本文選題：miR-101 + let-7a-1�。� 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的微小RNA(miRNA)參與不同的生物過程,包括胚胎發(fā)育,代謝,分化等生理和病理過程。它們主要是通過與靶mRNA的3'UTR中的互補序列結(jié)合在轉(zhuǎn)錄和轉(zhuǎn)錄后水平調(diào)控基因表達發(fā)揮生物學(xué)功能。Let-7靶定特異性的癌基因,通過多種信號通路參與腫瘤的發(fā)生發(fā)展等過程,功能上起到抑癌基因的作用。有報道表明,在乳腺癌細胞中,組蛋白去甲基化酶直接抑制let-7a的轉(zhuǎn)錄,多能性因子Lin28也可以結(jié)合其初級和前體轉(zhuǎn)錄本中的莖環(huán)結(jié)構(gòu)影響Dicer對let-7前體(pre-let-7a)的剪切,從而抑制let-7成熟體的生成。本研究的目的是探尋在宮頸癌細胞內(nèi),是否存在能夠靶定let-7a-1前體的莖環(huán)loop結(jié)構(gòu)的miRNAs及其對let-7a-1的成熟體、前體(pre-)和原始轉(zhuǎn)錄本(pri-)生成的影響,并確定參與這個過程的調(diào)控蛋白。本研究我們試圖闡明miRNAs間的調(diào)節(jié),以及miRNA成熟過程中前體loop結(jié)構(gòu)起到的重要作用。方法1.首先利用生物信息學(xué)預(yù)測能夠與pre-let-7a-1的loop結(jié)構(gòu)中27個堿基可能(不完全)互補匹配的miRNAs。構(gòu)建過表達和封閉上述miRNAs的質(zhì)�；蚝铣梢种苖iRNAs表達的寡核苷酸,RT-qPCR實驗檢測宮頸癌細胞中改變上述miRNAs表達水平對let-7a-1成熟體、前體及原始轉(zhuǎn)錄本的影響。2.選擇上述miRNAs中調(diào)控let-7a-1成熟體形成的miRNAs,通過EGFP熒光載體報告系統(tǒng)檢測該miRNAs與let-7a-1的loop結(jié)構(gòu)的靶定關(guān)系。3.Western blot檢測miR-101對let-7a-1下游直接靶基因Aurora蛋白表達水平的影響。4.RIP實驗檢測miR-101通過結(jié)合pre-let-7a-1的loop結(jié)構(gòu)對其成熟體形成調(diào)節(jié)依賴于Ago蛋白。結(jié)果1.生物信息學(xué)預(yù)測到miR-148a、miR-181b和miR-101是與pre-let-7a-1 loop在不同位點形成不完全互補配對,過表達和封閉上述miRNAs實驗表明僅miR-101能夠抑制let-7a-1成熟體表達,對其前體及原始轉(zhuǎn)錄本的表達水平無明顯影響。2.通過EGFP熒光報告載體系統(tǒng)發(fā)現(xiàn)miR-101可以抑制具有l(wèi)et-7a-1前體loop結(jié)構(gòu)的EGFP熒光水平和蛋白水平的表達。3.Western blot驗證了miR-101對let-7a-1下游直接靶基因Aurora的上調(diào)作用是通過let-7a-1發(fā)揮作用的。4.RIP實驗驗證miR-101結(jié)合pre-let-7a-1調(diào)節(jié)let-7a-1成熟體形成依賴于Ago蛋白結(jié)論本研究發(fā)現(xiàn)miR-101通過結(jié)合pre-let-7a-1的loop結(jié)構(gòu)抑制let-7a-1的成熟,該作用的發(fā)揮依賴Ago2蛋白。這對于miRNA形成的調(diào)控提供新的理論解釋及實驗依據(jù)。
[Abstract]:Objective small RNAs miRNAs are involved in various biological processes, including embryonic development, metabolism, differentiation and other physiological and pathological processes. They play a biological role in regulating gene expression at the transcriptional and post-transcriptional level by combining with complementary sequences in the 3'UTR of target mRNA, and by targeting specific oncogenes in Let-7, and participate in tumorigenesis and development through a variety of signal pathways. Function plays the role of tumor suppressor gene. It has been reported that histone demethylase directly inhibits the transcription of let-7a in breast cancer cells, and that the pluripotent factor Lin28 can also affect the splicing of let-7 precursor pre-let-7a by combining the stem ring structure in its primary and precursor transcripts. Thus, the formation of mature bodies of let-7 was inhibited. The aim of this study was to investigate the presence of miRNAs targeting the loop structure of the stem ring of let-7a-1 precursors and its effects on let-7a-1 maturation, precursor pre-formation and pripri-production in cervical cancer cells, and to identify the regulatory proteins involved in this process. In this study, we try to elucidate the regulation of miRNAs and the important role of precursor loop structure in the process of miRNA maturation. Method 1. First, bioinformatics was used to predict the possible (incomplete) matching miRNAs with 27 bases in the loop structure of pre-let-7a-1. Construction of plasmids that overexpressed and blocked the miRNAs or synthesis of oligonucleotide reverse transcriptase (RT-qPCR) to inhibit the expression of miRNAs were used to detect the effect of changes in the expression of miRNAs on let-7a-1 maturation, precursor and original transcripts in cervical cancer cells. Select miRNAss, which regulate the formation of let-7a-1 maturation in miRNAs, and detect the relationship between the loop structure of miRNAs and let-7a-1 by EGFP fluorescence vector reporting system. 3. Western blot detection of miR-101 on the expression level of Aurora protein downstream direct target gene of let-7a-1 .4.RIP experiment MiR-101 is dependent on Ago protein to regulate its maturation by binding to loop structure of pre-let-7a-1. Result 1. Bioinformatics predicted that miR-148a miR-181b and miR-101 formed incomplete complementary pairing with pre-let-7a-1 loop at different sites. Overexpression and blocking of the above miRNAs experiments showed that only miR-101 could inhibit the expression of let-7a-1 mature body, but had no significant effect on the expression level of its precursors and original transcripts. EGFP fluorescence report vector system showed that miR-101 could inhibit the expression of EGFP fluorescence level and protein level with let-7a-1 precursor loop structure. 3. Western blot confirmed that miR-101 upregulated let-7a-1 downstream direct target gene Aurora through let-7a-1. 4. Rip experiments demonstrated that miR-101 combined with pre-let-7a-1 regulates the formation of let-7a-1 maturation dependent on Ago protein. Conclusion in this study, miR-101 inhibits let-7a-1 maturation through loop structure binding to pre-let-7a-1. This role is dependent on Ago2 protein. This provides a new theoretical explanation and experimental basis for the regulation of the formation of miRNA.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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