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

发布时间：2018-06-17 11:50

本文选题：microRNA-195 + 胶质瘤　；参考：《山西医科大学》2014年博士论文

【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(P0.05)；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（P0.05）；（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（P0.05），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（P0.05）；（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（P0.05）；（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（P0.05）；（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（P0.05），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（P0.05）；（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（P0.05），而在SHG-44中，miR-195组穿过Matrigel膜的细胞数多于其余两组（P0.05）；(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（P0.05）；（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（P0.05）；（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（P0.05）。 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（P0.05），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（P0.05）；（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（P0.05），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（P0.05）；（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（P0.05），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（P0.05）；（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（P0.05），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（P0.05）。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。
[Abstract]:Malignant glioma is the most common malignant tumor of the central nervous system, which has the characteristics of high recurrence rate, death and high disability rate, which seriously endangers human health. Although the treatment of glioma is combined with radiotherapy at home and abroad, combined with the combination of temozolomide chemotherapy, high grade glioma especially glioblastoma is used. Not only is it still incurable, but the median survival time has not been significantly prolonged, which is closely related to the excessive proliferation of glioma cells, resistance to apoptosis, invasion and infiltration, and the biological behavior of chemotherapeutic drugs. Therefore, the molecular target, which can effectively inhibit the malignant biological behavior of glioma cells, is found for glioma. The treatment has important scientific significance and clinical application value.
MicroRNA (miRNA) is a small endogenous small molecular non coding RNA with 22 nucleotides long, mainly through the complete or incomplete combination with the target gene mRNA3 'UTR region, realizing the "one to many" mode of action of.MiRNA at the post transcriptional level of gene expression, regulating the network with the target gene, participating in the embryonic development and increasing the cell growth. There are many biological processes, such as apoptotic, neovascularization, and other biological processes. Because the target gene of miRNAs tends to be enriched in the same or some signal pathways, it also makes its regulation of cell function and phenotype more rapid and powerful.MiRNA expression is sometimes ordered, conserved and group specific, and it can be stable in various kinds of bodies. The fluid and even the formalin fixed paraffin tissue specimens (FFPE), therefore, become more and more ideal molecular markers for the diagnosis, treatment and prognosis of the tumor. A large number of recent studies have shown that the abnormal expression of specific miRNA and the infinite proliferation of glioma cells, the inhibition of apoptosis, the invasion and migration, and the chemotherapeutic resistance of chemotherapeutic agents. It is closely related to regulation.
MicroRNA-195 (miR-195) is an important member of the miR-15a/15b/16/195/497 family. The gene is located in the chromosome 17p13.1 region and is only 650kb from the tumor suppressor gene P53. The region often lacks heterozygosity. The study shows that miR-195 is widely used in a variety of malignant tumors, such as liver cancer, lung cancer, colon cancer, gastric cancer, breast cancer, primary peritoneal carcinoma, bladder tumor and so on. Down regulation plays the role of tumor suppressor genes by inhibiting many malignant biological behaviors of tumor cells. But the biological role and mechanism of miR-195 in glioma have not been clear. The subject group is pretested by the miRNA target gene database miRDB, TargetScan and PicTar predictions and the existing miR-195 target target at home and abroad. It is speculated that miR-195 plays an important role in regulating the G1/S phase transition of tumor cell cycle and the response to DNA damage response, and then affects the malignant biological behavior of tumor cells. Therefore, this study uses miR-195 as the research object to detect the table of miR-195 in glioma FFPE tissue, glioma cell line and glioma temozolomide resistant strain, respectively. By changing the expression level of miR-195, the effects of miR-195 on the proliferation and apoptosis of glioma cells, invasion and migration, cloning, in vivo tumorigenesis, and drug resistance were observed, and then the miRNA target gene was used to predict the results of the database, and the possible effects of miR-195 on the malignant biological behavior of glioma cells were studied at the molecular level. Molecular mechanisms, these studies will help to understand the biological function of miR-195 and the regulatory mechanism of malignant biological behavior of glioma cells, and provide new predictor and target for improving the diagnosis and treatment of glioma. It has important theoretical significance and application prospects.
