MicroRNA-132调控MMP-16表达抑制胶质瘤细胞侵袭的机制研究

发布时间：2018-01-30 20:32

本文关键词： 胶质瘤 MicroRNA 转移性侵袭性 MMP-16　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：背景和目的神经胶质瘤是最常见的原发性恶性脑肿瘤,具有恶性增值、血管生成和侵袭周围正常脑组织的特征。尽管经过积极的手术、放疗和化疗治疗,胶母细胞瘤患者的中位存活期仅仅有12-15个月。肿瘤侵袭是肿瘤治疗失败的主要原因。为延长患者生存期,提高患者生存质量,迫切需要新的临床生物标记和治疗靶点,从而提出抑制或减少肿瘤细胞侵袭能力的新治疗方案。MicroRNAs(miRNAs)是一类参与调控不同生物过程的异常非编码RNA(长度大约为22个核苷酸)。通过使用实时定量PCR发现microRNA-132(mi R-132)在胶质瘤组织中显著异常表达。基于miR-132靶基因的假设,我们预测miR-132和基质金属蛋白酶(MMP)家族中的一种蛋白MMP-16(MT-MP3)有着显著关联。在人源胶质瘤细胞株A172,SHG44和U87中过表达miR-132可以抑制细胞侵袭和转移。而且在A172,SHG44和U87细胞中通过使miR-132的过表达从而减少了MMP-16的表达。总结以上结果分析:miR-132通过MMP-16影响胶质瘤细胞侵袭和转移,并且在胶质瘤中miR-132作为一类抑制转移相关基因的miRNA而存在。MicroRNAs(mi RNAs)是较短的单链核苷酸RNA分子,通过结合它们靶基因的mRNA分子的3'非编码区来调控基因表达,从而抑制基因转录或诱导mRNA降解。miRNAs控制细胞生长、增殖、新陈代谢和凋亡。事实上,特殊类型的miRNA失调已经被证明与特定类型的肿瘤相关联。例如,miR-16在胶质瘤细胞低表达并且抑制Bcl-2表达;在转移性胶质瘤细胞中miR-145过表达,并且抑制ADAM17表达。基质金属蛋白酶16(Matrix metalloprotease 16)是一类膜型金属蛋白酶。作为酶原,通过激活proMMP-2(明胶酶A)活性来起作用。MMP-16是由细胞分泌产生。然而,酶谱显示明胶酶激活活化的MMP-2是激活MMP-16的间接机制。MMP-2可以裂开基底膜上的胶原蛋白IV,参与肿瘤转移过程。毫无疑问,在胃癌、肝细胞癌、前列腺癌和黑色素细胞瘤中mmp-16的高表达与肿瘤细胞侵袭力增加相关联。本研究不仅要探讨mir-132在非瘤脑组织、不同级别的人脑胶质瘤组织以及三个恶性胶质瘤细胞系(a172,shg44和u87)中的表达,而且进一步通过体外试验探讨mir-132与人脑胶质瘤细胞侵袭性的相关性。本研究还旨在为未来进一步深入研究mir-132在人类神经胶质瘤中的作用机制奠定基础。一、材料和方法1.通过使用microrna.orgdatabase、thedatabaseonpredicted和publishedmicrornas(mirwalk),我们预测mirna-132(mir-132)与恶性脑肿瘤具有较强关联性。2.化学合成的mir-132寡核苷酸随机序列通过脂质体2000被转染进恶性胶质瘤细胞株a172,shg44和u87中。3.人源胶质瘤细胞株a172,shg44和u87的转染效率通过流式细胞术和绿色荧光检测。4.使用实时定量pcr(qrt-pcr)分别检测非瘤脑组织和人类胶质瘤组织标本中mir-132表达情况。5.使用transwell实验检测mir-132对人源胶质瘤细胞株a172,shg44和u87中的侵袭能力的影响。6.使用westernblot检测mmp-2和mmp-16表达情况。7.荧光素酶报告实验证明mmp-16是mir-132直接靶向基因。8.显示的数据为均值±标准差。使用spss版本12.0windows版本软件。使用方差分析(anova)和student'st-test进行统计分析。经过统计分析p值0.05。二、结果1.mir-132在胶质瘤组织中的表达低于正常脑组织。本研究中使用实时定量pcr检测mir-132在11例低级别胶质瘤组织、12例高级别胶质瘤组织和8例正常脑组织中的表达情况。其实验结果显示:相对于正常脑组织,mir-132在胶质瘤组织中的表达显著降低;同时与正常脑组织相比,mir-132在胶质瘤细胞株(a172,shg44和u87)中的表达更低。mir-132在胶质瘤组织和胶质瘤细胞株的下调表明mir-132也许是胶质瘤治疗的潜在靶点。2.mir-132抑制胶质瘤细胞的侵袭能力。使用transwell实验检测mir-132对人源胶质瘤细胞株a172,shg44和u87中的侵袭能力的影响。本研究将mir-132模拟物和抑制剂被转染进人源胶质瘤细胞a172、shg44和u87。mir-132模拟物或干扰rna转染后的72小时,a172、shg44和u87被接种于上室,48小时后通过细胞外基质侵入的细胞被拍片和计数。在所有的细胞株中,相对于对照组,mir-132减少了侵入细胞的数量。实验数据表明mir-132是体外实验中细胞侵袭和迁移的调控分子。3.mmp-16mrna在胶质瘤组织的表达。实时荧光定量pcr分析表明:相对于8例正常脑组织,mmp-16mrna在11例低级别和12例高级别胶质瘤组织的表达显著增高。结果发现:mir-132在胶质瘤组织中的表达低于正常脑组织。由此说明在肿瘤组织中mmp-16mrna的表达量较mir-132表达高,表明在肿瘤组织中mmp-16mrna的表达与mir-132的表达呈负相关。4.mir-132下调mmp-16mrna的表达。为了进一步阐明mir-132在胶质瘤细胞中抑制细胞侵袭能力的机制,本研究使用targetscan5andmirbase预测mir-132的靶基因,这些软件显示:mmp-16可能作为mir-132直接靶向调节基因,在肿瘤的细胞转移和侵袭中扮演重要角色。同时运用rt-pcr实验和qrt-pcr实验检测发现:与对照组细胞相比,转染了mir-132模拟物的胶质瘤细胞(a172,shg44和u87)显著降低了mmp-16mrna的表达水平。以上的结果表明:mmp-16可能作为mir-132直接靶向调节基因,从而抑制胶质瘤细胞的迁移和侵袭能力。5.mir-132直接靶向调节mmp-16基因的表达。本研究运用westernblot分别检测在转染mir-132后a172,shg44和u87中mmp-16蛋白的表达水平,其结果表明:mmp-16的蛋白表达水平在被转染mir-132模拟物和抑制物后分别被下调和上调。进一步通过荧光素酶实验检测发现:mir-132直接抑制了mmp-16基因mrna的3’-utr表达,从而证实mmp-16是mir-132天然靶向调节基因。6.mmp-16作为mmp-2的活化剂激活mmp-2蛋白表达水平。mmp-16是一种膜结合型mmp,能激活mmp-2来增加侵袭能力。mmp-2被mmp-16激活,这是mmp-16功能的间接途径。mir-132被预测靶向作用mmp-16(mt3-mmp,membranetype3mmp)(www.microrna.org)。westernblot和荧光素酶实验被用来验证这一假设。结果表明:miR-132通过调节MMP-16的表达从而影响MMP-2蛋白的表达;miR-132的过度表达通过下调MMP-16的表达,从而抑制人类胶质瘤细胞(A172,SHG44和U87)的侵袭能力。三、结论Has-mi R-132基因是位于17号染色体(1953202-1953302),长为100bp的碱基对。在过去的几年里,许多研究发现miR-132异常表达参与了不同肿瘤的发生、发展过程。例如:miR-132作为肿瘤抑制器在乳腺癌中抑制细胞增殖。而且,miR-132异常表达在原发性骨肉瘤,阿尔兹海默病、前列腺癌和胰腺癌中被发现。然而,mi R-132在胶质瘤细胞迁移和侵袭中的作用仍有待探索。本研究发现miR-132的表达水平在胶质瘤组织中低于正常脑组织,而且mi R-132抑制胶质瘤细胞侵袭和迁移能力。被miR-132模拟物转染的胶质瘤细胞(A172,SHG44和U87)减少肿瘤细胞的迁移和侵袭能力,同时用miR-132抑制剂转染上述细胞则产生相反的结果。MMP-16是一种膜结合型MMP,能激活MMP-2来增加侵袭能力。MMP-2被MMP-16激活,这是MMP-16功能的间接途径。目前的研究结果表明了MMP-16与胶质瘤细胞侵袭和迁移能力密切相关。以前的研究结果显示减少的MMP-16的水平有效地抑制胶质瘤细胞的侵袭性。MMP-16同时被发现拥有抗细胞外基质组分的蛋白水解的活性,如III型胶原蛋白。在正常脑组织和星形胶质瘤组织中,MT3-MMP可能参与了细胞外基质更新。本研进一步证实miR-132通过减少基质金属蛋白酶基因MMP-16的表达从而抑制胶质瘤细胞(A172,SHG44和U87)侵袭和迁移能力。总而言之,本研究证实:在恶性胶质瘤细胞中,miR-132可能通过直接调节MMP-16蛋白的表达来影响胶质瘤细胞的迁移和侵袭性中起重要作用;miR-132也许是胶质瘤侵袭和转移的潜在治疗靶点。然而,在体内试验中,MMP-16的活性是否通过miR-132被影响需要更进一步的深入研究来确认。
