miR-195对人脑胶质瘤生物学行为的影响及相关机制的初步研究
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摘要
恶性胶质瘤是最常见的中枢神经系统恶性肿瘤，具有复发率高、致死、致残率高的特点，严重危害着人类的健康。尽管目前国内外治疗胶质瘤采用手术后辅以放疗、替莫唑胺化疗相结合的综合治疗方案，高级别胶质瘤尤其是胶质母细胞瘤不仅仍然无法治愈，而且中位生存期没有得到显著延长。这与胶质瘤细胞增殖过度、凋亡抵抗、侵袭浸润以及对化疗药物产生耐受的生物学行为特点密切相关。因此，寻找能够有效抑制胶质瘤细胞恶性生物学行为的分子靶点，对于胶质瘤的治疗具有重要的科学意义和临床应用价值。
MicroRNA (miRNA)是一种长约22个核苷酸的内源性小分子非编码RNA，主要通过与目的基因mRNA3’UTR区完全或者不完全结合，实现在转录后水平对基因表达的调控。miRNA的作用方式为“一对多”，与目标基因组成调控网络，参与胚胎发育，细胞增殖凋亡、新生血管生成等多种生物学过程。由于miRNAs的靶基因倾向富集于同一个或某几个信号通路的特点，也使得它对细胞功能和表型的调节更为迅速和有力。miRNA表达还具有时序性、保守性和组织特异性，同时它能够稳定存在于各种体液甚至福尔马林固定的石蜡组织标本（FFPE）中，因此，它越来越成为肿瘤诊断、治疗及预后相关的理想的分子标记物。近期大量研究表明，特异性miRNA的表达异常与胶质瘤细胞的无限增殖、凋亡受阻、侵袭迁移以及化疗耐药等恶性生物学行为调控密切相关。
MicroRNA-195（miR-195）是miR-15a/15b/16/195/497家族中的重要一员，基因定位于染色体17p13.1区域，距离抑癌基因P53仅650kb，该区域常发生杂合性缺失。研究显示，miR-195在肝癌、肺癌、结肠癌、胃癌、乳腺癌、原发性腹膜癌、膀胱肿瘤等多种恶性肿瘤中广泛下调，通过抑制肿瘤细胞的多种恶性生物学行为，发挥着抑癌基因的作用。但关于miR-195在胶质瘤中的生物学作用及相关机制尚未明确。课题组根据miRNA靶基因数据库miRDB，TargetScan和PicTar predictions预测和目前国内外已确认的miR-195作用靶点，推测miR-195在调控肿瘤细胞周期G1/S期转换和DNA损伤应答反应中发挥着重要作用，进而影响肿瘤细胞的恶性生物学行为。因此，本研究以miR-195为研究对象，分别检测miR-195在胶质瘤FFPE组织、胶质瘤细胞株及胶质瘤替莫唑胺耐药株中的表达情况，然后通过改变miR-195的表达水平，观察其对胶质瘤细胞增殖凋亡、侵袭迁移、克隆形成、体内成瘤以及耐药等生物学行为的影响，进而结合miRNA靶基因预测数据库的结果，从分子水平研究miR-195影响胶质瘤细胞恶性生物学行为可能的分子机制，这些研究将有助于深入理解miR-195的生物学功能和胶质瘤细胞恶性生物学行为的调控机制，为提高胶质瘤诊疗效果提供新的预测分子和干预靶点，具有重要的理论意义和应用前景。
本课题将从三个方面进行研究：
1. miR-195在人脑胶质瘤FFPE组织及细胞系中的表达及意义：
目的：研究miR-195在人脑胶质瘤福尔马林固定石蜡包埋组织及细胞系中的表达情况，分析其与胶质瘤临床病理分级之间的关系，为进一步探索miR-195在胶质瘤体内外中的生物学作用奠定基础。
方法：采用实时荧光定量逆转录-聚合酶链式反应（qRT-PCR）法检测miR-195在人脑胶质瘤细胞系SHG-44、U87、U251，人星形胶质细胞系，28例不同病理级别人脑胶质瘤福尔马林固定石蜡包埋的组织及2例正常脑组织中的表达情况。
结果：（1）qRT-PCR法检测结果显示，与正常人星形胶质细胞（HA）相比，三种细胞株SHG-44、U87、U251中miR-195含量均明显下调，其中SHG-44下调程度最高，而U251下调程度最小，差异具有统计学意义(P<0.05)；28例胶质瘤FFPE组织中miR-195含量均低于正常脑组织，差异有统计学意义（P<0.05）；（2）相关性分析：应用LSD法对四个不同病理级别组进行两两比较，结果显示：Ⅰ级、Ⅱ级之间miR-195表达差别无统计学意义（P>0.05），Ⅰ级、Ⅱ级分别和Ⅲ、Ⅳ级之间差异有统计学意义（P＜0.05），Ⅲ、Ⅳ级miR-195表达高于Ⅰ级、Ⅱ级。Spearman秩相关分析得出miR-195表达与胶质瘤患者的临床病理分级呈负相关（r=-0.798，P＜0.001）。
2. miR-195影响人脑胶质瘤细胞增殖凋亡和侵袭迁移能力的体内外研究：
目的：观察上调miR-195表达水平对人脑胶质瘤细胞增殖凋亡和侵袭迁移能力的影响。
方法：实验分为空白对照组（Blank）组，无义序列组（NC组）和miR-195过表达组（miR-195组）；首先利用阳离子脂质体LipofectamineTM2000将cy5标记的miR-195的模拟物(mimics)、无义序列分别转染入胶质瘤细胞株U251和SHG-44细胞中，激光共聚焦观察转染情况，流式细胞仪检测转染效率并筛选最佳转染浓度；实时荧光定量聚合酶链反应(qRT-PCR)法测定miR-195的表达情况；CCK-8法和平板克隆形成实验检测细胞增殖能力；AnnexinV-FITC/PI双染法和Hoechst33342/PI双染法检测细胞凋亡变化；流式细胞仪检测细胞周期；伤痕愈合实验检测细胞迁移能力；Transwell小室检测细胞侵袭能力；免疫细胞化学检测Bcl-2的表达；最后我们进一步评估了过表达miR-195对裸鼠皮下胶质瘤模型生长的影响。
结果:（1）miR-195mimics转入到胶质瘤细胞株U251和SHG-44后，在激光共聚焦下观察到较强的红色荧光，流式细胞仪筛选出miR-195mimics最佳转染浓度为50nM，转染效率达90%以上；（2）qRT-PCR结果显示与Blank组相比，U251、SHG-44miR-195组胶质瘤细胞内miR-195含量分别上调9.98、65.97倍（P<0.05）；（3）CCK-8法和平板克隆形成实验结果显示，与NC组和Blank组相比，两种细胞株过表达miR-195后，细胞增殖能力均明显减弱，克隆形成能力降低（P<0.05）；（4）流式细胞仪和Hoechst33342/PI双染法检测细胞凋亡，结果显示与Blank组及NC组相比，miR-195组细胞凋亡和坏死比例明显增高（P<0.05）；（5）伤痕愈合实验结果显示，在U251中，过表达miR-195可以抑制细胞迁移（P<0.05），而在SHG-44中，miR-195组的细胞迁移率与其余两组间差异无统计学意义（P>0.05）；（6）Transwell侵袭实验结果显示，在U251中，过表达miR-195组可以减少穿过Matrigel膜的细胞数（P<0.05），而在SHG-44中，miR-195组穿过Matrigel膜的细胞数多于其余两组（P<0.05）；(7)流式细胞仪检测细胞周期结果显示，与Blank组及NC组相比，miR-195组在G0/G1期细胞明显增加，而S期细胞显著减少（P<0.05）；（8）利用microRNA靶点预测网站及相关生物学信息软件，发现Bcl-2为miR-195的一个靶基因，免疫细胞化学结果显示过表达miR-195可下调Bcl-2的表达；（9）利用裸鼠皮下胶质瘤模型观察miR-195对胶质瘤形成的影响，结果显示miR-195组裸鼠瘤结节出现较晚，生长速度较慢，肿瘤剥离后重量和体积也明显低于NC组和Blank组（P<0.05）；（10）裸鼠肿瘤标本免疫组化分析结果显示，与NC组和Blank组相比，miR-195组Ki67表达下调（P<0.05）。
3. miR-195在人脑胶质瘤对替莫唑胺形成耐药中的作用及机制研究：
目的：研究miR-195在人脑胶质瘤对替莫唑胺形成耐药中的作用及机制。
方法：使用TMZ浓度递增的方法诱导构建胶质瘤耐药株，qRT-PCR法明确胶质瘤耐药株中miR-195的表达变化情况，然后改变耐药株中miR-195的表达水平，CCK-8检测胶质瘤细胞对TMZ耐药性、增殖能力的改变情况；流式细胞仪检测细胞凋亡和细胞周期变化；Western blot检测MGMT、P21、Bcl-2及Cyclin D1的表达；进一步构建裸鼠皮下胶质瘤模型，观察肿瘤生长情况以及对替莫唑胺的敏感性变化。
结果：（1）历时6个月成功建立了对TMZ耐药的U251、SHG44耐药株，分别命名为U251R、SHG-44R；（2）qRT-PCR结果显示，胶质瘤TMZ耐药株U251R、SHG-44R中miR-195含量分别下调至亲本细胞的40%、48%，SHG-44R下调更为显著（P<0.05），因此选择SHG-44行后续实验；（3）CCK-8法结果显示上调miR-195增加SHG-44R对TMZ的敏感性，而敲低miR-195则一定程度上增加SHG44R对TMZ的耐药性（P<0.05）；（4）流式细胞仪检测细胞凋亡结果显示过表达miR-195可明显增加胶质瘤耐药株的总凋亡率（P<0.05），敲低miR-195可略降低总凋亡率，但与对照组相比，差异无统计学意义（P>0.05）；（5）细胞周期结果显示上调SHG-44R细胞中的miR-195表达水平，胶质瘤细胞生长减慢，传代时间延长，G0/G1期细胞增加，S期细胞下降（P<0.05），而敲低miR-195对胶质瘤耐药细胞株的细胞周期影响与对照组差异不明显（P>0.05）；（6）Western blot结果显示过表达miR-195可以下调P21、Cyclin D1、Bcl-2的表达，而敲低miR-195后蛋白表达与之相反（P<0.05），MGMT表达水平不受miR-195表达变化影响；（7）裸鼠皮下胶质瘤模型和TUNEL染色结果显示miR-195联合TMZ组抑制胶质瘤生长及促进耐药的胶质瘤细胞凋亡最为显著（P<0.05）。
结论:（1）miR-195在所有已检测的胶质瘤FFPE标本及细胞系中均表达下调，并且表达程度与胶质瘤病理分级呈负相关；提示我们可以将其作为胶质瘤I-IV级新的有效的诊断标记物，同时作为常规组织病理学的分级标准的补充；（2）过表达miR-195能够减弱胶质瘤细胞体内外的增殖能力，诱导其G0/G1期阻滞，下调Bcl-2表达，促进胶质瘤细胞凋亡和坏死；但是miR-195对胶质瘤细胞侵袭迁移能力的影响与细胞类型有关，尚需进一步研究；总体上讲，miR-195能够负性调控胶质瘤细胞的恶性生物学行为；（3）miR-195在胶质瘤耐药株中低表达，上调miR-195含量，可以诱导耐药细胞凋亡和增殖受限，下调P21、Cyclin D1、Bcl-2的表达，一定程度上恢复体内外胶质瘤细胞对替莫唑胺的化疗敏感性，而这一作用与MGMT表达变化无关。综上述，miR-195有望成为胶质瘤基因治疗新的靶点。
