MicroRNA 145在视网膜母细胞瘤的增殖和凋亡中的机制研究

发布时间：2018-06-19 10:49

本文选题：视网膜母细胞瘤 + 胰岛素样生长因子受体1　；参考：《武汉大学》2015年博士论文

【摘要】：视网膜母细胞瘤(Retinoblastoma, RB)是最常见的婴幼儿眼内恶性肿瘤,全世界发病率大约1：20000,我国每年新病例约有1000人,占全世界每年新病例的20%,而存活率仅为63%,对患儿的视力和生命造成极大的威胁和危害,己成为医患双方共同关注的重点。视网膜母细胞瘤多发生于5岁以前,可单眼或双眼发病,常因患儿瞳孔区发白(白瞳症)或斜视就诊,也有晚期出现视力下降、眼球突出、青光眼等才引起家长注意,此时已到晚期,治疗效果较差。根据分期不同,往往采用手术治疗或保存治疗。目前国际上推荐采用化学减容法治疗取得了良好的效果,是目前RB治疗的新趋势。但由于其不容忽视的毒副作用,众多学者开始探索寻求新的生物治疗方法,基因治疗成为热点。微小RNA (micro-RNA)是一类长度为20-24个核苷酸的非编码单链小分子RNA,广泛分布于组织和细胞中,参与调控个体发育、细胞凋亡、增殖和分化等生命活动,与多种肿瘤的发生和发展密切相关,其作用靶点可为抑癌基因,可为癌基因。miRNA-145定位于染色体5q32-33,已经在乳腺癌、结肠癌和淋巴癌等相关疾病研究中证实其几乎在所有肿瘤组织中表达下调,能够抑制肿瘤细胞的增殖和凋亡。但其在视网膜母细胞瘤中的作用及相关机制研究尚未见报道。胰岛素样生长因子1受体(IGF-1R)属于酪氨酸蛋白激酶家族,能够促进细胞增生、转移并抑制细胞凋亡,与肿瘤细胞侵袭、转移、促进新生血管形成等密切相关。有研究证明,其被配体活化后可通过p-Akt的磷酸化激活PI3K/Akt通路促进脉络膜黑色素瘤的侵袭和转移；脉络膜黑色素瘤组织高表达IGF-1R也与其发生转移及预后呈正相关。我们推测其在视网膜母细胞瘤中也可能起到了相似作用,但尚无相关研究证实。本课题首先采用脂质体介导的基因转染技术,观察miRNA-145对视网膜母细胞瘤细胞增殖和凋亡的影响；随后采用免疫组化方法研究了临床视网膜母细胞瘤标本中IGF-1R的表达,并通过体外培养的视网膜母细胞瘤细胞细胞系-Y79细胞,对IGF-1R与细胞的增殖和凋亡的关系进行研究,初步探讨了IGF-1R成为视网膜母细胞瘤的治疗新靶点的可能性。最后对miR-145和IGF-1R对人视网膜母细胞瘤细胞的调控机制进行了进一步的探索,为探索治疗视网膜母细胞瘤的新手段提供实验依据。本课题包括以下三部分内容：第一部分Mir-145对视网膜母细胞瘤Y79细胞增殖和凋亡的影响目的：探讨MicroRNA 145(miR-145)对视网膜母细胞瘤Y79细胞的增殖和凋亡的影响。方法：将人视网膜母细胞瘤细胞株Y79分为四组：miR-145干预组、阴性miRNA对照组、空脂质体组、空白对照组。脂质体转染法将miR-145转入Y79细胞；荧光定量PCR检测转染后48h成熟miR-145的表达；CCK-8法检测转染48h后的细胞增殖抑制率；流式细胞术检测细胞周期；Annexin V/PI免疫荧光双染色和流式细胞术检测细胞凋亡。通过单因素方差分析分析比较各组数据。结果：采用脂质体转染方法,成功将miR-145转染至Y79细胞；荧光定量PCR显示：miR-145干预组成熟miR-145表达(79.06±3.45)明显增加,与阴性miRNA对照组(1.06±0.03)、空脂质体组(0.93±0.02)和空白组(1.00±0.02)比较,差异有统计学意义(F=229.853,P0.05)；miR-145干预组的增殖抑制率(21.64%)明显高于阴性miRNA对照组(2.57%)、空脂质体组(3.97%)(F=34.13,P0.05)；miR-145抑制Y79细胞周期于G1期(P0.01)；Annexin V/PI免疫荧光双染色和流式细胞术显示miR-145干预组Annexin V阳性和Annexin V/PI双阳性细胞明显增多,阳性率明显高于其他三组,差异有统计学意义(F=35.434,P0.05)。结论：miR-145可以抑制视网膜母细胞瘤细胞Y79的增殖,诱导细胞凋亡。第二部分IGF-1R在视网膜母细胞瘤中的表达和作用目的：研究IGF-1R对人视网膜母细胞瘤细胞的增殖和凋亡的影响。方法：免疫组化法检测IGF-1R在人视网膜母细胞瘤组织中的表达。培养人视网膜母细胞瘤细胞Y79后并分为三组：IGF-1R siRNA干预组、阴性siRNA对照组、空白对照组。以脂质体为载体把小干扰RNA (IGF-1R siRNA)导入Y79细胞；Real-Time PCR分析RNA干扰后IGF-1R mRNA的水平；Western blot检测IGF-1R蛋白表达水平；CCK-8法检测转染24h后的细胞增殖抑制率；Annexin V/PI免疫荧光双染色和流式细胞术检测细胞凋亡。通过单因素方差分析分析比较各组数据。结果：免疫组化法证实IGF-1R在人视网膜母细胞瘤细胞中有表达；小干扰RNA (IGF-1R siRNA)靶向阻止IGF-1R基因在Y79细胞中的表达；Real-Time PCR提示：siRNA组(0.426)的IGF-1R mRNA的基因表达明显下降,与阴性对照组(0.983)和空白对照组(1.000)相比,差异有统计学意义(P0.01); Western blot检测显示siRNA组的IGF-1R蛋白表达水平分别为阴性siRNA对照组的22.4%,空白对照组的24.0%；CCK-8检测细胞提示siRNA组的A值(0.30)低于CSI组(0.40)和NC(0.42),差异有显著差异(P0.05)。Annexin V/PI双染色结合流式检测细胞凋亡结果显示：siRNA组Annexin V率为28.9%,明显高于阴性对照组(6.4%)和空白对照组(6.6%),差异具有统计学意义(P0.01)。结论：IGF-1R在人视网膜母细胞瘤细胞中表达,且通过特异性降低IGF-1R蛋白表达,能够抑制Y79细胞的增殖。第三部分 miR-145通过抑制IGF-1R的表达来调控视网膜母细胞瘤细胞的增殖和凋亡目的：研究miR-145调控视网膜母细胞瘤细胞的增殖和凋亡的机制。方法：培养人视网膜母细胞瘤细胞株Y79后,将实验分为三组：实验分为三组：miR-145mimics干预组、阴性miRNA对照组、空白对照组。Real-Time PCR分析IGF-1R的mRNA的水平；Western blot检测IGF-1R蛋白的表达水平。构建pMIR-REPORT-IGF-1R 3'-UTR荧光素酶质粒。荧光素酶活性检测法检测miR-145与IGF-1R3'-UTR之间的相互作用。各组数据的比较采用单因素方差分析。结果：Real-Time PCR结果提示miR-145mimics干预组的IGF-1R mRNA的量明显下降(0.498),与阴性miRNA对照组为(0.951)和空白对照组(1)相比,差异有显著意义(P0.05)。Western blot检测显示miR-145组的IGF-1R蛋白表达水平明下降,与阴性miRNA对照组为(0.951)和空白对照组(1)相比,差异具有统计学意义(P0.01)。荧光素酶活性检测显示miR-145干预组的萤火虫荧光素酶的活性明显降低(P0.01),提示miR-145与IGF-1R的发生相互作用。结论：miR-145可以通过降低IGF-1R的表达来抑制人网膜母细胞瘤细胞Y79的增殖。
