. For the majority of samples, clinical and biological information was available. The study has been approved by the Hospitals'Ethical Review Committee.
Cell culture: The HBE (Human Bronchiolar Epithelium) cell line and the human lung cancer cell lines A549 and SPC-A-1 (adenocarcinoma), LH7 and NCI-H460 (large cell cancinoma) were propagated in RPMI1640 (Gibco). The A549 (adenoca-rcinoma) cell line was propagated in Dulbecco's Modifed Eagle Medium (Gibco). In both cases, the medium was supplemented with 10% fetal bovine serum (FBS),100 U/ml penicillin, and 100 U/ml streptomycin. Cells were cultured at 37℃in 5% CO2 until they reached a confluency of 75%.
Transfection:Depletion of hsa-miR-125a-3p/5p in the A549 and SPC-A-1 cell lines was achieved through transfection with antisense 2'-O-methyl oligonucleotides directed against hsa-miR-125a-3p/5p. Cells were transfected using LipofetamineTM 2000 (Invitrogen) according to the manufacturer's protocol. Briefly, complexes con-taining the oligonucleotides were prepared according to the recommended protocol and added directly to cells at a final oligonucleo tide concentration of 0.4 nmol/ml. Oligonucleotides composed entirely of 2'-O-methyl bases were chemically synthe-sized by Integrated DNA Technologies (GeneChem) and were comprised of the following sequences:2'-O-Me-sense-3p:5'-ACA GGU GAG GUU CUU GGG AGCC-3'2'-O-Me-antisense-3p:5'-GGC UCC CAA GAA CC U CAC CUGU-3', 2'-O-Me-scramble-3p:5'-GGU CGG UGC UCG AUG CAG GUAA-3',2'-O-Me-sense-5p:5'-UCC CUG AGA CCC UUU AAC CUG UGA-3',2'-O-Me-antisense-5p:5'-UCA CAG GUU AAA GGG UCU CAG GGA-3',2'-O-Me-scramble-5p: 5'-GGA CG G CGA UCA GAU AAG AGU UCU-3'. Cells were divided into four groups:an untreated group incubated only in the normal media (untreated), a group transfected with the scrambled 2'-O-methyl oligonucleotide (scramble); a group transfected with the sense 2'-O-methyl oligonucleotide (sense) and a group transfected with the antisense 2'-O-methyl oligonucleotide (antisense). The untreated and scramble groups served as negative controls.
Quantitative real-time polymerase chain reaction (qRT-PCR):Expression of mature miRNAs was assayed using the TaqMan MicroRNA Assay in accordance with the manufacturer's instructions (Applied Biosystems). All reactions, including no-template controls and RT-minus controls, were run in an ABI Prism 7900HT Sequence detection system (Applied Biosystems). Specific RT primers and TaqMan probes were used to quantify the expression of hsa-miR-125a-3p (PN:4395310) and hsa-miR-125a-5p (PN:4395309). Samples were normalized to RNU6B (PN:4373381) or U18 (PN:4380904) as indicated. For quantification of tissue samples, RT-PCR analysis was performed in two independent experiments, each using two independent samples. For quantification of cell samples, RT-PCR analysis was performed in three independent experiments, each using three independent samples. MiRNA expression data is presented as fold difference relative to either RNU6B or U18 based on the following equation:RQ=2-△△Ct.
Semi-quantitative RT-PCR: Total RNA was prepared using Trizol (Invitrogen, USA). cDNA was generated by reverse transcription of 500ng total RNA using PrimeScript RT reagent Kit (TaKaRa, Japan). Primer sequences of RhoA were 5'-TAT GTG CCC ACA GTG TTT GA-3'(forward) and 5'-CAT TCC GAA GAT CCT CCT TCT TATT-3'(reverse); Rockl primer sequences were 5'-GCA ACT ATG ATG TGC CTG AA-3'(forward) and 5'-CAC CAT TTC GCC CTA ACC-3'(reverse), and that ofβ-actin were 5'-AAA TCG TGC GTG ACA TTA A-3'(forward) and 5'-CTC GTC ATA CTC CTG CTTG-3'(reverse). The lengths of RhoA, Rockl andβ-actin PCR products were 270 bp,489 bp and 513 bp respectively. PCR was carried out by an initial denaturation step at 94℃for 5min, followed by 35 cycles of denaturation at 94℃for 40s, annealing at 56℃(RhoA) or 57.2℃(Rock1) for 40s, and elongation at 72℃for 40s. Cycling was completed by a final elongation step at 72℃for 5min. The PCR forβ-actin was performed in the same manner as above, except that 30 cycles of annealing at 50℃was used. The EC3 Imaging system (UVP Inc.) was used to catch up the specific bands, and the optical density of each band was measured using the Image J software.
