microRNA-19促进人肺腺癌细胞株A549向间充质细胞转化
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摘要
肺癌是当今世界上对人类健康与生命危害最大的恶性肿瘤,为全球癌症死亡的主要原因。近年来,我国肺癌的死亡率增加趋势明显,在所有癌症致死中上升最快,已经成为第一位的癌症死因。传统的肺癌治疗方法通常是依据肺癌的组织病理类型来决定,主要分为两种类型,即小细胞型肺癌和非小细胞型肺癌(non-small cell lung cancer, NSCLC),其中NSCLC由鳞状细胞癌、大细胞癌以及腺癌等三种亚型组成,肺腺癌是目前最常见的病理类型,约占肺癌总数的50%。手术切除仍然是NSCLC最佳的治疗方法,处于Ⅰ期NSCLC患者术后的5年生存率可以达到73%。但是,大部分肺癌获得诊断时已经处于晚期。尽管近年来在肺癌治疗上取得了一些新进展,但总体上来看在NSCLC的治疗效果未获得明显改善,其5年生存率也仅为16%。肺癌转移是肺癌治疗失败及肺癌患者致死的主要原因之一。目前大量的研究表明恶性肿瘤中上皮向间充质细胞转化(epithelial-to-mesenchymal transition, EMT)是引起肿瘤远处转移的重要步骤。深入认识EMT在肺癌进展中的调控机制是有效防治肺癌的关键。
在基因的表达调控中,功能性蛋白质被认为是基因功能的执行者,mRNA是介于基因与蛋白质之间的中间产物。随着人类基因组计划的完成,发现编码蛋白质的基因数目仅占整个基因组序列的2%,占基因组序列98%的非蛋白质编码区编码了大量的非编码RNA (noncoding RNA, nc-RNA)。Nc-RNA,特别是microRNA等小nc-RNA的发现,使基因表达调控进入RNA时代。
目前认为microRNA是一类含18-24个核糖核苷酸的小分子RNA家族,由基因组DNA编码,通过RNA聚合酶Ⅱ协助转录产生初始的miRNA,经过细胞核内和胞浆中一系列加工处理后在胞浆中形成成熟microRNA。microRNA通过识别靶向mRNA3'UTR (Untranslated Regions)与靶mRNA完全或不完全互补结合,促使靶mRNA降解或抑制其翻译,即在转录后水平与翻译水平调节靶基因的表达。首先,microRNA与其靶基因的互补程度决定其作用模式：①DmicroRNA与靶mRNA完全互补结合,降解靶mRNA;②icroRNA与靶mRNA不完全互补结合,抑制靶蛋白的翻译而不影响mRNA稳定性；③同时具有以上两种作用模式。根据这一作用机制,以及microRNA序列5'端2-8位核苷酸均与靶mRNA3'端-UTR完全互补这一重要特点,应用生物信息学方法可预测到在同一靶基因上存在多个microRNA结合位点,提示microRNA可能通过多元化途径调控靶基因表达。除了上述负向调控作用之外,少数microRNA还具有正向调控作用。其次,一个microRNA可以调控多个基因的表达,也可以通过几个microRNA的组合来精细调控某个基因的表达。由于存在于人类基因组内的microRNA基因预计达1000多种,但仅少数在功能上与特定的生物学途径相联系。最后,microRNA调控基因的表达变化依赖于细胞和组织的环境以及机体的生理与病理状态。可见microRNA的数量较多,调控过程复杂。
microRNA较为稳定,广泛表达于机体内的细胞、组织及器官,参与调控细胞增殖、分化和凋亡以及机体的生长、发育等生理进程。临床研究表明microRNA与肺癌的发生发展密切相关,如miR-146b、miR-155、let-7e、miR-34a、 miR-34c-5p、miR-25、miR-191等与NSCLC的预后密切相关。microRNA的生物合成异常可能会导致肺癌的发生。Dicer是microRNA形成过程中的关键蛋白,敲除Dicer可抑制成熟microRNA的生成,肺癌组织中的Dicer的表达水平与患者的预后密切相关。microRNA具有组织特异性,与肺组织相关的miRNA包括let-7a、miR-17-92、miR-34、miR-21、miR-29、miR-155、miR-31、miR-200等40余种,它们在肺癌组织表达中降低或升高,具有癌基因或抑癌基因样作用,调节转录后翻译,促进或抑制肿瘤发生发展。目前,在肺癌中具有代表性的抑癌microRNA有let-7、miR-29、miR-34a。
miR-17-92基因簇就是一个高度保守的多顺反子miRNA,编码miR-17、miR-18a、miR-19a、miR-20a、miR-19b-1和miR-92a等6个miRNAs,它位于人类第13号染色体上C1orf25基因初级转录本的第3个内含子区。现已发现,miR-17-92基因簇在肺癌、B细胞淋巴瘤、肝癌等多种肿瘤细胞中均高表达。miR-17-92基因簇可靶基因作用于促凋亡蛋白基因B／m、抑癌基因P21、PTEN、转化生长因子β(TGFβ)、转化生长因子p受体Ⅱ(TGFBR2)、视网膜母细胞瘤1(Rb1)、凝血酶敏感蛋白1(TSP-1)、结缔组织生长因子(CTGF)和转录因子E2F1等促进肿瘤发生发展。在miR-17-92家族成员中miR-19是促进肿瘤形成的主要成分,通过抑制PTEN以及Bim表达,促进肿瘤细胞的增殖。
miR-17-92家族成员在肺癌中发挥重要作用。抑制miR-17-5p和miR-20a的表达能够选择性诱导过表达miR-17-92基因簇的肺癌细胞凋亡。那么,作为miR-17-92致瘤形成的关键,miR-19如何促进肺腺癌的发展,值得深入研究。
为深入探讨miR-19对肺腺癌细胞株A549的作用及其潜在机制,本课题将从以下几个方面进行研究：(1)检测miR-19在不同肺癌细胞中的表达,体内外实验观察miR-19对A549细胞株形态及功能(增殖、迁移)的影响；(2)基因芯片检测miR-19对A549细胞基因表达谱的影响；(3)miR-19调控A549发生EMT的潜在机制。通过上述研究可能会获得miR-19在A549细胞中的作用以及相关调控机制。研究结果如下：
1miR-19对A549细胞形态及迁移增殖功能的影响
1.1荧光定量RT-PCR检测5种肺癌细胞株与肺上皮细胞株中miR-19的表达
miR-19在5种肺癌细胞株中的表达均高于BEAS-2B细胞,在非小细胞肺癌细胞H23中表达最高,而在肺腺癌A549及A549-Luc中最低。单因素方差分析的结果表明,6种细胞株中miR-19的表达差异具有显著性(F=797.865,P<0.001)。通过2-△△ct法,与BEAS-2B细胞相比,A549中miR-19的表达增高了约1.941倍,A549-Luc中增高了约1.541倍,H446中增高了约2.458倍,H460中增高了约2.696倍,H23中增高了约4.419倍。
1.2pm19H2BmRFP和pm19(-)H2BmRFP慢病毒表达载体的构建
1.2.1miR-19a/19b和miR-19a(-)19b(-)序列的扩增
以质粒MIG19a/19b. MIG19a(-)19b(-)为模板扩增miR-19a/19b (784bp)、 miR-19a(-)19b(-)(981bp)两个序列,PCR产物进行1%琼脂糖凝胶电泳,在784bp、981bp左右可见一清晰条带。
1.2.2重组载体PCR鉴定
miR-19a/19b. miR-19a(-)19b(-)PCR产物纯化后与载体pHIV-H2BmRFP进行酶切、连接、转化,各挑克隆,小量摇菌后进行菌液PCR鉴定,结果显示pm19H2BmRFP有8个克隆可扩增出784bp的目的片段；pm19(-)H2BmRFP有9个克隆均可扩增出981bp的目的片段。
1.2.3重组载体测序鉴定
重组载体经测序鉴定与模板MIG19a/19b、MIG19a(-)19b(-)序列一致,说明重组载体pm19H2BmRFP. pm19(-)H2BmRFP构建成功。
1.2.4pm19H2BmRFP、pm19(-)H2BmRFP过表达载体及其空载慢病毒的包装及滴度测定
分别将三质粒包装系统(空载pHIV-H2BmRFP或pm19H2BmRFP或pm19(-)H2BmRFP. psPAX2和pMD2.G)共转染293T细胞,包装产生三种慢病毒,即空白对照慢病毒(pHIV-H2BmRFP)和过表达miR-19a/19b. miR-19a(-)19b(-)的慢病毒(pm19H2BmRFP. pm19(-)H2BmRFP)。转染后48h,荧光显微镜下观察293T细胞发出红色荧光,RFP定位在核。经293T包装产生的慢病毒,采用逐孔稀释法感染293T细胞来测定病毒滴度,按公式计算得到病毒滴度。其中,空白对照慢病毒(pHIV-H2BmRFP)的病毒滴度为6.1x106TU/ml,过表达miR-19a/19b. miR-19a(-)19b(-)的慢病毒(pm19H2BmRFP. pm19(-)H2BmRFP)滴度为5.2x106TU/ml、5.0x106TU/ml.
