MicroRNAs Located in the Hox Gene Clusters Are Implicated in Huntington's Disease Pathogenesis

Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington's disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value<0.05). Three of these, miR-196a-5p, miR-196b-5p and miR-615-3p, were expressed at near zero levels in control brains. Expression was verified for all five miRNAs using reverse transcription quantitative PCR and all but miR-1247-5p were replicated in an independent sample (8HD/8C). Ectopic miR-10b-5p expression in PC12 HTT-Q73 cells increased survival by MTT assay and cell viability staining suggesting increased expression may be a protective response. All of the miRNAs but miR-1247-5p are located in intergenic regions of Hox clusters. Total mRNA sequencing in the same samples identified fifteen of 55 genes within the Hox cluster gene regions as differentially expressed in HD, and the Hox genes immediately adjacent to the four Hox cluster miRNAs as up-regulated. Pathway analysis of mRNA targets of these miRNAs implicated functions for neuronal differentiation, neurite outgrowth, cell death and survival. In regression models among the HD brains, huntingtin CAG repeat size, onset age and age at death were independently found to be inversely related to miR-10b-5p levels. CAG repeat size and onset age were independently inversely related to miR-196a-5p, onset age was inversely related to miR-196b-5p and age at death was inversely related to miR-615-3p expression. These results suggest these Hox-related miRNAs may be involved in neuroprotective response in HD. Recently, miRNAs have shown promise as biomarkers for human diseases and given their relationship to disease expression, these miRNAs are biomarker candidates in HD.

长期以来，转录失调（Transcriptional dysregulation）被认为是亨廷顿病（Huntington's disease, HD）发病机制的核心。微小RNA（MicroRNAs, miRNAs）是一类主要的转录后调控系统，可通过阻断翻译起始或靶向转录本进行储存与降解发挥调控作用。本研究对12例亨廷顿病患者与9例健康对照的前额叶皮层（prefrontal cortex, 布罗德曼9区Brodmann Area 9）开展下一代微RNA测序（next-generation miRNA sequencing），鉴定出5个在亨廷顿病中呈现全基因组显著性上调的miRNAs（错误发现率q值<0.05，FDR q-value<0.05），分别为miR-10b-5p、miR-196a-5p、miR-196b-5p、miR-615-3p及miR-1247-5p。其中3个miRNAs（miR-196a-5p、miR-196b-5p与miR-615-3p）在健康对照脑组织中的表达水平接近零。本研究通过逆转录定量PCR（reverse transcription quantitative PCR）验证了全部5个miRNAs的表达情况，除miR-1247-5p外，其余4个均在独立样本（8例HD患者/8例健康对照）中得到重复验证。在PC12 HTT-Q73细胞中异位过表达miR-10b-5p后，经MTT法检测（MTT assay）与细胞活力染色（cell viability staining）证实细胞存活率显著升高，提示该miRNA的上调可能是一种保护性应答反应。除miR-1247-5p外，其余4个miRNAs均定位于同源盒基因簇（Hox clusters）的基因间区（intergenic regions）。对同一批样本进行总mRNA测序，鉴定出同源盒基因簇区域内55个基因中的15个在亨廷顿病中呈差异表达（differentially expressed），且与这4个miRNAs紧邻的同源盒基因呈现上调趋势。对这些miRNAs的mRNA靶标进行通路分析（pathway analysis），发现其富集的生物学功能包括神经元分化（neuronal differentiation）、神经突起生长（neurite outgrowth）、细胞死亡与存活调控等。在亨廷顿病患者脑组织的回归模型中，研究人员发现亨廷汀CAG重复序列长度（huntingtin CAG repeat size）、发病年龄（onset age）与miR-10b-5p的表达水平呈独立负相关；CAG重复序列长度和发病年龄与miR-196a-5p的表达水平呈独立负相关；发病年龄与miR-196b-5p的表达水平呈负相关；死亡年龄（age at death）与miR-615-3p的表达水平呈负相关。上述结果提示，这些与同源盒基因簇相关的miRNAs可能参与了亨廷顿病的神经保护性应答反应。近年来，miRNAs已被证实可作为人类疾病的潜在生物标志物（biomarkers），结合其与疾病表型的关联，这些miRNAs有望成为亨廷顿病的候选生物标志物。