Malignant glioma, as the most common malignant tumor of the central nervoussystem, features a high rate of recurrence, mortality and disability, which is severelyharmful to human health. Despite intense efforts have gone into finding effectivetreatments, high-grade malignant gliomas, especially glioblastoma (GBM), arepresently still incurable. Temozolomide (TMZ) as a first-line chemotherapeuticagent, in addition to radiotherapy and surgical resection, improved both the overallsurvival and progression-free survival in patients with newly diagnosed GBM. Butmedian survival of GBM patients has not been significantly prolonged, which isclosely related to the biological behavior characteristics of glioma cells such asunlimited proliferation, apoptosis obstacle, enhanced invasion and resistance tochemotherapy drugs. Therefore, trying to find a molecular target that can effectivelyinhibit malignant biological behavior of glioma cells, will have great scientificsignificance and clinical application value in the treatment of glioma.
MicroRNAs (miRNA) are small, non-coding21-23nucleotide RNAs thatregulate gene expression by binding to the untranslated regions of their targetmRNA molecules, then repressing transcription or inducing mRNA degradation. AmiRNA can regulate hundreds of target genes with which it forms a complexnetwork. Through the network, a miRNA is widely involved in embryonicdevelopment, cell proliferation, differentiation, apoptosis, cell cycle, angiogenesisand other biological processes. Moreover, target genes of miRNAs often focus onone or several signal pathways, which makes them regulate cell function and phenotype more rapidly and forcefully. MiRNAs have the characteristics of timesequence, conservatism and tissue specificity. Besides, they can exist stably invarious body fluids, even in formalin-fixed and paraffin-embedded (FFPE) tissues.Therefore, it is expected to be an ideal molecular marker for tumor diagnosis,treatment and prognosis. Recently, a large number of studies show that theexpression of specific miRNA is closely linked to the regulation of malignantbiological behaviors of glioma cells, such as unrestrained proliferation, abnormalapoptosis, migration, invasion and drug resistance.
MicroRNA-195(miR-195) is an essential member of themiR-15a/15b/16/195/497family, located on chromosome17p13.1. The distancebetween miR-195and tumor suppressor gene P53is only650kb, where loss ofheterozygosity frequently occurs. Researches show that down-regulation of miR-195exists widely in various malignant tumors, including liver cancer, lung cancer, coloncancer, gastric cancer, breast cancer, primary peritoneal cancer, bladder cancer,tongue squamous cell carcinoma and so on. It plays a critical role in suppressingtumor growth through regulating a variety of biological behaviors of malignanttumor cells. Nevertheless, the biological role and the related mechanism of miR-195in glioma were not yet clear. According to the prediction based on the databases ofmiRNA target genes： miRDB, TargetScan and PicTar and domestically andinternationally confirmed miR-195target genes, we speculated that miR-195playeda crucial role in the regulation of G1/S transition of tumor cell cycle and the DNAdamage response, thereby affecting the malignant biological behavior of tumor cells.Therefore, our present study chose miR-195as the research object. The expressionlevels of miR-195in FFPE glioma tissues, glioma cells and TMZ-resistant strainwere examined. Then by changing the expression of miR-195, we observed its effect on the biological behaviors of glioma cells, including proliferation, migration,invasion, clone formation, tumor formation in vivo and chemotherapy resistance. Inlight of the findings above and the information collected from the database used forpredicting miRNA target gene, the possible molecular mechanism of the miR-195regulating the malignant biological behaviors of glioma cells was explored. Theresearch will provide an in-depth understanding of the biological functions ofmiR-195and the regulatory mechanisms of malignant biological behaviors ofglioma cells. It is of theoretical value and practical significance.
The current study was performed from three aspects as follows.
1Expression and clinical significance of miR-195in FFPE human gliomatissues and cell lines:
Objective: To investigate the expression of miR-195in human brain gliomaFFPE tissues and cell lines and analyze the correlation between miR-195expressionand pathological grades, laying a foundation for further exploring the biologicalfunction of miR-195in glioma in vitro and in vivo.
Methods: The expression of miR-195was detected in human glioma cell linesSHG-44, U87, U251, human astrocyte cell line,28cases human differentpathological grades glioma FFPE samples and2cases normal brain tissues byreal-time fluorescent quantitative reverse transcriptase polymerase chain reaction(qRT-PCR).