1.3稳定过表达miR-19a/19b和miR-19a(-)19b(-)的肺癌细胞株的建立
将含有miR-19a/19b.miR-19a(-)19b(-)片段的慢病毒pm19H2BmRFP. pm19(-)H2BmrfP和空载对照慢病毒pHIV-H2BmRFP分别感染A549-Luc细胞,感染48h后荧光显微镜下观察A549-Luc细胞发出红色荧光,RFP定位在核。
1.4荧光定量RT-PCR鉴定过表达miR-19.miR-19(一)以及空载的A549细胞中miR-17,miR-18a, miR-19(19a和19b),miR-20a,miR.92a的表达
相对于未感染细胞A549-Luc,A549/RFP+/m19中miR-19的表达倍数约升高1.893倍(P=0.027)；A549/RFP+/m19(-)中miR-17的表达水平约升高0.839倍(P<0.001),miR-18a的表达水平相对降低了0.116(P=O.020),miR-20a的表达水平约升高6.938倍(P=0.005),miR-92a的表达水平约升高1.546倍(P<0.001)。
1.5miR-19a/19b.miR-19a(-)19b(-)对A549-Luc细胞形态的影响
过表达miR-19a/19b.miR-19a(-)19b(-)的A549-Luc细胞经过传代,细胞的形态发生了明显改变。与A549/RFP+/H2B组细胞相比,A549/RFP+/m19组细胞形态瘦长,呈纺锤样、成纤维细胞样形态,播散分布；A549/RFP+/m19(-)组细胞突起,聚集成团。经过多次传代后,各组细胞仍保持上述形态。
1.6miR-19a/19b.miR-19a(-)19b(-)对A549-Luc细胞体外增殖能力的影响
采用MTT法检测A549/RFP+/m19.A549/RFP+/m19(-)以及空载细胞的体外增殖能力。采用单因素重复测量因素的方差分析,结果表明A549/RFP+/m19和A549/RFP+/m19(-)组均较空载组A549/RFP+/H2B增殖减慢(P<0.001)。
平板克隆形成实验的结果同样显示A549/RFP+/H2B.A549/RFP+/m19. A549/RFP+/m19(-)三组细胞的增殖速度具有显著性差异(F=76.155,P<0.001)。以上两个实验结果均表明过表达miR-19.miR-19(-)后,抑制了肺腺癌A549细胞的体外增殖。
1.7miR-19a/19b.miR-19a(-)19b(-)过表达对A549-Luc细胞周期的影响
采用流式细胞术分析过表达miR-19a/19b.miR-19a(-)19b(-)的A549-Luc细胞周期的分布,结果显示,与空载A549/RFP+/H2B相比,细胞G1期细胞比例在A549/RFP+/m19(P=0.011)和A549/RFP+/m19(-)(P<0.001)中显著升高,S期细胞比例在A549/RFP+/m19(P=0.001)和A549/RFP+/ml9(-)(P<0.001)中显著降低,即在过表达miR-19a/19b、miR-19a(-)19b(-)的A549-Luc细胞中,细胞出现了G1向S期转化障碍,细胞被阻滞在G1期。
1.8miR-19a/19b、miR-19a(-)19b(-)-过表达对A549-Luc细胞体外迁移运动能力的影响
采用Transwell小室检测空载细胞A549/RFP+/H2B和A549/RFP+/ml9. A549/RFP+/ml9(-)细胞体外迁移运动能力的变化,结果发现三组细胞运动能力的差异具有显著性(F=58.135,P<0.001)。与A549/RFP+/H2B细胞相比,A549/RFP+/m19细胞穿过膜的细胞数显著增多(P<0.001),其运动能力显著升高；A549/RFP+/m19(-)穿过膜的细胞数显著减少(P<0.01),其运动能力显著降低。
1.9荧光定量RT-PCR检测3种肺癌细胞株中E-Cadherin、Vimentin的表达
与A549/RFP+/H2B相比,E-Cadherin在A549/RFP+/m19中表达降低(P<0.05),Vimentin在A549/RFP+/m19中表达升高(P<0.05),在A549/RFP+/ml9(-)中表达降低(P<0.05),均具有统计学意义。
1.10Western blot检测3种肺癌细胞株中E-Cadherin、Vimentin的表达
与A549/RFP+/H2B组相比,A549/RFP+/m19组的E-cadherin表达降低、vimentin表达升高；A549/RFP+/m19(-)组的E-cadherin降低不明显,vimentin表达下调明显。
1.11可视化观察A549/RFP+/H2B, A549/RFP+/m19肺癌细胞株的皮下成瘤
皮下移植A549/RFP+/H2B细胞的小鼠,生物发光信号的强度均随着时间的推移而明显增强(F=17.037,P<0.001)；皮下移植A549/RFP+/m19细胞的小鼠,生物发光信号的强度均随着时间的推移轻度增强(F=3.850,P<0.024),增殖速度明显低于移植A549/RFP+/H2B细胞的小鼠(P<0.001)
2miR-19对A549细胞基因表达谱的影响
2.1基因芯片表达谱的数据分析
共检测了47,000个基因的表达情况,聚类分析结果提示A549/RFP+/ml9和A549/RFP+/H2B两组细胞间基因表达存在明显差异。与空载组相比,miR-19过表达组上调表达的基因有586个,下调表达的基因有504个。
利用博奥生物有限公司MAS数据分析系统对A549/RFP+/ml9和A549/RFP+/H2B两种细胞间差异基因进行GO分析,结果显示显著富集的GOTerm主要涉及Signal transduction、Cell adhesion、Transcription、Oxidation reduction、EMT等方面。与A549/RFP+/H2B细胞相比,在A549/RFP+/m19细胞中与EMT相关的差异表达基因有31个上调表达,10个下调表达；其中,上调表达的有：CDH2(N-cadherin)、CDH11(OB-cadherin)、ITGA5、ITGB6、COL3A1、 COL1A1、COL5A1、FN1、CALD1、MMP1、MMP2、MMP9等；下调表达的有CDH1(E-cadherin), KRT19等。两种细胞中差异表达的EMT相关基因的GO功能分类分析结果显示这些差异基因主要参与了细胞的生理发育、代谢与生理调控等过程；Pathway分析显示这些基因相互间存在多条信号通路。
2.2荧光定量RT-PCR检测A549/RFP+/ml9和A549/RFP+/H2B细胞中E-Cadherin、N-cadherin、Vimentin、Fibronectin、snail、MMP1和MMP10的表达
与A549/RFP+/H2B相比,E-Cadherin在A549/RFP+/m19中表达降低,N-cadherin、Vimentin、FN1、Snail、MMP1和MMP10在A549/RFP+/m19中表达明显升高。
2.3Western blot检测A549/RFP+/ml9和A549/RFP+/H2B细胞中E-cadherin、 Vimentin、Fibronectin、snail、MMP1和MMP10的表达
与空载组相比,过表达miR-19的A549细胞中E-cadherin表达明显下调,Vimentin表达轻度上调,Fibronectin、snail、MMP1和MMP10表达明显上调。
3过表达miR-19诱导A549细胞发生EMT的分子机制初步研究
Western blot检测A549/RFP+/H2B和A549/RFP+/m19肺腺癌细胞株中Pten、 STAT3. p-STAT3. ITGA5. P53以及c-myc的表达水平。与A549/RFP+/H2B组相比,A549/RFP+/m19组的Pten. P53以及c-myc的表达下调、STAT3. p-STAT3以及ITGA5表达升高。
通过上述三个部分的实验,我们能够得出以下结论：
(1)过表达miR-19可诱导A549细胞向间充质细胞转化。
(2)miR-19可能通过抑制Pten表达,激活STAT3以及Snail,抑制了E-cadherin表达、上调了Vimentin及ITGA5的表达,最终诱导A549细胞向间充质细胞转化。
Lung cancer, the leading cancer-related cause of death, is the most dangerous cancer to human health and life worldwide. In recent years, the death rate of lung cancer was obviously increased and became the the fastest growth rate and leading cancer-related cause of death in China. The traditional method of lung cancer treatment is usually based on the histopathological types of lung cancer, which mainly divided into two types, namely small cell lung cancer and non-small cell lung cancer(NSCLC). NSCLC consists of squamous-cell carcinoma, large cell carcinoma, and adenocarcinoma. Lung adenocarcinoma is the most common histological subtypes of lung cancer, accounting for70%of global lung cancer. The best treatment for NSCLC is surgical resection. Postoperative5-year survival rate for NSCLC patients in stage I can reach73%. Sadly, most lung cancer is diagnosed too late for curative treatment. Although great progresses have been made in understanding the disease, the therapeutic effect not gained significant improvement and the5-year survival rate was only16%. Metastasis was the leading cause of treatment failure and death of patients. Previous studies demonstrated that epithelial-to-mesenchymal transition was the important process of distant metastasis of cancer. Deeply investigating the role of EMT in the development of lung cancer was the key for lung cancer prevention and remedy.
Functional proteins are considered as the executor of the functional gene, mRNA is the intermediay. With the completion of the human genome project, the genome sequence encoding protein only accounts for2%of the entire genome sequence, but the rest of98%of genome sequence encodes a lot of the noncoding RNA. Nc-RNAs, especially small Nc-RNAs, such as microRNAs, make the gene regulation into RNA era.