Western blot: Cells were washed twice with ice-cold phosphate buffered saline (PBS) and lysed in M-PER reagent (Pierce Biotechnology) containing 1 mM PMSF and phosphatase inhibitors for 1h at 4℃. The supernatants were centrifuged at 12 OOO×g for 30 min at 4℃. The supernatant containing total protein was harvested. Aliquots containing 80μg of proteins were separated on 12% (RhoA) or 8% (Rockl) or 10%(β-actin) SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk, respectively incubated with mouse anti-RhoA, mouse anti-Rockl and rabbit anti-β-actin (1:200, Santa Cruz Biotechnology, USA) at 4℃overnight, and followed by each corresponding second antibody (1:4000, Chemicon, USA) at room temperature for 2 h. Immunoreactive straps were identified using Super ECL reagent (Pierce Biotechnology) according to the manufacturer's protocol. Specific bands for RhoA, Rockl andβ-actin were identified by pre-stained protein molecular weight marker (SM0441, MBI Fermentas). The EC3 Imaging System (UVP Inc.) was used to catch up the specific bands, and the optical density of each band was measured using the Image J software.
Cell migration and invasion assays:For the migration assay,5×104 cells were trypsinised, washed, resuspended in serum-free RPMI1640 or DMEM, and placed in the top portion of the chamber. The lower portion of the chamber contained 10% FBS as a chemo-attractant. The chambers were incubated at 37℃,5% CO2 for 12 h, and then cells on the membrane were washed with PBS, and fixed in 100% methanol, stained with Haematoxylin, photographed, and counted. For the invasion assay, pre-cooled serum-free RPMI1640 or DMEM was mixed with Matrigel (1:7 dilution, BD Biosciences, USA). The upper compartments were filled with 100μl of the mixture, and the Matrigel was allowed to solidify at room temperature for 4h. Other procedures followed the migration protocol, except that the chambers were incubated for 24h. Five random fields for each chamber were counted. Chambers were conducted in duplicate in three separate experiments.
Statistical analysis:The statistical package SPSS 13.0 was used for all analyses. For real-time PCR data, the statistical analysis of hsa-miR-125a-3p expression level between NSCLC tissues and corresponding LAC tissues was log2 transformed. Paired-Samples T Test was used to analyze the significant differences of hsa-miR-125a-3p expression between NSCLCs and LACs. All values were expressed as mean±SD. The Chi-square test and Two-sided fisher's exact test were used to determine the relationship between hsa-miR-125a-3p expression and clinicopathological factors. Mann-whitney test was used for pathological grade and pathological stage ranked data analysis, and Spearman correlation analysis was determined the correlation between hsa-miR-125a-3p expression and clinical stage and lymph node metastasis status. Other results were analyzed using Independent-Samples T Test. Results were considered statistically significant at the values of p<0.05.
Results
1、Hsa-miR-125a-3p and hsa-miR-125a-5p are downregulated in non-small cell lung cancer and have inverse effects on invasion and migration of lung cancer cells.
(1) Downregulation of hsa-miR-125a-3p/-5p expression in NSCLC tissues:The results, the relative expression of hsa-miR-125a-3p and hsa-miR-125a-5p in 52 NSCLCs and paired LACs by real-time PCR, showed that the expressions of hsa-miR-125a-3p and hsa-miR-125a-5p decreased in NSCLCs in comparison to matched LACs (p<0.05). The mean expression level of hsa-miR-125a-3p in NSCLCs was decreased about 4.5 folds (p<0.001) and hsa-miR-125a-5p was reduced about 6 folds (p<0.001) in NSCLCs of that in LACs.
(2) Correlation between hsa-miR-125a-3p/5p expression and clinicopathological variables of NSCLC:To determine the effects of hsa-miR-125a-3p/5p expression on tumor initiation and progression, lung cancer patients were divided into two groups based on the mean level of hsa-miR-125a-3p and hsa-miR-125a-5p expression in 52 NSCLCs. The two groups were defined as follows:hsa-miR-125a-3p low expression and high expression (1.2352 of the log2 value) and hsa-miR-125a-5p low expression and high expression (1.8594 of the log2 value). The results indicated that statistically significant associations between hsa-miR-125a-3p and hsa-miR-125a-5p expression and pathological stage were observed (p=0.012 and p=0.002, respectively). Changes in expression of hsa-miR-125a-3p and hsa-miR-125a-5p were also statistically significantly associated with lymph node metastasis in lung cancer (p=0.034 and p=0.042, respectively).No correlation was observed between hsa-miR-125a-3p/5p expression and gender, histology type, or pathological grade. However, the hsa-miR-125a-3p expression level was correlated with age (p=0.031), in contrast to that of hsa-miR-125a-5p. Spearman correlation test results showed a negative correlation between hsa-miR-125a-3p expression and pathological stage (r=-0.352, p=0.011), as well as lymph node metastasis (r=-0.326, p=0.018). However the correlations between hsa-miR-125a-5p expression and pathological stage (r=0.439, p=0.001) and lymph node metastasis (r=0.300, p=0.031) were positive.