[Abstract]:Retinoblastoma (RB) is the most common intraocular malignant tumor in infants and infants. The incidence of the disease is about 1:20000 in the world. There are about 1000 new cases in China each year, accounting for 20% of the new cases in the world, and the survival rate is only 63%. It has made a great threat and harm to the children's vision and life. It has become a common relationship between the doctors and patients. The focal point of note. Retinoblastoma occurs more than 5 years old, can monocular or binocular disease, often because of children's pupil white (white pupil) or strabismus treatment, also have late appearance of vision decline, eye protrusion, glaucoma, and so on to the parents attention, this time to the late, treatment effect is poor. According to different stages, often use surgical treatment or Conservation therapy is a new trend in RB therapy at present. However, because of its toxic and side effects that can not be ignored, many scholars have begun to explore new biotherapy methods. Gene therapy has become a hot spot. Small RNA (micro-RNA) is a class of 20-24 nucleotides. RNA, a small single strand of small molecule, is widely distributed in tissues and cells. It participates in the regulation of individual development, apoptosis, proliferation and differentiation. It is closely related to the occurrence and development of various tumors. The target can be the tumor suppressor gene and the oncogene.MiRNA-145 is located in chromosome 5q32-33. It has been found in breast, colon and lymphatic cancer. In the study of related diseases, it is confirmed that it is almost down-regulated in all tumor tissues and can inhibit the proliferation and apoptosis of tumor cells. However, the study of its role in retinoblastoma and its related mechanism have not yet been reported. The insulin like growth factor 1 receptor (IGF-1R) belongs to the tyrosine protein kinase family, which can promote cell proliferation and transfer. Migration and inhibition of apoptosis are closely related to the invasion, metastasis and angiogenesis of tumor cells. Studies have shown that the ligand activation can activate the PI3K/Akt pathway through the phosphorylation of p-Akt to promote the invasion and metastasis of choroidal melanoma, and the high expression of IGF-1R in the choroidal melanoma tissue is also associated with its metastasis and prognosis. We speculate that it may also play a similar role in retinoblastoma, but there is no related research yet. Firstly, the effect of miRNA-145 on the proliferation and apoptosis of retinoblastoma cells was observed by liposome mediated gene transfection, and then the clinical retina was studied by immunohistochemical method. The expression of IGF-1R in the tumor of the mother cell tumor and the relationship between the proliferation and apoptosis of IGF-1R and cell by -Y79 cells cultured in vitro, the possibility of IGF-1R as