MiRNAs are a class of small RNAs (21-24nt) that regulate the expression of target genes at the post-transcriptional level by base pairing with the3'untranslated regions (UTRs) of mRNAs and promoting mRNA unstability. They are first transcribed from miRNA genes in the genome as primary miRNAs (pri-miRNAs) which are processed into the mature miRNAs through two sequential cleavage steps and exported from the nuclei to cytoplasm. Firstly, depending on the complementarity between miRNA and3'untranslated region (UTR) of target mRNA there are three known mechanisms of miRNAs action on mRNAs:1) target mRNA degradation when miRNA is near-perfectly complementary with target mRNA,2) translational inhibition with little or no influence on mRNA levels when miRNA is only partially complementary to its target mRNA and3) the above two ways coexist. Application of bioinformatics method can predict the same target genes exist in multiple microRNA binding site based on the above mechanism and characteristics of the sequence of microRNA5'2-8bits nucleotide should fully complementary to the target mRNA3'UTR, indicating regulation of microRNA target gene expression possibly through diverse ways. In addition to the negative regulation function, minority microRNA also can positive regulate gene expression. Secondly, it is evident that single miRNAs may regulate translation of numerous downstream mRNAs and each mRNA is likely to be regulated by several miRNAs simultaneously. At last, microRNA regulate gene expression depending on the context of cells and tissues and the physiological and pathological state. It is obvious that the regulatory process of microRNA is complicated.
miRNAs are implicated in cell differentiation, proliferation, apoptosis, and various physiologic and pathologic processes, including growth and development. Clinical studies showed that microRNAs were closely related to the occurrence of lung cancer, for example, miR-146b, miR-155, let-7e, miR-34a, miR-34c-4p, miR-25, miR-191were closely related to the prognosis of NSCLC. Defects in the biogenesis of microRNAs are contributed to the development of lung cancer. Dicer is the best-established regulator of miRNA processing. Deletion of Dicer abrogates the production of mature miRNAs. Reduction of Dicer expression levels was correlated with poor survival of NSCLC patients. Specical microRNAs are existed in lung cancer. More than40microRNAs are involvd in the pathogenesis of lung cancer, including let-7a, miR-17-92, miR-34, miR-21, miR-29, miR-155, miR-31, and miR-200et al, they decreased or increased in lung cancer and function as oncogene or anti-oncogene. At present, the representatives of tumor suppressor microRNA in lung cancer are let-7, miR-29and miR-34a.
MiR-17-92, a polycistronic microRNA Cluster, comprises six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a) and resides in intron3of the C13orf25gene at13q31.3. miR-17-92is overexpressed in cancer, such as lung cancer, B-cell lymphomas and liver cancer et al. MiR-17-92promotes the development of cancer by targeting proapoptotic gene Bim, anti-oncogene p21and pten, TGFβ, TGFBR2, Rbl, TSP-1, CTGF and E2F1. MiR-19, a key oncogenic component for miR-17-92, promotes the proliferation of cancer cells by suppressing the expression of pten and Bim.
MiR-17-92family members play an important role in lung cancer. Supression of miR-17-5p and miR-20a can lead lung cancer cells overexpressing miR-17-92to apoptosis. Thus, it is valuable for studying the effect of miR-19on the development of lung adenocarcinoma.
To further investigate the effect and the potential mechanism of miR-19on the lung adenocarcinoma cell line A549, the following several aspects will be studied:(1) determining the expression level of miR-19in several lung cancer cell lines, observing the effect of miR-19on morphology, proliferation and migration of A549 cells in vivo and vitro;(2) determining the effect of miR-19on gene expression pattern of A549cells;(3) the regulation mechanism of miR-19on A549. The effect and potential mechanism of miR-19on A549will be obtained though above research.
The results are as follows:
1Effect of miR-19on the morphology, proliferation and migration of A549cells
1.1Determining the expression of miR-19in5lung cancer cell line using quantitative RT-PCR
The relative expression quantity are calculated by2-△△Ct. The expression of miR-19are higher in5lung cancer cell lines than that in BEAS-2B. The expression of ml9is the highest in non small cell lung cancer cell line H23, but the lowest in lung adenocarcinoma cell line A549and A549-Luc-CM-1. The statistics of One Way Anova showed that the expression of miR-19were significantly different in6lung cell line(F=797.865, P<0.001). Using2-△△Ct method, compared with BEAS-2B cell line, miR-19increased1.941,1.541,2.458,2.696,4.419-fold in A549, A549-luc, H446, H460and H23, respectively.
1.2Generation of lentivirus vector of pm19H2BmRFP, pm19(-)H2BmRFP
1.2.1Amplification of the sequence of miR-19a/19b; miR-19a(-)19b(-)
Amplification of the sequence of miR-19a/19b (784bp); miR-19a(-)19b(-)(981bp) based on MIG19a/19b; MIG19a(-)19b(-) plasmid. The PCR products were electrophoresed in1%agarose gel, and distinct bands were appeared at784bp and981bp, respectively.
