MEF2D促进骨肉瘤发生发展及其分子机制研究

发布时间：2018-05-14 04:36

本文选题：骨肉瘤 + 肌细胞增强因子2D　；参考：《吉林大学》2017年博士论文

【摘要】：骨肉瘤是青少年及成人中最常见的原发性恶性骨肿瘤,进展迅速,早期即出现转移。每年全世界的发病率约为4/105,易发生于生长较快的青年人,严重威胁着患者的身心健康。当前骨肉瘤的治疗手段仍以手术治疗联合系统放化疗为主,基因治疗、免疫治疗、分子靶向治疗等研究虽取得了一定进展,但在临床应用前仍需建立可靠的动物模型同时进行标准化的临床试验,因此疗效尚不明确。再者,已有大量研究表明许多因素均会影响骨肉瘤的发生发展,如基因突变、染色体异常、环境因素、微小RNA(microRNA,miRNA)等;还有粘附因子、凋亡异常、耐药性等也会影响骨肉瘤的治疗与转移,使得骨肉瘤的彻底治疗、死亡率降低变得尤为困难。因此,明晰骨肉瘤发生发展的分子生物学机制并利用其发生发展过程中特征的分子标志物早期预警、诊断及治疗对改善预后至关重要。然而当今关于骨肉瘤的潜在分子发病机制仍知之甚少,有待进一步探究。最近,转录因子-肌细胞增强因子2(Myocyte enhancer factor 2,MEF2)家族被发现与人类多种肿瘤的发生发展密切相关。肌细胞增强因子2D(Myocyte enhancer factor 2D,MEF2D)能够对许多类型癌症细胞的致瘤性发挥一定的促进作用,已明确为白血病、肝癌、恶性胶质瘤、结直肠癌和肺癌等恶性肿瘤的致癌基因。然而,它在骨肉瘤的产生和发展过程中是否也能发挥相同或类似的作用尚不明确,国内外研究未见相关报道。我们特别感兴趣的一点是,在肯定MEF2D过度表达在加快恶性肿瘤进程中发挥的作用之后,某些能够控制MEF2D基因过表达的miRNA也意外地被发现。因此,在我们通过体内外研究验证了MEF2D过表达对骨肉瘤的体内生长及骨肉瘤细胞的增殖活性和细胞周期都有一定的促进作用后,继而深入探究了能够作用于MEF2D表达并对其具有抑制作用的miR-144,以期为骨肉瘤患者寻找新的治疗策略提供指导方向。研究目的:观察MEF2D在骨肉瘤中的表达变化和功能特点,探讨MEF2D表达水平对骨肉瘤细胞及正常成骨细胞增殖活性与细胞周期的影响,明晰MEF2D促进骨肉瘤细胞致瘤的具体机制,并进一步探索可作用于mef2d基因并抑制其表达的mirna,从而为临床工作中寻找骨肉瘤新的治疗靶点提供方向。研究方法:1.mef2d在人骨肉瘤组织中的表达变化研究收集吉林大学第一医院等三家医院12例临床骨肉瘤组织及少量癌旁正常组织标本,提取肿瘤组织和对照组总rna和蛋白质,利用qrt-pcr、免疫组化和免疫印迹检测标本中mef2d基因和蛋白的表达情况。2.mef2d表达水平对骨肉瘤细胞及正常成骨细胞增殖能力的影响研究针对相应u2os、saos2骨肉瘤细胞和hfob1.19正常成骨细胞,利用rna干扰技术或基因过表达技术沉默或者过表达mef2d,通过mtt法观察mef2d表达水平对不同细胞增殖活性的影响。3.mef2d表达水平与骨肉瘤生长相关性的体内研究按前述方法分别用慢病毒lv-shmef2d-1(5×107pfu/mlin200μl),lv-shmef2d-2(5×107pfu/mlin200μl),lv-scrambled(5×107pfu/mlin200μl)感染u2os骨肉瘤细胞,同时分别用腺病毒ad-mef2d(5×107pfu/mlin200μl),ad-gfp(5×107pfu/mlin200μl)感染hfob1.19正常成骨细胞。随机选取30只6周龄的雄性balb/c裸鼠,移植部位选择右侧前肢腋窝处,定位后皮下注射总体积约为100μl的5×105个慢病毒感染的u2os细胞或者腺病毒感染的hfob1.19正常成骨细胞。在注射后35天内定期测量肿瘤直径并计算肿瘤体积。整个动物实验严格按照实验动物保护指南进行。所有动物实验均经吉林大学第二医院动物研究伦理审查委员会审查通过。4.mef2d表达水平影响骨肉瘤细胞及正常成骨细胞周期进程的机制研究我们进一步利用流式细胞术探究了感染慢病毒lv-shmef2d-1和lv-scrambled的u2os和saos2骨肉瘤细胞的细胞周期进程以明确mef2d过表达加速骨肉瘤细胞增殖的具体机制。同时,我们还检测了腺病毒ad-mef2d和ad-gfp感染正常成骨细胞hfob1.19后的细胞周期情况,对过表达mef2d成骨细胞的细胞周期进程进行了评估。5.mir-144通过调控mef2d表达对骨肉瘤的抑制作用研究5.1通过文献检索和mirna及靶基因数据库targetscanhuman的生物信息学分析,最终发现mef2dmrna的3'utr存在mir-144的mirna识别元件(mirnarecognitionelement,mre),它可能会抑制骨肉瘤的进程。5.2应用qrt-pcr法分别检测不同骨肉瘤细胞(u2os、saos2、hos、khos、p1、p2)和正常成骨细胞hfob1.19中mef2d和相应mir-144的表达水平。继而以mef2d表达水平为横坐标,mir-144表达水平为纵坐标描绘散点图,用以评估mir-144表达水平与mef2d表达水平之间是否存在线性关系。5.3依次以mir-144模拟剂转染u2os骨肉瘤细胞,mir-144抑制剂转染hfob1.19正常成骨细胞。24h之后,用westernblot检测u2os骨肉瘤细胞和hfob1.19正常成骨细胞中mef2d蛋白的表达情况。5.4分别将野生型和突变型mef2d3'utr插入到荧光素酶表达载体pmir-report中,分别为pmir-report-mef2d-3utr和pmir-report-mef2d-3utr-m,然后将其分别转染u2os骨肉瘤细胞系和hfob1.19正常成骨细胞系。之后,我们再用mir-144mimics转染u2os骨肉瘤细胞、mir-144inhibitors转染hfob1.19正常成骨细胞,通过检测其表达荧光素酶的情况,明确mir-144是否能够抑制mef2d的表达。研究结果:1.mef2d基因和蛋白在骨肉瘤组织中呈过度表达。通过qrt-pcr、免疫组化及免疫印迹证实在12例人骨肉瘤组织及癌旁正常组织标本中,mef2d基因和蛋白在其中10例骨肉瘤组织标本中的表达量显著高于瘤旁正常组织,差异有统计学意义。2.mef2d表达水平与骨肉瘤细胞的增殖活性密切相关。利用mtt法分析研究了mef2d表达水平对骨肉瘤细胞及正常成骨细胞增殖能力的影响。结果表明,mef2d表达水平越高,骨肉瘤细胞增殖率越高,并且mef2d表达水平和细胞增殖率呈正相关。与此同时,沉默mef2d能够降低u2os和saos2细胞的增殖率,mef2d过表达能够升高hfob1.19细胞的增殖率。3.体内改变mef2d表达水平影响骨肉瘤裸鼠移植瘤的生长。建立移植有u2os骨肉瘤细胞或hfob1.19正常成骨细胞的异种动物模型,通过lv-shmef2d-1和lv-shmef2d-2转染沉默裸鼠mef2d基因的表达,通过ad-mef2d转染高表达mef2d基因,用游标卡尺定期测量肿瘤大小并计算体积。结果表明在u2os细胞中,mef2d沉默能延缓骨肉瘤裸鼠移植瘤的生长;而在hfob1.19细胞中,mef2d过表达能够加速骨肉瘤裸鼠移植瘤的生长。4.mef2d表达水平和骨肉瘤细胞的周期进展密切相关。在明晰了过表达mef2d对骨肉瘤的细胞增殖与体内生长的促进作用之后,利用流式细胞术研究mef2d表达水平对骨肉瘤细胞及正常成骨细胞细胞周期的影响。流式细胞术结果表明在u2os和saos2细胞中,下调mef2d表达水平增加了细胞在g2/m期中的百分比,诱导了细胞周期的g2/m期阻滞。