Results:(1) qRT-PCR results showed that compared to normal humanastrocytes (HA), miR-195levels of three cell lines SHG-44, U87, U251weresignificantly reduced, with the highest degree of reduction in SHG-44and the lowestin U251(P<0.05).(2) The expression level of miR-195in28cases of glioma FFPEtissues was lower than that in normal brain tissues (P<0.05). There was no significant difference between grade I and grade II (P>0.05). There were differencesbetween grade I and grade III or grade IV (P<0.05). The difference also existedbetween grade II and grade III or grade IV (P<0.05). The expression of miR-195ingrade III or IV was significantly more than that in grade I or grade II (P<0.05). Theanalysis of correlation showed that the negative correlation existed betweenmiR-195expression level and pathological grades of human gliomas (r=0.798,P<0.001).
2Effects of miR-195on proliferation, apoptosis, migration and invasion ofhuman glioma cells in vitro and in vivo:
Objective: To observe the effects of upregulation of miR-195on proliferation,apoptosis, invasion and migration of human glioma cells in vitro and in vivo.
Methods: Control group (Blank group), nonsense sequence group (NC group)and miR-195over expression group (miR-195group) were designed in theexperiment. First, Cy5labeled miR-195mimics and nonsense sequences weretransfected into human glioma cell lines U251and SHG-44respectively by cationicliposome LipofectamineTM2000. Transfected cells were observed with confocallaser microscopy. The transfection efficiency was screened by flow cytometry andthe optimal concentration for transfection was identified. The expression of miR-195was determined by qRT-PCR. Second, cell proliferation was detected by CCK-8assay and colony formation assay. Apoptosis rate was assessed byAnnexinV-FITC/PI assay and Hoechst33342/PI staining. Cell cycle was analyzed byflow cytometry. Cell migratory ability was evaluated by wound healing assay. Cellinvasiveness was gauged by Transwell Matrigel invasion assay. The expressionlevel of Bcl-2protein was detected by immunocytochemical assay. Finally, we further investigated whether these effects could also be detected in subcutaneousglioma models of nude mice.
Results: After miR-195mimics were transfected into U251and SHG-44,strong red fluorescence can be observed by using the laser confocal. The besttransfection concentration of miR-195mimics was50nm, whose transfectionefficiency can be reached above90%. qRT-PCR results showed that compared withBlank group, miR-195contents in miR-195group of U251and SHG-44were up9.98,65.97times respectively (P<0.05). CCK-8assay and clone formationexperiment results showed that compared with the NC group and the Blank group,by overexpressing miR-195, the ability of cell proliferation was significantlydecreased with colony forming ability attenuated (P<0.05). Flow cytometry andHoechst33342/PI double staining were used to detect apoptosis. The results showedthat compared with the Blank group and the NC group, apoptosis and necrosis cellsin miR-195group rose sharply.(P<0.05). Wound healing assay results showed that,in U251, overexpression of miR-195can inhibit cell migration (P<0.05), but inSHG-44, there were no statistical differences on migration rate among three groups(P>0.05). Transwell invasion assay results showed that, in U251, overexpression ofmiR-195group can reduce the number of cells through the Matrigel membrane(P<0.05), but in SHG-44, the number of cells through the Matrigel membrane inmiR-195group was more than that in the other two groups (P<0.05). Cell cycleanalysis by flow cytometry showed that, in comparison with Blank group and NCgroup, the G0/G1phase cells markedly increased, while the S phase cells decreasedsignificantly in miR-195group (P<0.05). By microRNA target prediction websitesand related biological information software, we found that Bcl-2was an importanttarget gene of miR-195. And immunocytochemistry results indicated that overexpression of miR-195can downregulate the expression of Bcl-2. The effects ofmiR-195on the formation of glioma subcutaneous model in nude mice suggestedthat tumor nodules in miR-195group appeared later and grew at a slower rate. Bothtumor weight and tumor volume decreased markedly in miR-195group as comparedwith those of the NC group and the Blank group (P<0.05). Subsequently, tumorspecimens immunohistochemical analysis showed that, compared with the NC groupand the Blank group, the expression of Ki67reduced remarkably in miR-195group(P<0.05).
3Role and mechanism of miR-195in temozolomide-acquired resistance ofglioblastoma:
Objective: To study the role and mechanism of microRNA-195intemozolomide-acquired resistance of glioblastoma.
Methods: TMZ-resistant variants were developed by stepwise exposure toincreasing concentration of TMZ. The expression levels of miR-195in twoestablished resistant variants were detected by qRT-PCR. Then by changing theexpression level of miR-195in the resistant cells, we further examined the effects onproliferation and drug resistance of glioma cells by CCK-8assay. Cell apoptosis andcell cycle were analyzed by flow cytometry. The expressions of MGMT, P21, Bcl-2and Cyclin D1were detected by Western blot assay. Finally we establishedsubcutaneous glioma models in nude mice for observing the effects of up-regulatingmiR-195on the tumor growth and glioma susceptibility to TMZ.
Results: For6months we finally obtained the stable TMZ-resistant variants bystepwise increasing concentration of TMZ, named as U251R, SHG-44R. qRT-PCRresults showed that the expression levels of miR-195in U251R and SHG-44R wereless than that in the parental cells. Since SHG-44R reduced more significantly (P<0.05), SHG-44was chosen for subsequent experiments. CCK-8results showedthat the upregulation of miR-195increased the sensitivity to TMZ in SHG-44R,knockdown of miR-195increased the resistance to TMZ in SHG44R (P<0.05). Cellapoptosis was detected by flow cytometry. The results showed that overexpressionof miR-195can notably increase the total apoptosis rate of glioma resistant strain(P<0.05). Downregulation of miR-195can reduce the total apoptosis rate, but therewas no significant difference on total apoptosis rate in all three groups (P>0.05).Cell cycle analysis suggested that when the level of miR-195in SHG-44R wasupregualted, glioma cells grew more slowly and had a longer passage time withG0/G1phase cells increased and S phase cells decreased (P<0.05). There was nosignificant difference between miR-195inhibitor group and control group (P>0.05).Western blot assay showed that overexpression of miR-195can downregulate theexpression of P21, Cyclin D1, Bcl-2. Conversely, knockdown of miR-195increasedthese protein expressions(P<0.05). Notably the expression level of MGMT was notaffected by the changes of miR-195level. Subcutaneous glioma model in nude miceand TUNEL staining showed that miR-195combined with TMZ can inhibit gliomagrowth and promote glioma cell apoptosis significantly (P<0.05).
Conclusion:(1) miR-195was down-regulated in all the detected glioma FFPEspecimens and cell lines. And the expression level was negatively correlated withpathological grades of glioma. We may take it as a new effective glioma I-IV gradediagnostic marker and as a supplement to conventional histopathological gradingstandards.(2) Over expression of miR-195can attenuate the glioma cellsproliferation in vitro and in vivo, induce G0/G1arrest and downregulate Bcl-2expression. It also can increase apoptosis or necrosis of glioma cells. Nevertheless,its influence on migration and invasion of glioma cells may be associated with glioma cell types, and further studies were needed. In general, miR-195cannegatively regulate the malignant biological behaviors of glioma cells.(3) Theexpression of miR-195in glioblastoma resistant strains was much lower than that intheir parental cell lines. Upregulation of miR-195can inhibit proliferation, promotecell death and downregulate the expressions of P21, Cyclin D1and Bcl-2. MiR-195can restore in vitro and in vivo glioma cell sensitivity to TMZ treatment to a certainextent, which was not involved with MGMT. In summary, miR-195is expected tobecome a new potential gene therapeutic target of glioma.