1.2.2Identification the recombinant vector by PCR
The PCR products of miR-19a/19b, miR-19a(-)19b(-) were cut by Enzymatic digestion and purified, ligated, transformed, then colonies were selected to be identified. The result showed that the target fragment located at784bp,981bp were amplified from eight pm19H2BmRFP colonies and nine pm19(-)H2BmRFP colonies.
1.2.3Identificating sequence of the recombinant vector
The sequence of the recombinant vectors are in accordance with MIG19a/19b; MIG19a(-)19b(-), indicating the success of generation of pm19H2BmRFP and pm19(-)H2BmRFP.
1.2.4Lentivirus package and titer determination of control vector and overexpression vectors of pm19H2BmRFP and pm19(-)H2BmRFP
Cotransfection293T cells with three plasmid package system (Control pHIV-H2BmRFP or pm19H2BmRFP or pm19(-)H2BmRFP, psPAX2and pMD2.G) produced three lentiviruses, including pHIV-H2BmRFP, pm19H2BmRFP and pm19(-)H2BmRFP.48h later, red fluorescence were observed in the nucleus of293T cells. Assaying virus titers by gradually diluting the virus to infect293T cells. The titers of virus of pHIV-H2BmRFP, pm19H2BmRFP and pm19(-)H2BmRFP are6.1x106TU/ml,5.2x106TU/ml and5.0x106TU/ml, respectively.
1.3Generation of lung cancer cell line overexpressing miR-19a/19b and miR-19a(-)19b(-)
The lentivirus of control PHIV-H2BmRFP, pm19H2BmRFP and pml9(-)H2BmRFP, encoding miR-19a/19b, miR-19a(-)19b(-) were used to infect A549-Luc cells, respectively.48h later, red fluorescence were observed in the nucleus of293T cells.
1.4Determining the miR-17, miR-18a, miR-19, miR-20a and miR-92a in A549-Luc cells overexpressing miR-19, miR-19(-) using quantitative PCR.
Compared with A549-Luc, miR-19increased1.893times in A549/RFP+/m19; miR-17, miR-20a, miR-92a increased0.839,6.938and1.546times respectively; miR-18a are not obviously changed in A549/RFP+/m19(-) cell.
1.5Effect of miR19a/19b and miR19a(-)19b(-) on the morphology of A549-Luc cell
Several passages later, the morphology of A549cells overexpressing miR-19a/19b and miR-19a(-)19b(-) changed. Compared with A549/RFP+/H2B cells, the morphology of A549/RFP+/m19showed elongation, spindle-like, fibroblast shape, sporadic distribution; A549/RFP+/m19(-) showed cell gibbosity, clump together.
1.6Effect of miR-19a/19b, miR-19a(-)19b(-) on the proliferation of A549cells.
Determining the ability of proliferation of A549/RFP+/m19, A549/RFP+/m19(-) and A549/RFP+/H2B using MTT assay. Single repeated measure ANOVA showed that the ability of proliferation was significantly different in three groups. Compare to A549/RFP+/H2B, the speed of proliferation of A549/RFP+/m19and A549/RFP+/m19(-) cells slowed down(P<0.001).
Flat colony formation assay experiments also showed that the ability of proliferation was significantly different in three groups(F=76.155, P<0.001). The above mentioned experiments showed overexpression of miR-19and miR-19(-) suppressed the proliferation of A549-Luc cell in vitro.
1.7Effect of overexpression of miR-19a/19b and miR-19a(-)19b(-) on cell cycle of A549-Luc
The cell cycle of A549-Luc overexpressing miR-19a/19b and miR-19a(-)19b(-) were determined using FACS. Compare to A549/RFP+/H2B, the ratio of G1phase in A549/RFP+/m19(P=0.011) and A549/RFP+/m19(-)(P<0.001) were significantly upregulated, the ratio of S phase in A549/RFP+/m19(P=0.001) and A549/RFP+/m19(-)(P<0.001) were significantly downregulated. This result showed that A549/RFP+/ml9and A549/RFP+/ml9(-) cells were blocked in G1phase.
1.8Overexpression of miR-19a/19b, miR-19a(-)19b(-) on the migration of A549cells.
Detection the migration ability of A549/RFP+/H2B, A549/RFP+/ml9and A549/RFP+/ml9(-) cells using Transwell chamber showed that the migration ability was different in three groups (F=57.284, P<0.001). Compared with A549/RFP+/H2B cells, the migration ability of A549/RFP+/m19cells obviously increased(P<0.01), but in A549/RFP+/m19(-) cells decreased significantly(P<0.01).
1.9Determined the expression of E-cadherin and Vimentin using quantitative RT-PCR
Compared with A549/RFP+/H2B, the expression of E-cadherin decreased in A549/RFP+/m19cell line(P<0.05), and increased in A549/RFP+/m19(-)(P<0.05), respectively; the expression of Vimentin increased in A549/RFP+/m19cell line(P<0.05), and decreased in A549/RFP+/m19(-)(P<0.05).
1.10Determined the expression of E-cadherin and Vimentin using Western blot
Compared with A549/RFP+/H2B, the expression of E-cadherin and Vimentin were downregulated and upregulated in A549/RFP+/m19cell line, respectively; the expression of E-cadherein was not significantly downregulated in A549/RFP+/ml9cell line, but the expression of Vimentin decreased in A549/RFP+/m19(-).
1.11In vivo optical imaging of subcutaneous xenografts of A549/RFP+/H2B and A549/RFP+/m19cells
For subcutaneous transplantation of A549/RFP+/H2B cells, the photons of bioluminescence had significantly increased over time(F=17.037, P<0.001), For subcutaneous transplantation of A549/RFP+/m19cells, we found that the photons of bioluminescence had also increased over time(F=3.850, P<0.024), but the proliferation speed significantly lowered than that of A549/RFP+/H2B cells(P<0.001).
2Effect of miR-19on the gene expression pattern of A549cells
2.1Data analysis on the gene expression pattern
47,000genes were determined. Cluster analysis showed significant difference of gene expression between A549/RFP+/ml9and A549/RFP+/H2B cells. Compared with control vector group,586genes were up expressed and504genes were down expressed in A549cell with overexpression of miR-19(A549/RFP+/m19).
Using MAS data analysis system derived from Boao corporation, GO was used to analyze the differentially expressed genes between A549/RFP+/ml9and A549/RFP+/H2B cells. The result showed that GO Term were significantly enriched in Signal transduction, Cell adhesion, Transcription, Oxidation reduction and EMT. Compared with A549/RFP+/H2B cells, the marker of EMT, such as CDH2(N-cadherin), CDH11(OB-cadherin), ITGA5, ITGB6, COL3A1, COL1A1, COL5A1, FN1, CALD1, MMP1, MMP2and MMP9were upregulated, while CDHl(E-cadherin) and KRT19were downregulated in A549/RFP+/H2B cells. Furthermore, differentially expressed genes related to EMT were analyzed by GO functional classification. The result showed that these gene were involved in the process of physiogenesis, metabolism and physiology regulation. Pathway analysis showed these genes had multiple signal pathway between each other.
2.2Expression of E-Cadherin, N-cadherin, Vimentin, Fibronectin, snail, MMP1and MMP10was examined in A549/RFP+/H2B and A549/RFP+/ml9using quantitative PCR.
Compared with A549/RFP+/H2B, E-cadherin expression decreased in A549/RFP+/m19, while the expression of N-cadherin, Vimentin, FN1, Snail, MMP1and MMP10significantly increased in A549/RFP+/m19.
2.3Expression of E-Cadherin, Vimentin, Fibronectin, snail, MMP1and MMP10were detected in A549/RFP+/H2B and A549/RFP+/m19using western blot.
Compared with A549/RFP+/H2B, E-cadherin expression was obviously downregulated in A549/RFP+/m19. Vimentin expression was slightly upregulated, while expression of Fibronectin、snail、MMP1and MMP10were obviously upregulated in A549/RFP+/m19.