[Abstract]:Background and objective: glioma is the most common primary malignant brain tumors, with malignant proliferation, angiogenesis and invasion of surrounding normal brain tissue. Despite aggressive surgery, radiotherapy and chemotherapy in the treatment of patients with glioblastoma, the median survival is only 12-15 months. Tumor invasion is the main reason the failure of cancer treatment. To prolong the survival time of the patients, improve the quality of life of patients, urgent need for clinical biomarkers and new therapeutic targets, so as to put forward a new treatment scheme to suppress or reduce the invasiveness of.MicroRNAs (miRNAs) is a kind of different biological processes involved in the regulation of the abnormal RNA (encoding length is about 22 nucleotide). By using real-time quantitative PCR microRNA-132 (MI R-132) in glioma tissues significantly. Abnormal expression of miR-132 target genes based on the hypothesis, we predicted that miR-132 and matrix metalloproteinase The enzyme (MMP) a family of protein MMP-16 (MT-MP3) has a significant correlation. In human glioma cell lines A172, SHG44 and U87 over expression of miR-132 can inhibit the invasion and metastasis of cells. But in A172, SHG44 and U87 cells by overexpression of MMP-16 reduced the expression of miR-132. Summing up the above analysis results: miR-132 through the invasion and metastasis of MMP-16 glioma cells, and miR-132 as a kind of anti metastasis related gene miRNA in glioma and the presence of.MicroRNAs (MI RNAs) is a single nucleotide RNA molecular short, mRNA molecules through the combination of their target gene 3'encoding region of controlling gene the expression of proliferation, inhibit gene transcription or mRNA induced degradation of.MiRNAs in cell growth, apoptosis, and The new supersedes the old.. In fact, a special type of miRNA disorders has been proved with a specific type of tumor associated. For example, miR-16 Low expression in glioma cells and inhibit the expression of Bcl-2 in miR-145; metastasis of glioma cells by overexpression, and inhibit the expression of ADAM17. Matrix metalloproteinase 16 (Matrix metalloprotease 16) is a kind of membrane type metalloproteinase. As a zymogen, through the activation of proMMP-2 (gelatin A enzyme) activity is produced by.MMP-16 cell secretion. However, enzyme activation of gelatinase spectrum shows that the MMP-2 is.MMP-2 indirect mechanism activation of MMP-16 can cleave the basement membrane collagen IV in the metastatic process. There is no doubt that in gastric cancer, hepatocellular carcinoma, and the high expression of mmp-16 in tumor cells of prostate cancer and melanoma invasion is associated with increased the present study not only to explore mir-132 in non tumor brain tissues and glioma tissue of different levels and three malignant glioma cell lines (A172, SHG44 and U87) in the form of, and further By in vitro test on the invasion of the relationship between mir-132 and human glioma cells. This study also aims to lay the foundation for the future further research on the mechanism of mir-132 in human gliomas. Materials and methods, 1. by using microrna.orgdatabase, thedatabaseonpredicted and