a new target for the treatment of retinoblastoma is preliminarily discussed. The latter is the latter to the retinoblastoma of human retinoblastoma and the human retinoblastoma of the human retinoblastoma. The mechanism of cell regulation has been further explored to provide experimental basis for the exploration of the novice segment of retinoblastoma. This topic includes the following three parts: the first part: the effect of Mir-145 on the proliferation and apoptosis of Y79 cells in retinoblastoma: To explore the Y79 fine of MicroRNA 145 (miR-145) for retinoblastoma Effect of cell proliferation and apoptosis. Methods: the human retinoblastoma cell line Y79 was divided into four groups: miR-145 intervention group, negative miRNA control group, empty liposome group, blank control group. MiR-145 was transferred into Y79 cells by liposome transfection; fluorescence quantitative PCR was used to detect the expression of 48h mature miR-145 after transfection; CCK-8 method was used to detect the transfection of 48h. Cell proliferation inhibition rate; flow cytometry to detect cell cycle; Annexin V/PI immunofluorescence double staining and flow cytometry to detect cell apoptosis. The data were compared and analyzed by single factor analysis of variance. Results: the transfection method of liposome was used to transfect miR-145 to Y79 cells successfully; fluorescence quantitative PCR showed miR-145 intervention composition The expression of mature miR-145 (79.06 + 3.45) was significantly increased, compared with negative miRNA control group (1.06 + 0.03), empty liposome group (0.93 + 0.02) and blank group (1 + 0.02), the difference was statistically significant (F=229.853, P0.05), and the proliferation inhibition rate (21.64%) in miR-145 intervention group was significantly higher than that of negative miRNA control group (2.57%), and air liposome group (3.97%) (F=34.13, P0.05). MiR-145 inhibited Y79 cell cycle in G1 phase (P0.01), Annexin V/PI immunofluorescence double staining and flow cytometry showed that Annexin V positive and Annexin V/PI double positive cells increased significantly in miR-145 intervention group, and the positive rate was significantly higher than those of the other three groups, the difference was statistically significant (F= 35.434). The proliferation and apoptosis of cell Y79, inducing apoptosis. Second part IGF-1R expression in retinoblastoma and its purpose: To study the effect of IGF-1R on the proliferation and apoptosis of human retinoblastoma cells. Methods: immunohistochemical method was used to detect the expression of IGF-1R in human retinoblastoma tissue. The tumor cell Y79 was divided into three groups: the IGF-1R siRNA intervention group, the negative siRNA control group and the blank control group. The small interference RNA (IGF-1R siRNA) was introduced into Y79 cells with liposome as the carrier; Real-Time PCR was used to analyze the level of IGF-1R protein after RNA interference; The rate of colonization, Annexin V/PI immunofluorescence double staining