3Preliminary study on the molecular mechanism of induction EMT of A549by miR-19
The expression of Pten、STAT3、p-STAT3、ITGA5. P53and c-myc was detected in A549/RFP+/H2B cells and A549/RFP+/m19cells using western blot. Compared with A549/RFP+/H2B, Pten, P53and c-myc were downexpressed, while STAT3, P-STAT3and ITGA5was upexpressed in A549/RFP+/m19cells.
From the above3sections experiments, we can conclude that:
(1) miR-19can induce the transformation of human lung adenocarcinoma cell line A549to the mesenchymal cell.
(2) miR-19promotes the transformation of A549cells to mesenchymal cells perhaps via suppression of Pten, following activation of STAT3and Snail, and suppression of E-cadherin, upregulation of Vimentin and ITGA5.
在基因的表达调控中,功能性蛋白质被认为是基因功能的执行者,mRNA是介于基因与蛋白质之间的中间产物。随着人类基因组计划的完成,发现编码蛋白质的基因数目仅占整个基因组序列的2%,占基因组序列98%的非蛋白质编码区编码了大量的非编码RNA (noncoding RNA, nc-RNA)。Nc-RNA,特别是microRNA等小nc-RNA的发现,使基因表达调控进入RNA时代。
目前认为microRNA是一类含18-24个核糖核苷酸的小分子RNA家族,由基因组DNA编码,通过RNA聚合酶Ⅱ协助转录产生初始的miRNA,经过细胞核内和胞浆中一系列加工处理后在胞浆中形成成熟microRNA。microRNA通过识别靶向mRNA3'UTR (Untranslated Regions)与靶mRNA完全或不完全互补结合,促使靶mRNA降解或抑制其翻译,即在转录后水平与翻译水平调节靶基因的表达。首先,microRNA与其靶基因的互补程度决定其作用模式：①DmicroRNA与靶mRNA完全互补结合,降解靶mRNA;②icroRNA与靶mRNA不完全互补结合,抑制靶蛋白的翻译而不影响mRNA稳定性；③同时具有以上两种作用模式。根据这一作用机制,以及microRNA序列5'端2-8位核苷酸均与靶mRNA3'端-UTR完全互补这一重要特点,应用生物信息学方法可预测到在同一靶基因上存在多个microRNA结合位点,提示microRNA可能通过多元化途径调控靶基因表达。除了上述负向调控作用之外,少数microRNA还具有正向调控作用。其次,一个microRNA可以调控多个基因的表达,也可以通过几个microRNA的组合来精细调控某个基因的表达。由于存在于人类基因组内的microRNA基因预计达1000多种,但仅少数在功能上与特定的生物学途径相联系。最后,microRNA调控基因的表达变化依赖于细胞和组织的环境以及机体的生理与病理状态。可见microRNA的数量较多,调控过程复杂。
microRNA较为稳定,广泛表达于机体内的细胞、组织及器官,参与调控细胞增殖、分化和凋亡以及机体的生长、发育等生理进程。临床研究表明microRNA与肺癌的发生发展密切相关,如miR-146b、miR-155、let-7e、miR-34a、 miR-34c-5p、miR-25、miR-191等与NSCLC的预后密切相关。microRNA的生物合成异常可能会导致肺癌的发生。Dicer是microRNA形成过程中的关键蛋白,敲除Dicer可抑制成熟microRNA的生成,肺癌组织中的Dicer的表达水平与患者的预后密切相关。microRNA具有组织特异性,与肺组织相关的miRNA包括let-7a、miR-17-92、miR-34、miR-21、miR-29、miR-155、miR-31、miR-200等40余种,它们在肺癌组织表达中降低或升高,具有癌基因或抑癌基因样作用,调节转录后翻译,促进或抑制肿瘤发生发展。目前,在肺癌中具有代表性的抑癌microRNA有let-7、miR-29、miR-34a。
miR-17-92基因簇就是一个高度保守的多顺反子miRNA,编码miR-17、miR-18a、miR-19a、miR-20a、miR-19b-1和miR-92a等6个miRNAs,它位于人类第13号染色体上C1orf25基因初级转录本的第3个内含子区。现已发现,miR-17-92基因簇在肺癌、B细胞淋巴瘤、肝癌等多种肿瘤细胞中均高表达。miR-17-92基因簇可靶基因作用于促凋亡蛋白基因B／m、抑癌基因P21、PTEN、转化生长因子β(TGFβ)、转化生长因子p受体Ⅱ(TGFBR2)、视网膜母细胞瘤1(Rb1)、凝血酶敏感蛋白1(TSP-1)、结缔组织生长因子(CTGF)和转录因子E2F1等促进肿瘤发生发展。在miR-17-92家族成员中miR-19是促进肿瘤形成的主要成分,通过抑制PTEN以及Bim表达,促进肿瘤细胞的增殖。
miR-17-92家族成员在肺癌中发挥重要作用。抑制miR-17-5p和miR-20a的表达能够选择性诱导过表达miR-17-92基因簇的肺癌细胞凋亡。那么,作为miR-17-92致瘤形成的关键,miR-19如何促进肺腺癌的发展,值得深入研究。
为深入探讨miR-19对肺腺癌细胞株A549的作用及其潜在机制,本课题将从以下几个方面进行研究：(1)检测miR-19在不同肺癌细胞中的表达,体内外实验观察miR-19对A549细胞株形态及功能(增殖、迁移)的影响；(2)基因芯片检测miR-19对A549细胞基因表达谱的影响；(3)miR-19调控A549发生EMT的潜在机制。通过上述研究可能会获得miR-19在A549细胞中的作用以及相关调控机制。研究结果如下：
1miR-19对A549细胞形态及迁移增殖功能的影响
1.1荧光定量RT-PCR检测5种肺癌细胞株与肺上皮细胞株中miR-19的表达
miR-19在5种肺癌细胞株中的表达均高于BEAS-2B细胞,在非小细胞肺癌细胞H23中表达最高,而在肺腺癌A549及A549-Luc中最低。单因素方差分析的结果表明,6种细胞株中miR-19的表达差异具有显著性(F=797.865,P<0.001)。通过2-△△ct法,与BEAS-2B细胞相比,A549中miR-19的表达增高了约1.941倍,A549-Luc中增高了约1.541倍,H446中增高了约2.458倍,H460中增高了约2.696倍,H23中增高了约4.419倍。
1.2pm19H2BmRFP和pm19(-)H2BmRFP慢病毒表达载体的构建
1.2.1miR-19a/19b和miR-19a(-)19b(-)序列的扩增
以质粒MIG19a/19b. MIG19a(-)19b(-)为模板扩增miR-19a/19b (784bp)、 miR-19a(-)19b(-)(981bp)两个序列,PCR产物进行1%琼脂糖凝胶电泳,在784bp、981bp左右可见一清晰条带。
1.2.2重组载体PCR鉴定
miR-19a/19b. miR-19a(-)19b(-)PCR产物纯化后与载体pHIV-H2BmRFP进行酶切、连接、转化,各挑克隆,小量摇菌后进行菌液PCR鉴定,结果显示pm19H2BmRFP有8个克隆可扩增出784bp的目的片段；pm19(-)H2BmRFP有9个克隆均可扩增出981bp的目的片段。
1.2.3重组载体测序鉴定
重组载体经测序鉴定与模板MIG19a/19b、MIG19a(-)19b(-)序列一致,说明重组载体pm19H2BmRFP. pm19(-)H2BmRFP构建成功。
1.2.4pm19H2BmRFP、pm19(-)H2BmRFP过表达载体及其空载慢病毒的包装及滴度测定
分别将三质粒包装系统(空载pHIV-H2BmRFP或pm19H2BmRFP或pm19(-)H2BmRFP. psPAX2和pMD2.G)共转染293T细胞,包装产生三种慢病毒,即空白对照慢病毒(pHIV-H2BmRFP)和过表达miR-19a/19b. miR-19a(-)19b(-)的慢病毒(pm19H2BmRFP. pm19(-)H2BmRFP)。转染后48h,荧光显微镜下观察293T细胞发出红色荧光,RFP定位在核。经293T包装产生的慢病毒,采用逐孔稀释法感染293T细胞来测定病毒滴度,按公式计算得到病毒滴度。其中,空白对照慢病毒(pHIV-H2BmRFP)的病毒滴度为6.1x106TU/ml,过表达miR-19a/19b. miR-19a(-)19b(-)的慢病毒(pm19H2BmRFP. pm19(-)H2BmRFP)滴度为5.2x106TU/ml、5.0x106TU/ml.
