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.

中文翻译：

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平滑肌细胞表型转变中 lncRNA-miRNA-mRNA 相互作用的综合分析

目的：我们之前发现多能因子 OCT4 在人类和小鼠动脉粥样硬化斑块的平滑肌细胞 (SMC) 中重新激活，并发挥动脉粥样硬化保护作用。 SMC 中 OCT4 丢失体外与 SMC 迁移的减少有关。然而，负责动脉粥样硬化保护的 SMC-OCT4 依赖性作用的分子机制仍然未知。方法：由于胚胎干细胞研究表明 OCT4 调节长非编码 RNA (lncRNA) 和 microRNA (miRNA)，使它们成为 OCT4 效应介质的候选者，我们应用了体外方法研究 SMC 中 OCT4 调节的 lncRNA、mRNA 和 miRNA 之间的相互作用。我们使用 OCT4 缺陷型小鼠主动脉 SMC (MASMC)，并用促动脉粥样硬化氧化磷脂 POVPC 进行处理，正如我们之前所证明的，POVPC 可以抑制 SMC 收缩标记物并诱导 SMC 迁移。通过lncRNA/mRNA表达阵列和小RNA微阵列获得lncRNA、mRNA和miRNA的差异表达。长非编码 RNA 与 mRNA 的关联是根据它们的基因组邻近性以及与血管疾病的关联来预测的。鉴于最近发现的 miRNA 和 lncRNA 之间的串扰，我们还研究了 miRNA 与上调/下调的 lncRNA-mRNA 对之间的关​​联。结果：SMC 中的 POVPC 处理导致与轴突引导和粘着斑通路相关的基因上调。击倒10月4日导致与吞噬作用相关的途径的差异调节。重要的是，这些结果与我们的数据一致，表明 OCT4 缺陷减弱了 POVPC 诱导的 SMC 迁移并导致吞噬作用增加。接下来，我们确定了与OCT4缺陷SMC特有的特定mRNA上调相关的几种上调或下调lncRNA，包括ENSMUST00000140952-Hoxb5/6和ENSMUST00000155531-Zfp652的上调以及ENSMUST00000173605-Parp9和ENSMUST00000137的下调， 236-Zmym1。最后，我们发现许多下调的miRNA与细胞迁移相关，包括上调的ENSMUST00000140952的靶标miR-196a-1和miR-10a，以及上调的ENSMUST00000155531的靶标miR-155和miR-122。相反，上调的 miRNA 具有抗迁移和促吞噬作用，例如下调的 ENSMUST00000173605 的靶标 miR-10a/b 和 miR-15a/b，以及 ENSMUST00000137236 的 miR-146a/b 和 miR-15b 靶标。对 SMC 中 lncRNA-miRNA-mRNA 相互作用的综合分析表明，新的潜在 OCT4 依赖性机制可能在 SMC 表型转变中发挥作用。