MicroRNA (miRNA)是一种长约22个核苷酸的内源性小分子非编码RNA，主要通过与目的基因mRNA3’UTR区完全或者不完全结合，实现在转录后水平对基因表达的调控。miRNA的作用方式为“一对多”，与目标基因组成调控网络，参与胚胎发育，细胞增殖凋亡、新生血管生成等多种生物学过程。由于miRNAs的靶基因倾向富集于同一个或某几个信号通路的特点，也使得它对细胞功能和表型的调节更为迅速和有力。miRNA表达还具有时序性、保守性和组织特异性，同时它能够稳定存在于各种体液甚至福尔马林固定的石蜡组织标本（FFPE）中，因此，它越来越成为肿瘤诊断、治疗及预后相关的理想的分子标记物。近期大量研究表明，特异性miRNA的表达异常与胶质瘤细胞的无限增殖、凋亡受阻、侵袭迁移以及化疗耐药等恶性生物学行为调控密切相关。
MicroRNA-195（miR-195）是miR-15a/15b/16/195/497家族中的重要一员，基因定位于染色体17p13.1区域，距离抑癌基因P53仅650kb，该区域常发生杂合性缺失。研究显示，miR-195在肝癌、肺癌、结肠癌、胃癌、乳腺癌、原发性腹膜癌、膀胱肿瘤等多种恶性肿瘤中广泛下调，通过抑制肿瘤细胞的多种恶性生物学行为，发挥着抑癌基因的作用。但关于miR-195在胶质瘤中的生物学作用及相关机制尚未明确。课题组根据miRNA靶基因数据库miRDB，TargetScan和PicTar predictions预测和目前国内外已确认的miR-195作用靶点，推测miR-195在调控肿瘤细胞周期G1/S期转换和DNA损伤应答反应中发挥着重要作用，进而影响肿瘤细胞的恶性生物学行为。因此，本研究以miR-195为研究对象，分别检测miR-195在胶质瘤FFPE组织、胶质瘤细胞株及胶质瘤替莫唑胺耐药株中的表达情况，然后通过改变miR-195的表达水平，观察其对胶质瘤细胞增殖凋亡、侵袭迁移、克隆形成、体内成瘤以及耐药等生物学行为的影响，进而结合miRNA靶基因预测数据库的结果，从分子水平研究miR-195影响胶质瘤细胞恶性生物学行为可能的分子机制，这些研究将有助于深入理解miR-195的生物学功能和胶质瘤细胞恶性生物学行为的调控机制，为提高胶质瘤诊疗效果提供新的预测分子和干预靶点，具有重要的理论意义和应用前景。
本课题将从三个方面进行研究：
1. miR-195在人脑胶质瘤FFPE组织及细胞系中的表达及意义：
目的：研究miR-195在人脑胶质瘤福尔马林固定石蜡包埋组织及细胞系中的表达情况，分析其与胶质瘤临床病理分级之间的关系，为进一步探索miR-195在胶质瘤体内外中的生物学作用奠定基础。
方法：采用实时荧光定量逆转录-聚合酶链式反应（qRT-PCR）法检测miR-195在人脑胶质瘤细胞系SHG-44、U87、U251，人星形胶质细胞系，28例不同病理级别人脑胶质瘤福尔马林固定石蜡包埋的组织及2例正常脑组织中的表达情况。
结果：（1）qRT-PCR法检测结果显示，与正常人星形胶质细胞（HA）相比，三种细胞株SHG-44、U87、U251中miR-195含量均明显下调，其中SHG-44下调程度最高，而U251下调程度最小，差异具有统计学意义(P<0.05)；28例胶质瘤FFPE组织中miR-195含量均低于正常脑组织，差异有统计学意义（P<0.05）；（2）相关性分析：应用LSD法对四个不同病理级别组进行两两比较，结果显示：Ⅰ级、Ⅱ级之间miR-195表达差别无统计学意义（P>0.05），Ⅰ级、Ⅱ级分别和Ⅲ、Ⅳ级之间差异有统计学意义（P＜0.05），Ⅲ、Ⅳ级miR-195表达高于Ⅰ级、Ⅱ级。Spearman秩相关分析得出miR-195表达与胶质瘤患者的临床病理分级呈负相关（r=-0.798，P＜0.001）。
2. miR-195影响人脑胶质瘤细胞增殖凋亡和侵袭迁移能力的体内外研究：
目的：观察上调miR-195表达水平对人脑胶质瘤细胞增殖凋亡和侵袭迁移能力的影响。
方法：实验分为空白对照组（Blank）组，无义序列组（NC组）和miR-195过表达组（miR-195组）；首先利用阳离子脂质体LipofectamineTM2000将cy5标记的miR-195的模拟物(mimics)、无义序列分别转染入胶质瘤细胞株U251和SHG-44细胞中，激光共聚焦观察转染情况，流式细胞仪检测转染效率并筛选最佳转染浓度；实时荧光定量聚合酶链反应(qRT-PCR)法测定miR-195的表达情况；CCK-8法和平板克隆形成实验检测细胞增殖能力；AnnexinV-FITC/PI双染法和Hoechst33342/PI双染法检测细胞凋亡变化；流式细胞仪检测细胞周期；伤痕愈合实验检测细胞迁移能力；Transwell小室检测细胞侵袭能力；免疫细胞化学检测Bcl-2的表达；最后我们进一步评估了过表达miR-195对裸鼠皮下胶质瘤模型生长的影响。
结果:（1）miR-195mimics转入到胶质瘤细胞株U251和SHG-44后，在激光共聚焦下观察到较强的红色荧光，流式细胞仪筛选出miR-195mimics最佳转染浓度为50nM，转染效率达90%以上；（2）qRT-PCR结果显示与Blank组相比，U251、SHG-44miR-195组胶质瘤细胞内miR-195含量分别上调9.98、65.97倍（P<0.05）；（3）CCK-8法和平板克隆形成实验结果显示，与NC组和Blank组相比，两种细胞株过表达miR-195后，细胞增殖能力均明显减弱，克隆形成能力降低（P<0.05）；（4）流式细胞仪和Hoechst33342/PI双染法检测细胞凋亡，结果显示与Blank组及NC组相比，miR-195组细胞凋亡和坏死比例明显增高（P<0.05）；（5）伤痕愈合实验结果显示，在U251中，过表达miR-195可以抑制细胞迁移（P<0.05），而在SHG-44中，miR-195组的细胞迁移率与其余两组间差异无统计学意义（P>0.05）；（6）Transwell侵袭实验结果显示，在U251中，过表达miR-195组可以减少穿过Matrigel膜的细胞数（P<0.05），而在SHG-44中，miR-195组穿过Matrigel膜的细胞数多于其余两组（P<0.05）；(7)流式细胞仪检测细胞周期结果显示，与Blank组及NC组相比，miR-195组在G0/G1期细胞明显增加，而S期细胞显著减少（P<0.05）；（8）利用microRNA靶点预测网站及相关生物学信息软件，发现Bcl-2为miR-195的一个靶基因，免疫细胞化学结果显示过表达miR-195可下调Bcl-2的表达；（9）利用裸鼠皮下胶质瘤模型观察miR-195对胶质瘤形成的影响，结果显示miR-195组裸鼠瘤结节出现较晚，生长速度较慢，肿瘤剥离后重量和体积也明显低于NC组和Blank组（P<0.05）；（10）裸鼠肿瘤标本免疫组化分析结果显示，与NC组和Blank组相比，miR-195组Ki67表达下调（P<0.05）。
3. miR-195在人脑胶质瘤对替莫唑胺形成耐药中的作用及机制研究：
目的：研究miR-195在人脑胶质瘤对替莫唑胺形成耐药中的作用及机制。
方法：使用TMZ浓度递增的方法诱导构建胶质瘤耐药株，qRT-PCR法明确胶质瘤耐药株中miR-195的表达变化情况，然后改变耐药株中miR-195的表达水平，CCK-8检测胶质瘤细胞对TMZ耐药性、增殖能力的改变情况；流式细胞仪检测细胞凋亡和细胞周期变化；Western blot检测MGMT、P21、Bcl-2及Cyclin D1的表达；进一步构建裸鼠皮下胶质瘤模型，观察肿瘤生长情况以及对替莫唑胺的敏感性变化。
结果：（1）历时6个月成功建立了对TMZ耐药的U251、SHG44耐药株，分别命名为U251R、SHG-44R；（2）qRT-PCR结果显示，胶质瘤TMZ耐药株U251R、SHG-44R中miR-195含量分别下调至亲本细胞的40%、48%，SHG-44R下调更为显著（P<0.05），因此选择SHG-44行后续实验；（3）CCK-8法结果显示上调miR-195增加SHG-44R对TMZ的敏感性，而敲低miR-195则一定程度上增加SHG44R对TMZ的耐药性（P<0.05）；（4）流式细胞仪检测细胞凋亡结果显示过表达miR-195可明显增加胶质瘤耐药株的总凋亡率（P<0.05），敲低miR-195可略降低总凋亡率，但与对照组相比，差异无统计学意义（P>0.05）；（5）细胞周期结果显示上调SHG-44R细胞中的miR-195表达水平，胶质瘤细胞生长减慢，传代时间延长，G0/G1期细胞增加，S期细胞下降（P<0.05），而敲低miR-195对胶质瘤耐药细胞株的细胞周期影响与对照组差异不明显（P>0.05）；（6）Western blot结果显示过表达miR-195可以下调P21、Cyclin D1、Bcl-2的表达，而敲低miR-195后蛋白表达与之相反（P<0.05），MGMT表达水平不受miR-195表达变化影响；（7）裸鼠皮下胶质瘤模型和TUNEL染色结果显示miR-195联合TMZ组抑制胶质瘤生长及促进耐药的胶质瘤细胞凋亡最为显著（P<0.05）。
结论:（1）miR-195在所有已检测的胶质瘤FFPE标本及细胞系中均表达下调，并且表达程度与胶质瘤病理分级呈负相关；提示我们可以将其作为胶质瘤I-IV级新的有效的诊断标记物，同时作为常规组织病理学的分级标准的补充；（2）过表达miR-195能够减弱胶质瘤细胞体内外的增殖能力，诱导其G0/G1期阻滞，下调Bcl-2表达，促进胶质瘤细胞凋亡和坏死；但是miR-195对胶质瘤细胞侵袭迁移能力的影响与细胞类型有关，尚需进一步研究；总体上讲，miR-195能够负性调控胶质瘤细胞的恶性生物学行为；（3）miR-195在胶质瘤耐药株中低表达，上调miR-195含量，可以诱导耐药细胞凋亡和增殖受限，下调P21、Cyclin D1、Bcl-2的表达，一定程度上恢复体内外胶质瘤细胞对替莫唑胺的化疗敏感性，而这一作用与MGMT表达变化无关。综上述，miR-195有望成为胶质瘤基因治疗新的靶点。
Malignant glioma, as the most common malignant tumor of the central nervoussystem, features a high rate of recurrence, mortality and disability, which is severelyharmful to human health. Despite intense efforts have gone into finding effectivetreatments, high-grade malignant gliomas, especially glioblastoma (GBM), arepresently still incurable. Temozolomide (TMZ) as a first-line chemotherapeuticagent, in addition to radiotherapy and surgical resection, improved both the overallsurvival and progression-free survival in patients with newly diagnosed GBM. Butmedian survival of GBM patients has not been significantly prolonged, which isclosely related to the biological behavior characteristics of glioma cells such asunlimited proliferation, apoptosis obstacle, enhanced invasion and resistance tochemotherapy drugs. Therefore, trying to find a molecular target that can effectivelyinhibit malignant biological behavior of glioma cells, will have great scientificsignificance and clinical application value in the treatment of glioma.
