SNORD47调控胶质瘤生物学行为的研究
发布时间：2018-08-16 12:27
【摘要】：目的研究各WHO分级的脑胶质瘤组织中SNORD47的表达水平,WHO分级IV级胶质瘤组织中SNORD47与HOTAIR的表达相关性,以及SNORD47对胶质瘤患者生存、预后的影响。进一步研究SNORD47对脑胶质瘤增殖、周期、侵袭等生物学行为的影响,从而为抑制胶质瘤增殖、降低胶质瘤侵袭提供新的思路。方法1.第一步运用qRT-PCR检测124例脑胶质瘤组织及其临近的正常脑组织的SNORD47的表达水平,初步探究SNORD47在胶质瘤中的表达水平。2.第二步运用q RT-PCR检测124例不同WHO分级脑胶质瘤标本中SNORD47、GAS5、HO TAIR的表达水平,并分析WHO分级IV级中SNORD47与HOTAIR的表达相关性、SNORD47与胶质瘤患者临床病理特征之间的关系以及SNORD47对胶质瘤患者生存、预后的影响。3.第三步通过在U87-MG、U251细胞中转染lenti-SNORD47,检测其对胶质瘤细胞生物学行为的影响a.采用lenti-SNORD47转染U87-MG及U251胶质瘤细胞,采用qRT-PCR检测SNORD47及GAS5的表达;b.CCK-8、平板克隆、Edu细胞增殖实验检测过表达SNORD47后细胞增殖能力的变化;c.流式细胞术、细胞免疫荧光实验检测转染lenti-SNORD47后对细胞周期的影响,Western blot检测细胞周期相关蛋白的表达水平;d.Transwell实验检测过表达SNORD47后细胞侵袭能力的变化,划痕实验检测过表达SNORD47后细胞迁移能力的变化,细胞免疫荧光实验检测转染lenti-SNORD47后细胞骨架及EMT的改变,Western blot检测转染lenti-SNORD47后EMT相关蛋白的改变。4.第四步研究在U87-MG、U251细胞中转染lenti-SNORD47后对细胞耐药性的影响a.用逐渐升高浓度的替莫唑胺(TMZ)处理U87-MG、U251胶质瘤细胞72h,筛选出最适浓度的替莫唑胺;b.CCK-8、流式细胞术分别检测lenti-NC、lenti-SNORD47、lenti-NC+TMZ、lenti-SNORD47+TMZ各组细胞增殖能力及细胞周期的改变。5.第五步利用体内原位裸鼠模型研究SNORD47及替莫唑胺对裸鼠颅内肿瘤的影响,以及二者间的协同作用。结果1、脑胶质瘤组织中SNORD47的表达水平及其对胶质瘤患者生存、预后的影响a.SNORD47在肿瘤组织中的表达明显低于相邻的正常脑组织(P0.01);b.WHO IV级脑胶质瘤组织中SNORD47的表达明显低于WHO II级及III级脑胶质瘤组织(P0.05);GAS5的表达在各分级肿瘤中没有明显的统计学差异;HOTAIR的表达随着肿瘤级别的增高而升高,差异具有统计学意义;在WHO IV级脑胶质瘤组织中SNORD47与HOTAIR表达呈负相关(R=-0.5819,P0.01);c.临床病理特征分析结果提示SNORD47的低表达与WHO III/IV级相关。d.患者生存分析结果提示SNORD47高表达患者生存期明显长于SNORD47低表达患者,差异显著(P=0.0262)。2、用慢病毒转染过表达SNORD47后发现U87-MG、U251细胞SNORD47表达水平明显增高;CCK-8、平板克隆、Edu增殖等实验结果提示SNORD47能显著抑制胶质瘤细胞的增殖能力。3、转染lenti-SNORD47后发现胶质瘤细胞G2期比例明显升高;细胞免疫荧光结果提示过表达SNORD47后p H3阳性细胞数明显降低;cell cycle相关蛋白表达明显改变。4、转染lenti-SNORD47后,细胞侵袭、迁移能力显著下降;细胞免疫荧光及Western blot结果提示EMT明显受到抑制。5、用替莫唑胺处理慢病毒稳转的细胞发现,相较对照组和单一替莫唑胺处理或单一lenti-SNORD47处理,lenti-SNORD47和替莫唑胺联合处理对于细胞增殖的抑制效果及细胞周期的阻滞效果更明显。6、体内原位裸鼠成瘤实验发现,SNORD47能够显著抑制肿瘤的生长,增强肿瘤对替莫唑胺的敏感性,并且显著延长裸鼠的生存时间。结论1、SNORD47在脑胶质瘤组织,特别是WHO IV级脑胶质瘤组织中低表达,并且与HOTAIR表达呈负相关,并能显著延长患者生存时间;2、SNORD47能显著抑制U87-MG、U251细胞增殖、侵袭能力;3、SNORD47能增强替莫唑胺的疗效;4、SNORD47可能作为胶质瘤治疗的潜在靶点。
[Abstract]:Objective To study the expression of SNORD47 in gliomas of different WHO grades, the correlation between SNORD47 and HOTAIR in gliomas of WHO grades IV, and the effect of SNORD47 on survival and prognosis of gliomas. Methods 1. The expression of SNORD47 in 124 glioma tissues and adjacent normal brain tissues was detected by qRT-PCR, and the expression level of SNORD47 in glioma was preliminarily explored. 2. The second step was to detect SNORD47 and GA in 124 glioma specimens of different WHO grades by qRT-PCR. The expression levels of S5 and HO-TAIR, and the correlation between SNORD47 and HOTAIR in WHO grade IV, the relationship between SNORD47 and clinicopathological characteristics of glioma patients, and the influence of SNORD47 on survival and prognosis of glioma patients were analyzed. 3. The third step was to detect the biological behavior of lenti-SNORD47 in glioma cells by transfecting U87-MG and U251 cells. The expression of SNORD47 and GAS5 in U87-MG and U251 glioma cells transfected with lenti-SNORD47 was detected by qRT-PCR; the proliferation of cells after overexpression of SNORD47 was detected by b.CCK-8, plate cloning and Edu cell proliferation assay; and the effect of lenti-SNORD47 on cell cycle was detected by flow cytometry and immunofluorescence assay. Western blot was used to detect the expression level of cell cycle-related proteins; D. Transwell assay was used to detect the changes of cell invasion ability after overexpression of SNORD47; scratch test was used to detect the changes of cell migration ability after overexpression of SNORD47; immunofluorescence assay was used to detect the changes of cytoskeleton and EMT after transfection of lenti-SNORD47; Western blot was used to detect the changes of cell skeleton and EMT after transfection. Changes of EMT-related proteins in U87-MG and U251 cells after transfection of lenti-SNORD47 were studied. Fourthly, the effects of transfection of lenti-SNORD47 on cell resistance were studied. U87-MG and U251 glioma cells were treated with TMZ for 72 hours, and the