PDF(Pubmed)
Abstract：
UNASSIGNED: Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms.
UNASSIGNED: A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1.
UNASSIGNED: Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis.
UNASSIGNED: FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.
UNASSIGNED: A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1.
UNASSIGNED: Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis.
UNASSIGNED: FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.
摘要：
■载脂蛋白A1（APOA1）是人类病理性近视的潜在关键蛋白质和治疗目标。本研究旨在发现APOA1在近视巩膜重塑中的功能及其潜在机制。
■使用缺氧诱导近视细胞模型。在功能损耗和增益实验之后，肌成纤维细胞转分化相关因子和胶原产生相关因子的表达ForkheadboxM1(FOXM1),通过定量逆转录聚合酶链反应（RT-qPCR）和蛋白质印迹法检查近视细胞模型中的APOA1和甲基转移酶样3（METTL3）。细胞计数试剂盒-8法和流式细胞术检测细胞增殖和凋亡,分别。染色质免疫沉淀（ChIP）用于检查METTL3启动子中的FOXM1富集，甲基化RNA免疫沉淀（Me-RIP）以检查APOA1的N6-甲基腺苷（m6A）修饰水平，以及可光活化的核糖核苷增强的交联和免疫沉淀（PAR-CLIP）以检查METTL3和APOA1之间的结合。
■缺氧诱导的人巩膜成纤维细胞（HSF）具有高的APOA1和FOXM1表达和低的METTL3表达。FOXM1敲低METTL3表达升高，APOA1表达下调。FOXM1在METTL3启动子中富集。APOA1或FOXM1敲低或METTL3过表达逆转了缺氧诱导的黏珠蛋白升高，paxillin,和α-平滑肌肌动蛋白（α-SMA）水平和细胞凋亡和胶原蛋白的减少，I型,α1（COL1A1）水平与HSF细胞增殖的关系。METTL3或YTHN6-甲基腺苷RNA结合蛋白F2（YTHDF2）敲低或APOA1过表达逆转了FOXM1敲低对黏珠蛋白的影响，paxillin,α-SMA,和COL1A1表达与细胞增殖和凋亡。
■FOXM1通过抑制METTL3转录提高了APOA1的m6A甲基化水平，并通过减少YTHDF2识别的m6A甲基化转录本提高了APOA1mRNA的稳定性和转录。
■使用缺氧诱导近视细胞模型。在功能损耗和增益实验之后，肌成纤维细胞转分化相关因子和胶原产生相关因子的表达ForkheadboxM1(FOXM1),通过定量逆转录聚合酶链反应（RT-qPCR）和蛋白质印迹法检查近视细胞模型中的APOA1和甲基转移酶样3（METTL3）。细胞计数试剂盒-8法和流式细胞术检测细胞增殖和凋亡,分别。染色质免疫沉淀（ChIP）用于检查METTL3启动子中的FOXM1富集，甲基化RNA免疫沉淀（Me-RIP）以检查APOA1的N6-甲基腺苷（m6A）修饰水平，以及可光活化的核糖核苷增强的交联和免疫沉淀（PAR-CLIP）以检查METTL3和APOA1之间的结合。
■缺氧诱导的人巩膜成纤维细胞（HSF）具有高的APOA1和FOXM1表达和低的METTL3表达。FOXM1敲低METTL3表达升高，APOA1表达下调。FOXM1在METTL3启动子中富集。APOA1或FOXM1敲低或METTL3过表达逆转了缺氧诱导的黏珠蛋白升高，paxillin,和α-平滑肌肌动蛋白（α-SMA）水平和细胞凋亡和胶原蛋白的减少，I型,α1（COL1A1）水平与HSF细胞增殖的关系。METTL3或YTHN6-甲基腺苷RNA结合蛋白F2（YTHDF2）敲低或APOA1过表达逆转了FOXM1敲低对黏珠蛋白的影响，paxillin,α-SMA,和COL1A1表达与细胞增殖和凋亡。
■FOXM1通过抑制METTL3转录提高了APOA1的m6A甲基化水平，并通过减少YTHDF2识别的m6A甲基化转录本提高了APOA1mRNA的稳定性和转录。
