MiR-9在胶质瘤恶性行为中的作用及其机制的研究

发布时间：2018-05-01 23:37

本文选题：胶质瘤 + MicroRNA　；参考：《第四军医大学》2014年硕士论文

【摘要】：【背景】胶质瘤是最常见的原发性中枢神经系统肿瘤。根据胶质瘤组织病理学特点和临床标准，世界卫生组织（WHO）将其分为I-IV级。I级胶质瘤在儿童多发，且一般认为I级胶质瘤为良性肿瘤，可通过手术切除治愈，很少发生恶性进展；相比之下II、III级胶质瘤具有一定侵袭性且恶性程度更高，预后较差；恶性程度最高且预后最差的是IV级胶质母细胞瘤(Glioblastoma multiforme,GBM)，根据其是否由低级别胶质瘤进展而来，可将其分为原发性胶质母细胞瘤和继发性胶质母细胞瘤。 MicroRNAs(miRNA)为普遍存在于动植物体内的内源性小片段非编码RNA分子，可通过与多个靶基因信使RNA(mRNA)结合从而调控基因的表达。miRNAs在进化中高度保守性，提示其可能在生命功能的诸多方面发挥重要作用。与肿瘤相关的miRNAs可根据其对肿瘤发生发展的调节作用，分为促癌miRNAs和抑癌miRNAs。目前的研究主要集中于挖掘与正常组织或细胞系相比，肿瘤中miRNAs的差异表达，进而研究其功能及寻找下游调控靶基因。已有大量研究表明miRNAs在包括胶质瘤在内的肿瘤中发生差异表达，这些miRNAs在肿瘤的发生和进展中发挥重要作用。 MiR-9在动植物中高度保守，且已被证实在多种肿瘤的发生与发展中发挥了重要作用。然而，其在胶质瘤中的功能却不明确。MiR-9在胶质瘤中的表达有何特点？其异常表达对肿瘤的发生发展有何影响？MiR-9又是通过调节哪些重要靶基因来影响胶质瘤的进展过程？在胶质瘤中miR-9异常表达的机制是什么？上述问题的研究将有助于探索胶质瘤的恶性行为机制，探究将miR-9作为治疗靶点的可行性，为胶质瘤的临床治疗提供了新策略。【目的】探究胶质瘤临床样本及其胶质瘤细胞系中miR-9的表达情况；研究miR-9在胶质瘤恶性进展中的生物学作用；寻找及鉴定miR-9调节的功能相关靶基因；寻找调控miR-9的转录因子和表观遗传学调节因素，为胶质瘤的预防、早期诊断以及治疗靶点选择提供新的理论依据。【方法】 1.通过qRT-PCR检测临床组织样本和胶质瘤细胞系中miR-9的表达水平；2.体外A172细胞转染miR-9mimic和U251细胞转染miR-9inhibitor分别上调和下调miR-9的表达，通过MTT实验、流式细胞术、划痕实验、Transwell实验、血管生成实验和粘附实验分析miR-9对胶质瘤细胞多种生物学行为的影响；3、运用生物信息学分析软件预测miR-9潜在的功能靶基因，分子克隆技术将预测靶基因3’UTR结合区域连接至PGL3报告基因质粒；4、利用荧光素酶报告基因实验鉴定miR-9直接调控靶基因，并用qRT-PCR、Western blot和ELISA实验在mRNA和蛋白水平上进行验证；5、利用生物信息学网站预测对miR-9调节的转录因子及H3K27组蛋白甲基化，qRT-PCR检测转录因子与H3K27组蛋白甲基化状态；6.采用siRNA干扰技术和瞬时转染过表达质粒下调/上调转录因子和组蛋白去甲基化酶，分析miR-9的表达情况。【结果】 1.qRT-PCR分析结果显示，与正常脑组织相比，在胶质瘤组织样本中miR-9的表达水平增高；与正常胶质细胞HEB相比，miR-9在胶质瘤细胞系中均高表达，其中在胶质瘤细胞A172中相对表达较低，在U251细胞系中表达较高；与正常肠上皮细胞相比，miR-9在结直肠癌中表达较高；而与正常肾上皮细胞相比，在肾癌中miR-9呈现低表达，表明miR-9的表达具有组织特异性；2.A172细胞转染mimic后可有效上调miR-9的表达水平，MTT结果显示miR-9促进胶质瘤细胞增殖能力，细胞周期分析过表达miR-9组细胞进入S期细胞增多，G1期细胞数减少；上调miR-9表达后，A172胶质瘤细胞迁移和侵袭细胞数明显增高，过表达miR-9组条件培养基培养内皮细胞HUVEC，增强了HUVEC细胞体外成管能力；而同时对U251细胞系转染inhibitor后，细胞增殖能力下降、细胞周期S期细胞数量显著减少，G1期细胞增多，Transwell结果可见下调miR-9组穿过小室细胞数量明显减少，条件培养液培养HUVEC细胞，，成管能力显著减弱；3.外源转染miR-9mimic可显著提高HUVEC细胞miR-9的表达水平，Transwell结果显示过表达miR-9组较多细胞穿至小室膜下，过表达miR-9HUVEC细胞成管数量相比NC组显著增多；且miR-9组内皮细胞粘附力增强；4.生物信息学预测提示，THBS2、COL18A1、PTCH1和PHD3可能为潜在miR-9的下游靶基因，从而影响胶质瘤增殖能力、迁移和侵袭、血管生成等生物学行为；5.qRT-PCR、Western blot、ELISA和荧光素酶报告基因实验进一步证实THBS2、COL18A1、PTCH1和PHD3确实为miR-9直接调控的下游靶基因；6、胶质瘤细胞A172中过表达C-MYC后，qRT-PCR分析发现miR-9表达增高；在U251细胞中应用siRNA技术下调C-MYC后，miR-9表达下调，且均主要影响miR-9-2的表达；7、在胶质瘤细胞A172中瞬时上调OCT4的表达，qRT-PCR结果显示miR-9表达上调，而且siRNA干扰U251细胞中OCT4的表达，miR-9表达下调，但是主要调节miR-9-1/3的表达；8.在胶质瘤细胞A172中过表达组蛋白去甲基化酶KDM6A/B，qRT-PCR结果显示miR-9的表达增高，而在U251中干扰KDM6A/B表达则miR-9表达呈现降低，均主要调节miR-9-2的表达。【结论】 1. MiR-9在胶质瘤中高表达，且具有组织特异性；2. MiR-9促进胶质瘤细胞增殖能力，促进细胞周期进入S期，增强了胶质瘤细胞迁移和侵袭能力，促进胶质瘤血管生成；3. MiR-9增强内皮细胞迁移和侵袭能力，增强了血管生成能力和细胞粘附力；4.THBS2、COL18A1、PTCH1和PHD3为miR-9直接调控的下游靶基因；5. MiR-9的表达受转录因子C-MYC和OCT4调控；6.组蛋白H3K27的甲基化状态参与对miR-9的表达调控。
[Abstract]:Background background