1.3稳定过表达miR-19a/19b和miR-19a(-)19b(-)的肺癌细胞株的建立
将含有miR-19a/19b.miR-19a(-)19b(-)片段的慢病毒pm19H2BmRFP. pm19(-)H2BmrfP和空载对照慢病毒pHIV-H2BmRFP分别感染A549-Luc细胞,感染48h后荧光显微镜下观察A549-Luc细胞发出红色荧光,RFP定位在核。
1.4荧光定量RT-PCR鉴定过表达miR-19.miR-19(一)以及空载的A549细胞中miR-17,miR-18a, miR-19(19a和19b),miR-20a,miR.92a的表达
相对于未感染细胞A549-Luc,A549/RFP+/m19中miR-19的表达倍数约升高1.893倍(P=0.027)；A549/RFP+/m19(-)中miR-17的表达水平约升高0.839倍(P<0.001),miR-18a的表达水平相对降低了0.116(P=O.020),miR-20a的表达水平约升高6.938倍(P=0.005),miR-92a的表达水平约升高1.546倍(P<0.001)。
1.5miR-19a/19b.miR-19a(-)19b(-)对A549-Luc细胞形态的影响
过表达miR-19a/19b.miR-19a(-)19b(-)的A549-Luc细胞经过传代,细胞的形态发生了明显改变。与A549/RFP+/H2B组细胞相比,A549/RFP+/m19组细胞形态瘦长,呈纺锤样、成纤维细胞样形态,播散分布；A549/RFP+/m19(-)组细胞突起,聚集成团。经过多次传代后,各组细胞仍保持上述形态。
1.6miR-19a/19b.miR-19a(-)19b(-)对A549-Luc细胞体外增殖能力的影响
采用MTT法检测A549/RFP+/m19.A549/RFP+/m19(-)以及空载细胞的体外增殖能力。采用单因素重复测量因素的方差分析,结果表明A549/RFP+/m19和A549/RFP+/m19(-)组均较空载组A549/RFP+/H2B增殖减慢(P<0.001)。
平板克隆形成实验的结果同样显示A549/RFP+/H2B.A549/RFP+/m19. A549/RFP+/m19(-)三组细胞的增殖速度具有显著性差异(F=76.155,P<0.001)。以上两个实验结果均表明过表达miR-19.miR-19(-)后,抑制了肺腺癌A549细胞的体外增殖。
1.7miR-19a/19b.miR-19a(-)19b(-)过表达对A549-Luc细胞周期的影响
采用流式细胞术分析过表达miR-19a/19b.miR-19a(-)19b(-)的A549-Luc细胞周期的分布,结果显示,与空载A549/RFP+/H2B相比,细胞G1期细胞比例在A549/RFP+/m19(P=0.011)和A549/RFP+/m19(-)(P<0.001)中显著升高,S期细胞比例在A549/RFP+/m19(P=0.001)和A549/RFP+/ml9(-)(P<0.001)中显著降低,即在过表达miR-19a/19b、miR-19a(-)19b(-)的A549-Luc细胞中,细胞出现了G1向S期转化障碍,细胞被阻滞在G1期。
1.8miR-19a/19b、miR-19a(-)19b(-)-过表达对A549-Luc细胞体外迁移运动能力的影响
采用Transwell小室检测空载细胞A549/RFP+/H2B和A549/RFP+/ml9. A549/RFP+/ml9(-)细胞体外迁移运动能力的变化,结果发现三组细胞运动能力的差异具有显著性(F=58.135,P<0.001)。与A549/RFP+/H2B细胞相比,A549/RFP+/m19细胞穿过膜的细胞数显著增多(P<0.001),其运动能力显著升高；A549/RFP+/m19(-)穿过膜的细胞数显著减少(P<0.01),其运动能力显著降低。
1.9荧光定量RT-PCR检测3种肺癌细胞株中E-Cadherin、Vimentin的表达
与A549/RFP+/H2B相比,E-Cadherin在A549/RFP+/m19中表达降低(P<0.05),Vimentin在A549/RFP+/m19中表达升高(P<0.05),在A549/RFP+/ml9(-)中表达降低(P<0.05),均具有统计学意义。
1.10Western blot检测3种肺癌细胞株中E-Cadherin、Vimentin的表达
与A549/RFP+/H2B组相比,A549/RFP+/m19组的E-cadherin表达降低、vimentin表达升高；A549/RFP+/m19(-)组的E-cadherin降低不明显,vimentin表达下调明显。
1.11可视化观察A549/RFP+/H2B, A549/RFP+/m19肺癌细胞株的皮下成瘤
皮下移植A549/RFP+/H2B细胞的小鼠,生物发光信号的强度均随着时间的推移而明显增强(F=17.037,P<0.001)；皮下移植A549/RFP+/m19细胞的小鼠,生物发光信号的强度均随着时间的推移轻度增强(F=3.850,P<0.024),增殖速度明显低于移植A549/RFP+/H2B细胞的小鼠(P<0.001)
2miR-19对A549细胞基因表达谱的影响
2.1基因芯片表达谱的数据分析
共检测了47,000个基因的表达情况,聚类分析结果提示A549/RFP+/ml9和A549/RFP+/H2B两组细胞间基因表达存在明显差异。与空载组相比,miR-19过表达组上调表达的基因有586个,下调表达的基因有504个。
利用博奥生物有限公司MAS数据分析系统对A549/RFP+/ml9和A549/RFP+/H2B两种细胞间差异基因进行GO分析,结果显示显著富集的GOTerm主要涉及Signal transduction、Cell adhesion、Transcription、Oxidation reduction、EMT等方面。与A549/RFP+/H2B细胞相比,在A549/RFP+/m19细胞中与EMT相关的差异表达基因有31个上调表达,10个下调表达；其中,上调表达的有：CDH2(N-cadherin)、CDH11(OB-cadherin)、ITGA5、ITGB6、COL3A1、 COL1A1、COL5A1、FN1、CALD1、MMP1、MMP2、MMP9等；下调表达的有CDH1(E-cadherin), KRT19等。两种细胞中差异表达的EMT相关基因的GO功能分类分析结果显示这些差异基因主要参与了细胞的生理发育、代谢与生理调控等过程；Pathway分析显示这些基因相互间存在多条信号通路。
2.2荧光定量RT-PCR检测A549/RFP+/ml9和A549/RFP+/H2B细胞中E-Cadherin、N-cadherin、Vimentin、Fibronectin、snail、MMP1和MMP10的表达
与A549/RFP+/H2B相比,E-Cadherin在A549/RFP+/m19中表达降低,N-cadherin、Vimentin、FN1、Snail、MMP1和MMP10在A549/RFP+/m19中表达明显升高。
2.3Western blot检测A549/RFP+/ml9和A549/RFP+/H2B细胞中E-cadherin、 Vimentin、Fibronectin、snail、MMP1和MMP10的表达
与空载组相比,过表达miR-19的A549细胞中E-cadherin表达明显下调,Vimentin表达轻度上调,Fibronectin、snail、MMP1和MMP10表达明显上调。
3过表达miR-19诱导A549细胞发生EMT的分子机制初步研究
Western blot检测A549/RFP+/H2B和A549/RFP+/m19肺腺癌细胞株中Pten、 STAT3. p-STAT3. ITGA5. P53以及c-myc的表达水平。与A549/RFP+/H2B组相比,A549/RFP+/m19组的Pten. P53以及c-myc的表达下调、STAT3. p-STAT3以及ITGA5表达升高。
通过上述三个部分的实验,我们能够得出以下结论：
(1)过表达miR-19可诱导A549细胞向间充质细胞转化。
(2)miR-19可能通过抑制Pten表达,激活STAT3以及Snail,抑制了E-cadherin表达、上调了Vimentin及ITGA5的表达,最终诱导A549细胞向间充质细胞转化。
Lung cancer, the leading cancer-related cause of death, is the most dangerous cancer to human health and life worldwide. In recent years, the death rate of lung cancer was obviously increased and became the the fastest growth rate and leading cancer-related cause of death in China. The traditional method of lung cancer treatment is usually based on the histopathological types of lung cancer, which mainly divided into two types, namely small cell lung cancer and non-small cell lung cancer(NSCLC). NSCLC consists of squamous-cell carcinoma, large cell carcinoma, and adenocarcinoma. Lung adenocarcinoma is the most common histological subtypes of lung cancer, accounting for70%of global lung cancer. The best treatment for NSCLC is surgical resection. Postoperative5-year survival rate for NSCLC patients in stage I can reach73%. Sadly, most lung cancer is diagnosed too late for curative treatment. Although great progresses have been made in understanding the disease, the therapeutic effect not gained significant improvement and the5-year survival rate was only16%. Metastasis was the leading cause of treatment failure and death of patients. Previous studies demonstrated that epithelial-to-mesenchymal transition was the important process of distant metastasis of cancer. Deeply investigating the role of EMT in the development of lung cancer was the key for lung cancer prevention and remedy.
