肿瘤抑制蛋白p53通过microRNAs调控B7-H1表达的研究

发布时间：2018-07-01 15:28

  本文选题：CRISPR/CAS9 + p53　； 参考：《华中科技大学》2016年博士论文

【摘要】：第一部分P53敲除A375细胞系的建立目的：利用CRISPR/CAS9系统建立p53敲除的细胞系方法：设计两条针对人的p53基因的gRNA,分别将两条gRNA插入pBT-U6-Cas9-2A-GFP表达质粒,然后将含有不同gRNA的质粒分别转染进入K562细胞,用SUVEYOR突变检测试剂盒,对两条设计的gRNA的剪切效率进行比较,选取效率较高gRNA进行后续p53敲除。在A375细胞中转染GFP标记含有gRNA和CAS9核酸酶的质粒,验证突变,同时流式分选出GFP阳性的细胞。将分选出的细胞,分别单个接种于96孔板,待其克隆形成。部分克隆形成之后,将其扩增传代,形成单克隆细胞系。为从中筛选出p53敲除细胞,首先我们使用x-ray处理所有获得克隆,然后免疫电泳检测所有克隆中p53的表达情况,选出p53表达阴性的克隆进行测序,以验证p53敲除的可靠性。同时也对X射线刺激细胞,然后对p53下游基因p21进行检测,以在功能上确认p53的敲除。结果：SUVEYOR结果显示设计的两条gRNA均可以造成突变,编辑效率第一条gRNA较好。使用gRNA 1转染A375细胞,SUVEYOR检测到突变发生。Western检测p53在各个克隆中的表达发现,在获得的23个克隆中,有3个克隆在p53位置出现了缺失,敲除效率为3/23.同时使用PCR对敲除位点进行了扩增,相比正常WT p53条带,所获得疑似敲除克隆的p53条带位置均发生了变化。测序结果显示,所获得三个阳性克隆p53基因段均发生了明显的基因插入或者缺失,导致了p53表达的失调。最后对p53下游基因p21的表达进行检测,发现所获克隆中,在p53已被激活的情况下,p21的表达全部缺失。结论：利用CRISPR/CAS9系统,成功敲除p53基因,获得了稳定遗传的p53完全敲除和p53变异细胞株。第二部分p53通过microRNAs抑制B7-H1的表达目的：研究p53敲除后是否对细胞B7-H1的表达造成影响以及通过何种机制影响其表达方法：分别用流式检测A375.HCT116和其p53敲除前后的B7-H1表达；使用RT-PCR检测B7-H1的信使RNA在A375, HCT116和其p53敲除细胞系中的表达：使用microRNA芯片检测A375和A375Δp53细胞之间的microRNA表达差异,并结合B7-H1靶点预测,从而筛选出可能具有对B7-H1具有调控功能的microRNAs;将筛选出来的microRNAs mimics转染A375Δp53细胞,观察B7-H1在各个不同mimics转染后的表达情况,选择出调控B7-H1表达的microRNA;使用特异性inhibitors同mimics一同转染A375Δp53细胞,以确认microRNA作用的特异性;使用qRT-PCR检测A375,HCT116和其p53敲除前后以及使用siRNA敲除p53之后的microRNA-34a以及microRNA-200b表达,以验证p53对microRNA-34a以及microRNA-200b表达的调控作用,并使用免疫电泳对p53以及B7-H1的表达进行检测。构建双荧光报告载体验证microRNA-34a以及microRNA-200b靶向作用于B7-Hl的3UTR。结果：当p53缺失时,通过流式检测发现B7-H1的表达在A375, HCT116细胞中均明显上升；之后,对B7-H1的信使RNA表达水平进行检测,发现p53的敲除,并不影响B7-H1 mRNA的表达,结果无显著性差异；通过筛选,并结合靶向预测,我们发现在p53敲除前后,有8个候选microRNAs可能调控B7-H1表达。转染mimics后发现,microRNA-34a和microRNA-200b可下调B7-H1在A375Δp53细胞中的表达,差异明显。在加入microRNA-34a和microRNA-200b inhibitors共转染时,这种下调效果可被消除。实时定量PCR检测发现,在p53敲除或敲低的情况下,microRNA-34a和microRNA-200b表达明显下降,同时B7-H1的表达上调。双荧光素报告载体检测发现,microRNA-34a和microRNA-200b可特异性结合域B7-H1的3'UTR端调控转录。结论：p53的敲除下调microRNA-34a和microRNA-200b的表达,从而抑制microRNA-34a和microRNA-200b对B7-H1信使RNA的作用,导致B7-H1蛋白表达上升。第三部分microRNA-34a和microRNA-200b调控Jurkat增殖和细胞因子分泌目的：研究microRNA-34a和microRNA-200b通过调控B7-H1分子从而影响Jurkat细胞的功能,介导免疫抑制。方法：培养Jurkat细胞,用TPA刺激,诱导其表达PD-1受体,免疫电泳检测诱导表达效果;将诱导表达PD-1的Jurkat细胞同A375Δp53细胞共培养,利用B7-H1阻断性抗体抑制B7-H1/PD-1通路的激活,观察Jurkat细胞的增殖情况。在使用microRNA-34a和microRNA-200b的mimics和inhibitors分别转染A375Ap53细胞后同Jurkat细胞共培养,以观察microRNA-34a和microRNA-200b对细胞造成的增殖改变。同时收集共培养细胞的上清培养液,检测microRNA-34a和microRNA-200b对细胞分泌IFN-gamma, IL-2细胞因子的影响。结果：jurkat细胞在TPA刺激后,其PD-1的表达明显增强。将刺激后的细胞同A375Δp53细胞共培养,其增殖受到明显抑制,在用B7-H1抗体阻断后,此种增值抑制效应可以得到解除；同时,结果显示,A375Ap53细胞相对于A375细胞更能明显抑制Jurkat细胞增殖。在A375Δp53细胞中转染进microRNA-34a和microRNA-200b的mimics后可以部分消除对Jurkat细胞的增殖抑制效应,加入对应inhibitors,可以恢复对Jurkat细胞的抑制。同时对细胞因子的检测发现,A375Δp53细胞表达B7-H1抑制Jurkat细胞的细胞因子分泌,microRNA-34a和microRNA-200b的mimics转染可以有效解除抑制效果,共转染对应inhibitors可以使得分泌再次被抑制。结论：PD-1/B7-H1通路的激活可以抑制Jurkat细胞的增殖。A375Ap53细胞相比A375细胞具有更强的免疫抑制能力,此种抑制效应同microRNA-34a和microRNA-200b对B7-H1表达的调控具有联系。microRNA-34a和microRNA-200b的表达可以明显抑制对Jurkat细胞功能的抑制。