与此相对应的是,在hfob1.19细胞中,mef2d过表达降低了细胞在g2/m期中的百分比,加快细胞周期的g2/m期过渡。通过qrt-pcr定量分析发现,在u2os和saos2细胞中,mef2d沉默可引起g2/m细胞周期调控蛋白-rprm和cdkn1a明显升高。相反,在hfob1.19细胞中,mef2d过度表达则引起g2/m细胞周期调控蛋白-rprm和cdkn1a明显降低。5.mir-144通过下调mef2d表达对骨肉瘤的生长发挥抑制作用。鉴于mef2d过表达对骨肉瘤细胞增殖活性和细胞周期的抑制作用,进一步研究mef2d在其中的调控机制。通过在线数据库的筛选,提示mir-144可能是调控mef2d表达的关键mirna。通过qrt-pcr定量分析得出mir-144表达水平在u2os骨肉瘤细胞中减少,并与mef2d表达水平呈负相关。免疫印迹结果显示mir-144mimics降低了u2os细胞中mef2d蛋白的表达水平,而mir-144inhibitors上调了hfob1.19细胞中mef2d蛋白的表达水平。荧光素酶检测显示,将合成的mir-144mimics转染u2os细胞后,极大地抑制了pmir-report-mef2d-3utr荧光素酶的表达,但不会影响pmir-report-mef2d-3utr-m(p0.01);而mir-144inhibitors则增加了正常成骨细胞中pmir-report-mef2d-3utr荧光素酶的表达,但对突变株没有影响(p0.01)。这说明mir-144是通过下调mef2d表达来抑制骨肉瘤的生长。研究结论:1.mef2d在骨肉瘤组织及细胞中均呈高表达,与骨肉瘤发生发展具有密切相关性,可作为骨肉瘤的候选致癌基因。2.mef2d在mir-144的调控下通过抑制rprm和cdkn1a加速骨肉瘤细胞的G2/M期过渡而促进骨肉瘤的发展。因此,mi R-144减少可能是骨肉瘤中MEF2D高表达的原因,或可作为治疗骨肉瘤的新靶点。创新点:1.首次对MEF2D在骨肉瘤临床标本中的表达特点进行了探讨,验证了MEF2D与骨肉瘤发生发展之间的关系,证实MEF2D是骨肉瘤的候选致癌基因。2.首次研究了MEF2D促进骨肉瘤发生发展的分子机制,并明确了其与肿瘤抑制因子miR-144的关系,证实miR-144是通过调控MEF2D表达对骨肉瘤发挥抑制作用。这为骨肉瘤的早期诊断及治疗提供了一个新思路,值得进一步探究。
[Abstract]:Osteosarcoma is the most common primary malignant bone tumor in adolescents and adults, with rapid progress and early metastasis. The incidence of osteosarcoma is about 4/105 every year. It is easy to occur in young people with faster growth, and it is a serious threat to the physical and mental health of the patients. Although some progress has been made in the study of treatment, immunotherapy and molecular targeting therapy, a reliable animal model still needs to be established and standardized clinical trials are still needed before clinical application. Therefore, the curative effect is not clear. Often, environmental factors, small RNA (microRNA, miRNA), and adhesion factors, abnormal apoptosis and drug resistance also affect the treatment and metastasis of osteosarcoma, which makes the radical treatment of osteosarcoma and the reduction of mortality become particularly difficult. Therefore, the molecular biological mechanism of osteosarcoma development is clarifying and the characteristics of the development process are used. Early warning of submarkers, diagnosis and treatment are essential to improve the prognosis. However, little is known about the potential molecular pathogenesis of osteosarcoma today. It remains to be further explored. Recently, the transcription factor - the Myocyte enhancer factor 2 (MEF2) family has been found to be closely related to the development of a variety of human tumors. 2D (Myocyte enhancer factor 2D, MEF2D) can play a certain role in the tumorigenicity of many types of cancer cells. It has been identified as the oncogene of leukemia, liver cancer, malignant glioma, colorectal cancer and lung cancer. However, it can also play a role in the process of the production and development of osteosarcoma. The role of the same or similar is not clear, and there is no related report at home and abroad. One of our particular interest is that some miRNA, which can control the overexpression of MEF2D genes, have been discovered unexpectedly after affirming the role of overexpression of MEF2D in accelerating the process of malignant tumor. Therefore, we have verified the MEF2D in our body and body. Over expression can promote the growth of osteosarcoma in vivo and the proliferation and cell cycle of osteosarcoma