摘要
脑胶质瘤是最常见的原发性颅内肿瘤，约占颅内肿瘤的46％。恶性胶质瘤的发病率为5-8／100，000，世界卫生组织1998年公布按死亡率顺序排位，恶性胶质瘤是34岁以下肿瘤患者的第2位死亡原因，是35～54岁患者的第4位死亡原因。由于胶质瘤的侵袭性生长，手术难以完全切除；化疗和放疗既不能高度特异性地杀伤胶质瘤细胞，治疗效果差，又可能产生中枢神经系统的毒、副作用。上述情况在近30年的临床实践中未得到根本改善，因此脑胶质瘤仍是神经外科领域的难治性疾病，阐明发病机理、寻找新的治疗手段的仍然是神经外科的热点课题。
目前分子病理学认为胶质瘤本质上是一种多基因异常疾病，通过一种／多种癌基因的过表达、同时伴随抑癌基因的突变缺失从而使信号传导通路异常，肿瘤细胞逃避了正常生长调控机制，自主进行增殖和侵袭、出现恶性表型。近年来研究发现，微小RNA(microRNA或miRNA)为长度约21～25核苷酸的小型非编码RNA，这些miRNA通过与靶mRNA的3`非编码区结合，可在转录后水平通过促进靶mRNA降解和／或抑制其翻译而发挥负调控基因表达的作用。已有研究证明超过50％的miRs定位在癌相关基因区域或在脆弱区域，尤其是发现有些miRs在肿瘤细胞与相应正常细胞中的表达截然不同，这使人们有理由相信miRs与肿瘤生成有关。同样，在研究胶质瘤miRs表达谱时发现一些miRs表达与正常星形细胞不同，其中miR-221和miR-222的表达明显高于正常星形细胞。miR-221和miR-222是通过调控哪些靶基因的表达来促进胶质瘤的生成，敲低胶质瘤miR-221和miR-222表达是否可以逆转胶质瘤的恶性表型，从而成为胶质瘤新的治疗靶点，这些都是值得关注的重要课题。为此，本研究将着重探索miR221和miR222在胶质瘤恶性表型中所起的作用，通过体内外研究来证实敲低胶质瘤miR-221和miR-222表达对胶质瘤生长的抑制作用。通过鉴定miR221和miR222的靶基因来探讨敲低胶质瘤miR-221和miR-222表达对胶质瘤抑制作用的机制。通过联合放射治疗来探讨敲低胶质瘤miR-221和miR-222与放疗是否具有抑制胶质瘤生长的协同作用。
本研究分为五个部分进行：
1．分析胶质瘤中miRNA表达数据，并通过人工合成2’-O-methyl(OMe)修饰的反义寡核苷酸(As-miR-221和As-miR-222)敲低胶质母细胞瘤细胞系U251细胞中miR-221和miR-222的表达来系统分析miR-221和miR-222对胶质母细胞瘤发生、进展所起的作用。
2用脂质体介导转染As-miR-221／222于胶质母细胞瘤细胞系U251，敲低miR-221和miR-222表达，观察胶质瘤细胞增殖活性、细胞周期、侵袭能力及细胞凋亡等生物学性状的变化。
3通过生物信息学的方法获得miR-221和miR-222的潜在靶基因，并通过荧光素酶报告载体策略进行鉴定，初步探讨敲低胶质瘤miR-221和miR-222表达抑制胶质瘤生长的作用机制。
4构建U251裸鼠皮下荷瘤模型，进行反义miRNA221／222的体内治疗，观察肿瘤生长速度与体积，将肿瘤组织进行免疫组化染色和原位杂交分析，进一步验证反义miRNA221／222治疗后肿瘤靶基因的表达变化以及与细胞增殖，细胞周期，侵袭及凋亡的关系。
5通过平板克隆形成实验证实敲低胶质瘤细胞系U251细胞的miR-221和miR-222表达对放射治疗是否具有增敏效果。
通过本实验，可以得出以下几个结论：
1．胶质瘤中存在一系列miRNA表达异常，miR-221和miR-222是其中表达上调的成簇miRs，miR-221过表达可以被认为是人脑胶质瘤新的分子标签。
2．p27kip1、PUMA、PTEN、TIMP3、Cx43基因mRNA的3’UTR是miR-221和miR-222直接作用靶点。
3．Lipofectamine 2000介导转染2’-O-methyl(OMe)修饰的反义寡核苷酸-As-miR-221／222可有效敲低miR-221和miR-222在U251人胶质母细胞瘤细胞系的表达，同时上调靶基因的表达，从而其恶性表型得以逆转，包括细胞的增殖和侵袭能力受到抑制，出现G0／G1期阻滞，并诱导细胞凋亡。
4．裸鼠皮下胶质瘤模型应用Lipofectamine 2000介导的As-miR-221／222治疗，同样可有效敲低miR-221和miR-222的表达，增加靶基因的表达(p27kip1、PUMA、PTEN、TIMP3、Cx43)，从而抑制肿瘤的生长，诱导细胞凋亡，与体外试验结果一致。
5．敲低胶质瘤细胞系U251细胞的miR-221和miR-222后对其放射治疗有明显的增敏效果。
6．miR-221和miR-222种子序列相同，为了达到最佳治疗效果，我们在敲低种子序列相同的miRs簇时，必须同时抑制两者的表达。
Gliomas are the most common intracranial tumors which account for 46% of primarybrain tumors.The incidence of malignant gliomas is approximately 5～8 per 10,0000individuals per year.According to the statistics of World Health Organization(WHO),the mortality rate of malignant gliomas is the second in all tumor patients that areunder 34 years old and the fourth in all tumor patients that are 35～54 years old.Because of invasive growth,malignant gliomas can not be totally resected.Moreover,malignant gliomas are resistant to radiotherapy and chemotherapy.In addition,chemo-radiotherapy usually generates toxic and side-effects to normal central nervoussystem,so malignant glioma is a refractory disease in neurosurgery,the prognosis ofpatients with malignant glioma has not been changed significantly over the past threedecades.The study on the pathogenesis and new therapeutic approaches of malignantgliomas is imperative in neurosurgical field..
