Pirogov Russian National Research Medical University, 117997 Moscow, Russia.
Pirogov Russian National Research Medical University, 117997 Moscow, Russia.
Gene. 2022 Aug 20;836:146676. doi: 10.1016/j.gene.2022.146676. Epub 2022 Jun 14.
The role of miRNAs, small non-coding regulatory RNAs, in the molecular mechanisms of multiple sclerosis (MS) development has been intensively studied. MiRNAs tend to be clustered within imprinted regions, and the largest number of miRNA genes is observed in the DLK1-DIO3 locus. Earlier using RNA-seq we identified sex-specific upregulation of the set of miRNA genes from this locus in peripheral blood mononuclear cells (PBMC) of treatment-naive relapsing-remitting MS (RRMS) patients. In the present study we set up to independently investigate the expression of a vast array of genes present in the DLK1-DIO3 imprinted locus. First, we analyzed the expression of miRNA genes, which levels in RRMS were mostly inconsistent based on RNA-seq data and not previously explored using qPCR. We identified that all selected miRNAs - miR-337-3p and -665 from 14q32.2 cluster and miR-370c, -380, -494, -654-3p, -300, -539, -668, and -323b-5p - were upregulated in MS men, but not women when compared to controls, regardless of conflicting RNA-seq data. The expression of miRNAs from the DLK1-DIO3 locus was highly correlated, indicating the existence of a common regulatory mechanism(s) that controls miRNA expression, regardless of the position of their genes within this region. Second, we performed the expression analysis of non-miRNA genes within the locus. The genes encoding proteins (DLK1, DIO3, RTL1), long non-coding RNAs (MEG3, MEG8, and MEG9) and small nucleolar RNAs (SNORD112, SNORD113-5, SNORD113-7, SNORD114-3, SNORD114-8, SNORD114-19) were not dysregulated in RRMS both in men and women. DNA methylation analysis of selected CpG sites within the differentially methylated regions IG-DMR, MEG3-DMR, and MEG8-DMR of the DLK1-DIO3 imprinted locus pointed out that they were not involved in the regulation of miRNA gene expression in RRMS, at least in PBMC population. The question of whether the observed changes in expression of miRNA genes (given that there is a constant expression of other non-miRNA genes of the DLK1-DIO3 locus) are involved in the development of RRMS or are they a consequence of the disease progress, remains open and needs further investigation.
微小 RNA（miRNA）是一类小的非编码调节 RNA，其在多发性硬化症（MS）发展的分子机制中的作用已得到深入研究。miRNA 倾向于聚集在印记区域内，并且在 DLK1-DIO3 基因座中观察到最多数量的 miRNA 基因。早期我们使用 RNA-seq 技术在未经治疗的复发缓解型 MS（RRMS）患者的外周血单核细胞（PBMC）中鉴定出该基因座的一组 miRNA 基因的性别特异性上调。在本研究中，我们独立研究了 DLK1-DIO3 印记基因座中存在的大量基因的表达。首先，我们分析了 miRNA 基因的表达水平，这些基因的表达水平在 RRMS 中主要与 RNA-seq 数据不一致，并且以前未使用 qPCR 进行过探索。我们发现，在所选择的 miRNA 中，miR-337-3p 和 -665 来自 14q32.2 簇，miR-370c、-380、-494、-654-3p、-300、-539、-668 和 -323b-5p 在 MS 男性中上调，但在女性中没有上调，与对照组相比，无论 RNA-seq 数据是否存在冲突，均如此。来自 DLK1-DIO3 基因座的 miRNA 的表达高度相关，表明存在一个共同的调节机制（多个），可以控制 miRNA 的表达，而不受其基因在该区域内位置的影响。其次，我们对该基因座内的非 miRNA 基因进行了表达分析。编码蛋白（DLK1、DIO3、RTL1）、长非编码 RNA（MEG3、MEG8 和 MEG9）和小核仁 RNA（SNORD112、SNORD113-5、SNORD113-7、SNORD114-3、SNORD114-8、SNORD114-19）的基因在 RRMS 中未发生失调男性和女性。对 DLK1-DIO3 印记基因座中差异甲基化区域 IG-DMR、MEG3-DMR 和 MEG8-DMR 内选定 CpG 位点的 DNA 甲基化分析表明，它们不参与 RRMS 中 miRNA 基因表达的调节，至少在 PBMC 群体中不参与调节。观察到的 miRNA 基因表达变化（鉴于 DLK1-DIO3 基因座的其他非 miRNA 基因的表达是恒定的）是否参与 RRMS 的发展，或者它们是否是疾病进展的结果，这一问题仍然存在，需要进一步研究。