Gliomas are the most common primary central nervous system tumors . According to the pathological characteristics and clinical criteria of glioma , the World Health Organization ( WHO ) divides it into grade I - IV . Grade I glioma is multi - hair in children .
in contrast , grade II and grade III glioma have certain invasive and malignant degree and poor prognosis ;
The highest degree of malignancy and the worst prognosis is Glioblastoma multiforme , which can be divided into primary and secondary Glioblastoma , depending on whether it is progressing from low grade glioma .

MicroRNAs ( miRNA ) are non - coding RNA molecules of endogenous small fragments which are ubiquitous in animals and plants , and can regulate the expression of genes by combining RNA ( mRNA ) with multiple target genes .

MiR - 9 is highly conserved in animal and plant , and has been proved to play an important role in the genesis and development of various tumors . However , the function of MiR - 9 in glioma is not clear . What is the mechanism of abnormal expression of MiR - 9 in glioma ? What is the mechanism of abnormal expression of miR - 9 in glioma ? The research of the above - mentioned problems will help to explore the malignant behavior mechanism of glioma , and explore the feasibility of using miR - 9 as therapeutic target , and provide a new strategy for clinical treatment of glioma .

Purpose of the project

To investigate the expression of miR - 9 in glioma clinical samples and their glioma cell lines ;
To study the biological effects of miR - 9 in malignant progression of glioma ;
finding and identifying the function - related target gene regulated by miR - 9 ;
To find the transcriptional factor and epigenetics regulation factor of miR - 9 , provide a new theoretical basis for the prevention , early diagnosis and treatment target selection of glioma .

