重编程相关IncRNA（lincRNA-ROR）在脑胶质瘤中的表达及功能的研究

发布时间：2018-06-29 05:04

本文选题：胶质瘤 + 长链非编码　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：目的：明确lincRNA-ROR在胶质瘤组织及瘤旁正常脑组织中的表达水平。在此基础上进一步研究lincRNA-ROR在胶质瘤细胞及胶质瘤干细胞中的生物学功能，探索其在胶质瘤发生发展中可能的分子作用机制。方法： 1. q-PCR法检测lincRNA-ROR在胶质瘤组织及瘤旁正常脑组织中的表达水平。收集26例胶质瘤患者肿瘤及瘤旁正常脑组织手术标本，运用q-PCR技术检测lincRNA-ROR在胶质瘤组织及瘤旁脑组织的表达水平。并检测与lincRNA-ROR相关的两个基因SOX11及KLF4的表达相关性情况。 2.构建LincRNA-RoR shRNA质粒，将其转染至胶质瘤细胞系中，q-PCR技术检测lincRNA-ROR表达情况，下调lincRNA-RoR的表达后检测lincRNA-RoR对胶质瘤细胞及胶质瘤干细胞生物学特性的影响。胶质瘤细胞系培养48小时后使用CD133标记肿瘤干细胞，用流式细胞仪检测CD133+的胶质瘤干细胞数量；MTS分析法检测U87细胞增殖情况，连续3天检测细胞增殖能力；胶质瘤细胞系培养7天后，干细胞成球实验检测下调lincRNA-RoR后干细胞成球能力。 3.构建lincRNA-RoR真核表达载体，将其转染至胶质瘤细胞系中，q-PCR技术检测lincRNA-ROR表达情况。上调lincRNA-RoR的表达后检测lincRNA-RoR对胶质瘤细胞及胶质瘤干细胞生物学特性的影响。胶质瘤细胞系培养48小时后使用CD133标记肿瘤干细胞，用流式细胞仪检测CD133+的胶质瘤干细胞数量；MTS分析法检测U87细胞增殖情况，连续3天检测细胞增殖能力；胶质瘤细胞系培养7天后，干细胞成球实验检测过表达lincRNA-RoR后干细胞成球能力。4.检测lincRNA-RoR表达下调及上调后KLF4的表达情况。结果： 1. lincRNA-RoR在胶质瘤组织中的表达明显低于在瘤旁脑组织中的表达。 2. lincRNA-RoR与KLF4mRNA在胶质瘤组织中的表达呈负相关。与SOX11mRNA在胶质瘤组织中的表达呈正相关。 3.成功的构建LincRNA-RoR shRNA质粒，将LincRNA-RoR shRNA质粒稳定的转染到U87胶质瘤细胞。下调LincRNA-RoR表达后流式细胞仪检测CD133+的胶质瘤干细胞数量明显增加；MTS分析法检测显示U87胶质瘤细胞的增殖能力明显增强，且随着时间的推移，，增殖程度越加明显；成球实验显示胶质瘤干细胞成球数量明显增加。 4.成功的构建了PcDNA-lincRNA-RoR质粒，将PcDNA-lincRNA-RoR质粒稳定的转染到U87胶质瘤细胞。上调LincRNA-RoR表达后流式细胞仪检测CD133+的胶质瘤干细胞数量明显下降；MTS分析法检测显示U87胶质瘤细胞的增殖能力受到抑制，且随着时间的推移，增殖程度逐渐下降；成球实验显示胶质瘤干细胞成球数量明显减少。 5.下调lincRNA-RoR表达后U87胶质瘤细胞KLF4mRNA表达明显降增高。上调lincRNA-RoR表达后U87胶质瘤细胞KLF4mRNA表达明显降下降。结论：本研究初步证实了重编程相关的基因lincRNA-RoR可能是一个新的胶质瘤抑制基因。在胶质瘤组织及胶质瘤细胞系中的表达降低，在胶质瘤细胞中过表达lincRNA-RoR可以抑制胶质瘤细胞增殖及胶质瘤干细胞自我更新能力；部分的抑制了的KLF4mRNA表达，可能与KLF4相互调节而起作用。
[Abstract]:Objective: to clarify the expression level of lincRNA-ROR in glioma and normal brain tissue, and to further study the biological function of lincRNA-ROR in glioma cells and glioma stem cells, and to explore the possible molecular mechanism of the action in the development of glioma.