cells, and then explore the miR-144 which can play a role in MEF2D expression and have inhibitory effect on the osteosarcoma, in order to provide guidance for the osteosarcoma patients to find new therapeutic strategies. Objective: To observe the MEF2D in bone. The expression changes and functional characteristics in sarcomas are used to explore the effect of MEF2D expression on the proliferation and cell cycle of osteosarcoma cells and normal osteoblasts, and to clarify the specific mechanism of MEF2D to promote osteosarcoma cells, and to further explore the function of the MEF2D gene and inhibit the miRNA of the osteosarcoma, so as to find bone meat for clinical work. Research methods: study methods: study methods: 1.mef2d expression in human osteosarcoma tissue, 12 cases of clinical osteosarcoma and a small number of normal tissue specimens were collected from three hospitals, such as No.1 Hospital of Jilin University, and the total RNA and protein in the tumor tissue and control group were extracted, and qRT-PCR, immunohistochemistry and Western blotting were used. The effect of the expression of MEF2D gene and protein in the samples on the proliferation of osteosarcoma cells and normal osteoblasts by.2.mef2d expression level, the study aimed at corresponding U2OS, saos2 osteosarcoma cells and hfob1.19 normal osteoblasts. The RNA interference technique or gene overexpression technique was used to silence or overexpress MEF2D, and the MEF2D table was observed by MTT method. In vivo studies on the effects of levels of.3.mef2d on the growth of different cell proliferation and osteosarcoma growth in vivo, respectively, using lentivirus lv-shmef2d-1 (5 x 107pfu/mlin200 Mu L), lv-shmef2d-2 (5 * 107pfu/mlin200 Mu L), lv-scrambled (5 * 107pfu/mlin200 micron L) infected with U2OS osteosarcoma cells, and adenovirus ad-me, respectively. F2D (5 * 107pfu/mlin200 Mu L), Ad-GFP (5 * 107pfu/mlin200 Mu L) infected normal osteoblasts of hfob1.19. 30 6 week old male balb/c nude mice were randomly selected. The transplant site selected the axillary fossa of the right forelimb, and then subcutaneous injection of 5 x 105 lentivirus infected U2OS cells or adenovirus infected hfob1.19 normal osteogenesis after localization. Cells. The diameter of the tumor and the volume of the tumor were regularly measured within 35 days after the injection. The whole animal experiment was carried out strictly in accordance with the guide of experimental animal protection. All animal experiments were examined by the animal research ethics committee of the second hospital of Jilin University to examine the effects of the.4.mef2d expression level on the osteosarcoma cells and normal osteoblast cycles. We further explored the cell cycle process of U2OS and saos2 osteosarcoma cells infected with lentivirus lv-shmef2d-1 and lv-scrambled by flow cytometry to clarify the specific mechanism of MEF2D overexpression to accelerate the proliferation of osteosarcoma cells. At the same time, we also detected HF of adenosis ad-mef2d and Ad-GFP infection normal osteoblast HF. The cell cycle status after ob1.19, evaluation of the cell cycle process over expression of MEF2D osteoblasts and the inhibitory effect of.5.mir-144 on osteosarcoma by regulating MEF2D expression 