miR-372对细胞周期的调控

发布时间：2018-06-09 23:34

本文选题：microRNA + 细胞周期　；参考：《天津医科大学》2008年硕士论文

【摘要】：研究目的：为了确定AFP静默后细胞周期停滞的HepG2细胞中是否有microRNA参与了细胞周期停滞的调节,并确定此nicroRNA对细胞周期的影响和靶基因,完善对AFP信号调节通路的理解,史加深入地明了肝癌的发生机制,为发病率和死亡率都甚高危害国人和人类健康的肝癌提供更多的分子机制和治疗依据。研究内容：在本实验室的前期工作中,我们发现AFP静默后,HepG2细胞出现了细胞周期的停滞。通过michip分析,在AFP静默的单克隆HepG2-B5细胞中有1个microRNA上调7个microRNA下调。这些差异microRNA中,可能存在有一个或多个microRNA,通过改变一系列细胞周期相关靶基因的丰度来阻滞AFP静默的HepG2的细胞周期。在本课题中,我们首先筛选出对细胞周期具有调节作用的microRNA: miR-372,然后在HeLa229、OVCAR3等更多的细胞系中进一步验证了miR-372的不同表达水平对细胞周期的调节作用。为了研究miR-372对细胞周期的调控机制,我们预测并验证出CDK2、CCNA1、PPP6C等十余种细胞周期相关蛋白为miR-372的靶基因。研究方法：一、AFP静默后HepG2中细胞周期效应microRNA的筛选与miR-372的过度表达后S期比例增加G2/M期比例减少 1.通过反义寡核苷酸(ASO)技术封闭相应的microRNA,通过生长曲线筛选差异microRNA中可以改变细胞生长速度的microRNA。发现miR-372为关键效应microRNA。 2.研究miR-372过表达后对HepG2(-)、HeLa229等细胞生长速度的影响。 3.使用流式细胞术(FACS)检测miR-372水平升高后细胞周期的改变。 4.通过克隆形成实验验证miR-372对HeLa229、OVCAR3克隆形成能力的影响。二、封闭miR-372后不改变S期与G2/M期的细胞分布 1.瞬时转染miR-372ASO,研究封闭miR-372后对A549、LO2、HepG2、 HeLa229、HeLa229-933等细胞生长速度的影响。 2.通过流式细胞术(FACS)检测miR-372的水平降低后细胞周期的改变。三、miR-372的细胞周期调控靶基因的预测与验证 1.使用生物信息学方法结合mRNA cDNA芯片的结果,预测出miR-372的靶基因。 2.通过半定量RT-PCR和western分别检测miR-372表达水平不同的细胞中靶基因RNA和蛋白水平的差异。 3.绿色荧光蛋白融合技术验证miR-372对靶基因的直接作用。结果和结论：通过反义核苷酸ASO封闭michip中所筛选的microRNA后,发现miR-372ASO可以使AFP静默后发生周期停滞的HepG2-B5细胞生长速度加快。过表达miR-372后,HepG2(-)、HeLa229、OVCAR3等细胞的生长速度均明显受到抑制。FACS检测细胞周期的改变后发现miR-372的过量表达后S期比例增加G2/M期比例减少。通过克隆形成实验发现,过表达miR-372可以明显抑制HeLa229、OVCAR3的克隆形成能力。封闭miR-372后A549、LO2、HepG2、HeLa229、HeLa229-933的生长同样减速,FACS检测提示封闭miR-372后没有引起S期和G2/M期之间细胞分布比例的变化。 Targetscan、pictar、MIRBASE、mirnaviewer四个网站预测出的靶基因结合AmiGO给出的细胞周期相关基因(见附录)以及在HepG2-B5中经mRNA cDNA芯片检测的下调的基因,寻找出CDK2、CCNA1、PPP6C等17个细胞周期相关蛋白。对其中的6个进行验证,发现6个靶基因在半定量PCR和绿色荧光报告载体的实验中的表达量均与miR-372的含量不同程度的负相关,同时CCNA1和PPP6C经过western blot验证其蛋白表达与miR-372也高度负相关。从6个靶基因与miR-372的负相关程度上看,CDK2、CCNA1和PPP6C是miR-372影响最强的靶基因。WEE1和RAD17的影响程度稍弱,p21的负相关程度最弱。以上结果表明,在AFP静默的HepG2细胞中,miR-372是通过靶定CDK2等靶基因引起细胞周期的停滞的关键效应microRNA。miR-372表达水平的高低变化对细胞生长速度均具有负性调节作用。miR-372高表达时由于S期合成减速和M期加速使得S期比例增加和G2/M期比例减少。miR-372封闭时各时期的抑制性因子同时升高,使细胞周期不改变S期和G2/M期的分布但周期时间延长。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R3416

【共引文献】