本文選題：microRNA + 細(xì)胞周期��； 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】：研究目的：為了確定AFP靜默后細(xì)胞周期停滯的HepG2細(xì)胞中是否有microRNA參與了細(xì)胞周期停滯的調(diào)節(jié),并確定此nicroRNA對細(xì)胞周期的影響和靶基因,完善對AFP信號調(diào)節(jié)通路的理解,史加深入地明了肝癌的發(fā)生機(jī)制,為發(fā)病率和死亡率都甚高危害國人和人類健康的肝癌提供更多的分子機(jī)制和治療依據(jù)。 研究內(nèi)容：在本實(shí)驗(yàn)室的前期工作中,我們發(fā)現(xiàn)AFP靜默后,HepG2細(xì)胞出現(xiàn)了細(xì)胞周期的停滯。通過michip分析,在AFP靜默的單克隆HepG2-B5細(xì)胞中有1個microRNA上調(diào)7個microRNA下調(diào)。這些差異microRNA中,可能存在有一個或多個microRNA,通過改變一系列細(xì)胞周期相關(guān)靶基因的豐度來阻滯AFP靜默的HepG2的細(xì)胞周期。 在本課題中,我們首先篩選出對細(xì)胞周期具有調(diào)節(jié)作用的microRNA: miR-372,然后在HeLa229、OVCAR3等更多的細(xì)胞系中進(jìn)一步驗(yàn)證了miR-372的不同表達(dá)水平對細(xì)胞周期的調(diào)節(jié)作用。為了研究miR-372對細(xì)胞周期的調(diào)控機(jī)制,我們預(yù)測并驗(yàn)證出CDK2、CCNA1、PPP6C等十余種細(xì)胞周期相關(guān)蛋白為miR-372的靶基因。 研究方法： 一、AFP靜默后HepG2中細(xì)胞周期效應(yīng)microRNA的篩選與miR-372的過度表達(dá)后S期比例增加G2/M期比例減少 1.通過反義寡核苷酸(ASO)技術(shù)封閉相應(yīng)的microRNA,通過生長曲線篩選差異microRNA中可以改變細(xì)胞生長速度的microRNA。發(fā)現(xiàn)miR-372為關(guān)鍵效應(yīng)microRNA。 2.研究miR-372過表達(dá)后對HepG2(-)、HeLa229等細(xì)胞生長速度的影響。 3.使用流式細(xì)胞術(shù)(FACS)檢測miR-372水平升高后細(xì)胞周期的改變。 4.通過克隆形成實(shí)驗(yàn)驗(yàn)證miR-372對HeLa229、OVCAR3克隆形成能力的影響。 二、封閉miR-372后不改變S期與G2/M期的細(xì)胞分布 1.瞬時轉(zhuǎn)染miR-372ASO,研究封閉miR-372后對A549、LO2、HepG2、 HeLa229、HeLa229-933等細(xì)胞生長速度的影響。 2.通過流式細(xì)胞術(shù)(FACS)檢測miR-372的水平降低后細(xì)胞周期的改變。 三、miR-372的細(xì)胞周期調(diào)控靶基因的預(yù)測與驗(yàn)證 1.使用生物信息學(xué)方法結(jié)合mRNA cDNA芯片的結(jié)果,預(yù)測出miR-372的靶基因。 2.通過半定量RT-PCR和western分別檢測miR-372表達(dá)水平不同的細(xì)胞中靶基因RNA和蛋白水平的差異。 3.綠色熒光蛋白融合技術(shù)驗(yàn)證miR-372對靶基因的直接作用。 結(jié)果和結(jié)論： 通過反義核苷酸ASO封閉michip中所篩選的microRNA后,發(fā)現(xiàn)miR-372ASO可以使AFP靜默后發(fā)生周期停滯的HepG2-B5細(xì)胞生長速度加快。過表達(dá)miR-372后,HepG2(-)、HeLa229、OVCAR3等細(xì)胞的生長速度均明顯受到抑制。FACS檢測細(xì)胞周期的改變后發(fā)現(xiàn)miR-372的過量表達(dá)后S期比例增加G2/M期比例減少。通過克隆形成實(shí)驗(yàn)發(fā)現(xiàn),過表達(dá)miR-372可以明顯抑制HeLa229、OVCAR3的克隆形成能力。 封閉miR-372后A549、LO2、HepG2、HeLa229、HeLa229-933的生長同樣減速,FACS檢測提示封閉miR-372后沒有引起S期和G2/M期之間細(xì)胞分布比例的變化。 Targetscan、pictar、MIRBASE、mirnaviewer四個網(wǎng)站預(yù)測出的靶基因結(jié)合AmiGO給出的細(xì)胞周期相關(guān)基因(見附錄)以及在HepG2-B5中經(jīng)mRNA cDNA芯片檢測的下調(diào)的基因,尋找出CDK2、CCNA1、PPP6C等17個細(xì)胞周期相關(guān)蛋白。對其中的6個進(jìn)行驗(yàn)證,發(fā)現(xiàn)6個靶基因在半定量PCR和綠色熒光報(bào)告載體的實(shí)驗(yàn)中的表達(dá)量均與miR-372的含量不同程度的負(fù)相關(guān),同時CCNA1和PPP6C經(jīng)過western blot驗(yàn)證其蛋白表達(dá)與miR-372也高度負(fù)相關(guān)。從6個靶基因與miR-372的負(fù)相關(guān)程度上看,CDK2、CCNA1和PPP6C是miR-372影響最強(qiáng)的靶基因。WEE1和RAD17的影響程度稍弱,p21的負(fù)相關(guān)程度最弱。 以上結(jié)果表明,在AFP靜默的HepG2細(xì)胞中,miR-372是通過靶定CDK2等靶基因引起細(xì)胞周期的停滯的關(guān)鍵效應(yīng)microRNA。miR-372表達(dá)水平的高低變化對細(xì)胞生長速度均具有負(fù)性調(diào)節(jié)作用。miR-372高表達(dá)時由于S期合成減速和M期加速使得S期比例增加和G2/M期比例減少。miR-372封閉時各時期的抑制性因子同時升高,使細(xì)胞周期不改變S期和G2/M期的分布但周期時間延長。
[Abstract]:Purpose: to determine whether there is microRNA in the HepG2 cells with the stagnant cell cycle of AFP after the silent cell cycle is involved in the regulation of cell cycle stagnation, and the effect of the nicroRNA on the cell cycle and the target gene are determined, and the understanding of the AFP signaling pathway is perfected. High risk human liver cancer and human health provide more molecular mechanisms and therapeutic evidence.