optimum concentration of temozolomide was screened out; B. CCK-8, lenti-NC, lenti-SNORD47 were detected by flow cytometry, respectively. The effects of SNORD47 and temozolomide on intracranial tumors in nude mice were studied by in situ nude mice model. Results 1. The expression of SNORD47 in glioma tissues and the survival of glioma patients. The expression of SNORD47 in tumor tissues was significantly lower than that in adjacent normal brain tissues (P 0.01); the expression of SNORD47 in b.WHO grade IV glioma tissues was significantly lower than that in WHO grade II and III glioma tissues (P 0.05); the expression of GAS5 in tumor tissues was not significantly different with tumor grade; The expression of SNORD47 was negatively correlated with the expression of HOTAIR (R = - 0.5819, P 0.01) in WHO grade IV gliomas. C. The results of clinicopathological analysis showed that the low expression of SNORD47 was correlated with WHO grade III/IV. D. Survival analysis of patients with high expression of SNORD47 indicated that the survival time of patients with SNORD47 was significantly longer than that of patients with low expression of SNORD47. The expression level of SNORD47 in U87-MG and U251 cells was significantly increased after lenti-SNORD47 was transfected. CCK-8, plate cloning, Edu proliferation and other experimental results indicated that SNORD47 could significantly inhibit the proliferation of glioma cells. 3. After lenti-SNORD47 transfection, the proportion of glioma cells in G2 phase was significantly increased. The results of immunofluorescence showed that the number of P H3-positive cells decreased significantly after overexpression of SNORD47; the expression of cell cycle-related protein changed significantly. 4. After transfection of lenti-SNORD47, the invasion and migration ability of cells decreased significantly; the results of immunofluorescence and Western blot indicated that EMT was inhibited significantly. 5. Lentiviruses were treated with temozolomide. Cell growth inhibition and cell cycle arrest of lenti-SNORD47 combined with temozolomide were more obvious than those of control group and single temozolomide treatment or single lenti-SNORD47 treatment. 6. In vivo in nude mice tumorigenesis test showed that SNORD47 could significantly inhibit tumor growth and enhance tumorigenesis to temozolomide. Conclusion1, SNORD47 is low expressed in glioma tissues, especially in grade IV glioma tissues, and is negatively correlated with the expression of HOTAIR, and can significantly prolong the survival time of patients; 2, SNORD47 can significantly inhibit the proliferation and invasion of U87-MG, U251 cells; 3, SNORD47 can enhance the ability to replace U87-MG, U251 cells. The efficacy of mozolomide is 4; SNORD47 may be a potential target for glioma treatment.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
本文编号：2185982
[Abstract]:Objective To study the expression of SNORD47 in gliomas of different WHO grades, the correlation between SNORD47 and HOTAIR in gliomas of WHO grades IV, and the effect of SNORD47 on survival and prognosis of gliomas. Methods 1. The expression of SNORD47 in 124 glioma tissues and adjacent normal brain tissues was detected by qRT-PCR, and the expression level of SNORD47 in glioma was preliminarily explored. 2. The second step was to detect SNORD47 and GA in 124 glioma specimens of different WHO grades by qRT-PCR. The expression levels of S5 and HO-TAIR, and the correlation between SNORD47 and HOTAIR in WHO grade IV, the relationship between SNORD47 and clinicopathological characteristics of glioma patients, and the influence of SNORD47 on survival and prognosis of glioma patients were analyzed. 