5.1 through bibliographic information analysis of literature retrieval and miRNA and target gene database targetscanhuman, it is found that mef2dmrna 3'UTR exists mir-144 MI. The RNA identification element (mirnarecognitionelement, MRE), which may inhibit the process of osteosarcoma,.5.2 use qRT-PCR to detect the expression level of different osteosarcoma cells (U2OS, saos2, HOS, khos, P1, P2) and the normal osteoblast hfob1.19 and corresponding expressions. The standard portrayed the scatter plot to assess whether there is a linear relationship between mir-144 expression level and MEF2D expression level.5.3 transfection of U2OS osteosarcoma cells with mir-144 simulant, mir-144 inhibitor transfected to hfob1.19 normal osteoblast.24h, and Westernblot detection of U2OS osteosarcoma cells and hfob1.19 normal osteoblasts by Westernblot The expression situation.5.4 inserted wild and mutant mef2d3'utr into the luciferase expression vector pmir-report, respectively pmir-report-mef2d-3utr and pmir-report-mef2d-3utr-m, and then transfected them to U2OS osteosarcoma cell lines and hfob1.19 normal osteoblast lines respectively. Then, we transfected the U2OS osteosarcoma cells with mir-144mimics. Mir-144inhibitors transfected hfob1.19 normal osteoblasts. By detecting its expression of luciferase, it is clear whether mir-144 can inhibit the expression of MEF2D. The results: the 1.mef2d gene and protein were overexpressed in the osteosarcoma tissue. Through qRT-PCR, immunohistochemistry and immunization were confirmed in 12 human osteosarcoma tissues and adjacent to cancer. In the normal tissue specimens, the expression of MEF2D gene and protein in 10 cases of osteosarcoma was significantly higher than that of normal tissue adjacent to the tumor. The difference was statistically significant between the expression level of.2.mef2d and the proliferation activity of osteosarcoma cells. The proliferation of MEF2D expressed by MTT was analyzed and the proliferation of osteosarcoma cells and normal osteoblasts was studied. The results showed that the higher the expression level of MEF2D, the higher the proliferation rate of osteosarcoma cells, and the positive correlation between the MEF2D expression level and the cell proliferation rate. At the same time, silent MEF2D can reduce the proliferation rate of U2OS and saos2 cells, and MEF2D overexpression can increase the proliferation rate of fob1.19 cells in.3. and change the level of MEF2D expression to influence bone. The growth of sarcoma transplanted tumor in nude mice. A xenograft model of U2OS osteosarcoma cells or hfob1.19 normal osteoblasts was established. The expression of MEF2D gene in nude mice was transfected by lv-shmef2d-1 and lv-shmef2d-2. The high expression of MEF2D gene was transfected by ad-mef2d. The size of the tumor was measured with the vernier caliper and the volume was calculated. The results showed that the tumor size was measured and the volume was calculated. In U2OS cells, MEF2D silencing can delay the growth of osteosarcoma xenografts in nude mice. In hfob1.19 cells, MEF2D overexpression can accelerate the growth of.4.mef2d expression level of osteosarcoma xenografts and the progression of osteosarcoma cells. The promotion of cell proliferation and growth in