2、Hsa-miR-125a-3p and hsa-miR-125a-5p regulate migration and invasion in lung cancer cell lines by Rho/Rockl pathway.
(1) Downregulation of hsa-miR-125a-3p/5p expression in NSCLC cell lines:In order to identify suitable cell lines for further studies, we examined the relative expression levels of hsa-miR-125a-3p/5p in four lung cancer cell lines (LH7, A549, SPC-A-1, and NCI-H460). Expression of hsa-miR-125a-3p/5p in the lung cancer cell lines was normalized to that of a control human bronchiolar epithelium (HBE) cell line. U18 was used as an internal standard for real-time PCR. We found that the expression levels of hsa-miR-125a-3p and hsa-miR-125a-5p were lower in each of the four lung cancer cell lines than in the HBE cell line (p<0.01). The mean level of hsa-miR-125a-3p expression was moderately decreased in A549 cells (p<0.001), and the mean level of hsa-miR-125a-5p expression was moderately decreased in SPC-A-1 cells (p<0.001).
(2) Effects of 2'-O-methyl oligonucleotides on the expressions of hsa-miR-125a-3p and hsa-miR-125a-5p:Real-time PCR results ensured that transfection with sense 2'-O-methyl oligonucleotides increased hsa-miR-125a-3p (p<0.001) and hsa-miR-125a-5p expressions (p<0.001), and antisense 2'-O-methyl oligonucleotides decreased hsa-miR-125a-3p (p<0.001) and hsa-miR-125a-5p expressions after 24 hours (p<0.001). Expression in transfected cells was normalized to that of untreated cells, and U18 expression was used as an internal standard.
(3) Effects of hsa-miR-125a-3p on migratory and invasive capabilities of A549 cells:For analysis of hsa-miR-125a-3p, the number of A549 cells in the untreated group that migrated through a microporous membrane was 34.40±2.47. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (34.40±2.38, p=1.000). However, the number of migrating cells was significantly decreased when cells were transfected with the sense-3p oligonucleotide (25.33±3.15, p<0.001), and the number of migrating cells was significantly increased when cells were transfected with the antisense-3p oligonucleotide(48.80±2.65, p<0.001). In the hsa-miR-125a-3p invasion analysis, the number of A549 cells in the untreated group that invaded through the Matrigel was 24.33±2.06. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (24.00±2.54, p=0.696). The number of invading cells decreased significantly when cells were transfected with the sense-3p oligonucleotide (11.13±1.60, p<0.001), and the number of invading cells was significantly increased when cells were transfected with the antisense-3p oligonucleotide (34.80±2.88, p<0.001).
(4) Effects of hsa-miR-125a-5p on migratory and invasive capabilities of SPC-A-1:For analysis of hsa-miR-125a-5p, the number of A549 cells in the untreated group that migrated through a microporous membrane was 24.20±1.78. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (28.80±2.70, p=0.445). However, the number of migrating cells was significantly increased when cells were transfected with the sense-3p oligonucleotide (45.27±2.71, p<0.001), and the number of migrating cells was significantly decreased when cells were transfected with the antisense-3p oligonucleotide (16.93±3.24, p<0.001). In the hsa-miR-125a-5p invasion analysis, the number of A549 cells in the untreated group that invaded through the Matrigel was 24.20±1.78. There was no difference between untreated cells and cells transfected with the scramble-3p oligonucleotide (23.80±2.00, p=0.568). The number of invading cells increased significantly when cells were transfected with the sense-3p oligonucleotide (35.80±1.74, p<0.001), and the number of invading cells was significantly decreased when cells were transfected with the antisense-3p oligonucleotide (17.47±2.07, p<0.001).
(5) Predicted and confirmed hsa-miR-125a-3p target:We proceeded to identify potential targets known to play a role in cell mobility and invasion by using microrna.org and TargetScanHuman 5.1 webservers. Among the candidates surveyed, we found that the 3'UTR of the RhoA gene contains two highly conserved regions that may serve as a binding site for hsa-miR-125a-3p as determined by the miRanda and TargetScan algorithm.
