miR-101调节STMN-1的表达并参与调控GBM细胞增殖，迁移及侵袭的初步研究

发布时间：2018-09-14 10:08

【摘要】：成人胶质母细胞瘤（GBM）是颅内最常见的恶性肿瘤之一，其患病率及死亡率较高，尽管现在已有标准的手术及辅助放化疗治疗方法，但患者的中位生存期仍小于14个月。目前认为GBM的高复发率是导致其预后较差的主要原因，GBM极少出现远隔转移且多为原位复发，故该种高复发率可能暗示了肿瘤细胞对瘤旁组织固有的侵袭性及其自身极强的增殖，侵袭能力。现有治疗GBM的方式主要为手术切除肿瘤的实质部分及瘤周的胶质增生带，以期有效缓解颅高压症状及降低载瘤负荷再辅助以术后标准放化疗，但该种治疗方式并不能保证清除已侵袭至周围正常脑组织中的肿瘤细胞，肿瘤仍有复发可能。临床上不同级别胶质瘤的术后复发时间不同，甚至同一级别的胶质瘤经同种方案治疗后复发时间仍可能不同，这可能与肿瘤细胞不同的侵袭能力有关。因此抑制胶质瘤细胞的增殖、迁移及侵袭能力将有可能成为一种辅助现有治疗措施的新方式。 MicroRNAs是一种内源性非编码单链核苷酸，其长度约为19-25个核苷酸。microRNAs广泛调控细胞增殖、迁移、侵袭、凋亡、自噬及衰老等多种细胞学行为。现已证实microRNA的种子序列（seed sequence）能通过完全或不完全碱基互补配对的方式与目标mRNA3’UTR的特定序列相结合从而影响mRNA的稳定性或抑制其翻译过程，进而在转录后水平调节基因的表达。目前越来越多的证据显示microRNA表达水平的变化与人类肿瘤性疾病的关系极为紧密，约50%microRNA定位于染色体上的脆性位点，这些位点在肿瘤细胞中通常缺失或者扩增，而且microRNA的异常表达与临床患者的预后有一定的关系。miR-101是众多microRNAs中的一员，近年来多有文献报道其在肿瘤细胞中的表达量下降并能在体内或体外抑制肿瘤的生长。综合分析miRbase、TargetScanHuman等数据库及文献可知miR-101的靶基因包括EZH2、COX-2、SOX-9、MCL-2、APP等上千种，通过负性调控这些基因的表达miR-101可能参与多种和肿瘤发生、发展等相关的信号通路。 2011年在乳腺癌组织中发现了miR-101的三个新靶标，，STMN-1是其中之一。STMN-1(stathmin-1)又名OP（oncoprotein-18），是一种微管解聚磷蛋白，最早见于Sobel等人在1983年的报道。本课题组前期使用基因芯片技术分析了72例胶质瘤组织标本并结合生物信息分析技术筛选出29个耐药相关基因，STMN-1是其中之一。在细胞有丝分裂的前期，STMN-1蛋白第25、38号丝氨酸位点发生磷酸化，辅助纺锤体的正确形成及姐妹染色体的分离，这与肿瘤细胞的增殖能力有关；STMN-1还参与细胞凋亡及自噬等过程，我们前期的实验结果表明干扰STMN-1表达后U251细胞对TMZ的敏感性增强且该现象可能与细胞凋亡、自噬有关；此外，在细胞迁移的过程中STMN-1第16位丝氨酸位点发生磷酸化并通过Rac1-Pak1途径调控微管功能进而影响细胞运动。故STMN-1表达水平或磷酸化水平的改变在检测肿瘤的发生、恶性演进及评估预后等方面有潜在意义。本次实验通过分析四中胶质母细胞瘤细胞系中miR-101的表达差异以筛选实验细胞系；随后构建miR-101、STMN-1过表达及干扰慢病毒载体，检测转染后细胞的迁移及侵袭能力；进一步建立包含STMN-1的3’UTR野生型及突变型的双荧光慢毒载体，与miR-101过表达病毒共转染GBM细胞后检测荧光强度验证在胶质瘤细胞中miR-101与STMN-1的3’UTR直接结合；最后得出在胶质瘤细胞中存在miR-101—STMN-1—细胞增殖、迁移、侵袭这一调控通路。实验分为两部分：第一部分，（1）收集胶质瘤组织标本检测其中miR-101的表达量并分析表达量与肿瘤级别的关系，（2）构建相关慢病毒载体利用Q-PCR、WB、双荧光靶标验证等技术验证在胶质瘤细胞中存在miR-101与STMN-1直接负性调节关系；第二部分，胶质瘤细胞株中分别转染miR-101过表达及STMN-1干扰等慢病毒，随后检测细胞增殖、迁移及侵袭能力。第一部分胶质瘤组织标本中miR-101表达量的检测，验证胶质瘤细胞株存在miR-101对STMN-1的直接负性调控关系研究目的：检测胶质瘤组织标本中miR-101表达量与肿瘤级别的关系；分析四中胶质瘤细胞系中miR-101的表达量；验证胶质瘤细胞中存在miR-101对STMN-1的直接负性调控关系。研究内容及结果：方法：应用荧光定量PCR技术分析37例胶质瘤组织标本中及四种胶质瘤细胞系中miR-101的表达量；使用real-time PCR和western-blot技术检测miR-101过表达或抑制后U251细胞内STMN-1的mRNA水平及蛋白水平的变化；构建pL/TO/IRES/Luc-STMN1-3’UTR和pL/TO/IRES/Luc-STMN1-3’UTR-mut慢病毒载体，利用双荧光素酶报告系统技术验证在胶质瘤细胞中miR-101的种子序列能与STMN-1的3’UTR直接结合。结果：备选的四种GBM细胞系中miR-101的表达量依次为：U251＞A172＞U87＞T98；37例胶质瘤组织经实时荧光定量PCR分析后显示miR-101的表达量在正常脑组织、低级别胶质瘤（WHOⅠ-Ⅱ级）和高级别胶质瘤（WHOⅢ-Ⅳ级）之间有明显的统计学差异（P＜0.01），且随着肿瘤级别的增高，细胞内miR-101表达量逐渐下降，但WHOⅠ级与Ⅱ级之间或WHOⅢ级与Ⅳ级之间则无明显统计学差异（P＞0.05）。