MicroRNAs (miRNA) are small, non-coding21-23nucleotide RNAs thatregulate gene expression by binding to the untranslated regions of their targetmRNA molecules, then repressing transcription or inducing mRNA degradation. AmiRNA can regulate hundreds of target genes with which it forms a complexnetwork. Through the network, a miRNA is widely involved in embryonicdevelopment, cell proliferation, differentiation, apoptosis, cell cycle, angiogenesisand other biological processes. Moreover, target genes of miRNAs often focus onone or several signal pathways, which makes them regulate cell function and phenotype more rapidly and forcefully. MiRNAs have the characteristics of timesequence, conservatism and tissue specificity. Besides, they can exist stably invarious body fluids, even in formalin-fixed and paraffin-embedded (FFPE) tissues.Therefore, it is expected to be an ideal molecular marker for tumor diagnosis,treatment and prognosis. Recently, a large number of studies show that theexpression of specific miRNA is closely linked to the regulation of malignantbiological behaviors of glioma cells, such as unrestrained proliferation, abnormalapoptosis, migration, invasion and drug resistance.
MicroRNA-195(miR-195) is an essential member of themiR-15a/15b/16/195/497family, located on chromosome17p13.1. The distancebetween miR-195and tumor suppressor gene P53is only650kb, where loss ofheterozygosity frequently occurs. Researches show that down-regulation of miR-195exists widely in various malignant tumors, including liver cancer, lung cancer, coloncancer, gastric cancer, breast cancer, primary peritoneal cancer, bladder cancer,tongue squamous cell carcinoma and so on. It plays a critical role in suppressingtumor growth through regulating a variety of biological behaviors of malignanttumor cells. Nevertheless, the biological role and the related mechanism of miR-195in glioma were not yet clear. According to the prediction based on the databases ofmiRNA target genes： miRDB, TargetScan and PicTar and domestically andinternationally confirmed miR-195target genes, we speculated that miR-195playeda crucial role in the regulation of G1/S transition of tumor cell cycle and the DNAdamage response, thereby affecting the malignant biological behavior of tumor cells.Therefore, our present study chose miR-195as the research object. The expressionlevels of miR-195in FFPE glioma tissues, glioma cells and TMZ-resistant strainwere examined. Then by changing the expression of miR-195, we observed its effect on the biological behaviors of glioma cells, including proliferation, migration,invasion, clone formation, tumor formation in vivo and chemotherapy resistance. Inlight of the findings above and the information collected from the database used forpredicting miRNA target gene, the possible molecular mechanism of the miR-195regulating the malignant biological behaviors of glioma cells was explored. Theresearch will provide an in-depth understanding of the biological functions ofmiR-195and the regulatory mechanisms of malignant biological behaviors ofglioma cells. It is of theoretical value and practical significance.
The current study was performed from three aspects as follows.
1Expression and clinical significance of miR-195in FFPE human gliomatissues and cell lines:
Objective: To investigate the expression of miR-195in human brain gliomaFFPE tissues and cell lines and analyze the correlation between miR-195expressionand pathological grades, laying a foundation for further exploring the biologicalfunction of miR-195in glioma in vitro and in vivo.
Methods: The expression of miR-195was detected in human glioma cell linesSHG-44, U87, U251, human astrocyte cell line,28cases human differentpathological grades glioma FFPE samples and2cases normal brain tissues byreal-time fluorescent quantitative reverse transcriptase polymerase chain reaction(qRT-PCR).
Results:(1) qRT-PCR results showed that compared to normal humanastrocytes (HA), miR-195levels of three cell lines SHG-44, U87, U251weresignificantly reduced, with the highest degree of reduction in SHG-44and the lowestin U251(P<0.05).(2) The expression level of miR-195in28cases of glioma FFPEtissues was lower than that in normal brain tissues (P<0.05). There was no significant difference between grade I and grade II (P>0.05). There were differencesbetween grade I and grade III or grade IV (P<0.05). The difference also existedbetween grade II and grade III or grade IV (P<0.05). The expression of miR-195ingrade III or IV was significantly more than that in grade I or grade II (P<0.05). Theanalysis of correlation showed that the negative correlation existed betweenmiR-195expression level and pathological grades of human gliomas (r=0.798,P<0.001).
2Effects of miR-195on proliferation, apoptosis, migration and invasion ofhuman glioma cells in vitro and in vivo:
Objective: To observe the effects of upregulation of miR-195on proliferation,apoptosis, invasion and migration of human glioma cells in vitro and in vivo.
Methods: Control group (Blank group), nonsense sequence group (NC group)and miR-195over expression group (miR-195group) were designed in theexperiment. First, Cy5labeled miR-195mimics and nonsense sequences weretransfected into human glioma cell lines U251and SHG-44respectively by cationicliposome LipofectamineTM2000. Transfected cells were observed with confocallaser microscopy. The transfection efficiency was screened by flow cytometry andthe optimal concentration for transfection was identified. The expression of miR-195was determined by qRT-PCR. Second, cell proliferation was detected by CCK-8assay and colony formation assay. Apoptosis rate was assessed byAnnexinV-FITC/PI assay and Hoechst33342/PI staining. Cell cycle was analyzed byflow cytometry. Cell migratory ability was evaluated by wound healing assay. Cellinvasiveness was gauged by Transwell Matrigel invasion assay. The expressionlevel of Bcl-2protein was detected by immunocytochemical assay. Finally, we further investigated whether these effects could also be detected in subcutaneousglioma models of nude mice.