(6) Hsa-miR-125a-3p inhibited migration and invasion by targeting RhoA: Western blotting and RT-PCR analyses revealed that RhoA protein (p=0.001) but not RhoA mRNA (p=0.482) was considerably decreased in sense-3p transfected cells. As expected, Rockl protein (p=0.001) and mRNA (p=0.003) were also reduced clearly. Transfection with 20μM of antisense-3p resulted in an increase in RhoA protein measured by Western blotting (p<0.001), however, RhoA mRNA remained unchanged (p=0.374). As expected, Rockl mRNA and protein were also increased obviously (p<0.001). These results indicated that hsa-miR-125a-3p could post-transcriptionally regulate RhoA mRNA and repressed its translation and contribute to mediate Rockl.
To further demonstrate that RhoA is a potential target of hsa-miR-125a-3p, we generated luciferase reporters that contained the full length of the RhoA gene 3'UTR. Results from triplicate experiments showed that reporter activity was reduced by the ectopic expression of hsa-miR-125a-3p (p<0.001). Taken together, these results indicated that hsa-miR-125a-3p inhibited cell migration and invasion in SPC-A-1 cells by targeting RhoA.
To further validate whether inhibitor of hsa-miR-125a-3p regulate migration and invasion via the Rho pathway, we blocked Rho with Rho inhibitor CT04 at a final concentration of 2.0μg/ml according to the previous studies of our laboratory in A549 cell line. The number of migratory cells and invasive cells were decreased clearly after treatment with CT04 when compared with the untreated group (p<0.001). The number of migratory cells and invasive cells were not significantly decreased in cells transfected with sense-3p (p>0.05), and also not increased obviously in cells transefected with antisense-3p (p>0.05), when compared with CT04 group. These results suggest that hsa-miR-125a-3p regulate migration and invasion in Rho-dependent manner.
(7) Hsa-miR-125a-5p enhanced migration and invasion by targeting RhoA: Western blotting and RT-PCR analyses revealed that RhoA protein (p=0.001) and mRNA (p=0.263) remained unchanged (p=0.146). Interestingly, RhoC protein and mRNA were considerably increased in sense-3p transfected cells (p<0.001). As expected, Rockl protein (p<0.001) and mRNA (p<0.001) were also increased clearly. Transfection with 20μM of antisense-3p resulted in a decrease in RhoC protein measured by Western blotting (p<0.001), however, RhoA mRNA (p=0.087) and protein (p=0.147) remained unchanged. As expected, Rockl mRNA and protein were also decreased obviously (p<0.001).
To further validate whether inhibitor of hsa-miR-125a-5p induce migration and invasion via the Rho pathway, we blocked Rho with Rho inhibitor CT04 in SPC-A-1 cell line. The number of migratory cells and invasive cells were decreased clearly after treatment with CT04 when compared with the untreated group (p<0.001). The number of migratory cells and invasive cells were not significantly decreased in cells transfected with antisense-5p (p>0.05), and also not increased obviously in cells transefected with sense-5p (p>0.05), when compared with CT04 group. These results suggest that hsa-miR-125a-5p regulate migration and invasion in Rho-dependent manner.
Conclusions
1、Hsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC.
2、Hsa-miR-125a-3p inhibited migration and invasion by RhoA/Rockl pathway.
3、Hsa-miR-125a-5p enhanced migration and invasion by RhoC/Rockl pathway.
引文
1 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell.1993; 75:843-854.
2 Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature.2003; 425:415-419.
3 Kong W, Zhao JJ, He L, et al. Strategies for profiling microRNA expression. J Cell Physiol. 2009; 218:22-25.
4 Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science.2001; 293:834-838.
5 Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell.2001; 106:23-34.
6 Schwarz DS, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell.2003; 115:199-208.
7 (?)rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008; 30:460-471.
8 Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol. 2005; 3:85.
9 Bartel DP. MicroRNAs genomics, biogenesis, mechanism, and function. Cell.2004; 116: 281-297.
10 Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA.2004; 101: 2999-3004.
11 Crawford M, Brawner E, Batte K, et al. MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines. Biochem Biophys Res Commun.2008; 373:607-612.
12 Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res.2008; 18:350-359.
13 Tavazoie SF, Alarcon C, Oskarsson T, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature.2008; 451:147-152.
14 Nicoloso MS, Spizzo R, Shimizu M, et al. MicroRNAs — the micro steering wheel of tumour metastases. Nat Rev Cancer.2009; 9:293-302.
15 Valastyan S, Reinhardt F, Benaich N, et al. A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell.2009; 137:1032-1046.
16 Kong W, Yang H, He L, et al. MicroRNA-155 is regulated by the transforming growth factor beta/Smad pathway and contributes to epithelial cell plasticity by targeting RhoA. Mol Cell Biol.2008; 28:6773-6784.