U251细胞转染miR-101过表达及miR-101干扰病毒后细胞内STMN-1的mRNA水平及蛋白表达水平均明显下降，显示出miR-101对STMN-1明显的负性调节作用；构建好的双荧光慢病毒，miR-101过表达及阴性对照慢病毒按照实验分组转染U251细胞后测定萤火虫荧光酶素RLU和海肾荧光素酶素RLU，数据显示miR-101能够与STMN1-3’UTR直接作用。结论：miR-101的表达量与肿瘤级别的增高呈负相关，在低级别胶质瘤和高级别胶质瘤之间有明显的差异；miR-101的种子序列能与STMN1-3’UTR直接结合并降低STMN-1mRNA及蛋白的表达水平。第二部分验证miR-101能通过STMN-1调控U251细胞的增殖、迁移及侵袭能力研究目的：证明miR-101能调控U251细胞的增殖、迁移能力并通过STMN-1来调控侵袭能力。研究内容及结果：方法：分别用构建好的miR-101过表达、miR-101干扰、STMN-1-siRNA及相应的阴性对照（NC）病毒转染U251细胞株；用CCK-8法检测细胞增殖能力并绘制增殖曲线；用细胞划痕实验评估细胞迁移能力；用Transwell法检测细胞的侵袭能力。结果：加入CCK-8试剂后用酶标仪检测的结果显示U251+miR-101过表达组的细胞增殖能力较U251+miR-101干扰组，U251+miR-101过表达NC组，U251+miR-101干扰NC组及U251组明显下降且有显著地统计学差异（P＜0.05），但后三组之间没有统计学差异。U251细胞分别转染miR-101干扰，miR-101过表达NC及miR-101干扰NC慢病毒72h后做细胞划痕实验，24h后结果显示U251+miR-101过表达组的细胞划痕面积的变化值要明显小于其余三组并存在统计学差异（P＜0.05）；U251细胞转染miR-101过表达，NC，siRNA-STMN-1慢病毒后利用Transwell技术检测细胞侵袭能力，18h后结果显示U251+miR-101过表达组及U251+siRNA-STMN-1组细胞的侵袭能力下降，两者分别与NC组及正常U251组比较均有统计学差异（P＜0.05），二者之间亦存在明显统计学差异（P＜0.05）。结论：miR-101能在表观遗传学水平抑制STMN-1的表达，从而抑制U251细胞的增殖，迁移及侵袭能力。
[Abstract]:Adult glioblastoma (GBM) is one of the most common intracranial malignancies. Its morbidity and mortality are high. Although there are standard surgical and adjuvant chemoradiotherapy methods, the median survival time of the patients is still less than 14 months. Because septal metastasis is mostly in situ recurrence, this high recurrence rate may indicate the intrinsic invasiveness of tumor cells to adjacent tissues and their own strong proliferation and invasiveness. Re-adjuvant radiotherapy and chemotherapy are standard, but this treatment does not guarantee the removal of tumor cells that have invaded the surrounding normal brain tissue, and tumor recurrence is still possible. Inhibition of proliferation, migration and invasion of glioma cells may be a new approach to adjuvant therapy.
MicroRNAs are endogenous non-coding single-stranded nucleotides with a length of about 19-25 nucleotides. MicroRNAs regulate cell proliferation, migration, invasion, apoptosis, autophagy and senescence. It has been demonstrated that microRNA seed sequences can complement target mRNA 3 through complete or incomplete base complementary pairing. More and more evidences show that changes in microRNA expression levels are closely related to human cancerous diseases. About 50% of microRNAs are located at fragile sites on chromosomes. MicroRNA-101 is a member of many microRNAs. In recent years, it has been reported that the expression of microRNAs in tumor cells decreases and can inhibit tumor growth in vivo or in vitro. According to uman and other databases and literatures, the target genes of microRNAs-101 include EZH2, COX-2, SOX-9, MCL-2, APP and so on. Through negative regulation of these genes, microRNAs-101 may participate in a variety of signaling pathways related to tumorigenesis and development.
