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,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。