基于microRNAs鉴别同卵双生子的法医学探索研究

Individual discrimination of monozygotic twins (MZ) who are widely regarded as genetically identical is a worldwide forensic problem. Although some researchers at home and abroad had made some progress in identifying MZ by detecting epigenetic markers, yet the problem still have not been solved completely. So, it is very essential to find new biomarkers for the individual discrimination of MZ. In recent years, with the development of molecular biology, microRNAs gradually become a research hotspot. microRNAs are a class of small, endogenous, non-protein-coding RNA molecules of approximately 22 nucleotides in length, and exist extensively in a variety of eukaryotic cells. Known as 'dimmer switches' of gene expression, microRNAs regulate gene expression and play fundamental roles in multiple biological processes, including cell differentiation, proliferation and apoptosis as well as aging and disease processes. Since microRNAs play important regulatory roles in human body, then, whether microRNAs are differentially expressed between MZ is the focus of our attention, and this hypothesis was confirmed by our preliminary study. Based on this, in this study, microRNA microarray will be selected to screen the differentially expressed microRNAs between MZ in the genome-wide, and then the quantitative analysis of difference of these target microRNA markers between MZ will be performed systematically by using the TaqMan MicroRNA Assays. The purpose of this study is to explore the effectiveness and application value of these differential microRNA target markers in identifying MZ, and expect to solve the forensic problem of individual discrimination of MZ.

基因组完全相同的同卵双生子（monozygotic twins, MZ）个体的甑别是一个世界性难题。国内外部分学者尝试利用表观遗传标记鉴别MZ个体，虽然取得了一定的进展，但仍未能彻底有效解决MZ的个体识别问题，故探寻新的MZ鉴别指标仍具有重要意义。作为近年来的研究热点，microRNAs广泛参与调节细胞生长发育、分化、凋亡、衰老及疾病的发生，被誉为基因表达的"调节器"。由此，在体内具有重要调节功能的microRNAs在MZ个体间有否表达差异是我们关注的焦点并得到预试验的初步证实。基于此，本课题拟采用microRNA芯片全面筛选MZ个体间具有显著表达差异的microRNAs，再应用TaqMan MicroRNA Assays技术系统定量研究其在MZ个体间的表达差异程度，旨在探讨以这些差异表达的microRNAs分子作为新指标甑别MZ个体的有效性和应用价值，以期解决MZ个体鉴别这一法医学难题。

基因组完全相同的MZ个体的甑别是一个世界性难题。已有学者尝试利用表观遗传标记鉴别MZ个体，虽取得了一定的进展，但仍未能彻底有效解决MZ的个体识别问题，故探寻新的MZ鉴别指标仍具有重要意义。本课题旨在探索MZ个体间microRNAs表达谱差异，寻找甑别MZ个体的新指标。我们首先采用microRNA芯片对7对不同年龄和性别的MZ个体血样的microRNAs表达谱进行了检测分析，并采用定量PCR对差异表达的microRNAs进行了验证。结果发现在≥4对MZ间具有共同表达差异的microRNAs 29个，从中挑选了11个采用定量PCR进行验证，除miR-339-5p外，其余microRNAs检测结果与芯片结果完全一致。结合定量PCR验证结果及microRNAs的绝对表达量，最终确定miR-142-3p、miR-4325、miR-3653-3p等20个microRNAs作为靶标记用于MZ个体的鉴别。然后采用qRT-PCR对64对MZ样本进行上述20个靶microRNAs标记检测，以MZ两个体间存在≥1个microRNAs具有显著表达差异者记为有效甑别。结果在64对MZ中，有38对可有效甑别，甑别率为59.4%。38对可甑别的MZ中，差异表达的靶microRNAs个数最少仅1个，最多的为13个，平均5.8个。所有20个靶microRNAs中，以let-7i-5p鉴别能力最强，38对可有效甑别的MZ中，有20对存在该靶标记的表达差异，miR-222-3p次之，第三为miR-151a-3p。此外，从38对MZ的靶microRNAs定量检测结果发现存在一定的年龄相关性，即年龄越大的MZ个体间差异表达的靶microRNAs个数相对越多，年龄越小的MZ差异表达的靶microRNAs个数相对较少。本研究证实了MZ个体间存在microRNAs表达谱差异，可用作甑别MZ个体的新的生物学指标。