and flow cytometry were used to detect cell apoptosis. The data were compared and analyzed by single factor analysis of variance. Results: immunohistochemistry showed that IGF-1R was expressed in human retinoblastoma cells; small interference RNA (IGF-1R siRNA) targeting the expression of IGF-1R gene in Y79 cells; Real-Time PCR indicated that the gene expression of IGF-1R mRNA in group siRNA (0.426) decreased significantly, compared with negative control group (0.983) and blank control group (1), the difference was statistically significant (P0.01); Western blot detection showed that the expression level of IGF-1R protein in siRNA group was divided into 22.4% of negative siRNA control group and 24% in blank control group; Detection cells suggested that the A value of siRNA group (0.30) was lower than that of group CSI (0.40) and NC (0.42). The difference was significant (P0.05).Annexin V/PI double staining combined flow cytometry showed that the Annexin V rate of siRNA group was 28.9%, which was significantly higher than that of negative control group (6.4%) and blank control group (6.6%). The difference was statistically significant (P0.01). Conclusion: IGF- 1R is expressed in human retinoblastoma cells and can inhibit the proliferation of Y79 cells by specific reduction of the expression of IGF-1R protein. Third part miR-145 regulates the proliferation and apoptosis of retinoblastoma cells by inhibiting the expression of IGF-1R: the study of miR-145 regulating the proliferation and apoptosis of retinoblastoma cells of retinoblastoma Methods: after training the human retinoblastoma cell line Y79, the experiment was divided into three groups: the experiment was divided into three groups: the miR-145mimics intervention group, the negative miRNA control group, the blank control group.Real-Time PCR analysis of IGF-1R mRNA level; Western blot detected the expression level of IGF-1R protein. Construction pMIR-REPORT-IGF-1R 3'-UTR luciferase The interaction between miR-145 and IGF-1R3'-UTR was detected by the assay of luciferase activity. The data of each group were compared by single factor variance analysis. Results: the results of Real-Time PCR showed that the amount of IGF-1R mRNA in the miR-145mimics intervention group decreased significantly (0.498), compared with the negative miRNA control group (0.951) and the blank control group (1). The significant (P0.05).Western blot detection showed that the expression level of IGF-1R protein in the miR-145 group decreased obviously, compared with the negative miRNA control group (0.951) and the blank control group (1), the difference was statistically significant (P0.01). The luciferase activity in miR-145 intervention group was significantly decreased (P0.01), suggesting miR-145 (P0.01), suggesting miR-145. Conclusion: miR-145 can inhibit the proliferation of human omentoma cell line Y79 by decreasing the expression of IGF-1R. IGF-1R
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.7

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