[Abstract]:The first part of the P53 knockout A375 cell line: using the CRISPR/CAS9 system to establish the p53 knockout cell line method: design two gRNA for human p53 gene, insert two gRNA into pBT-U6-Cas9-2A-GFP expression plasmid, and then transfer the plasmid containing different gRNA into K562 cells, and use SUVEYOR mutation to detect the reagent. In the box, the shear efficiency of the two designed gRNA was compared, and the efficient gRNA was selected for subsequent p53 knockout. The plasmid containing gRNA and CAS9 nuclease was transfected in the A375 cell to verify the mutation, and the flow formula was used to separate the GFP positive cells. After the formation of the lung, the PCR was amplified to form a monoclonal cell line. In order to screen out the p53 knockout cells, we first used X-ray to treat all the clones, and then the expression of p53 in all the clones was detected by immunoelectrophoresis, and the clones with negative p53 expression were sequenced to verify the reliability of the p53 knockout. At the same time, the X ray stimulation was also used. Cells, then detection of the downstream gene p21 of p53 to confirm the knockout of p53 in function. Results: SUVEYOR results showed that the two gRNA of the designed gRNA could cause the mutation, the efficiency of the first gRNA was better. GRNA 1 was used to transfect A375 cells, and SUVEYOR detected the expression of.Western detection p53 in the individual clones. Of the 23 clones, 3 clones were missing in the p53 position, the knockout efficiency was 3/23. and the PCR knockout loci were amplified. Compared to the normal WT p53 strip, the position of the suspected knockout clone p53 bands had changed. The results showed that the three positive clones p53 gene segments had obvious genes. The insertion or deletion resulted in the maladjustment of p53 expression. Finally, the expression of the p53 downstream gene p21 was detected. In the clones, the expression of p21 was completely missing in the case of p53 being activated. Conclusion: the p53 gene was successfully knocked out by the CRISPR/CAS9 system, and the stable hereditary p53 knockout and p53 variant cell lines were obtained. Second parts were obtained. P53 suppression of the expression of B7-H1 through microRNAs: the study of whether p53 knockout has an impact on the expression of B7-H1, and the mechanism to affect its expression: A375.HCT116 and B7-H1 expression before and after the p53 knockout, respectively, and the messenger and its knockout cells using RT-PCR to detect B7-H1. Expression in the system: microRNA chip was used to detect the difference of microRNA expression between A375 and A375 delta p53 cells, and the B7-H1 target prediction was used to screen the microRNAs that may have regulatory function to B7-H1, and the selected microRNAs mimics transfected to A375 delta p53 cells. In addition, microRNA was selected to regulate the expression of B7-H1, and A375 delta p53 cells were transfected with mimics with specific inhibitors to confirm the specificity of microRNA action, and qRT-PCR was used to detect A375, HCT116 and its p53 knockout, as well as the expression after the use of siRNA knockout. The expression of croRNA-200b and the expression of p53 and B7-H1 were detected by immunoelectrophoresis. A double fluorescent report vector was constructed to verify the 3UTR. results of microRNA-34a and microRNA-200b targeting B7-Hl. When p53 was missing, the expression of B7-H1 was obviously increased in A375, HCT116 cells by flow detection; after that, the expression of B7-H1 was obviously increased. To detect the expression level of B7-H1's messenger RNA, it is found that p53 knockout does not affect the expression of B7-H1 mRNA, and there is no significant difference in the results. Through screening and targeting prediction, we found that 8 candidate microRNAs