publishedmicrornas (mirwalk), mirna-132 (mir-132) and we predict malignant brain tumors has the strong association of.2. chemical synthesis of mir-132 by Lipofectamine 2000 random oligonucleotide sequences were transfected into glioma cell line A172,.3. human glioma cell line A172 source SHG44 and U87, SHG44 and U87 transfection efficiency by flow cytometry and green fluorescence detection of.4. using real-time quantitative PCR (qRT-PCR) were detected respectively. Non tumor brain tissue and mir-132 in human glioma tissues the expression of.5. using Transwell assay on human mir-132 glue Glioma cell lines A172, SHG44 and U87 in the invasion effect of.6. using Westernblot to detect MMP-2 and mmp-16 expression of.7. luciferase reporter experiments prove that mmp-16 is the direct target gene.8. to display mir-132 data as mean standard deviation. Use the SPSS version of the 12.0windows version of the software. Using analysis of variance (ANOVA) and student'st-test statistics through statistical analysis analysis. The p value of 0.05. two, the expression of 1.mir-132 in glioma tissues than in normal brain tissues. Mir-132 was detected in 11 cases of low grade glioma tissues using real-time quantitative PCR in this study, the expression of 12 cases of high grade glioma tissues and 8 cases of normal brain tissues. The experimental results show that compared to the normal brain tissue, the expression of mir-132 in glioma tissues was significantly decreased; compared with normal brain tissue, mir-132 in glioma cell lines (A172, SHG44 and U87) in the table .mir-132 was lower in down regulated glioma tissues and glioma cell lines revealed that mir-132 may be a potential target for the treatment of glioma.2.mir-132 inhibits glioma cells invasion. On human glioma cell line A172 using Transwell assay, mir-132, SHG44 and U87 in the effect of invasion. This study analogue of mir-132 and inhibitor were transfected into human glioma cell line A172, SHG44 and u87.mir-132 mimic or interfere with RNA 72 hours after transfection, A172, SHG44 and U87 were seeded in the upper chamber, 48 hours after the invasion through the extracellular matrix cells were filming and counting. In all of the cell lines, compared with the control group, mir-132 reduce the number of invasive cells. The experimental data show that mir-132 is the expression and regulation of molecular.3.mmp-16mrna invasion and migration of cells in vitro in glioma tissue. Real time fluorescence quantitative PCR analysis showed that: In 8 cases of normal brain tissue, the expression of mmp-16mrna in 11 cases of low grade and 12 cases of high grade glioma tissues increased significantly. The results showed that the expression of mir-132 in glioma tissues was lower than that in normal tissues. The expression of mmp-16mrna in tumor tissues with high expression of mir-132, expression of mir-132 and mmp-16mrna showed that the expression of in tumor tissues was negatively correlated with the down-regulation of.4.mir-132 mmp-16mrna. In order to further elucidate the mechanism of mir-132 inhibition of cell invasion in glioma cells, this study used targetscan5andmirbase target gene prediction of mir-132, the software shows that the mmp-16 could be used to directly target the mir-132 regulatory gene, plays an important role in the invasion and metastasis of tumor cells at the same time. By using the RT-PCR test and qRT-PCR test showed that: compared with the control group transfected glioma cells, mir-132 mimics fine The cell (A172, SHG44 and U87) significantly reduced the