This topic will be studied from three aspects:
Expression and significance of 1. miR-195 in human glioma FFPE tissues and cell lines:
Objective: To study the expression of miR-195 in the paraffin embedded tissues and cell lines of human glioma, formalin, and to analyze the relationship with the clinicopathological classification of glioma, and to lay a foundation for the further exploration of the biological role of miR-195 in the internal and external glioma.
Methods: real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-195 in human glioma cell lines SHG-44, U87, U251, human astrocyte system, 28 cases of different pathological grades of human glioma, formalin, fixed paraffin embedded tissues and 2 normal brain tissues.
Results: (1) the results of qRT-PCR assay showed that compared with normal astrocytes (HA), the content of miR-195 in SHG-44, U87 and U251 of the three cell lines decreased obviously, and the downregulation of SHG-44 was the highest, but the decrease of U251 was the least, and the difference was statistically significant (P0.05). The miR-195 content of FFPE tissues in 28 cases of glioma was lower than that of the normal brain group. The difference was statistically significant (P0.05); (2) correlation analysis: LSD method was used to compare 22 different pathological grade groups. The results showed that there was no significant difference in miR-195 expression between grade I and class II (P0.05), grade I, grade II and grade III, IV (P < 0.05), and high expression of grade IV miR-195. At rank I and rank II.Spearman rank correlation analysis, the expression of miR-195 was negatively correlated with the clinicopathological grade of glioma patients (r=-0.798, P < 0.001).
2. the effect of miR-195 on proliferation, apoptosis and invasion and migration of human glioma cells in vivo and in vitro:
Objective: To investigate the effect of up regulation of miR-195 expression on proliferation, apoptosis, invasion and migration of human glioma cells.
Methods: the experiment was divided into the blank control group (Blank) group, the non sense sequence group (group NC) and the miR-195 overexpression group (group miR-195). First, the cationic liposome LipofectamineTM2000 was used to transfect the Cy5 labeled miR-195 analogue (mimics) and the nonsense sequence into the glioma cell line U251 and SHG-44 cells respectively. Laser confocal observation was used to observe the transfection situation. The transfection efficiency was detected by flow cytometry and the optimal transfection concentration was screened. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of miR-195; CCK-8 and flat clones were used to detect cell proliferation; AnnexinV-FITC/PI double staining and Hoechst33342/PI double staining were used to detect cell apoptosis; flow cytometry was used. The cell cycle was detected, the scar healing test was used to detect the cell migration ability, the Transwell chamber was used to detect the cell invasiveness, and the expression of Bcl-2 was detected by immunocytochemistry. Finally, we further evaluated the effect of overexpression of miR-195 on the growth of the subcutaneous glioma model in nude mice.
Results: (1) after miR-195mimics was transferred into the glioma cell line U251 and SHG-44, the strong red fluorescence was observed under the laser confocal laser. The optimal transfection concentration of miR-195mimics was 50nM and the transfection efficiency was more than 90%. (2) qRT-PCR results showed that the miR-195 contained in U251 and SHG-44miR-195 group glioma cells compared with the Blank group. The amount of 9.98,65.97 times increased (P0.05); (3) the results of CCK-8 and flat clones showed that compared with the NC group and the Blank group, the cell proliferation ability of the two cell lines decreased significantly and the clone formation ability decreased (P0.05). (4) the flow cytometry and Hoechst33342/PI double staining were used to detect the apoptosis, and the result showed that the cell apoptosis was with Bl. The proportion of cell apoptosis and necrosis in group miR-195 was significantly higher in group ank than in group NC (P0.05); (5) the result of scar healing experiment showed that overexpression of miR-195 in U251 could inhibit cell migration (P0.05), but in SHG-44, there was no significant difference between the cell migration rate of the miR-195 group and the other two groups (P0.05); (6) Transwell invasion experiment results. In U251, over expression of miR-195 group could reduce the number of cells passing through the Matrigel membrane (P0.05), while in SHG-44, the number of cells passing through the Matrigel membrane in the miR-195 group was more than the other two groups (P0.05). (7) the cell cycle results of the flow cytometry showed that the miR-195 group was significantly increased in the G0/G1 phase compared with the Blank and NC groups. Significantly decreased (P0.05); (8) using the microRNA target prediction site and related biological information software, Bcl-2 was found as a target gene of miR-195. Immunocytochemical results showed that miR-195 could down regulate the expression of Bcl-2; (9) the effects of miR-195 on the formation of glioma were observed by subcutaneous glioma model in nude mice, and the results showed miR-195 group. The nodule of nude mice appeared late and the growth rate was slow, and the weight and volume of the tumor were significantly lower than that of the NC group and Blank group (P0.05). (10) the immunohistochemical analysis of the tumor specimens in nude mice showed that the Ki67 expression in the miR-195 group was lower than that of the NC group and the Blank group (P0.05).