Research content: in our previous work in the laboratory, we found that after AFP silence, HepG2 cells were stagnant in cell cycle. Through michip analysis, 1 microRNA up regulated 7 microRNA downgrades in AFP silent monoclonal HepG2-B5 cells. In these differences, there may be one or more microRNA, by changing one system. The abundance of cell cycle related target genes block the cell cycle of AFP silent HepG2.
In this subject, we first screened the microRNA: miR-372 that regulates the cell cycle, and then further verified the regulation of the different expression levels of miR-372 on the cell cycle in more cell lines such as HeLa229, OVCAR3 and so on. In order to study the regulation mechanism of miR-372 on the cell cycle, we predict and verify CDK2, CCNA1 More than ten cell cycle related proteins such as PPP6C are the target genes of miR-372.
Research methods:
1. After AFP silence, the cell cycle effect microRNA in HepG2 was increased and the proportion of S phase increased after miR-372 overexpression, and the proportion of G2/M phase decreased.
1. the corresponding microRNA was closed by antisense oligonucleotide (ASO) technology, and the growth curve was used to screen the microRNA. that can change the cell growth rate in microRNA and found that miR-372 was the key effect microRNA.
2. to study the effect of over expression of miR-372 on the growth rate of HepG2 (-), HeLa229 and other cells.
3. using flow cytometry (FACS) to detect changes in cell cycle after elevated miR-372.
4. the effect of miR-372 on the clone formation ability of HeLa229 and OVCAR3 was confirmed by clonogenic test.
Two, after blocking miR-372, the cell distribution of S and G2/M phases did not change.
1. transient transfection of miR-372ASO was used to study the effects of miR-372 on A549, LO2, HepG2, HeLa229, HeLa229-933 and other cell growth rates.
2. flow cytometer (FACS) was used to detect the change of cell cycle after miR-372 level was lowered.
Three, prediction and validation of target genes for cell cycle regulation in miR-372
1. bioinformatics combined with the results of mRNA cDNA chip, predicted the target gene of miR-372.
2. semi quantitative RT-PCR and Western were used to detect the difference of target gene RNA and protein levels in miR-372 cells with different expression levels.
3. green fluorescent protein fusion technology verified the direct effect of miR-372 on target genes.
Results and conclusions:
After blocking the microRNA screened in michip by antisense nucleotide ASO, it was found that miR-372ASO could accelerate the growth rate of HepG2-B5 cells that stagnate after the silence of AFP. After the expression of miR-372, the growth rate of HepG2 (-), HeLa229, OVCAR3 and other cells was obviously inhibited by.FACS detection of cell cycle changes and the discovery of miR-372 excess. The proportion of S phase increased and the proportion of G2/M phase decreased after expression. By cloning experiment, it was found that over expression of miR-372 could significantly inhibit HeLa229 and OVCAR3 clonogenic ability.
After blocking miR-372, the growth of A549, LO2, HepG2, HeLa229, HeLa229-933 was also slowed down. FACS detection showed no change in the proportion of cell distribution between the S phase and the G2/M phase after the closed miR-372.
The target genes predicted by four sites of Targetscan, pictar, MIRBASE, and mirnaviewer combined with the cell cycle related genes (see Appendix) given by AmiGO and down regulated genes detected by mRNA cDNA chips in HepG2-B5 to find out 17 cell cycle related proteins, CDK2, CCNA1, PPP6C and so on. 6 of them were verified and 6 target genes were found. The expression in the semi quantitative PCR and the green fluorescent report carrier was negatively correlated with the content of miR-372, while CCNA1 and PPP6C passed Western blot to verify that the protein expression was also highly negatively correlated with miR-372. From the negative correlation between the 6 target genes and miR-372, CDK2, CCNA1 and PPP6C were the strongest targets for miR-372. The negative correlation between p21 and.WEE1 is the weakest because of the slight influence of RAD17 and the degree of influence.
The above results show that in the AFP silent HepG2 cells, miR-372 is a key effect of the stagnation of cell cycle by targeting CDK2 and other target genes, the level of microRNA.miR-372 expression has a negative regulation of the cell growth speed, and the.MiR-372 high expression is high when the S phase synthesis deceleration and M phase acceleration make the S phase increase. The ratio of G2/M to.MiR-372 decreased and the inhibitory factors increased at the same time when S was blocked. The cell cycle did not change the distribution of S and G2/M phases, but the cycle time prolonged.
【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】：2008
【分類號】：R3416

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