17 Asangani IA, Rasheed SA, Nikolova DA, et al. MicroRNA-21 (miR-21) posttranscriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene.2008; 27:2128-2136.
18 Gabriely G, Wurdinger T, Kesari S, et al. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol.2008; 28:5369-5380.
19 Yang J, Mani SA, Weinberg RA. Exploring a new twist on tumor metastasis. Cancer Res.2006; 66:4549-4552.
20 Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell.2006; 9:189-198.
21 Yan LX, Huang XF, Shao Q, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA.2008; 14:2348-2360.
22 Seguin RM, Ferrari N. Emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease. Expert Opin Investig Drugs.2009; 18:1505-1517.
23 Cheng AM, Byrom MW, Shelton J, et al. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 2005; 33:1290-1297.
24 O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature.2005; 435:839-843.
25 Fabani MM, Gait MJ. miR-122 targeting with LNA/2(?)-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates. RNA.2008; 14:336-346.
26 Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer.2009; 100:1002-1011.
27 Deng S, Calin GA, Croce CM, et al. Mechanisms of microRNA deregulation in human cancer. Cell Cycle.2008; 7:2643-2646.
28 Zhang B, Pan X, Cobb GP, et al. microRNAs as oncogenes and tumor suppressors. Dev Biol. 2007; 302:1-12.
29 Inamura K, Togashi Y, Nomura K, et al. let-7 microRNA expression is reduced in bronchioloalveolar carcinoma, a non-invasive carcinoma, and is not correlated with prognosis. Lung Cancer.2007; 58:392-396.
30 Calin GA, Cimmino A, Fabbri M, et al. MiR-15a and miR-16-1 cluster functions in human leukemia. Proc Natl Acad Sci U S A.2008; 105:5166-5171.
31 Markou A, Tsaroucha EG, Kaklamanis L, et al. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR. Clin Chem.2008; 4:1696-1704.
32 Eis PS, Tam W, Sun L, et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA.2005; 102:3627-3632.
33 Habbe N, Koorstra JB, Mendell JT, et al. MicroRNA miR-155 is a biomarker of early pancreatic neoplasia. Cancer Biol Ther.2009; 8:340-346.
34 Caldas C, Brenton JD. Sizing up miRNAs as cancer genes. Nat Med.2005; 11:712-714.
35 Iorio MV, Casalini P, Tagliabue E, et al. MicroRNA profiling as a tool to understand prognosis, therapy response and resistance in breast cancer. Eur J Cancer.2008; 44:2753-2759.
36 Tavazoie SF, Alarcon C, Oskarsson T, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature.2008; 451:147-152.
37 Geiger TR, Peeper DS. Metastasis mechanisms. Biochim Biophys Acta.2009; 1796:293-308.
38 Hedley BD, Chambers AF. Tumor dormancy and metastasis. Adv Cancer Res.2009; 102: 67-101.
39 Ma L, Weinberg RA. Micromanagers of malignancy:role of microRNAs in regulating metastasis. Trends Genet.2008; 24:448-456.
40 Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res.2009; 19:1175-1183.
41 Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature.2008; 455:1124-1128.
42 Raftopoulou M, Hall A. Cell migration: Rho GTPases lead the way. Dev Biol.2004; 265: 23-32.
43 Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer.2002; 2:133-142.
44 Kamai T, Tsujii T, Arai K, et al. Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin Cancer Res.2003; 9:2632-2641.
45 Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J.2000; 348:241-255.
46 Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003; 4:446-456.
47 Salhia B, Rutten F, Nakada M, et al. Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Racl. Cancer Res.2005; 65:8792-8800.
48 Ming J, Liu N, Gu Y, et al. PRL-3 facilitates angiogenesis and metastasis by increasing ERK phosphorylation and up-regulating the levels and activities of Rho-A/C in lung cancer. Pathology.2009; 41:118-126.
49 Itoh K, Yoshioka K, Akedo H, et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat. Med.1999; 5:221-225.
50 Imamura F, Mukai M, Ayaki M, et al. Y-27632, an inhibitor of Rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res.2000; 91:811-816.
51 Myers C, Charboneau A, Cheung I, et al. Sustained expression of homeobox D10 inhibits angiogenesis. Am J Pathol.2002; 161:2099-2109.
52 Walker K, Olson MF. Targeting Ras and Rho GTPases as opportunities for cancer therapeutics. Curr Opin Genet Dev.2005,15:62-68.
1 Shabalina SA, Spiridonov NA. The mammalian transcriptome and the function of non-coding DNA sequences. Genome Biol.2004; 5:105.
2 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell.1993; 75:843-854.