STMN-1, also known as oncoprotein-18, is a microtubule depolymerized phosphoprotein. It was first reported by Sobel et al. in 1983. Seventy-two glioma specimens were analyzed by gene chip technique and bioinformatics. STMN-1 is one of the 29 drug-resistance-related genes screened by information analysis. At the early stage of cell mitosis, STMN-1 protein phosphorylated at the 25th and 38th serine sites, assisted spindle formation and sister chromosome isolation, which is related to the proliferation of tumor cells; STMN-1 is also involved in apoptosis and autophagy. In addition, the phosphorylation of the 16th serine site of STMN-1 during cell migration and the regulation of microtubule function via Rac1-Pak1 pathway affect cell movement. Changes in levels of phosphorylation or reaching levels are potentially useful in detecting tumorigenesis, malignant progression, and evaluating prognosis.
In this experiment, we screened the experimental cell lines by analyzing the expression difference of microRNAs-101 in the four glioblastoma cell lines; then constructed microRNAs-101, STMN-1 overexpression and interfering lentivirus vector to detect the migration and invasiveness of the transfected cells; further established the wild-type and mutant dual-fluorescent lentivirus vector containing STMN-1. After co-transfection with the virus, the fluorescence intensity was detected to verify the direct binding of the RNA-101 to the 3'UTR of STMN-1 in glioma cells. Finally, it was concluded that there was a regulation pathway of proliferation, migration and invasion in glioma cells. The experiment was divided into two parts: the first part, (1) collecting glioma tissue. The expression of microRNAs-101 in glioma cells was detected and the relationship between microRNAs-101 expression and tumor grade was analyzed. (2) Construction of lentiviral vectors was used to verify the direct negative regulatory relationship between microRNAs-101 and STMN-1 in glioma cells by Q-PCR, WB, double fluorescence target assay and other techniques. N-1 interference such as lentivirus, then detect cell proliferation, migration and invasion ability.
