本文選題：骨肉瘤 + miRNA表達(dá)譜�。� 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：目的：探索miRNA在不同骨肉瘤細(xì)胞系中的表達(dá)特征及差異,明確骨肉瘤細(xì)胞miRNA的共同表達(dá)譜,并為后續(xù)的機(jī)制研究提供方向指導(dǎo)。 方法：收集四種人骨肉瘤細(xì)胞系(U-20S, Saos-2, MNNG/HOS, MG63)和人成骨細(xì)胞系(hFOB),提取細(xì)胞中的總RNA,應(yīng)用miRNA表達(dá)譜芯片進(jìn)行掃描篩查分析,建立骨肉瘤細(xì)胞系的miRNA表達(dá)譜。 結(jié)果：應(yīng)用miRNA表達(dá)譜芯片檢測四種人骨肉瘤細(xì)胞系(U-2OS, Saos-2, MNNG/HOS, MG63)和人成骨細(xì)胞系(hFOB)之間具有差異性表達(dá)特點(diǎn)的miRNA。我們發(fā)現(xiàn)：相對于人成骨細(xì)胞系,在四種人骨肉瘤細(xì)胞系中表達(dá)差異達(dá)2倍及以上的miRNA共有27個(gè),其中表達(dá)水平上調(diào)的miRNA共有23個(gè)(let-7e-5p, miR-20b-5p, miR-92a-3p, miR-125a-5p, miR-196a-5p, miR-214-3p, miR-20a-5p, miR-181a-5p, miR-29b-3p, miR-10b-5p, miR-301a-3p, miR-181d, miR-21-5p, miR-206, miR-7-5p, miR-29a-3p, miR-17-5p, miR-210, miR-25-3p, miR-18a-5p, miR-181c-5p, miR-32-5p, miR-27a-3p),表達(dá)水平下調(diào)的miRNA共有4個(gè)(miR-142-5p, miR-133b, miR-16, miR-144-3p)。 結(jié)論：通過miRNA表達(dá)譜芯片的檢測與篩選,我們發(fā)現(xiàn)與人成骨細(xì)胞系相對比,在四種入骨肉瘤細(xì)胞系(U-2OS, Saos-2, MNNG/HOS, MG63)共有27個(gè)miRNA呈差異性表達(dá)的特點(diǎn)。這些具有差異性表達(dá)的miRNA可以為進(jìn)一步研究miRNA在骨肉瘤中的作用與功能提供一定的實(shí)驗(yàn)基礎(chǔ)及方向指導(dǎo)。 目的：觀察miR-27a對骨肉瘤細(xì)胞增殖、凋亡和侵襲影響及其調(diào)控機(jī)制。 方法：利用Lipofectamine(?)2000將miR-27a inhibitor、miR-27a negative control、 miR-27a mimics和miR-27a mimics control轉(zhuǎn)染于骨肉瘤細(xì)胞系。利用Caspase-3、8和9酶活性試劑盒檢測檢測Caspase-3、8和9酶活性,流式細(xì)胞檢測各組細(xì)胞的凋亡情況,MTT實(shí)驗(yàn)檢測對細(xì)胞增殖的影響,Transwell遷移與侵襲實(shí)驗(yàn)評價(jià)細(xì)胞遷移侵襲能力,細(xì)胞克隆形成實(shí)驗(yàn)評價(jià)骨肉瘤細(xì)胞克隆形成的能力,qRT-PCR和Western blot檢測骨肉瘤細(xì)胞PUMA、BAK和BAX表達(dá)情況。建立裸鼠皮下骨肉瘤瘤模型,繪制腫瘤生長曲線,測量腫瘤體積以及腫瘤重量,qRT-PCR和Western blotting檢測PUMA、BAK和BAX表達(dá)情況。 結(jié)果：與其他組相比,miR-27a inhibitor組骨肉瘤細(xì)胞的Caspase-3、9酶活性顯著增加(P0.01),Caspase-8酶活性也有增加(與無轉(zhuǎn)染組對比P0.05),細(xì)胞的凋亡率顯著增加(P0.01),增殖明顯受到抑制(P0.01),遷移顯著減少(P0.01),細(xì)胞的穿膜細(xì)胞數(shù)顯著減少(P0.01),細(xì)胞的克隆形成數(shù)目顯著減少(P0.01)。miR-27a的表達(dá)水平與PUMA蛋白表達(dá)呈顯著的負(fù)相關(guān)性,熒光素酶報(bào)告法證實(shí)PUMA基因的3'UTR可以被miR-27a結(jié)合。動物實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),miR-27a inhibitor組裸鼠的腫瘤大小、體積及生長速度比miR-27a inhibitor negative組顯著減小(P0.01)。 結(jié)論：PUMA是miR-27a的靶基因,抑制miR-27a表達(dá)引起PUMA表達(dá)上調(diào)使骨肉瘤細(xì)胞Caspase-3、8和9酶活性增加,凋亡增加,并且可以抑制骨肉瘤細(xì)胞增殖、減少骨肉瘤細(xì)胞侵襲與遷移,減少骨肉瘤細(xì)胞克隆的形成,而且抑制裸鼠的腫瘤生長。
[Abstract]:Aim: to explore the expression characteristics and differences of miRNA in different osteosarcoma cell lines, to clarify the co-expression profile of miRNA in osteosarcoma cells, and to provide guidance for the further study of the mechanism. Methods: four kinds of human osteosarcoma cell lines were collected, including human osteosarcoma cell lines, such as U-20S, Saos-2, MNNG-HOSand MG63. the total RNAs were extracted from human osteosarcoma cell lines, and the miRNA expression profiles of osteosarcoma cell lines were established by scanning and screening with miRNA microarray. Results: four human osteosarcoma cell lines, namely U-2OS, Saos-2, MNNG-HOSMG63) and human osteoblast cell line hFOBwere detected by miRNA microarray. We found that compared with human osteoblast cell lines, there were 27 miRNA in four human osteosarcoma cell lines. 鍏朵腑琛ㄨ揪姘村鉤涓婅皟鐨刴iRNA鍏辨湁23涓，

本文編號：1912952