3 Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature.2000; 403:901-906.
4 Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science.2001; 294:858-862.
5 Ambros V, Bartel B, Bartel DP, et al. A uniform system for microRNA annotation. RNA.2003; 9:277-279.
6 Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science.2001; 294:853-862.
7 Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature.2003; 425:415-419.
8 Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature.2001; 409:363-366.
9 Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science.2001; 293:834-838.
10 Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell.2001; 106:23-34.
11 Schwarz DS, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell.2003; 115:199-208.
12 Khvorova A, Reynolds A, Jayasena SD, et al. Functional siRNAs and miRNAs exhibit strand bias. Cell.2003; 115:209-216.
13 (?)rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008; 30:460-471.
14 Liu J, Carmell MA, Rivas FV, et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science.2004; 305:1437-1441.
15 Song JJ, Smith SK, Hannon GJ,et al. Crystal structure of Argonaute and its implications for RISC slicer activity. Science.2004; 305:1434-1437.
16 Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol. 2005; 3:85.
17 Ruvkun G. Molecular biology. Glimpses of a tiny RNA world. Science.2001; 294:797-799.
18 Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science.2001; 294:862-864.
19 Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature.2000; 408: 86-89.
20 Mourelatos Z, Dostie J, Paushkin S, et al. miRNPs:a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev.2002; 16:720-728.
21 Abbott AL, Alvarez-Saavedra E, Miska EA, et al. The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev Cell.2005; 9:403-414.
22 Neilson JR, Zheng GX, Burge CB, et al. Dynamic regulation of miRNA expression in ordered stages of cellular development. Genes Dev.2007; 21:578-589.
23 Ason B, Darnell DK, Wittbrodt B, et al. Differences in vertebrate microRNA expression. Proc Natl Acad Sci U S A.2006; 103:14385-14389.
24 Reinhart BJ, Weinstein EG, Rhoades MW, et al. MicroRNAs in plants. Genes Dev.2002; 16: 1616-1626.
25 Moss EG, Tang L. Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites. Dev Biol.2003; 258:432-442.
26 Eis PS, Tam W, Sun L,et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA.2005; 102:3627-3632.
27 Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res.2005; 65:6029-6033.
28 Takamizawa J, Konishi H, Yanagisawa K,et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res.2004; 64: 3753-3756.
29 Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA.2005; 102:13944-13949.
30 Yu SL, Chen HY, Chang GC, et al. MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell.2008; 13:48-57.
31 Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res.2008; 18:350-359.
32 Nam EJ, Yoon H, Kim SW, et al. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res.2008; 14:2690-2695.
33 Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell.2006; 9:189-198.
34 Ferretti E, De Smaele E, Po A, et al. MicroRNA profiling in human medulloblastoma. Int J Cancer.2008; 124:568-577.
35 Wu L, Belasco JG. Micro-RNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells. Mol Cell Biol.2005; 25:9198-9208.
36 Duan R, Pak C, Jin P. Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA. Hum Mol Genet.2007; 16:1124-1131.
37 Laneve P, Di Marcotullio L, Gioia U, et al. The interplay between microRNAs and the neurotrophin receptor tropomyosin-related kinase C controls proliferation of human neuroblastoma cells. Proc Natl Acad Sci USA.2007; 104:7957-7962.
38 Scott GK, Goga A, Bhaumik D, et al. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J Biol Chem.2007; 282: 1479-1486.
39 Bartel D P. MicroRNAs genomics, biogenesis, mechanism, and function. Cell.2004; 116: 281-297.
40 Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res.2009; 19:1175-1183.
41 (?) rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008; 30:460-471.
42 Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature.2008; 455:1124-1128.
2 Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature.2003; 425:415-419.
3 Kong W, Zhao JJ, He L, et al. Strategies for profiling microRNA expression. J Cell Physiol. 2009; 218:22-25.
4 Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science.2001; 293:834-838.
5 Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell.2001; 106:23-34.
6 Schwarz DS, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell.2003; 115:199-208.
7 (?)rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008; 30:460-471.
8 Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol. 2005; 3:85.
9 Bartel DP. MicroRNAs genomics, biogenesis, mechanism, and function. Cell.2004; 116: 281-297.
10 Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA.2004; 101: 2999-3004.
11 Crawford M, Brawner E, Batte K, et al. MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines. Biochem Biophys Res Commun.2008; 373:607-612.
12 Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res.2008; 18:350-359.
13 Tavazoie SF, Alarcon C, Oskarsson T, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature.2008; 451:147-152.
14 Nicoloso MS, Spizzo R, Shimizu M, et al. MicroRNAs — the micro steering wheel of tumour metastases. Nat Rev Cancer.2009; 9:293-302.