Results: After miR-195mimics were transfected into U251and SHG-44,strong red fluorescence can be observed by using the laser confocal. The besttransfection concentration of miR-195mimics was50nm, whose transfectionefficiency can be reached above90%. qRT-PCR results showed that compared withBlank group, miR-195contents in miR-195group of U251and SHG-44were up9.98,65.97times respectively (P<0.05). CCK-8assay and clone formationexperiment results showed that compared with the NC group and the Blank group,by overexpressing miR-195, the ability of cell proliferation was significantlydecreased with colony forming ability attenuated (P<0.05). Flow cytometry andHoechst33342/PI double staining were used to detect apoptosis. The results showedthat compared with the Blank group and the NC group, apoptosis and necrosis cellsin miR-195group rose sharply.(P<0.05). Wound healing assay results showed that,in U251, overexpression of miR-195can inhibit cell migration (P<0.05), but inSHG-44, there were no statistical differences on migration rate among three groups(P>0.05). Transwell invasion assay results showed that, in U251, overexpression ofmiR-195group can reduce the number of cells through the Matrigel membrane(P<0.05), but in SHG-44, the number of cells through the Matrigel membrane inmiR-195group was more than that in the other two groups (P<0.05). Cell cycleanalysis by flow cytometry showed that, in comparison with Blank group and NCgroup, the G0/G1phase cells markedly increased, while the S phase cells decreasedsignificantly in miR-195group (P<0.05). By microRNA target prediction websitesand related biological information software, we found that Bcl-2was an importanttarget gene of miR-195. And immunocytochemistry results indicated that overexpression of miR-195can downregulate the expression of Bcl-2. The effects ofmiR-195on the formation of glioma subcutaneous model in nude mice suggestedthat tumor nodules in miR-195group appeared later and grew at a slower rate. Bothtumor weight and tumor volume decreased markedly in miR-195group as comparedwith those of the NC group and the Blank group (P<0.05). Subsequently, tumorspecimens immunohistochemical analysis showed that, compared with the NC groupand the Blank group, the expression of Ki67reduced remarkably in miR-195group(P<0.05).
3Role and mechanism of miR-195in temozolomide-acquired resistance ofglioblastoma:
Objective: To study the role and mechanism of microRNA-195intemozolomide-acquired resistance of glioblastoma.
Methods: TMZ-resistant variants were developed by stepwise exposure toincreasing concentration of TMZ. The expression levels of miR-195in twoestablished resistant variants were detected by qRT-PCR. Then by changing theexpression level of miR-195in the resistant cells, we further examined the effects onproliferation and drug resistance of glioma cells by CCK-8assay. Cell apoptosis andcell cycle were analyzed by flow cytometry. The expressions of MGMT, P21, Bcl-2and Cyclin D1were detected by Western blot assay. Finally we establishedsubcutaneous glioma models in nude mice for observing the effects of up-regulatingmiR-195on the tumor growth and glioma susceptibility to TMZ.
Results: For6months we finally obtained the stable TMZ-resistant variants bystepwise increasing concentration of TMZ, named as U251R, SHG-44R. qRT-PCRresults showed that the expression levels of miR-195in U251R and SHG-44R wereless than that in the parental cells. Since SHG-44R reduced more significantly (P<0.05), SHG-44was chosen for subsequent experiments. CCK-8results showedthat the upregulation of miR-195increased the sensitivity to TMZ in SHG-44R,knockdown of miR-195increased the resistance to TMZ in SHG44R (P<0.05). Cellapoptosis was detected by flow cytometry. The results showed that overexpressionof miR-195can notably increase the total apoptosis rate of glioma resistant strain(P<0.05). Downregulation of miR-195can reduce the total apoptosis rate, but therewas no significant difference on total apoptosis rate in all three groups (P>0.05).Cell cycle analysis suggested that when the level of miR-195in SHG-44R wasupregualted, glioma cells grew more slowly and had a longer passage time withG0/G1phase cells increased and S phase cells decreased (P<0.05). There was nosignificant difference between miR-195inhibitor group and control group (P>0.05).Western blot assay showed that overexpression of miR-195can downregulate theexpression of P21, Cyclin D1, Bcl-2. Conversely, knockdown of miR-195increasedthese protein expressions(P<0.05). Notably the expression level of MGMT was notaffected by the changes of miR-195level. Subcutaneous glioma model in nude miceand TUNEL staining showed that miR-195combined with TMZ can inhibit gliomagrowth and promote glioma cell apoptosis significantly (P<0.05).
Conclusion:(1) miR-195was down-regulated in all the detected glioma FFPEspecimens and cell lines. And the expression level was negatively correlated withpathological grades of glioma. We may take it as a new effective glioma I-IV gradediagnostic marker and as a supplement to conventional histopathological gradingstandards.(2) Over expression of miR-195can attenuate the glioma cellsproliferation in vitro and in vivo, induce G0/G1arrest and downregulate Bcl-2expression. It also can increase apoptosis or necrosis of glioma cells. Nevertheless,its influence on migration and invasion of glioma cells may be associated with glioma cell types, and further studies were needed. In general, miR-195cannegatively regulate the malignant biological behaviors of glioma cells.(3) Theexpression of miR-195in glioblastoma resistant strains was much lower than that intheir parental cell lines. Upregulation of miR-195can inhibit proliferation, promotecell death and downregulate the expressions of P21, Cyclin D1and Bcl-2. MiR-195can restore in vitro and in vivo glioma cell sensitivity to TMZ treatment to a certainextent, which was not involved with MGMT. In summary, miR-195is expected tobecome a new potential gene therapeutic target of glioma.
引文
[1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell,2004,116:281-297.
[2] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinctsignatures in B cell chronic lymphocytic leukemias[J]. Proc Natl Acad Sci U S A,2004,101:11755-11760.
[3] Weber F, Teresi RE, Broelsch CE, et al. A limited set of human MicroRNAis deregulated in follicular thyroid carcinoma[J]. J Clin Endocrinol Metab,2006,91:3584-3591.
[4] Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouseand human[J]. RNA,2003,9:175-179.
[5] Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNAexpression atlas based on small RNA library sequencing[J]. Cell,2007,129:1401-1414.
[6] Flavin RJ, Smyth PC, Laios A, et al. Potentially important microRNAcluster on chromosome17p13.1in primary peritoneal carcinoma[J]. Mod Pathol,2009,22:197-205.
[7] Wong TS, Liu XB, Wong BY, et al. Mature miR-184as PotentialOncogenic microRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res,2008,14:2588-2592.
[8] Ichimi T, Enokida H, Okuno Y, et al. Identification of novel microRNAtargets based on microRNA signatures in bladder cancer[J]. Int J Cancer,2009,125:345-352.
[9]黄健清. miR-195对胃癌BCG823细胞株增殖与凋亡的影响[J].中华实验外科杂志,2011,28:1485-1487.
[10] Xu T, Zhu Y, Xiong Y, et al. MicroRNA-195suppresses tumorigenicityand regulates G1/S transition of human hepatocellular carcinoma cells[J].Hepatology,2009,50:113-121.
[11] Heneghan HM, Miller N, Lowery AJ, et al. Circulating microRNAs asnovel minimally invasive biomarkers for breast cancer[J]. Ann Surg,2010,251:499-505.
[1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell,2004,116:281-297.
[2] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinctsignatures in B cell chronic lymphocytic leukemias[J]. Proc Natl Acad Sci U S A,2004,101:11755-11760.
[3] Weber F, Teresi RE, Broelsch CE, et al. A limited set of human MicroRNAis deregulated in follicular thyroid carcinoma[J]. J Clin Endocrinol Metab,2006,91:3584-3591.