3. The third step was to detect the biological behavior of lenti-SNORD47 in glioma cells by transfecting U87-MG and U251 cells. The expression of SNORD47 and GAS5 in U87-MG and U251 glioma cells transfected with lenti-SNORD47 was detected by qRT-PCR; the proliferation of cells after overexpression of SNORD47 was detected by b.CCK-8, plate cloning and Edu cell proliferation assay; and the effect of lenti-SNORD47 on cell cycle was detected by flow cytometry and immunofluorescence assay. Western blot was used to detect the expression level of cell cycle-related proteins; D. Transwell assay was used to detect the changes of cell invasion ability after overexpression of SNORD47; scratch test was used to detect the changes of cell migration ability after overexpression of SNORD47; immunofluorescence assay was used to detect the changes of cytoskeleton and EMT after transfection of lenti-SNORD47; Western blot was used to detect the changes of cell skeleton and EMT after transfection. Changes of EMT-related proteins in U87-MG and U251 cells after transfection of lenti-SNORD47 were studied. Fourthly, the effects of transfection of lenti-SNORD47 on cell resistance were studied. U87-MG and U251 glioma cells were treated with TMZ for 72 hours, and the optimum concentration of temozolomide was screened out; B. CCK-8, lenti-NC, lenti-SNORD47 were detected by flow cytometry, respectively. The effects of SNORD47 and temozolomide on intracranial tumors in nude mice were studied by in situ nude mice model. Results 1. The expression of SNORD47 in glioma tissues and the survival of glioma patients. The expression of SNORD47 in tumor tissues was significantly lower than that in adjacent normal brain tissues (P 0.01); the expression of SNORD47 in b.WHO grade IV glioma tissues was significantly lower than that in WHO grade II and III glioma tissues (P 0.05); the expression of GAS5 in tumor tissues was not significantly different with tumor grade; The expression of SNORD47 was negatively correlated with the expression of HOTAIR (R = - 0.5819, P 0.01) in WHO grade IV gliomas. C. The results of clinicopathological analysis showed that the low expression of SNORD47 was correlated with WHO grade III/IV. D. Survival analysis of patients with high expression of SNORD47 indicated that the survival time of patients with SNORD47 was significantly longer than that of patients with low expression of SNORD47. The expression level of SNORD47 in U87-MG and U251 cells was significantly increased after lenti-SNORD47 was transfected. CCK-8, plate cloning, Edu proliferation and other experimental results indicated that SNORD47 could significantly inhibit the proliferation of glioma cells. 3. After lenti-SNORD47 transfection, the proportion of glioma cells in G2 phase was significantly increased. The results of immunofluorescence showed that the number of P H3-positive cells decreased significantly after overexpression of SNORD47; the expression of cell cycle-related protein changed significantly. 4. After transfection of lenti-SNORD47, the invasion and migration ability of cells decreased significantly; the results of immunofluorescence and Western blot indicated that EMT was inhibited significantly. 5. Lentiviruses were treated with temozolomide. Cell growth inhibition and cell cycle arrest of lenti-SNORD47 combined with temozolomide were more obvious than those of control group and single temozolomide treatment or single lenti-SNORD47 treatment. 6. In vivo in nude mice tumorigenesis test showed that SNORD47 could significantly inhibit tumor growth and enhance tumorigenesis to temozolomide. Conclusion1, SNORD47 is low expressed in glioma tissues, especially in grade IV glioma tissues, and is negatively correlated with the expression of HOTAIR, and can significantly prolong the survival time of patients; 2, SNORD47 can significantly inhibit the proliferation and invasion of U87-MG, U251 cells; 3, SNORD47 can enhance the ability to replace U87-MG, U251 cells. The efficacy of mozolomide is 4; SNORD47 may be a potential target for glioma treatment.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
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1 Jeff Boyd;;Implication of snoRNA U50 in human breast cancer[J];遗传学报;2009年08期
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