干扰TPD52基因表达对胶质瘤细胞的影响及miRNA-34a通过靶向TPD52调节胶质瘤U87细胞的周期变化

发布时间：2018-04-20 01:03

本文选题：胶质瘤 + miRNA-34a　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究目的:(1)探究TPD52基因在胶质瘤组织、正常脑组织及U87细胞中的表达水平;(2)探究干扰TPD52基因表达后对胶质瘤U87细胞增殖、周期及凋亡的影响;(3)探究干扰TPD52基因表达后对胶质瘤U87侵袭、迁移的影响;(4)探究mi RNA-34a在胶质瘤组织及U87细胞中的表达水平;(5)探究mi RNA-34a对胶质瘤细胞细胞周期的调节作用;(6)探究mi RNA-34a通过靶向TPD52调节胶质瘤U87细胞的周期变化。研究方法:(1)收集我院12例胶质瘤患者病例标本,12例脑外伤患者正常脑组织病理标本,应用荧光定量PCR检测TPD52 m RNA的表达量,应用Western blot检测TPD52蛋白的表达量。应用荧光定量PCR检测mi RNA-34a的表达量。(2)将携带着sh RNA-tpd52(抑制tpd52的核苷酸序列)、sh RNA-NC(阴性对照的核苷酸序列)的慢病毒体外转染胶质瘤细胞系U87。在转染48 h后荧光显微镜下观察表达GFP细胞数量,采用荧光定量PCR、Western blot分别检测TPD52 m RNA及蛋白表达量,MTT检测细胞增殖能力,流式细胞术检测细胞周期分布,Annexin V和PI双染流式细胞术检测细胞凋亡,划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力。(3)将mi RNA34a模拟物及mi RNA-34a抑制物转染入U87细胞,并用荧光定量PCR检测转然后细胞mi RNA-34a的表达水平。查阅相关文献并使用生物信息网分析预测TPD52的3,UTR是mi RNA-34a的作用靶点,采用荧光素酶报告实验检验TPD52是否为mi RNA-34a的靶基因。U87细胞经不同因素处理后,采用荧光定量PCR检测TPD52及相关蛋白的m RNA表达水平;采用Western blot实验检测TPD52及相关蛋白的蛋白表达水平;利用流式细胞仪检测U87细胞的细胞周期。研究结果:(1)荧光定量PCR检测结果显示tpd52m RNA在Ⅲ-Ⅳ级胶质瘤组织(ΔCT0.73±0.47)、U87细胞(ΔCT0.74±0.32)中的表达水平明显高于正常脑组织(ΔCT1.92±0.42)(P0.05);而U87细胞与Ⅲ-Ⅳ级胶质瘤组织中tpd52m RNA表达水平差异无统计学意义。Western blot检测结果显示TPD52蛋白表达量在在Ⅲ-Ⅳ级胶质瘤组织、U87细胞中的表达水平明显高于正常脑组织的表达水平。(2)U87细胞慢病毒转染率通过携带的GFP表达来评估,通过在荧光显微镜下细胞计数得出细胞转染率90%。与sh RNA-NC组及空白对照组相比较,sh RNA-tpd52组细胞TPD52 m RNA及蛋白表达量明显减少(P0.01),而且细胞增殖能力明显下降,差异有统计学意义(P0.05);细胞周期被阻滞在G0/G1期、细胞凋亡比例明显增加(P0.05),细胞迁移能力明显下降差异有统计学意义(P0.05);细胞侵袭能力明显下降差异有统计学意义(P0.05)。(3)荧光定量PCR检测结果显示mi RNA-34a在Ⅲ-Ⅳ级胶质瘤组织(ΔCT3.26±0.24)、U87细胞(ΔCT3.45±0.12)中的表达水平明显低于正常脑组织(ΔCT1.94±0.40)(P0.05);而U87细胞与Ⅲ-Ⅳ级胶质瘤组织中mi RNA-34a表达水平差异无统计学意义。(4)经转染后的U87细胞,经周期实验发现转染mi RNA-34a模拟物可明显使U87细胞停留在S期的细胞数量比例明显减少为16.12%,P0.05,而转染mi RNA-34a抑制物可明显使U87细胞停留在S期的细胞数量比例明显增加为43.56%,P0.05。双荧光素酶报告基因实验发现转染mi RNA-34a模拟物可明显使U87细胞中TPD52的3,UTR活性明显降低为32%,P0.05,而转染mi RNA-34a抑制物可明显使U87细胞中TPD52的3,UTR活性明显升高为58%,P0.05。结论:(1)TPD52在胶质瘤组织中表达水平比正常脑组织中较高。(2)干扰TPD52基因表达,可抑制胶质瘤细胞的增殖,促进细胞凋亡、阻滞细胞周期。(3)干扰TPD52基因表达,可抑制胶质瘤细胞迁移能力及侵袭能力。(4)mi RNA-34a在胶质瘤组织中表达水平比正常脑组织中较低。(5)mi RNA-34a可以调节胶质瘤细胞的细胞周期,抑制胶质瘤细胞S期细胞比例。(6)mi RNA-34a具有直接靶向TPD52调节胶质瘤细胞的周期变化。
[Abstract]:Objective: (1) to explore the expression level of TPD52 gene in glioma, normal brain and U87 cells; (2) to explore the effect of TPD52 gene expression on the proliferation, cycle and apoptosis of glioma U87 cells, and (3) to explore the influence of TPD52 gene expression on the invasion and migration of glioma U87, and (4) to explore the MI RNA-34a in glioma tissue and The expression level in U87 cells; (5) explore the regulation of MI RNA-34a on the cell cycle of glioma cells; (6) explore the periodic changes of MI RNA-34a through targeted TPD52 to regulate glioma U87 cells. (1) collect 12 cases of glioma cases in our hospital, 12 cases of normal brain tissue pathology specimens of patients with traumatic brain injury, and use fluorescent quantitative PCR The expression of TPD52 m RNA was detected and the expression of TPD52 protein was detected by Western blot. The expression of MI RNA-34a was detected by fluorescence quantitative PCR. (2) the transfection of the glioma cell line with SH RNA-tpd52 (inhibition of the nucleotide sequence of tpd52) and transfected to the glioma cell line in vitro The number of GFP cells was observed under microscopical microscope. TPD52 m RNA and protein expression were detected by fluorescence quantitative PCR and Western blot respectively. MTT was used to detect cell proliferation. Flow cytometry was used to detect cell cycle distribution. Annexin V and PI double dye flow cytometry was used to detect cell apoptosis. The migration ability of cells was detected by scratch test. Transwell experiment detected cell (3) mi RNA34a mimics and MI