[4] Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouseand human[J]. RNA,2003,9:175-179.
[5] Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNAexpression atlas based on small RNA library sequencing[J]. Cell,2007,129:1401-1414.
[6] Flavin RJ, Smyth PC, Laios A, et al. Potentially important microRNAcluster on chromosome17p13.1in primary peritoneal carcinoma[J]. Mod Pathol,2009,22:197-205.
[7] Wong TS, Liu XB, Wong BY, et al. Mature miR-184as PotentialOncogenic microRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res,2008,14:2588-2592.
[8] Ichimi T, Enokida H, Okuno Y, et al. Identification of novel microRNAtargets based on microRNA signatures in bladder cancer[J]. Int J Cancer,2009,125:345-352.
[9]黄健清. miR-195对胃癌BCG823细胞株增殖与凋亡的影响[J].中华实验外科杂志,2011,28:1485-1487.
[10] Xu T, Zhu Y, Xiong Y, et al. MicroRNA-195suppresses tumorigenicityand regulates G1/S transition of human hepatocellular carcinoma cells[J].Hepatology,2009,50:113-121.
[11] Heneghan HM, Miller N, Lowery AJ, et al. Circulating microRNAs asnovel minimally invasive biomarkers for breast cancer[J]. Ann Surg,2010,251:499-505.
[12] Ujifuku K, Mitsutake N, Takakura S, et al. miR-195, miR-455-3p andmiR-10a(*) are implicated in acquired temozolomide resistance in glioblastomamultiforme cells[J]. Cancer Lett,2010,296:241-248.
[13] Soon PS, Tacon LJ, Gill AJ, et al. miR-195and miR-483-5p Identifiedas Predictors of Poor Prognosis in Adrenocortical Cancer[J]. Clinical cancerresearch: an official journal of the American Association forCancer Research,2009,15:7684-7692.
[14] Zanette DL, Rivadavia F, Molfetta GA, et al. miRNA expressionprofiles in chronic lymphocytic and acute lymphocytic leukemia[J]. Braz J Med BiolRes,2007,40:1435-1440.
[15] Hui W, Yuntao L, Lun L, et al. MicroRNA-195inhibits theproliferation of human glioma cells by directly targeting cyclin D1and cyclin E1[J].PLoS One,2013,8:e54932.
[16] Zhang QQ, Xu H, Huang MB, et al. MicroRNA-195plays atumor-suppressor role in human glioblastoma cells by targeting signaling pathwaysinvolved in cellular proliferation and invasion[J]. Neuro Oncol,2012,14:278-287.
[17] Liu L, Chen L, Xu Y, et al. microRNA-195promotes apoptosis andsuppresses tumorigenicity of human colorectal cancer cells[J]. Biochem BiophysRes Commun,2010,400:236-240.
[18] Qi J, Yu JY, Shcherbata HR, et al. microRNAs regulate humanembryonic stem cell division[J]. Cell Cycle,2009,8:3729-3741.
[19] Mellios N, Huang HS, Grigorenko A, et al. A set of differentiallyexpressed miRNAs, including miR-30a-5p, act as post-transcriptional inhibitors ofBDNF in prefrontal cortex[J]. Hum Mol Genet,2008,17:3030-3042.
[20] Nishi H, Ono K, Iwanaga Y, et al. MicroRNA-15b modulates cellularATP levels and degenerates mitochondria via Arl2in neonatal rat cardiacmyocytes[J]. J Biol Chem,2010,285:4920-4930.
[21] Deng H, Guo Y, Song H, et al. MicroRNA-195and microRNA-378mediate tumor growth suppression by epigenetical regulation in gastric cancer[J].Gene,2013,518:351-359.
[22] Fei X, Qi M, Wu B, et al. MicroRNA-195-5p suppresses glucoseuptake and proliferation of human bladder cancer T24cells by regulating GLUT3expression[J]. FEBS Lett,2012,586:392-397.
[23] Muller PA, Vousden KH. p53mutations in cancer[J]. Nat Cell Biol,2013,15:2-8.
[24] Yang X, Yin J, Yu J, et al. miRNA-195sensitizes human hepatocellularcarcinoma cells to5-FU by targeting BCL-w[J]. Oncol Rep,2012,27:250-257.
[25] Mao JH, Zhou RP, Peng AF, et al. microRNA-195suppressesosteosarcoma cell invasion and migration in vitro by targeting FASN[J]. Oncol Lett,2012,4:1125-1129.
[26] Wang R, Zhao N, Li S, et al. MicroRNA-195suppresses angiogenesisand metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF,VAV2, and CDC42[J]. Hepatology,2013,58:642-653.
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[1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell,2004,116(2):281-297.
[2] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinctsignatures in B cell chronic lymphocytic leukemias[J]. Proc Natl Acad Sci U S A,2004,101(32):11755-11760.
[3] Weber F, Teresi RE, Broelsch CE, et al. A limited set of human MicroRNA isderegulated in follicular thyroid carcinoma[J]. J Clin Endocrinol Metab,2006,91(9):3584-3591. PMID:16822819
[4] Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouseand human[J]. RNA,2003,9(2):175-179.
[5] Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expressionatlas based on small RNA library sequencing[J]. Cell,2007,129(7):1401-1414.
[6] Flavin RJ, Smyth PC, Laios A, et al. Potentially important microRNA clusteron chromosome17p13.1in primary peritoneal carcinoma[J]. Mod Pathol,2009,22(2):197-205.
[7] Wong TS, Liu XB, Wong BY, et al. Mature miR-184as Potential OncogenicmicroRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res,2008,14(9):2588-2592.
[8] Ichimi T, Enokida H, Okuno Y, et al. Identification of novel microRNAtargets based on microRNA signatures in bladder cancer[J]. Int J Cancer,2009,125(2):345-352.
[9]黄健清. miR-195对胃癌BCG823细胞株增殖与凋亡的影响[J].中华实验外科杂志,2011,28(9):1485-1487.
[10] Xu T, Zhu Y, Xiong Y, et al. MicroRNA-195suppresses tumorigenicityand regulates G1/S transition of human hepatocellular carcinoma cells[J]. Hepatology,2009,50(1):113-121.
[11] Heneghan HM, Miller N, Lowery AJ, et al. Circulating microRNAs asnovel minimally invasive biomarkers for breast cancer[J]. Ann Surg,2010,251(3):499-505.
[12] Ujifuku K, Mitsutake N, Takakura S, et al. miR-195, miR-455-3p andmiR-10a(*) are implicated in acquired temozolomide resistance in glioblastomamultiforme cells[J]. Cancer Lett,2010,296(2):241-248.
[13] Soon PS, Tacon LJ, Gill AJ, et al. miR-195and miR-483-5p Identifiedas Predictors of Poor Prognosis in Adrenocortical Cancer[J]. Clinical cancer research:an official journal of the American Association for Cancer Research,2009,15(24):7684-7692.
[14] Zanette DL, Rivadavia F, Molfetta GA, et al. miRNA expression profilesin chronic lymphocytic and acute lymphocytic leukemia[J]. Braz J Med Biol Res,2007,40(11):1435-1440.
[15] Zhang QQ, Xu H, Huang MB, et al. MicroRNA-195plays atumor-suppressor role in human glioblastoma cells by targeting signaling pathwaysinvolved in cellular proliferation and invasion[J]. Neuro Oncol,2012,14:278-287.
[16] Liu L, Chen L, Xu Y, et al. microRNA-195promotes apoptosis andsuppresses tumorigenicity of human colorectal cancer cells[J]. Biochem Biophys ResCommun,2010,400:236-240.
[17] Qi J, Yu JY, Shcherbata HR, et al. microRNAs regulate humanembryonic stem cell division[J]. Cell Cycle,2009,8:3729-3741.
[18] Mellios N, Huang HS, Grigorenko A, et al. A set of differentiallyexpressed miRNAs, including miR-30a-5p, act as post-transcriptional inhibitors ofBDNF in prefrontal cortex[J]. Hum Mol Genet,2008,17(19):3030-3042.