Method:
1. q-PCR method was used to detect the expression level of lincRNA-ROR in glioma tissue and normal brain tissue adjacent to the tumor. 26 cases of glioma and normal brain tissue around the tumor were collected and the expression level of lincRNA-ROR in glioma tissue and paratatal tissue was detected by q-PCR technique. And two genes related to lincRNA-ROR were detected, SOX11 and KLF 4 of the correlation of expression.
2. LincRNA-RoR shRNA plasmid was constructed and transfected into glioma cell line, q-PCR technique was used to detect the expression of lincRNA-ROR, and the expression of lincRNA-RoR was downregulated to detect the effect of lincRNA-RoR on the biological characteristics of glioma cells and glioma stem cells. After the culture of glioma cell line, the CD133 was used to mark the tumor stem cells with the flow formula. The number of CD133+ glioma stem cells was detected by cytometer, and the proliferation of U87 cells was detected by MTS analysis, and the cell proliferation ability was detected for 3 days. After 7 days of glioma cell line culture, the ball formation test was used to detect the ability of down-regulation of lincRNA-RoR stem cells.
3. construct lincRNA-RoR eukaryotic expression vector, transfect it into glioma cell line and detect the expression of lincRNA-ROR by q-PCR technique. After up regulation of lincRNA-RoR expression, the effects of lincRNA-RoR on the biological characteristics of glioma cells and glioma stem cells were detected. After 48 hours culture of glioma cell line, the use of CD133 to mark tumor stem cells was used. The number of glioma stem cells in CD133+ was detected by flow cytometry; the proliferation of U87 cells was detected by MTS analysis, and cell proliferation was detected for 3 days. After 7 days of culture of glioma cell lines, the expression of lincRNA-RoR expression was detected and the expression of KLF4 after the expression of lincRNA-RoR was detected by.4. of stem cells after the expression of lincRNA-RoR in the stem cell formation test. Condition.
Result:
1. the expression of lincRNA-RoR in glioma tissue was significantly lower than that in tumor adjacent brain tissue.
2. the expression of lincRNA-RoR was negatively correlated with KLF4mRNA expression in glioma tissues, and positively correlated with the expression of SOX11mRNA in glioma tissues.
3. LincRNA-RoR shRNA plasmid was successfully constructed, and LincRNA-RoR shRNA plasmid was transfected into U87 glioma cells steadily. The number of CD133+ glioma stem cells increased significantly after the down-regulation of LincRNA-RoR expression, and MTS analysis showed that the proliferation energy of U87 glioma cells increased significantly and increased with time. The growth of glioma stem cells was significantly increased.
4. successfully constructed the PcDNA-lincRNA-RoR plasmid and transfected the PcDNA-lincRNA-RoR plasmid into U87 glioma cells steadily. After up regulation of LincRNA-RoR expression, the number of glioma stem cells in CD133+ was significantly decreased, and the proliferation ability of U87 glioma cells was inhibited by MTS analysis, and as time went on, The proliferation of glioma stem cells decreased significantly.
5. after downregulation of lincRNA-RoR expression, the expression of KLF4mRNA in U87 glioma cells increased significantly. After upregulated lincRNA-RoR expression, the expression of KLF4mRNA in glioma cells decreased significantly.
Conclusion: This study has preliminarily confirmed that reprogramming related gene lincRNA-RoR may be a new glioma suppressor gene. The expression in glioma and glioma cell lines is reduced. Overexpression of lincRNA-RoR in glioma cells can inhibit the proliferation of glioma cells and the self-renewal capacity of glioma stem cells; The expression of KLF4mRNA may play a role in regulating KLF4.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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