Liu Zequn, Jiang Yanmin, Fang Fu, Li Ru, Han Jin, Yang Xin, Deng Qiong, Li Lu-Shan, Lei Ting-Ying, Li Dong-Zhi, Liao Can
Department of Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, 510623, Guangdong, China.
Institute of Obstetrics and Gynecology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, 510623, Guangdong, China.
Biochem Biophys Rep. 2023 Jun 27;35:101505. doi: 10.1016/j.bbrep.2023.101505. eCollection 2023 Sep.
Congenital heart disease (CHD) is a serious condition with unknown etiology. In a recent study, a compound heterozygous mutation (c.3526C > T [p.Arg1176Trp] and c.4643A > G [p.Asp1548Gly]) in the ASXL3 gene was identified, which is associated with CHD. This mutation was overexpressed in HL-1 mouse cardiomyocyte cells, leading to increased cell apoptosis and decreased cell proliferation. However, whether this effect is mediated by long noncoding RNAs (lncRNAs) is yet to be determined. We identified the differences among lncRNA and mRNA profiles in mouse heart tissues using sequencing to explore this issue. We detected HL-1 cell proliferation and apoptosis through CCK8 and flow cytometry. Fgfr2, lncRNA, and Ras/ERK signaling pathway expressions were evaluated using quantitative real time polymerase chain reaction (qRT-PCR) and western blot (WB) assays. We also conducted functional investigations by silencing lncRNA NONMMUT063967.2. The sequencing revealed significant changes in lncRNA and mRNA profiles, with the expression of lncRNA NONMMUT063967.2 being significantly promoted in the ASXL3 gene mutations group (MT) while the expression of Fgfr2 being downregulated. The experiments showed that ASXL3 gene mutations inhibited the proliferation of cardiomyocytes and accelerated cell apoptosis by promoting the expression of lncRNAs (NONMMUT063967.2, NONMMUT063918.2, and NONMMUT063891.2), suppressing the formation of FGFR2 transcripts, and inhibiting the Ras/ERK signaling pathway. The decrease in FGFR2 had the same effect on the Ras/ERK signaling pathway, proliferation, and apoptosis in mouse cardiomyocytes as ASXL3 mutations. Further mechanistic studies revealed that suppression of lncRNA NONMMUT063967.2 and overexpression of FGFR2 reversed the effects of the ASXL3 mutations on the Ras/ERK signaling pathway, proliferation, and apoptosis in mouse cardiomyocytes. Therefore, ASXL3 mutation decreases FGFR2 expression by upregulating lncRNA NONMMUT063967.2, inhibiting cell proliferation and promoting cell apoptosis in mouse cardiomyocytes.
先天性心脏病（CHD）是一种病因不明的严重疾病。在最近的一项研究中，在ASXL3基因中鉴定出一种复合杂合突变（c.3526C>T [p.Arg1176Trp]和c.4643A>G [p.Asp1548Gly]），其与CHD相关。这种突变在HL-1小鼠心肌细胞中过表达，导致细胞凋亡增加和细胞增殖减少。然而，这种效应是否由长链非编码RNA（lncRNA）介导尚待确定。我们使用测序技术鉴定了小鼠心脏组织中lncRNA和mRNA谱的差异，以探讨这个问题。我们通过CCK8和流式细胞术检测HL-1细胞的增殖和凋亡。使用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹（WB）分析评估Fgfr2、lncRNA和Ras/ERK信号通路的表达。我们还通过沉默lncRNA NONMMUT063967.2进行了功能研究。测序结果显示lncRNA和mRNA谱有显著变化，lncRNA NONMMUT063967.2在ASXL3基因突变组（MT）中的表达显著升高，而Fgfr2的表达下调。实验表明，ASXL3基因突变通过促进lncRNA（NONMMUT063967.2、NONMMUT063918.2和NONMMUT063891.2）的表达、抑制FGFR2转录本的形成以及抑制Ras/ERK信号通路，从而抑制心肌细胞的增殖并加速细胞凋亡。FGFR2的减少对小鼠心肌细胞的Ras/ERK信号通路、增殖和凋亡的影响与ASXL3突变相同。进一步的机制研究表明，抑制lncRNA NONMMUT063967.2和过表达FGFR2可逆转ASXL3突变对小鼠心肌细胞Ras/ERK信号通路、增殖和凋亡的影响。因此，ASXL3突变通过上调lncRNA NONMMUT063967.2降低FGFR2表达，抑制小鼠心肌细胞的增殖并促进细胞凋亡。
