【摘要】：目的研究各WHO分級的腦膠質瘤組織中SNORD47的表達水平,WHO分級IV級膠質瘤組織中SNORD47與HOTAIR的表達相關性,以及SNORD47對膠質瘤患者生存、預后的影響。進一步研究SNORD47對腦膠質瘤增殖、周期、侵襲等生物學行為的影響,從而為抑制膠質瘤增殖、降低膠質瘤侵襲提供新的思路。方法1.第一步運用qRT-PCR檢測124例腦膠質瘤組織及其臨近的正常腦組織的SNORD47的表達水平,初步探究SNORD47在膠質瘤中的表達水平。2.第二步運用q RT-PCR檢測124例不同WHO分級腦膠質瘤標本中SNORD47、GAS5、HO TAIR的表達水平,并分析WHO分級IV級中SNORD47與HOTAIR的表達相關性、SNORD47與膠質瘤患者臨床病理特征之間的關系以及SNORD47對膠質瘤患者生存、預后的影響。3.第三步通過在U87-MG、U251細胞中轉染lenti-SNORD47,檢測其對膠質瘤細胞生物學行為的影響a.采用lenti-SNORD47轉染U87-MG及U251膠質瘤細胞,采用qRT-PCR檢測SNORD47及GAS5的表達;b.CCK-8、平板克隆、Edu細胞增殖實驗檢測過表達SNORD47后細胞增殖能力的變化;c.流式細胞術、細胞免疫熒光實驗檢測轉染lenti-SNORD47后對細胞周期的影響,Western blot檢測細胞周期相關蛋白的表達水平;d.Transwell實驗檢測過表達SNORD47后細胞侵襲能力的變化,劃痕實驗檢測過表達SNORD47后細胞遷移能力的變化,細胞免疫熒光實驗檢測轉染lenti-SNORD47后細胞骨架及EMT的改變,Western blot檢測轉染lenti-SNORD47后EMT相關蛋白的改變。4.第四步研究在U87-MG、U251細胞中轉染lenti-SNORD47后對細胞耐藥性的影響a.用逐漸升高濃度的替莫唑胺(TMZ)處理U87-MG、U251膠質瘤細胞72h,篩選出最適濃度的替莫唑胺;b.CCK-8、流式細胞術分別檢測lenti-NC、lenti-SNORD47、lenti-NC+TMZ、lenti-SNORD47+TMZ各組細胞增殖能力及細胞周期的改變。5.第五步利用體內原位裸鼠模型研究SNORD47及替莫唑胺對裸鼠顱內腫瘤的影響,以及二者間的協(xié)同作用。結果1、腦膠質瘤組織中SNORD47的表達水平及其對膠質瘤患者生存、預后的影響a.SNORD47在腫瘤組織中的表達明顯低于相鄰的正常腦組織(P0.01);b.WHO IV級腦膠質瘤組織中SNORD47的表達明顯低于WHO II級及III級腦膠質瘤組織(P0.05);GAS5的表達在各分級腫瘤中沒有明顯的統(tǒng)計學差異;HOTAIR的表達隨著腫瘤級別的增高而升高,差異具有統(tǒng)計學意義;在WHO IV級腦膠質瘤組織中SNORD47與HOTAIR表達呈負相關(R=-0.5819,P0.01);c.臨床病理特征分析結果提示SNORD47的低表達與WHO III/IV級相關。d.患者生存分析結果提示SNORD47高表達患者生存期明顯長于SNORD47低表達患者,差異顯著(P=0.0262)。2、用慢病毒轉染過表達SNORD47后發(fā)現(xiàn)U87-MG、U251細胞SNORD47表達水平明顯增高;CCK-8、平板克隆、Edu增殖等實驗結果提示SNORD47能顯著抑制膠質瘤細胞的增殖能力。3、轉染lenti-SNORD47后發(fā)現(xiàn)膠質瘤細胞G2期比例明顯升高;細胞免疫熒光結果提示過表達SNORD47后p H3陽性細胞數明顯降低;cell cycle相關蛋白表達明顯改變。4、轉染lenti-SNORD47后,細胞侵襲、遷移能力顯著下降;細胞免疫熒光及Western blot結果提示EMT明顯受到抑制。5、用替莫唑胺處理慢病毒穩(wěn)轉的細胞發(fā)現(xiàn),相較對照組和單一替莫唑胺處理或單一lenti-SNORD47處理,lenti-SNORD47和替莫唑胺聯(lián)合處理對于細胞增殖的抑制效果及細胞周期的阻滯效果更明顯。6、體內原位裸鼠成瘤實驗發(fā)現(xiàn),SNORD47能夠顯著抑制腫瘤的生長,增強腫瘤對替莫唑胺的敏感性,并且顯著延長裸鼠的生存時間。結論1、SNORD47在腦膠質瘤組織,特別是WHO IV級腦膠質瘤組織中低表達,并且與HOTAIR表達呈負相關,并能顯著延長患者生存時間;2、SNORD47能顯著抑制U87-MG、U251細胞增殖、侵襲能力;3、SNORD47能增強替莫唑胺的療效;4、SNORD47可能作為膠質瘤治療的潛在靶點。
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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