15 Valastyan S, Reinhardt F, Benaich N, et al. A pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell.2009; 137:1032-1046.
16 Kong W, Yang H, He L, et al. MicroRNA-155 is regulated by the transforming growth factor beta/Smad pathway and contributes to epithelial cell plasticity by targeting RhoA. Mol Cell Biol.2008; 28:6773-6784.
17 Asangani IA, Rasheed SA, Nikolova DA, et al. MicroRNA-21 (miR-21) posttranscriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene.2008; 27:2128-2136.
18 Gabriely G, Wurdinger T, Kesari S, et al. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol.2008; 28:5369-5380.
19 Yang J, Mani SA, Weinberg RA. Exploring a new twist on tumor metastasis. Cancer Res.2006; 66:4549-4552.
20 Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell.2006; 9:189-198.
21 Yan LX, Huang XF, Shao Q, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA.2008; 14:2348-2360.
22 Seguin RM, Ferrari N. Emerging oligonucleotide therapies for asthma and chronic obstructive pulmonary disease. Expert Opin Investig Drugs.2009; 18:1505-1517.
23 Cheng AM, Byrom MW, Shelton J, et al. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 2005; 33:1290-1297.
24 O'Donnell KA, Wentzel EA, Zeller KI, et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature.2005; 435:839-843.
25 Fabani MM, Gait MJ. miR-122 targeting with LNA/2(?)-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates. RNA.2008; 14:336-346.
26 Chen HC, Chen GH, Chen YH, et al. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer.2009; 100:1002-1011.
27 Deng S, Calin GA, Croce CM, et al. Mechanisms of microRNA deregulation in human cancer. Cell Cycle.2008; 7:2643-2646.
28 Zhang B, Pan X, Cobb GP, et al. microRNAs as oncogenes and tumor suppressors. Dev Biol. 2007; 302:1-12.
29 Inamura K, Togashi Y, Nomura K, et al. let-7 microRNA expression is reduced in bronchioloalveolar carcinoma, a non-invasive carcinoma, and is not correlated with prognosis. Lung Cancer.2007; 58:392-396.
30 Calin GA, Cimmino A, Fabbri M, et al. MiR-15a and miR-16-1 cluster functions in human leukemia. Proc Natl Acad Sci U S A.2008; 105:5166-5171.
31 Markou A, Tsaroucha EG, Kaklamanis L, et al. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR. Clin Chem.2008; 4:1696-1704.
32 Eis PS, Tam W, Sun L, et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA.2005; 102:3627-3632.
33 Habbe N, Koorstra JB, Mendell JT, et al. MicroRNA miR-155 is a biomarker of early pancreatic neoplasia. Cancer Biol Ther.2009; 8:340-346.
34 Caldas C, Brenton JD. Sizing up miRNAs as cancer genes. Nat Med.2005; 11:712-714.
35 Iorio MV, Casalini P, Tagliabue E, et al. MicroRNA profiling as a tool to understand prognosis, therapy response and resistance in breast cancer. Eur J Cancer.2008; 44:2753-2759.
36 Tavazoie SF, Alarcon C, Oskarsson T, et al. Endogenous human microRNAs that suppress breast cancer metastasis. Nature.2008; 451:147-152.
37 Geiger TR, Peeper DS. Metastasis mechanisms. Biochim Biophys Acta.2009; 1796:293-308.
38 Hedley BD, Chambers AF. Tumor dormancy and metastasis. Adv Cancer Res.2009; 102: 67-101.
39 Ma L, Weinberg RA. Micromanagers of malignancy:role of microRNAs in regulating metastasis. Trends Genet.2008; 24:448-456.
40 Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res.2009; 19:1175-1183.
41 Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature.2008; 455:1124-1128.
42 Raftopoulou M, Hall A. Cell migration: Rho GTPases lead the way. Dev Biol.2004; 265: 23-32.
43 Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer.2002; 2:133-142.
44 Kamai T, Tsujii T, Arai K, et al. Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin Cancer Res.2003; 9:2632-2641.
45 Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J.2000; 348:241-255.
46 Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003; 4:446-456.
47 Salhia B, Rutten F, Nakada M, et al. Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Racl. Cancer Res.2005; 65:8792-8800.
48 Ming J, Liu N, Gu Y, et al. PRL-3 facilitates angiogenesis and metastasis by increasing ERK phosphorylation and up-regulating the levels and activities of Rho-A/C in lung cancer. Pathology.2009; 41:118-126.
49 Itoh K, Yoshioka K, Akedo H, et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat. Med.1999; 5:221-225.
50 Imamura F, Mukai M, Ayaki M, et al. Y-27632, an inhibitor of Rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res.2000; 91:811-816.