[19] Nishi H, Ono K, Iwanaga Y, et al. MicroRNA-15b modulates cellularATP levels and degenerates mitochondria via Arl2in neonatal rat cardiac myocytes[J].J Biol Chem,2010,285(7):4920-4930.
[20] Hui W, Yuntao L, Lun L, et al. MicroRNA-195inhibits the proliferationof human glioma cells by directly targeting cyclin D1and cyclin E1[J]. PLoS One,2013,8(1):e54932.
[21] Deng H, Guo Y, Song H, et al. MicroRNA-195and microRNA-378mediate tumor growth suppression by epigenetical regulation in gastric cancer[J].Gene,2013,518(2):351-359.
[22] Fei X, Qi M, Wu B, et al. MicroRNA-195-5p suppresses glucose uptakeand proliferation of human bladder cancer T24cells by regulating GLUT3expression[J]. FEBS Lett,2012,586(4):392-397.
[23] Yang X, Yin J, Yu J, et al. miRNA-195sensitizes human hepatocellularcarcinoma cells to5-FU by targeting BCL-w[J]. Oncol Rep,2012,27(1):250-257.
[24] Muller PA, Vousden KH. p53mutations in cancer[J]. Nat Cell Biol,2013,15(1):2-8.
[25] Mao JH, Zhou RP, Peng AF, et al. microRNA-195suppressesosteosarcoma cell invasion and migration in vitro by targeting FASN[J]. Oncol Lett,2012,4(5):1125-1129.
[26] Wang R, Zhao N, Li S, et al. MicroRNA-195suppresses angiogenesisand metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF,VAV2, and CDC42[J]. Hepatology,2013,58(2):642-653.
[27] Qu J, Zhao L, Zhang P, et al. MicroRNA-195chemosensitizes coloncancer cells to the chemotherapeutic drug doxorubicin by targeting the first bindingsite of BCL2L2mRNA[J]. J Cell Physiol,2013.
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[30] Ilhan-Mutlu A, Wohrer A, Berghoff AS, et al. Comparison of microRNAexpression levels between initial and recurrent glioblastoma specimens[J]. JNeurooncol,2013,112(3):347-354.
[2] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinctsignatures in B cell chronic lymphocytic leukemias[J]. Proc Natl Acad Sci U S A,2004,101:11755-11760.
[3] Weber F, Teresi RE, Broelsch CE, et al. A limited set of human MicroRNAis deregulated in follicular thyroid carcinoma[J]. J Clin Endocrinol Metab,2006,91:3584-3591.
[4] Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouseand human[J]. RNA,2003,9:175-179.
[5] Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNAexpression atlas based on small RNA library sequencing[J]. Cell,2007,129:1401-1414.
[6] Flavin RJ, Smyth PC, Laios A, et al. Potentially important microRNAcluster on chromosome17p13.1in primary peritoneal carcinoma[J]. Mod Pathol,2009,22:197-205.
[7] Wong TS, Liu XB, Wong BY, et al. Mature miR-184as PotentialOncogenic microRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res,2008,14:2588-2592.
[8] Ichimi T, Enokida H, Okuno Y, et al. Identification of novel microRNAtargets based on microRNA signatures in bladder cancer[J]. Int J Cancer,2009,125:345-352.
[9]黄健清. miR-195对胃癌BCG823细胞株增殖与凋亡的影响[J].中华实验外科杂志,2011,28:1485-1487.
[10] Xu T, Zhu Y, Xiong Y, et al. MicroRNA-195suppresses tumorigenicityand regulates G1/S transition of human hepatocellular carcinoma cells[J].Hepatology,2009,50:113-121.
[11] Heneghan HM, Miller N, Lowery AJ, et al. Circulating microRNAs asnovel minimally invasive biomarkers for breast cancer[J]. Ann Surg,2010,251:499-505.
[1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function[J].Cell,2004,116:281-297.
[2] Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinctsignatures in B cell chronic lymphocytic leukemias[J]. Proc Natl Acad Sci U S A,2004,101:11755-11760.
[3] Weber F, Teresi RE, Broelsch CE, et al. A limited set of human MicroRNAis deregulated in follicular thyroid carcinoma[J]. J Clin Endocrinol Metab,2006,91:3584-3591.
[4] Lagos-Quintana M, Rauhut R, Meyer J, et al. New microRNAs from mouseand human[J]. RNA,2003,9:175-179.
[5] Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNAexpression atlas based on small RNA library sequencing[J]. Cell,2007,129:1401-1414.
[6] Flavin RJ, Smyth PC, Laios A, et al. Potentially important microRNAcluster on chromosome17p13.1in primary peritoneal carcinoma[J]. Mod Pathol,2009,22:197-205.
[7] Wong TS, Liu XB, Wong BY, et al. Mature miR-184as PotentialOncogenic microRNA of Squamous Cell Carcinoma of Tongue[J]. Clin Cancer Res,2008,14:2588-2592.
[8] Ichimi T, Enokida H, Okuno Y, et al. Identification of novel microRNAtargets based on microRNA signatures in bladder cancer[J]. Int J Cancer,2009,125:345-352.
[9]黄健清. miR-195对胃癌BCG823细胞株增殖与凋亡的影响[J].中华实验外科杂志,2011,28:1485-1487.
[10] Xu T, Zhu Y, Xiong Y, et al. MicroRNA-195suppresses tumorigenicityand regulates G1/S transition of human hepatocellular carcinoma cells[J].Hepatology,2009,50:113-121.
[11] Heneghan HM, Miller N, Lowery AJ, et al. Circulating microRNAs asnovel minimally invasive biomarkers for breast cancer[J]. Ann Surg,2010,251:499-505.
[12] Ujifuku K, Mitsutake N, Takakura S, et al. miR-195, miR-455-3p andmiR-10a(*) are implicated in acquired temozolomide resistance in glioblastomamultiforme cells[J]. Cancer Lett,2010,296:241-248.
[13] Soon PS, Tacon LJ, Gill AJ, et al. miR-195and miR-483-5p Identifiedas Predictors of Poor Prognosis in Adrenocortical Cancer[J]. Clinical cancerresearch: an official journal of the American Association forCancer Research,2009,15:7684-7692.
[14] Zanette DL, Rivadavia F, Molfetta GA, et al. miRNA expressionprofiles in chronic lymphocytic and acute lymphocytic leukemia[J]. Braz J Med BiolRes,2007,40:1435-1440.
[15] Hui W, Yuntao L, Lun L, et al. MicroRNA-195inhibits theproliferation of human glioma cells by directly targeting cyclin D1and cyclin E1[J].PLoS One,2013,8:e54932.
[16] Zhang QQ, Xu H, Huang MB, et al. MicroRNA-195plays atumor-suppressor role in human glioblastoma cells by targeting signaling pathwaysinvolved in cellular proliferation and invasion[J]. Neuro Oncol,2012,14:278-287.
[17] Liu L, Chen L, Xu Y, et al. microRNA-195promotes apoptosis andsuppresses tumorigenicity of human colorectal cancer cells[J]. Biochem BiophysRes Commun,2010,400:236-240.
[18] Qi J, Yu JY, Shcherbata HR, et al. microRNAs regulate humanembryonic stem cell division[J]. Cell Cycle,2009,8:3729-3741.
[19] Mellios N, Huang HS, Grigorenko A, et al. A set of differentiallyexpressed miRNAs, including miR-30a-5p, act as post-transcriptional inhibitors ofBDNF in prefrontal cortex[J]. Hum Mol Genet,2008,17:3030-3042.
[20] Nishi H, Ono K, Iwanaga Y, et al. MicroRNA-15b modulates cellularATP levels and degenerates mitochondria via Arl2in neonatal rat cardiacmyocytes[J]. J Biol Chem,2010,285:4920-4930.
[21] Deng H, Guo Y, Song H, et al. MicroRNA-195and microRNA-378mediate tumor growth suppression by epigenetical regulation in gastric cancer[J].Gene,2013,518:351-359.
[22] Fei X, Qi M, Wu B, et al. MicroRNA-195-5p suppresses glucoseuptake and proliferation of human bladder cancer T24cells by regulating GLUT3expression[J]. FEBS Lett,2012,586:392-397.
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