Abstract：
LncRNA-Atherosclerotic plaque pathogenesis-associated transcript (APPAT) could be detected in circulating blood and has been demonstrated to correlate with the development of atherosclerosis in our previous work. It could be a potential noninvasive biomarker for earlier diagnoses of clinical cardiovascular disease. Moreover, the expression of miR-647 increased in ox-LDL-treated vascular smooth muscle cells and peripheral blood of patients with coronary heart disease. A negative correlation between APPAT and miR-647 was confirmed, and FGF5 was screened as molecular target of miR-647. However, it is largely unclear how APPAT, miR-647, and FGF5 interact and function in disease development. Here, we aim to explore the underlying molecular mechanism in this progression.
APPAT, miR-647, and FGF5 expression levels were detected by quantitative reverse transcription polymerase chain reaction; cell proliferation was detected by EdU incorporation assay; cell migration was detected by wound-healing assay; the molecular interaction of APPAT/FGF5 with miR-647 was verified by dual-luciferase reporter assay; the western blot was performed to determine the gene expression at protein levels; subcellular localizations of APPAT and miR-647 were observed by fluorescence in situ hybridization; cytosolic and nucleus fractionation assay was performed to further detect the distribution of miR-647.
APPAT and miR-647 have inverse effects on human aortic smooth muscle cells\' (HASMCs) proliferation and migration. APPAT negatively regulated the cell activity, whereas miR-647 did it in a positive way (p<0.05). Three pairs of molecular interplay were found: mutual negative regulation between APPAT and miR-647, APPAT downregulated FGF5, miR-647 regulation on FGF5 (p<0.05). Subcellular location assay confirmed the molecular interaction of APPAT and miR-647.
APPAT could suppress the migration and proliferation of ox-LDL-treated HASMCs via interacting with miR-647 and FGF5. We revealed a nontypical competing endogenous RNA mechanism of long noncoding RNA in the progression of atherosclerosis.
APPAT, miR-647, and FGF5 expression levels were detected by quantitative reverse transcription polymerase chain reaction; cell proliferation was detected by EdU incorporation assay; cell migration was detected by wound-healing assay; the molecular interaction of APPAT/FGF5 with miR-647 was verified by dual-luciferase reporter assay; the western blot was performed to determine the gene expression at protein levels; subcellular localizations of APPAT and miR-647 were observed by fluorescence in situ hybridization; cytosolic and nucleus fractionation assay was performed to further detect the distribution of miR-647.
APPAT and miR-647 have inverse effects on human aortic smooth muscle cells\' (HASMCs) proliferation and migration. APPAT negatively regulated the cell activity, whereas miR-647 did it in a positive way (p<0.05). Three pairs of molecular interplay were found: mutual negative regulation between APPAT and miR-647, APPAT downregulated FGF5, miR-647 regulation on FGF5 (p<0.05). Subcellular location assay confirmed the molecular interaction of APPAT and miR-647.
APPAT could suppress the migration and proliferation of ox-LDL-treated HASMCs via interacting with miR-647 and FGF5. We revealed a nontypical competing endogenous RNA mechanism of long noncoding RNA in the progression of atherosclerosis.
摘要：
■LncRNA-动脉粥样硬化斑块发病机制相关转录物（APPAT）可以在循环血液中检测到，并且在我们以前的工作中已经证明与动脉粥样硬化的发展相关。它可能是临床心血管疾病早期诊断的潜在非侵入性生物标志物。此外，miR-647在ox-LDL处理的血管平滑肌细胞和冠心病患者外周血中的表达增加。APPAT与miR-647呈负相关,筛选FGF5作为miR-647的分子靶标。然而,目前还不清楚APPAT是如何，miR-647和FGF5在疾病发展中相互作用和功能。这里,我们的目的是探索这一进展的潜在分子机制。
■APPAT，通过定量逆转录聚合酶链反应检测miR-647和FGF5的表达水平；通过EdU掺入法检测细胞增殖；通过伤口愈合法检测细胞迁移；通过双荧光素酶报告基因法验证APPAT/FGF5与miR-647的分子相互作用；进行蛋白质印迹以确定蛋白水平的基因表达；进行细胞亚定位APPAT和miR-647的细胞分级分离，并进一步通过荧光检测细胞核47。
■APPAT和miR-647对人主动脉平滑肌细胞（HASMC）的增殖和迁移具有相反的作用。APPAT负调控细胞活性，而miR-647以积极的方式（p<0.05）。发现三对分子相互作用：APPAT与miR-647之间相互负调节，APPAT下调FGF5，miR-647对FGF5的调节（p<0.05）。亚细胞定位测定证实了APPAT和miR-647的分子相互作用。
■APPAT可以通过与miR-647和FGF5相互作用抑制ox-LDL处理的HASMC的迁移和增殖。我们揭示了长链非编码RNA在动脉粥样硬化进展中的非典型竞争性内源性RNA机制。
■APPAT，通过定量逆转录聚合酶链反应检测miR-647和FGF5的表达水平；通过EdU掺入法检测细胞增殖；通过伤口愈合法检测细胞迁移；通过双荧光素酶报告基因法验证APPAT/FGF5与miR-647的分子相互作用；进行蛋白质印迹以确定蛋白水平的基因表达；进行细胞亚定位APPAT和miR-647的细胞分级分离，并进一步通过荧光检测细胞核47。
■APPAT和miR-647对人主动脉平滑肌细胞（HASMC）的增殖和迁移具有相反的作用。APPAT负调控细胞活性，而miR-647以积极的方式（p<0.05）。发现三对分子相互作用：APPAT与miR-647之间相互负调节，APPAT下调FGF5，miR-647对FGF5的调节（p<0.05）。亚细胞定位测定证实了APPAT和miR-647的分子相互作用。
■APPAT可以通过与miR-647和FGF5相互作用抑制ox-LDL处理的HASMC的迁移和增殖。我们揭示了长链非编码RNA在动脉粥样硬化进展中的非典型竞争性内源性RNA机制。