RNA-34a inhibitors were transfected into U87 cells, and the expression level of MI RNA-34a was detected by fluorescence quantitative PCR and the expression level of MI RNA-34a was detected by using the related literature and the bioinformatics network was used to predict the 3 of TPD52. UTR was the target of MI RNA-34a. After the target gene.U87 cells of 34a were treated by different factors, the expression of M RNA expression of TPD52 and related proteins was detected by fluorescence quantitative PCR, the protein expression level of TPD52 and related proteins was detected by Western blot test, and the cell cycle of U87 cells was detected by flow cytometry. The results were as follows: (1) the fluorescence quantitative PCR detection results showed tpd52m The expression level of NA in grade III to IV glioma (delta CT0.73 + 0.47) and U87 cells (delta CT0.74 + 0.32) was significantly higher than that of normal brain tissue (delta CT1.92 + 0.42) (P0.05), while there was no significant difference in the RNA expression of tpd52m RNA in the tissues of U87 cells and grade III to IV glioma..Western blot detection results showed that the expression of TPD52 protein was in grade III - IV The expression level of U87 cells in glioma tissue was significantly higher than that in normal brain tissue. (2) U87 cell lentivirus transfection rate was evaluated by the expression of GFP, and the cell transfection rate was compared with the SH RNA-NC group and the blank control group by counting the cell count under the fluorescence microscope. TPD52 M RNA and protein of SH RNA-tpd52 group cells. The expression volume decreased significantly (P0.01), and the cell proliferation ability decreased significantly (P0.05), the cell cycle was blocked in G0/G1 phase, the percentage of cell apoptosis increased significantly (P0.05), the difference of cell migration ability was statistically significant (P0.05), and the difference of cell invasion ability was statistically significant (P0.05). (3) fluoret The results of optical quantitative PCR detection showed that MI RNA-34a was in grade III to IV glioma (delta CT3.26 + 0.24), U87 cells (delta CT3.45 + 0.12) were significantly lower than normal brain tissues (delta CT1.94 + 0.40) (P0.05), but there was no significant difference in the level of MI RNA-34a in U87 cells and grade III - IV glioma tissues. (4) the transfected U87 cells, It was found that transfection of MI RNA-34a mimics could obviously reduce the number of U87 cells in S phase to 16.12%, P0.05, and MI RNA-34a inhibitor could obviously increase the number of U87 cells in S phase to 43.56%, and P0.05. double fluoropenzyme reporter gene experiment found that MI RNA-34a model was transfected. The 3 and UTR activity of TPD52 in U87 cells were obviously reduced to 32%, P0.05, and MI RNA-34a inhibitor could significantly increase the 3 and UTR activity of TPD52 in U87 cells to 58%, P0.05. conclusion: (1) TPD52 in glioma tissues was higher than that in normal brain tissue. (2) interference of the gene expression of TPD52 genes and the inhibition of glioma cells Proliferation, promoting cell apoptosis and blocking cell cycle. (3) interference with TPD52 gene expression can inhibit the migration ability and invasion ability of glioma cells. (4) the expression level of MI RNA-34a in glioma tissues is lower than that in normal brain tissue. (5) mi RNA-34a can regulate the cell cycle of glioma cells and inhibit the proportion of S cells in glioma cells. (6) M I RNA-34a has direct targeting TPD52 to regulate the cell cycle of glioma cells.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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