Part I Detection of the expression of microRNAs-101 in glioma tissue specimens to verify the direct negative regulation of microRNAs-101 on STMN-1 in glioma cell lines
Objective: To investigate the relationship between the expression of microRNAs-101 and tumor grade in glioma tissues, analyze the expression of microRNAs-101 in glioma cell lines, and verify the direct negative regulation of microRNAs-101 on STMN-1 in glioma cells.
RESULTS: The expression of microRNAs-101 in 37 glioma tissues and four glioma cell lines was analyzed by fluorescence quantitative PCR, and the mRNA and protein levels of STMN-1 in U251 cells were detected by real-time PCR and Western-blot. C-STMN1-3'UTR and pL/TO/IRES/Luc-STMN1-3'UTR-mut lentiviral vectors were used to verify that the seed sequence of microRNAs-101 in glioma cells could bind directly to the 3'UTR of STMN-1. Results: The expression of microRNAs-101 in the four GBM cell lines was U251 > A172 > U87 > T98 in turn. Real-time fluorescence quantitative PCR analysis showed that the expression of microRNA-101 was significantly different among normal brain tissues, low-grade gliomas (WHO grade I-II) and high-grade gliomas (WHO grade III-IV) (P There was no significant difference between grade III and grade IV (P > 0.05). The overexpression of miR-101 in U251 cells and the expression of STMN-1 protein were significantly decreased after interfering with the virus, which indicated that the expression of STMN-1 was negatively regulated by the virus. The constructed double fluorescent lentivirus, the overexpression of microwave-101 and the negative control were observed. Luciferin RLU and nephron luciferin RLU in U251 cells transfected by lentiviruses were measured. The data showed that microRNA101 could directly interact with STMN1-3'UTR.
Conclusion: The expression of microRNAs-101 is negatively correlated with the increase of tumor grade, and there is a significant difference between low-grade glioma and high-grade glioma. The seed sequence of microRNAs-101 can directly bind to STMN1-3'UTR and reduce the expression of STMN-1 mRNA and protein.
The second part verifies that miR-101 can regulate the proliferation, migration and invasion ability of U251 cells through STMN-1.
OBJECTIVE: To demonstrate that microRNAs-101 can regulate the proliferation, migration and invasion of U251 cells through STMN-1.
RESULTS: U251 cells were transfected with constructed over-expression of microRNAs-101, interference of microRNAs-101, STMN-1-siRNA and corresponding negative control (NC) virus, the proliferation ability of U251 cells was detected by CCK-8 assay, the cell migration ability was evaluated by cell scratch test, and the invasive ability of U251 cells was detected by Transwell assay. Results: After adding CCK-8 reagent, the proliferation ability of U251 + Mi-101 overexpression group was significantly lower than that of U251 + Mi-101 interference group, U251 + Mi-101 overexpression NC group, U251 + Mi-101 interference NC group and U251 interference NC group (P < 0.05), but there was no significant difference between the latter three groups. Mi-101 interference, Mi-101 overexpression NC and Mi-101 interference NC lentivirus 72 hours after the scratch test, 24 hours after the results showed that U251 + Mi-101 overexpression group of cell scratch area was significantly smaller than the other three groups and there were statistical differences (P < 0.05); U251 cells overexpression of Mi-101, NC, siRNA-STMN-1 lentivirus after transfection. Transwell technique was used to detect the invasive ability of U251+microRNA-101 cells. After 18 hours, the invasive ability of U251+microRNA-101 overexpression group and U251+siRNA-STMN-1 group decreased, and there were significant differences between them (P<0.05) and NC group and normal U251 group, respectively.
Conclusion: Mi-101 can inhibit the expression of STMN-1 at the epigenetic level, thereby inhibiting the proliferation, migration and invasion of U251 cells.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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