The role and mechanism of 3. miR-195 in resistance to temozolomide in human gliomas:
Objective: To study the role and mechanism of miR-195 in the resistance of Tempe to drug resistance in human gliomas.
Methods: using the method of increasing the concentration of TMZ to induce the drug resistant strain of glioma, the expression of miR-195 in the drug resistant strain of glioma was determined by qRT-PCR, then the expression level of miR-195 in the drug resistant strain was changed. CCK-8 was used to detect the change of the drug resistance and proliferation of the glioma cells, and the flow cytometry was used to detect the apoptosis and the cells. Periodic changes; Western blot were used to detect the expression of MGMT, P21, Bcl-2 and Cyclin D1, and the subcutaneous glioma model of nude mice was further constructed to observe the tumor growth and the sensitivity to temozolomide.
Results: (1) TMZ resistant U251, SHG44 resistant strains were successfully established for 6 months, respectively named U251R, SHG-44R, and (2) qRT-PCR results showed that the TMZ resistance of glioma was U251R, miR-195 content in SHG-44R decreased to 40%, 48% and SHG-44R (P0.05) in SHG-44R (P0.05). The results showed that up regulation of miR-195 increased the sensitivity of SHG-44R to TMZ, while knocking low miR-195 increased the resistance of SHG44R to TMZ (P0.05). (4) flow cytometry showed that over expression of miR-195 could significantly increase the total apoptosis rate (P0.05) of the drug-resistant strain of glioma, and low miR-195 could decrease the total apoptosis rate. The difference was not statistically significant (P0.05), and (5) the cell cycle results showed that the expression level of miR-195 in SHG-44R cells was up, the growth of glioma cells slowed, the time of passage was prolonged, the cells of G0/G1 phase increased, and the cells of S phase decreased (P0.05), while the influence of the knock down miR-195 on the cell cycle of the glioma resistant cell lines was not distinct from the control group. (P0.05); (6) the results of Western blot showed that overexpression of miR-195 could downregulate the expression of P21, Cyclin D1, Bcl-2, and the expression of protein after knocking down miR-195 was opposite (P0.05) and MGMT expression level was not affected by miR-195 expression; (7) the subcutaneous glioma model in nude mice and the staining results showed that the combination group inhibited the growth and growth of glioma. Apoptosis of glioma cells was most significant (P0.05).
Conclusion: (1) miR-195 is down regulated in all detected glioma FFPE specimens and cell lines, and the expression level is negatively correlated with the pathological grade of glioma. It is suggested that we can use it as a new and effective diagnostic marker for glioma I-IV grade and supplement the classification standard of conventional histopathology; (2) overexpression of miR-1 95 can weaken the proliferation ability of glioma cells inside and outside of glioma cells, induce G0/G1 phase block, down regulation of Bcl-2 expression, promote apoptosis and necrosis of glioma cells, but the effect of miR-195 on the invasion and migration of glioma cells is related to cell types, and further research is needed. In general, miR-195 can negatively regulate the evil of glioma cells. Sexual biological behavior; (3) low expression of miR-195 in the drug resistant glioma and up regulation of miR-195 content, can induce apoptosis and proliferation restriction of drug-resistant cells, down regulate the expression of P21, Cyclin D1, Bcl-2, to a certain extent, to restore the chemensitivity of timozolamine by glioma cells in vivo and in vitro, and this effect is not related to the changes in MGMT expression. MiR above, miR -195 is expected to become a new target for gene therapy of glioma.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

【共引文献】