51 Myers C, Charboneau A, Cheung I, et al. Sustained expression of homeobox D10 inhibits angiogenesis. Am J Pathol.2002; 161:2099-2109.
52 Walker K, Olson MF. Targeting Ras and Rho GTPases as opportunities for cancer therapeutics. Curr Opin Genet Dev.2005,15:62-68.
1 Shabalina SA, Spiridonov NA. The mammalian transcriptome and the function of non-coding DNA sequences. Genome Biol.2004; 5:105.
2 Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell.1993; 75:843-854.
3 Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature.2000; 403:901-906.
4 Lau NC, Lim LP, Weinstein EG, et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science.2001; 294:858-862.
5 Ambros V, Bartel B, Bartel DP, et al. A uniform system for microRNA annotation. RNA.2003; 9:277-279.
6 Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science.2001; 294:853-862.
7 Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature.2003; 425:415-419.
8 Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature.2001; 409:363-366.
9 Hutvagner G, McLachlan J, Pasquinelli AE, et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science.2001; 293:834-838.
10 Grishok A, Pasquinelli AE, Conte D, et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell.2001; 106:23-34.
11 Schwarz DS, Hutvagner G, Du T, et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell.2003; 115:199-208.
12 Khvorova A, Reynolds A, Jayasena SD, et al. Functional siRNAs and miRNAs exhibit strand bias. Cell.2003; 115:209-216.
13 (?)rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008; 30:460-471.
14 Liu J, Carmell MA, Rivas FV, et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science.2004; 305:1437-1441.
15 Song JJ, Smith SK, Hannon GJ,et al. Crystal structure of Argonaute and its implications for RISC slicer activity. Science.2004; 305:1434-1437.
16 Brennecke J, Stark A, Russell RB, et al. Principles of microRNA-target recognition. PLoS Biol. 2005; 3:85.
17 Ruvkun G. Molecular biology. Glimpses of a tiny RNA world. Science.2001; 294:797-799.
18 Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science.2001; 294:862-864.
19 Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature.2000; 408: 86-89.
20 Mourelatos Z, Dostie J, Paushkin S, et al. miRNPs:a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev.2002; 16:720-728.
21 Abbott AL, Alvarez-Saavedra E, Miska EA, et al. The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev Cell.2005; 9:403-414.
22 Neilson JR, Zheng GX, Burge CB, et al. Dynamic regulation of miRNA expression in ordered stages of cellular development. Genes Dev.2007; 21:578-589.
23 Ason B, Darnell DK, Wittbrodt B, et al. Differences in vertebrate microRNA expression. Proc Natl Acad Sci U S A.2006; 103:14385-14389.
24 Reinhart BJ, Weinstein EG, Rhoades MW, et al. MicroRNAs in plants. Genes Dev.2002; 16: 1616-1626.
25 Moss EG, Tang L. Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites. Dev Biol.2003; 258:432-442.
26 Eis PS, Tam W, Sun L,et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA.2005; 102:3627-3632.
27 Chan JA, Krichevsky AM, Kosik KS. MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res.2005; 65:6029-6033.
28 Takamizawa J, Konishi H, Yanagisawa K,et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res.2004; 64: 3753-3756.
29 Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA.2005; 102:13944-13949.
30 Yu SL, Chen HY, Chang GC, et al. MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell.2008; 13:48-57.
31 Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res.2008; 18:350-359.
32 Nam EJ, Yoon H, Kim SW, et al. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res.2008; 14:2690-2695.
33 Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell.2006; 9:189-198.
34 Ferretti E, De Smaele E, Po A, et al. MicroRNA profiling in human medulloblastoma. Int J Cancer.2008; 124:568-577.
35 Wu L, Belasco JG. Micro-RNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells. Mol Cell Biol.2005; 25:9198-9208.
36 Duan R, Pak C, Jin P. Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA. Hum Mol Genet.2007; 16:1124-1131.
37 Laneve P, Di Marcotullio L, Gioia U, et al. The interplay between microRNAs and the neurotrophin receptor tropomyosin-related kinase C controls proliferation of human neuroblastoma cells. Proc Natl Acad Sci USA.2007; 104:7957-7962.
38 Scott GK, Goga A, Bhaumik D, et al. Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b. J Biol Chem.2007; 282: 1479-1486.
39 Bartel D P. MicroRNAs genomics, biogenesis, mechanism, and function. Cell.2004; 116: 281-297.
40 Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res.2009; 19:1175-1183.
41 (?) rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008; 30:460-471.
42 Tay Y, Zhang J, Thomson AM, et al. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature.2008; 455:1124-1128.