expression level of mmp-16mrna. The above results showed that mmp-16 could be used to directly target the mir-132 regulating gene expression and inhibition of migration and invasion of.5.mir-132 glioma cells to target the direct regulation of mmp-16 gene. In this study the use of Westernblot A172 were detected in mir-132 after transfection, expression the level of mmp-16 protein and SHG44 in U87, the results showed that the expression of mmp-16 in transfected mir-132 mimics and inhibitor respectively is downregulated and upregulated. Further by luciferase reporter assay found that mir-132 directly inhibits mmp-16 gene mRNA 3 -utr expression, thus confirming that mmp-16 is mir-132 natural targeting gene regulation.6.mmp-16 as an activator of MMP-2 activated MMP-2 Protein expression level of.Mmp-16 is a membrane-bound MMP, can activate MMP-2 to increase the invasion ability of.Mmp-2 was mmp-16 This is the.Mir-132 activation, mmp-16 function is predicted indirectly targeting mmp-16 (mt3-mmp, membranetype3mmp) (www.microrna.org).Westernblot and luciferase assays were used to test this hypothesis. The results show that miR-132 can affect the expression of MMP-2 protein by regulating the expression of MMP-16; overexpression of miR-132 by down regulating the expression of MMP-16, thus inhibiting human glioma cells (A172, SHG44 and U87) invasion. Three. Conclusion Has-mi R-132 gene is located on chromosome 17 (1953202-1953302), 100bp long base pairs. In the past few years, many studies have found that the abnormal expression of miR-132 in different tumors, such as tumor development process. Suppressor miR-132 cell proliferation inhibition in breast cancer. Moreover, the abnormal expression of miR-132 in primary osteosarcoma, Alzheimer's disease, prostate cancer and pancreatic cancer were found in natural. However, the role of MI R-132 in the invasion and migration of glioma cells remains to be explored. This study found that the expression level of miR-132 is lower than that of normal brain tissue in glioma tissues, and MI R-132 inhibited the invasion and migration of glioma cells. MiR-132 mimics transfected glioma cells (A172, SHG44 and U87) to reduce the migration and invasion of tumor cells, and the cells were transfected with miR-132 inhibitor nterproductive.MMP-16 is a membrane-bound MMP, can activate MMP-2 to increase the invasion ability of.MMP-2 activated by MMP-16, which is indirectly the function of MMP-16. The results indicated that the MMP-16 and the invasion and migration of glioma cells closely related. Previous studies showed decreased levels of MMP-16 inhibit glioma cell invasion of.MMP-16 was also found to have anti extracellular matrix components of protein water Solution of the activity, such as type III collagen. In normal brain tissues and glioma tissues, MT3-MMP may be involved in extracellular matrix update. This study further confirmed that miR-132 can inhibit glioma cells by decreasing the expression of matrix metalloproteinase gene MMP-16 (A172, SHG44 and U87) invasion and migration. In a word, this study confirmed: in malignant glioma cells, miR-132 may directly regulate the expression of MMP-16 protein by the effect plays an important role in migration and invasion of glioma cells; miR-132 may be a potential therapeutic target in the invasion and metastasis of glioma. However, in vivo experiment, the activity of MMP-16 through miR-132 is affected further further studies to confirm.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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