bone sarcomas by MEF2D is clarified. After use, flow cytometry was used to study the effect of MEF2D expression on the cell cycle of osteosarcoma cells and normal osteoblasts. Flow cytometry results showed that down regulation of MEF2D expression in U2OS and saos2 cells increased the percentage of cells in g2/m phase and induced the g2/m phase block in the cell cycle. This corresponds to that in hfob1 In.19 cells, the over expression of MEF2D reduces the percentage of cells in the g2/m phase and speeds up the g2/m transition in the cell cycle. Through qRT-PCR quantitative analysis, it is found that MEF2D silencing can cause the g2/m cell cycle regulation protein -rprm and CDKN1A to rise obviously in U2OS and saos2 cells. Cyclical regulatory proteins -rprm and CDKN1A significantly reduce the inhibitory effect of.5.mir-144 on the growth of osteosarcoma by down regulation of MEF2D. In view of the inhibitory effect of MEF2D overexpression on the proliferation and cell cycle of osteosarcoma cells, the regulatory mechanism of MEF2D is further studied. Through screening in a line database, it is suggested that mir-144 may be adjusted. The key mirna. for controlling MEF2D expression showed that the expression level of mir-144 decreased in U2OS osteosarcoma cells and was negatively correlated with the level of MEF2D expression. The results of immunoblotting showed that mir-144mimics decreased the expression level of MEF2D protein in U2OS cells, while mir-144inhibitors increased the expression of MEF2D protein in hfob1.19 cells. Level. Luciferase assay showed that the transfection of the synthesized mir-144mimics to U2OS cells greatly inhibited the expression of pmir-report-mef2d-3utr luciferase, but did not affect pmir-report-mef2d-3utr-m (P0.01), and mir-144inhibitors increased the expression of pmir-report-mef2d-3utr luciferase in normal osteoblasts, but to the mutant strain. It does not affect (P0.01). This indicates that mir-144 inhibits the growth of osteosarcoma by down regulation of MEF2D. Conclusion: 1.mef2d is highly expressed in osteosarcoma tissues and cells, and is closely related to the development of osteosarcoma, and can be used as a candidate oncogene of osteosarcoma by inhibiting RPRM and CDKN1A under the control of mir-144. The G2/M transition of the rapid osteosarcoma cells promotes the development of osteosarcoma. Therefore, the decrease of MI R-144 may be the cause of high expression of MEF2D in osteosarcoma, or can be used as a new target for the treatment of osteosarcoma. Innovation: 1. for the first time, the expression of MEF2D in the clinical specimens of osteosarcoma was discussed, which verified the development between MEF2D and osteosarcoma. It is confirmed that MEF2D is the candidate oncogene.2. of osteosarcoma for the first time to study the molecular mechanism of MEF2D to promote osteosarcoma development, and to clarify its relationship with the tumor suppressor factor miR-144, which confirms that miR-144 plays an inhibitory effect on osteosarcoma through the regulation of MEF2D expression, which provides a new diagnosis and treatment for osteosarcoma. The idea is worth further exploring.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R738.1

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