may regulate the expression of B7-H1 before and after p53 knockout. After transfection of mimics, microRNA-34a and microRNA-200b can downregulate B7-H1 The expression in A375 delta p53 cells was distinctly different. The down regulation effect could be eliminated when CO transfected with microRNA-34a and microRNA-200b inhibitors. Real-time quantitative PCR detection found that the expression of microRNA-34a and microRNA-200b decreased significantly in the case of p53 knockout or knock down, while the expression of B7-H1 was up. It is found that the 3'UTR terminal of the microRNA-34a and microRNA-200b specific binding domain B7-H1 controls the transcription. Conclusion: p53 knocks down the expression of microRNA-34a and microRNA-200b, thus inhibiting the effect of microRNA-34a and microRNA-200b on B7-H1 messenger RNA, leading to the increase of the expression of the B7-H1 protein. The purpose of Kat proliferation and cytokine secretion is to study the effect of microRNA-34a and microRNA-200b on the function of Jurkat cells by regulating B7-H1 molecules and mediated immunosuppression. Methods: Jurkat cells were cultured and stimulated by TPA to induce the expression of PD-1 receptors, and immunoelectrophoresis was used to detect the induction of expression; Jurkat cells expressing PD-1 were induced with A375 delta p53. Cell co culture was used to observe the proliferation of Jurkat cells by using B7-H1 blocking antibodies to inhibit the activation of the B7-H1/PD-1 pathway. The proliferation of A375Ap53 cells was co cultured with A375Ap53 cells using mimics and inhibitors in microRNA-34a and microRNA-200b, respectively, to observe the proliferation of microRNA-34a and microRNA-200b on the cells. The effect of microRNA-34a and microRNA-200b on the secretion of IFN-gamma and IL-2 cytokine was detected by the supernatant culture of co cultured cells. Results: the expression of PD-1 in Jurkat cells increased obviously after TPA stimulation. The stimulated cells were co cultured with A375 delta p53 cells, and the proliferation of Jurkat cells was significantly inhibited. This type of cells was blocked by B7-H1 antibodies. At the same time, the results showed that A375Ap53 cells could significantly inhibit the proliferation of Jurkat cells compared with A375 cells. The proliferation inhibition response to Jurkat cells could be partially eliminated after A375 delta p53 cells were transferred to microRNA-34a and microRNA-200b mimics, and the corresponding inhibitors could be restored to Jurkat. At the same time, the detection of cytokines showed that A375 delta p53 cells expressed B7-H1 to inhibit the secretion of cytokines in Jurkat cells, and mimics transfection of microRNA-34a and microRNA-200b could effectively relieve the inhibitory effect. Co transfection of corresponding inhibitors could cause the secretion of secretions to be suppressed again. Conclusion: the activation of PD-1/B7-H1 pathway can inhibit J. The proliferating.A375Ap53 cells of urkat cells have stronger immunosuppressive ability than A375 cells. This inhibitory effect is associated with the regulation of microRNA-34a and microRNA-200b on the expression of B7-H1 and the expression of.MicroRNA-34a and microRNA-200b can obviously inhibit the inhibition of Jurkat cell function.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2

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