【作者】 唐琳；

【导师】 辛洪波；

【作者基本信息】 南昌大学 ， 动物学， 2013， 硕士

【摘要】 立项依据：MicroRNA (miRNA)通过与靶基因mRNA的3’非翻译区(3’UTR)特异性结合诱导靶基因mRNA的降解或阻遏其翻译。c-Myc作为一种重要的癌症相关转录因子在调节细胞生长、分化、恶性转化和凋亡等过程中起重要作用。近期的研究表明,c-Myc也可通过调节miRNA的表达而参与肿瘤的发生发展过程。我们利用生物信息学方法从已知的实验数据中,预测到大量niRNA受c-Myc调控,其中受c-Myc直接调控的110个miRNA位于基因的内含子和107个miRNA位于基因之间。根据我们设定的筛选条件如niRNA表达水平较高(reads数大于4)、具有c-Myc的结合位点(motif>0)、TSS即E-box与niRNA结合位点距离较近(5kb以内)以及FDR值即转录因子对基因的调控强度较大(小于0.05)等,共选出5个位于基因内的miRNA和16个位于基因间的miRNA。最后经过Q-PCR验证以及miRNA的可能作用靶点分析,确定has-miR-365a作为实验对象,并系统探讨其对乳腺癌细胞MCF-7生长的作用及其分子机制。实验结果：1. c-Myc特异性siRNA可明显抑制乳腺癌细胞MCF-7的c-Myc表达,其抑制mRNA表达的强度达80%以上,对蛋白也有明显的抑制作用。血清依赖性细胞生长实验和饱和密度实验结果都表明,c-Myc特异性siRNA介导的c-Myc表达抑制可使MCF-7]增殖明显减缓。同时,实验结果还显示,抑制MCF-7的c-Myc表达可显著升高细胞内has-miR-365a水平。2.我们在血清依赖性细胞生长和饱和密度实验,以及活细胞检测等观测到,Has-miR-365a模拟物(mimic)可明显抑制MCF-7的恶性增殖,而其抑制剂(inhibitor)则可几乎完全取消has-miR-365a的作用。3. Has-miR-365a模拟物(mimic)和抑制物(inhibitor)可分别显著抑制和取消has-mir-365a的靶基因HSPA8(编码蛋白是Hsc70)的表达,luciferase报告基因实验也证明,has-mir-365a(模拟物)可特异性地作用于HSPA8的3’UTR区域,进而抑制靶基因的翻译。讨论与结论：c-Myc通过miRNA调节途径而参与肿瘤的发生发展过程已引起人们的高度关注。HSPA8是热休克蛋白70(HSP70)家族一员,编码73kDa的热休克同源蛋白Hsc70,后者在促进蛋白折叠及维持其正常的结构和功能中都扮演着重要的角色。以往的工作表明,Hsc70无论是正常状态或是应激状态下对维持蛋白的动态平衡——如转运、组装、降解等过程中起关键作用。我们的实验结果表明,c-Myc调控的has-mir-365a对乳腺癌细胞MCF-7的生长具有重要的调节作用。同时我们的结果还证明,has-mir-365a对MCF-7细胞的抑制作用是通过调节其靶基因HSPA8(Hsc70)而实现的。因此,我们得出结论:c-Myc调控的has-mir-365a-Hsc70途径在乳腺癌的发生发展过程中起重要作用。更多还原

【Abstract】 BackgroundmicroRNA (miRNA) was found to induce the mRNA degredation and translational repression by specific binding to the3’-UTR of target genes. As an important cancer related transcription factor, c-Myc involved in a variety of biological processes, such as cell proliferation, differentiation, transformation and apoptosis. Resent study indicate that c-Myc may participate in the development of cancer by mediating the expression of miRNAs. In our study, we use bioinformatics analysis predicted abundance miRNAs regulated by c-Myc through known experimental data. Among these result, there are110miRNAs located in the intron and107miRNAs located between different genes. According to the screening condition-high expression level (reads>4), containg c-Myc binding site(motif>0), close distance between E-box and miRNAs(TSS<5kb), and large regulation strength of transcription factor to genes (FDR value<0.05)and so on-we set, we chose5miRNAs located in and16miRNAs between the genes. By the last, we determined the has-miR-365a as the final study object after realtime-PCR verification and target analysis, and systematic investigated the effect to the breast cancer cell line MCF-7proliferation and the molecular mechanism.Result1. C-Myc specific siRNA could significant inhibit the protein expression of C-Myc in breast cancer cell line MCF-7, and the repression effect to mRNA could be up to80%. The result of Serum dependent cell growth experiment and saturation density detection showed, C-Myc specific siRNA mediated C-Myc repression could attenuate the MCF-7proliferation. At the same time, our result showed, the C-Myc repression could significantly increase the endogenous has-miR-365a gene level.2. In our Serum dependent cell growth experiment, saturation density detection and CCK8cell activity detection results, Has-miR-365a mimic could significantly repress the malignant proliferation of MCF-7, while the inhibitor could completely avoid the effect of has-miR-365a. 3. Has-miR-365a mimic and inhibitor could significant alternatively repress or avoid the expression of target gene HSPA8(coding protein Hsc70). Luciferase report assay proved that Has-miR-365a mimic could specific binding to the3’UTR of HSPA8, and then inhibit the target gene translation.Discussion and conclusionc-Myc participating to the cancer development through miRNA regulation pathway has cause great concern. HSPA8is a member of HSP70family, coding73kDa heat shock cognate protein70, which involved in an important part to the protein folding and maintain the normal construction and function. Our previous work indicate that Hsc70played an important role in maintain protein homeostasis (protein folding, translocation, assemble and degradation) in both normal and stress condition. Our study result showed, c-Myc regulated has-mir-365a has an great significance on the breast cancer cell line MCF-7proliferation, what’s more, the repression effect to MCF-7mediated by has-mir-365a was coursed by regulation its target gene HSPA8(Hsc70). So we get the conclusion:The regulatory pathway formed by c-Myc, hsa-miR-365a and Hsc70may play an important role in the regulation of proliferation of MCF-7.更多还原