Functional proteins are considered as the executor of the functional gene, mRNA is the intermediay. With the completion of the human genome project, the genome sequence encoding protein only accounts for2%of the entire genome sequence, but the rest of98%of genome sequence encodes a lot of the noncoding RNA. Nc-RNAs, especially small Nc-RNAs, such as microRNAs, make the gene regulation into RNA era.
MiRNAs are a class of small RNAs (21-24nt) that regulate the expression of target genes at the post-transcriptional level by base pairing with the3'untranslated regions (UTRs) of mRNAs and promoting mRNA unstability. They are first transcribed from miRNA genes in the genome as primary miRNAs (pri-miRNAs) which are processed into the mature miRNAs through two sequential cleavage steps and exported from the nuclei to cytoplasm. Firstly, depending on the complementarity between miRNA and3'untranslated region (UTR) of target mRNA there are three known mechanisms of miRNAs action on mRNAs:1) target mRNA degradation when miRNA is near-perfectly complementary with target mRNA,2) translational inhibition with little or no influence on mRNA levels when miRNA is only partially complementary to its target mRNA and3) the above two ways coexist. Application of bioinformatics method can predict the same target genes exist in multiple microRNA binding site based on the above mechanism and characteristics of the sequence of microRNA5'2-8bits nucleotide should fully complementary to the target mRNA3'UTR, indicating regulation of microRNA target gene expression possibly through diverse ways. In addition to the negative regulation function, minority microRNA also can positive regulate gene expression. Secondly, it is evident that single miRNAs may regulate translation of numerous downstream mRNAs and each mRNA is likely to be regulated by several miRNAs simultaneously. At last, microRNA regulate gene expression depending on the context of cells and tissues and the physiological and pathological state. It is obvious that the regulatory process of microRNA is complicated.
miRNAs are implicated in cell differentiation, proliferation, apoptosis, and various physiologic and pathologic processes, including growth and development. Clinical studies showed that microRNAs were closely related to the occurrence of lung cancer, for example, miR-146b, miR-155, let-7e, miR-34a, miR-34c-4p, miR-25, miR-191were closely related to the prognosis of NSCLC. Defects in the biogenesis of microRNAs are contributed to the development of lung cancer. Dicer is the best-established regulator of miRNA processing. Deletion of Dicer abrogates the production of mature miRNAs. Reduction of Dicer expression levels was correlated with poor survival of NSCLC patients. Specical microRNAs are existed in lung cancer. More than40microRNAs are involvd in the pathogenesis of lung cancer, including let-7a, miR-17-92, miR-34, miR-21, miR-29, miR-155, miR-31, and miR-200et al, they decreased or increased in lung cancer and function as oncogene or anti-oncogene. At present, the representatives of tumor suppressor microRNA in lung cancer are let-7, miR-29and miR-34a.
MiR-17-92, a polycistronic microRNA Cluster, comprises six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a) and resides in intron3of the C13orf25gene at13q31.3. miR-17-92is overexpressed in cancer, such as lung cancer, B-cell lymphomas and liver cancer et al. MiR-17-92promotes the development of cancer by targeting proapoptotic gene Bim, anti-oncogene p21and pten, TGFβ, TGFBR2, Rbl, TSP-1, CTGF and E2F1. MiR-19, a key oncogenic component for miR-17-92, promotes the proliferation of cancer cells by suppressing the expression of pten and Bim.
MiR-17-92family members play an important role in lung cancer. Supression of miR-17-5p and miR-20a can lead lung cancer cells overexpressing miR-17-92to apoptosis. Thus, it is valuable for studying the effect of miR-19on the development of lung adenocarcinoma.
To further investigate the effect and the potential mechanism of miR-19on the lung adenocarcinoma cell line A549, the following several aspects will be studied:(1) determining the expression level of miR-19in several lung cancer cell lines, observing the effect of miR-19on morphology, proliferation and migration of A549 cells in vivo and vitro;(2) determining the effect of miR-19on gene expression pattern of A549cells;(3) the regulation mechanism of miR-19on A549. The effect and potential mechanism of miR-19on A549will be obtained though above research.
The results are as follows:
1Effect of miR-19on the morphology, proliferation and migration of A549cells
1.1Determining the expression of miR-19in5lung cancer cell line using quantitative RT-PCR
The relative expression quantity are calculated by2-△△Ct. The expression of miR-19are higher in5lung cancer cell lines than that in BEAS-2B. The expression of ml9is the highest in non small cell lung cancer cell line H23, but the lowest in lung adenocarcinoma cell line A549and A549-Luc-CM-1. The statistics of One Way Anova showed that the expression of miR-19were significantly different in6lung cell line(F=797.865, P<0.001). Using2-△△Ct method, compared with BEAS-2B cell line, miR-19increased1.941,1.541,2.458,2.696,4.419-fold in A549, A549-luc, H446, H460and H23, respectively.
1.2Generation of lentivirus vector of pm19H2BmRFP, pm19(-)H2BmRFP
1.2.1Amplification of the sequence of miR-19a/19b; miR-19a(-)19b(-)
Amplification of the sequence of miR-19a/19b (784bp); miR-19a(-)19b(-)(981bp) based on MIG19a/19b; MIG19a(-)19b(-) plasmid. The PCR products were electrophoresed in1%agarose gel, and distinct bands were appeared at784bp and981bp, respectively.
1.2.2Identification the recombinant vector by PCR
The PCR products of miR-19a/19b, miR-19a(-)19b(-) were cut by Enzymatic digestion and purified, ligated, transformed, then colonies were selected to be identified. The result showed that the target fragment located at784bp,981bp were amplified from eight pm19H2BmRFP colonies and nine pm19(-)H2BmRFP colonies.
1.2.3Identificating sequence of the recombinant vector
The sequence of the recombinant vectors are in accordance with MIG19a/19b; MIG19a(-)19b(-), indicating the success of generation of pm19H2BmRFP and pm19(-)H2BmRFP.
