Table2_Integrative analysis of the lncRNA-miRNA-mRNA interactions in smooth muscle cell phenotypic transitions.XLS

Objectives: We previously found that the pluripotency factor OCT4 is reactivated in smooth muscle cells (SMC) in human and mouse atherosclerotic plaques and plays an atheroprotective role. Loss of OCT4 in SMC in vitro was associated with decreases in SMC migration. However, molecular mechanisms responsible for atheroprotective SMC-OCT4-dependent effects remain unknown.
Methods: Since studies in embryonic stem cells demonstrated that OCT4 regulates long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), making them candidates for OCT4 effect mediators, we applied an in vitro approach to investigate the interactions between OCT4-regulated lncRNAs, mRNAs, and miRNAs in SMC. We used OCT4 deficient mouse aortic SMC (MASMC) treated with the pro-atherogenic oxidized phospholipid POVPC, which, as we previously demonstrated, suppresses SMC contractile markers and induces SMC migration. Differential expression of lncRNAs, mRNAs, and miRNAs was obtained by lncRNA/mRNA expression array and small-RNA microarray. Long non-coding RNA to mRNA associations were predicted based on their genomic proximity and association with vascular diseases. Given a recently discovered crosstalk between miRNA and lncRNA, we also investigated the association of miRNAs with upregulated/downregulated lncRNA-mRNA pairs.
Results: POVPC treatment in SMC resulted in upregulating genes related to the axon guidance and focal adhesion pathways. Knockdown of Oct4 resulted in differential regulation of pathways associated with phagocytosis. Importantly, these results were consistent with our data showing that OCT4 deficiency attenuated POVPC-induced SMC migration and led to increased phagocytosis. Next, we identified several up- or downregulated lncRNA associated with upregulation of the specific mRNA unique for the OCT4 deficient SMC, including upregulation of ENSMUST00000140952-Hoxb5/6 and ENSMUST00000155531-Zfp652 along with downregulation of ENSMUST00000173605-Parp9 and, ENSMUST00000137236-Zmym1. Finally, we found that many of the downregulated miRNAs were associated with cell migration, including miR-196a-1 and miR-10a, targets of upregulated ENSMUST00000140952, and miR-155 and miR-122, targets of upregulated ENSMUST00000155531. Oppositely, the upregulated miRNAs were anti-migratory and pro-phagocytic, such as miR-10a/b and miR-15a/b, targets of downregulated ENSMUST00000173605, and miR-146a/b and miR-15b targets of ENSMUST00000137236.
Conclusion: Our integrative analyses of the lncRNA-miRNA-mRNA interactions in SMC indicated novel potential OCT4-dependent mechanisms that may play a role in SMC phenotypic transitions.

研究目的：既往研究发现，人与小鼠动脉粥样硬化斑块内的平滑肌细胞（smooth muscle cells，SMC）中可重新激活多能性因子OCT4（pluripotency factor OCT4），且该因子发挥抗动脉粥样硬化作用。体外实验中，平滑肌细胞内OCT4缺失会削弱细胞迁移能力，但介导OCT4发挥抗动脉粥样硬化平滑肌细胞相关作用的分子机制仍未阐明。

研究方法：由于胚胎干细胞（embryonic stem cells）中的研究表明，OCT4可调控长链非编码RNA（long non-coding RNAs，lncRNAs）与微小RNA（microRNAs，miRNAs）的表达，使其成为OCT4作用效应的潜在介导分子，因此本研究采用体外实验方法，探究平滑肌细胞内OCT4调控的长链非编码RNA、信使RNA（messenger RNAs，mRNAs）与微小RNA之间的相互作用。我们使用了经致动脉粥样硬化氧化磷脂POVPC处理的OCT4敲低小鼠主动脉平滑肌细胞（mouse aortic SMC，MASMC）——既往研究已证实，POVPC可抑制平滑肌细胞的收缩表型标志物表达并诱导细胞迁移。通过长链非编码RNA/信使RNA表达芯片与小RNA芯片，我们获取了差异表达的长链非编码RNA、信使RNA与微小RNA。基于基因组邻近性及与血管疾病的关联，我们预测了长链非编码RNA与信使RNA的调控关联。鉴于近期发现的微小RNA与长链非编码RNA之间的串扰现象，我们同时探究了微小RNA与上调/下调的长链非编码RNA-信使RNA配对的关联。

研究结果：在平滑肌细胞中经POVPC处理后，与轴突导向、黏着斑通路相关的基因出现上调。Oct4敲低会导致与吞噬作用相关的通路出现差异调控。值得注意的是，该结果与我们的实验数据一致：OCT4缺失会削弱POVPC诱导的平滑肌细胞迁移能力，并增强吞噬作用。随后，我们鉴定出数条与OCT4敲低平滑肌细胞中特异性信使RNA上调相关的差异表达长链非编码RNA，包括ENSMUST00000140952-Hoxb5/6与ENSMUST00000155531-Zfp652的上调，以及ENSMUST00000173605-Parp9与ENSMUST00000137236-Zmym1的下调。最后，我们发现大量下调的微小RNA与细胞迁移相关，包括上调的ENSMUST00000140952的靶标miR-196a-1与miR-10a，以及上调的ENSMUST00000155531的靶标miR-155与miR-122。与之相反，上调的微小RNA具有抗迁移与促吞噬作用，例如下调的ENSMUST00000173605的靶标miR-10a/b与miR-15a/b，以及ENSMUST00000137236的靶标miR-146a/b与miR-15b。

研究结论：我们对平滑肌细胞内长链非编码RNA-微小RNA-信使RNA相互作用的整合分析表明，存在潜在的新型OCT4依赖型调控机制，该机制可能参与平滑肌细胞的表型转化过程。