中文摘要
中文摘要
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qRT-PCR检测124例不同WHO分级脑胶质瘤标本中SNORD47、GAS5、HOTAIR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；
中文摘要
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（P<0.01）；
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（P<0.05）；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，P<0.01）；
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pH3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，
中文摘要
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可能作为胶质瘤的潜在靶点。
关键词：SNORD47；胶质瘤；细胞周期；增殖；侵袭
Abstract
ABSTRACT
Objective
proliferationTo investigate the expression of SNORD47 in 124 clinical glioma specimens and adjacent normal tissues. To measure the expression of SNORD47 in 124 clinical glioma specimens of different grades. To explore the relationship between SNORD47 and HOTAIR in WHO grade IV glioma specimens and the effect of SNORD47 on the survival of glioma patients. To further study the effects of SNORD47 on the proliferation, cell cycle, invasion of glioma cells and provide a new insight into suppressing the proliferation and invasion in glioma.
Methods
The first step: Quantitative Real-Time PCR was used to measure the expression level of SNORD47 in 124 clinical glioma specimens of different grades and adjacent normal tissues.
The second step: The expression of SNORD47, GAS5, HOTAIR in 124 clinical glioma specimens of different grades was detected by Quantitative Real-Time PCR. We study the correlation between HOTAIR and SNORD47, the relationship between the expression of SNORD47 and clinicopathological parameters in glioma patients and the effect of SNORD47 on the survival of glioma patients.
The third step: To explore the potential effects of SNORD47 on the biological behavior in glioma cells after transfected with lenti-SNORD47.
a. After transfected with lenti-SNORD47, the expression levels of SNORD47 and GAS5 was detected by Quantitative Real-Time PCR.
b. The CCK-8 assay, colony formation assay and Edu assay were used to evaluate the proliferation ability of glioma cells Cell after transfected with lenti-SNORD47.
c. The cell cycle was analyzed by flow cytometry and immunofluorescence assay. Western blot was used to detected the expression of cell cycle related proteins after transfected with lenti-SNORD47.
d. Transwell assay and wound-healing assay were used to evaluate the change of
Abstract
invasion ability and migration ability of glioma cells respectively after the transfection. Immunofluorescence assay was used to study the change of cytoskeleton and Epithelial-Mesenchymal Transition (EMT). EMT-related proteins was measured by western blot after the transfection.
The fourth step: To study the effect of SNORD47 on the sensitivity of glioma cells to temozolomide
a. We treated U87-MG and U251 cells with increasing concentrations of temozolomide for 72 h to screen optimal concentration.
b. The changes of invasion ability and cell cycle in lenti-NC、lenti-SNORD47、lenti-NC+TMZ、lenti-SNORD47+TMZ group were explored by CCK-8 assay and flow cytometry respectively.
The fifth group: to determine whether SNORD47 had an antitumor effect and whether SNORD47 could strengthen the temozolomide-induced suppression of tumorigenesis in vivo.
Results
1、The expression of SNORD47 in glioma specimens and the effect of SNORD47 on the survival of glioma patients
a. The results showed that the expression of SNORD47 was significantly lower in glioma specimens compared to the adjacent normal tissues (P<0.01).
b. The expression of SNORD47 was significantly decreased in grade IV glioblastoma compared with the lower-grade glioma specimens (P<0.05). However, there was no significant difference in the expression of SNORD47 between the grade I/II and grade III glioma specimens. There were no significant differences in the expression of GAS5 among the different grades of glioma specimens. The expression level of HOTAIR was positively associated the grades of the glioma specimens. And the correlation between HOTAIR and SNORD47 was significantly inverse (R=-0.5819, P<0.01).
c. The results of clinicopathological parameters in glioma patients showed that the lower expression of SNORD47 was significantly associated with the advanced WHO grade (III/IV).
d. Kaplan-Meier survival analysis indicated that patients with higher SNORD47
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