1.2.4Lentivirus package and titer determination of control vector and overexpression vectors of pm19H2BmRFP and pm19(-)H2BmRFP
Cotransfection293T cells with three plasmid package system (Control pHIV-H2BmRFP or pm19H2BmRFP or pm19(-)H2BmRFP, psPAX2and pMD2.G) produced three lentiviruses, including pHIV-H2BmRFP, pm19H2BmRFP and pm19(-)H2BmRFP.48h later, red fluorescence were observed in the nucleus of293T cells. Assaying virus titers by gradually diluting the virus to infect293T cells. The titers of virus of pHIV-H2BmRFP, pm19H2BmRFP and pm19(-)H2BmRFP are6.1x106TU/ml,5.2x106TU/ml and5.0x106TU/ml, respectively.
1.3Generation of lung cancer cell line overexpressing miR-19a/19b and miR-19a(-)19b(-)
The lentivirus of control PHIV-H2BmRFP, pm19H2BmRFP and pml9(-)H2BmRFP, encoding miR-19a/19b, miR-19a(-)19b(-) were used to infect A549-Luc cells, respectively.48h later, red fluorescence were observed in the nucleus of293T cells.
1.4Determining the miR-17, miR-18a, miR-19, miR-20a and miR-92a in A549-Luc cells overexpressing miR-19, miR-19(-) using quantitative PCR.
Compared with A549-Luc, miR-19increased1.893times in A549/RFP+/m19; miR-17, miR-20a, miR-92a increased0.839,6.938and1.546times respectively; miR-18a are not obviously changed in A549/RFP+/m19(-) cell.
1.5Effect of miR19a/19b and miR19a(-)19b(-) on the morphology of A549-Luc cell
Several passages later, the morphology of A549cells overexpressing miR-19a/19b and miR-19a(-)19b(-) changed. Compared with A549/RFP+/H2B cells, the morphology of A549/RFP+/m19showed elongation, spindle-like, fibroblast shape, sporadic distribution; A549/RFP+/m19(-) showed cell gibbosity, clump together.
1.6Effect of miR-19a/19b, miR-19a(-)19b(-) on the proliferation of A549cells.
Determining the ability of proliferation of A549/RFP+/m19, A549/RFP+/m19(-) and A549/RFP+/H2B using MTT assay. Single repeated measure ANOVA showed that the ability of proliferation was significantly different in three groups. Compare to A549/RFP+/H2B, the speed of proliferation of A549/RFP+/m19and A549/RFP+/m19(-) cells slowed down(P<0.001).
Flat colony formation assay experiments also showed that the ability of proliferation was significantly different in three groups(F=76.155, P<0.001). The above mentioned experiments showed overexpression of miR-19and miR-19(-) suppressed the proliferation of A549-Luc cell in vitro.
1.7Effect of overexpression of miR-19a/19b and miR-19a(-)19b(-) on cell cycle of A549-Luc
The cell cycle of A549-Luc overexpressing miR-19a/19b and miR-19a(-)19b(-) were determined using FACS. Compare to A549/RFP+/H2B, the ratio of G1phase in A549/RFP+/m19(P=0.011) and A549/RFP+/m19(-)(P<0.001) were significantly upregulated, the ratio of S phase in A549/RFP+/m19(P=0.001) and A549/RFP+/m19(-)(P<0.001) were significantly downregulated. This result showed that A549/RFP+/ml9and A549/RFP+/ml9(-) cells were blocked in G1phase.
1.8Overexpression of miR-19a/19b, miR-19a(-)19b(-) on the migration of A549cells.
Detection the migration ability of A549/RFP+/H2B, A549/RFP+/ml9and A549/RFP+/ml9(-) cells using Transwell chamber showed that the migration ability was different in three groups (F=57.284, P<0.001). Compared with A549/RFP+/H2B cells, the migration ability of A549/RFP+/m19cells obviously increased(P<0.01), but in A549/RFP+/m19(-) cells decreased significantly(P<0.01).
1.9Determined the expression of E-cadherin and Vimentin using quantitative RT-PCR
Compared with A549/RFP+/H2B, the expression of E-cadherin decreased in A549/RFP+/m19cell line(P<0.05), and increased in A549/RFP+/m19(-)(P<0.05), respectively; the expression of Vimentin increased in A549/RFP+/m19cell line(P<0.05), and decreased in A549/RFP+/m19(-)(P<0.05).
1.10Determined the expression of E-cadherin and Vimentin using Western blot
Compared with A549/RFP+/H2B, the expression of E-cadherin and Vimentin were downregulated and upregulated in A549/RFP+/m19cell line, respectively; the expression of E-cadherein was not significantly downregulated in A549/RFP+/ml9cell line, but the expression of Vimentin decreased in A549/RFP+/m19(-).
1.11In vivo optical imaging of subcutaneous xenografts of A549/RFP+/H2B and A549/RFP+/m19cells
For subcutaneous transplantation of A549/RFP+/H2B cells, the photons of bioluminescence had significantly increased over time(F=17.037, P<0.001), For subcutaneous transplantation of A549/RFP+/m19cells, we found that the photons of bioluminescence had also increased over time(F=3.850, P<0.024), but the proliferation speed significantly lowered than that of A549/RFP+/H2B cells(P<0.001).
2Effect of miR-19on the gene expression pattern of A549cells
2.1Data analysis on the gene expression pattern
47,000genes were determined. Cluster analysis showed significant difference of gene expression between A549/RFP+/ml9and A549/RFP+/H2B cells. Compared with control vector group,586genes were up expressed and504genes were down expressed in A549cell with overexpression of miR-19(A549/RFP+/m19).
Using MAS data analysis system derived from Boao corporation, GO was used to analyze the differentially expressed genes between A549/RFP+/ml9and A549/RFP+/H2B cells. The result showed that GO Term were significantly enriched in Signal transduction, Cell adhesion, Transcription, Oxidation reduction and EMT. Compared with A549/RFP+/H2B cells, the marker of EMT, such as CDH2(N-cadherin), CDH11(OB-cadherin), ITGA5, ITGB6, COL3A1, COL1A1, COL5A1, FN1, CALD1, MMP1, MMP2and MMP9were upregulated, while CDHl(E-cadherin) and KRT19were downregulated in A549/RFP+/H2B cells. Furthermore, differentially expressed genes related to EMT were analyzed by GO functional classification. The result showed that these gene were involved in the process of physiogenesis, metabolism and physiology regulation. Pathway analysis showed these genes had multiple signal pathway between each other.
2.2Expression of E-Cadherin, N-cadherin, Vimentin, Fibronectin, snail, MMP1and MMP10was examined in A549/RFP+/H2B and A549/RFP+/ml9using quantitative PCR.
Compared with A549/RFP+/H2B, E-cadherin expression decreased in A549/RFP+/m19, while the expression of N-cadherin, Vimentin, FN1, Snail, MMP1and MMP10significantly increased in A549/RFP+/m19.
2.3Expression of E-Cadherin, Vimentin, Fibronectin, snail, MMP1and MMP10were detected in A549/RFP+/H2B and A549/RFP+/m19using western blot.
Compared with A549/RFP+/H2B, E-cadherin expression was obviously downregulated in A549/RFP+/m19. Vimentin expression was slightly upregulated, while expression of Fibronectin、snail、MMP1and MMP10were obviously upregulated in A549/RFP+/m19.
3Preliminary study on the molecular mechanism of induction EMT of A549by miR-19
The expression of Pten、STAT3、p-STAT3、ITGA5. P53and c-myc was detected in A549/RFP+/H2B cells and A549/RFP+/m19cells using western blot. Compared with A549/RFP+/H2B, Pten, P53and c-myc were downexpressed, while STAT3, P-STAT3and ITGA5was upexpressed in A549/RFP+/m19cells.
From the above3sections experiments, we can conclude that:
(1) miR-19can induce the transformation of human lung adenocarcinoma cell line A549to the mesenchymal cell.
(2) miR-19promotes the transformation of A549cells to mesenchymal cells perhaps via suppression of Pten, following activation of STAT3and Snail, and suppression of E-cadherin, upregulation of Vimentin and ITGA5.
引文
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