By study on the molecular pathology of gliomas,it is considered that glioma is adisease of multiple gene aberration involved amplification and overexpression ofoncogenes and mutation or deletion of tumor suppressor genes,which results in thederegulation of signaling transduction pathways,then the tumor cells escape from thenormal growth control mechanism and present the malignant phenotype.Recentstudies have discovered microRNAs(miRNAs or miRs),21-25 nucleotides in length,being the largest family of noncoding RNA involved in gene silencing.MiRNAsnegatively regulate protein expression at the post-transcriptional level in a sequencespecific manner through binding to the target mRNA 3'UTRs(3'untranslated regions)and triggering translation inhibition or target mRNA degradation.Some studiesdemonstrated that more than 50% of miRNA genes are located in cancer-associatedgenomic regions or in fragile sites of chromosomal regions.In particular,Throughnegative regulation of gene expression,microRNAs(miRNAs)can function incancers as oncosuppressors,and they can show altered expression in various tumortypes.These evidences suggest that miRNAs may play an important role in thepathogenesis of human cancers.As the same with other cancers,The miRNAexpression profiles of gliomas also demonstrate that some miRNAs are expressed aberrantly in gliomas including upregulation of miR-221 and miR-222.However,thetarget genes regulated by miR-221 and miR-222 inducing the development of gliomasare not clear.Whether knocking down miR-221 and miR-222 is able to reversemalignant phenotype of gliomas or not and if miR-221 and miR-222 can be thepotential targets of glioma therapy.These problems need to be explored.In the presentstudy,the role ofmiR-221 and miR-222 may play in gliomagenesis is investigated.
This study is ivided to 5 parts:
1 The role of miR-221 and miR-222 playing in the development and progressionof glioblastoma is analyzed by 2′-O-methyl(OMe)-modified antisenseoligonucleotides for knocking down miR-221 and miR-222 in glioblastoma.
2 After transfection with As-miR-221/222 into glioblastoma(GBM)cells,thechange of biological characteristics of GBM cells including proliferation activity,cellcycle kinetics,invasive ability and apoptosis are investigated in vitro.
3 By bioinformatics analysis,some potential target genes of miR-221 andmiR-222 are obtained and identified by luciferase reporter assay.Then,themechanism of miR-221 and miR-222 regulating glioma cell growth and invasion maybe preliminary clarified.
4 The established U251 subcutaneous glioma model in nude mice is treated withAs-miR221/222 and the growth rate and volume of tumors in vivo are compared withthe controls.The correlation of expression of target genes with proliferation,invasionand apoptosis in xenograft tumor samples are investigated.
5 The effect of knocking down miR-221 and miR-222 on the radiosensitivity ofglioma cells is studied by Clonogenic assay.
The conclusion drawing from the present study is as follows:
1)Some miRs are altered expression in the four glioma cell lines includingupregulation of miR-221 and miR-222.Mir-221 overexpression is considered to be anovel molecular signature in gliomas.
2)Five genes including p27kipl、PUMA、PTEN、TIMP3、Cx43 are target genesofmiR-221 and miR-222.
3)By transfecting As-miR-221/222 into glioma cells,the expression of miR-221 and miR-222 are knocked down,and that results in reduced cell proliferationand invasive ability,arrested cell cycle and increased cell apoptosis.The reversedphenotype of glioma cells is correlated with the upregulation of the target genes ofmiR221/222.
4)The growth rate of established U251 subcutaneous glioma model in nude micetreated with As-miR221/222 is slowed down and the tumor volume of treated mice ismuch smaller than that of control mice.MiR-221/222 expression in tumor specimensis knocked down but the expression of their target genes is significantly upregulated.This result in vivo is in concordant with that of in vitro experiment.
5)Downregulation of miR-221 and miR-222 by transfection withAs-miR-221/222 can enhance radiosensitivity of glioma cells as determined byclonogenic assay.
6)Since miR-221 and miR-222 possess the similar seed sequence,it is importantto co-suppress the expression of miR-221 and miR-222 cluster for optimizing theantiglioma effect.
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