Methodology

1 . The expression level of miR - 9 in clinical tissue samples and glioma cell lines is detected by qRT - PCR ;
2 . The expression of miR - 9 was up - regulated and down - regulated by miR - 9 inhibitor transfected with miR - 9 inhibitor and U251 cells in vitro . The effects of miR - 9 on various biological behaviors of glioma cells were analyzed by MTT assay , flow cytometry , scratch test , Transwell experiment , angiogenesis experiment and adhesion experiment .
3 , using bioinformatics analysis software to predict the potential functional target gene of miR - 9 , and the molecular cloning technology is used for connecting the predicted target gene ' s 3 ' untranslated region to the PGL3 reporter plasmid ;
4 . Using luciferase reporter gene experiment , the target gene was identified directly by miR - 9 , and the mRNA and protein levels were verified by qRT - PCR , Western blot and ELISA .
5 , using bioinformatics website to predict the transcription factor of miR - 9 and histone methylation of H3K27 , and detecting the methylation state of transcription factor and H3K27 histone by qRT - PCR ;
6 . The expression of miR - 9 was analyzed by using siRNA interference technique and transiently transfected expression plasmid to downregulate / regulate transcription factor and histone demethylase .

The result is not valid .

Compared with normal intestinal epithelial cells , miR - 9 has high expression in colorectal cancer ;
Compared with normal glial cell HEB , miR - 9 is highly expressed in glioma cell line , wherein the relative expression of miR - 9 in glioma cell A172 is lower , and the expression of miR - 9 is higher in U251 cell line ;
1 . The results of qRT - PCR showed that miR - 9 expression was increased in glioma tissue samples compared with normal brain tissue ;
Compared with normal renal epithelial cells , miR - 9 exhibits low expression in renal cancer , suggesting that miR - 9 expression has tissue specificity ;
2 . After transfection with A172 cells , the expression level of miR - 9 can be increased effectively , and the MTT results show that miR - 9 promotes the proliferation of glioma cells , and the cell cycle analysis expresses that the miR - 9 group cells enter the S phase cell to increase , and the number of G1 phase cells is reduced ;
After up - regulation of miR - 9 expression , the number of migration and invasion cells of A172 glioma cells was obviously increased , and the cultured endothelial cells were cultured in the conditioned medium of miR - 9 group , thus enhancing the in vitro tube forming ability of HUVEC cells ;
At the same time , after transfection of the U251 cell line , the cell proliferation ability decreased , the number of cell cycle S phase cells decreased significantly , the number of G1 phase cells increased , Transwell results showed that the reduction of miR - 9 group through the small cell number was obviously reduced , the conditioned medium cultured HUVEC cells , and the ability of forming the tube was significantly reduced ;
3 . The expression level of miR - 9 in HUVEC was significantly increased by exogenous transfection of miR - 9 cells , and the results of Transwell showed that the expression of miR - 9 group was significantly increased compared with NC group compared with NC group .
and the adhesion of the endothelial cells of the miR - 9 group is enhanced ;
4 . Bioinformatic prediction suggests that THBS2 , COL18A1 , PTCH1 and PHD3 may be downstream target genes of potential miR - 9 , thus affecting the biological behavior of glioma proliferation , migration and invasion and angiogenesis ;
5 . qRT - PCR , Western blot , ELISA and luciferase reporter gene experiments further confirmed that THBS2 , COL18A1 , PTCH1 and PHD3 were indeed the downstream target genes which were directly regulated by miR - 9 ;
6 . After overexpression of C - MYC in glioma cells A172 , the expression of miR - 9 was increased by qRT - PCR analysis .
After downregulating the C - MYC by siRNA in U251 cells , the expression of miR - 9 was downregulated , and the expression of miR - 9 - 2 was mainly influenced ;
7 . The expression of OCT4 was transiently increased in glioma cells A172 , and the expression of miR - 9 was up - regulated by qRT - PCR , and the expression of OCT4 and miR - 9 expression in U251 cells were downregulated by siRNA , but the expression of miR - 9 - 1 / 3 was mainly regulated ;
8 . In the glioma cells A172 , the expression of miR - 9 was increased , and the expression of miR - 9 was decreased in U251 , and the expression of miR - 9 - 2 was mainly regulated by the expression of miR - 9 in U251 .

Conclusion

1.MiR - 9 is highly expressed in glioma , and has tissue specificity ;
2 . MiR - 9 promotes glioma cell proliferation ability , promotes cell cycle to enter S phase , enhances glioma cell migration and invasion ability , promotes glioma angiogenesis ;
3 . MiR - 9 enhanced endothelial cell migration and invasion ability , enhanced angiogenesis and cell adhesion ;
4 . THBS2 , COL18A1 , PTCH1 and PHD3 are downstream target genes which are directly regulated by miR - 9 ;
5 . Expression of MiR - 9 is regulated by transcription factor C - MYC and OCT4 ;
6 . The methylation status of histone H3K27 is involved in the expression regulation of miR - 9 .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

【共引文献】