Department of Pediatrics, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, No. 123 Tian Fei Xiang, Mo Chou Road, Nanjing 210004, Jiangsu Province, China.
Department of Pediatrics, Women's Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, No. 123 Tian Fei Xiang, Mo Chou Road, Nanjing 210004, Jiangsu Province, China.
Gene. 2019 Apr 30;694:83-92. doi: 10.1016/j.gene.2018.12.080. Epub 2019 Feb 1.
Each year, cardiac diseases may cause a high morbidity and mortality worldwide. Long non-coding RNAs (lncRNAs) that contained ultra-conserved elements (UCEs) may play important roles on cardiomyocytes differentiation. Further investigations underlying mechanisms of lncRNA-UC regulating embryonic heart development are necessary. In this study, we investigated the effects of lnc-uc.245 on proliferation, migration, apoptosis, and cardiomyocyte-like differentiation in P19 cells with DMSO stimulation, and hypothesized that lnc-uc.245 would influence cardiomyocytes differentiation via FOG2. Lentiviral vectors of pGPU6/GFP/Neo-uc.245 and pGPU6/GFP/Neo-shRNA-uc.245 were respectively transfected into P19 cells to overexpress or silence uc.245. MTT assay, Annexin V-FITC/PI double-staining, scratch test and transwell assay were performed and the results showed that uc.245 overexpression could significantly suppress P19 cell proliferation, migration, cardiomyocyte-like differentiation but promote cell apoptosis. Contrarily, sh-uc.245 treatment caused the opposite changes. Uc.245 overexpression obviously downregulated the expression of cardiomyogenic-specific molecular markers (cTnI, ANP, α-MHC, Nkx2.5, GATA4, MEF2C) but remarkably upregulated the expression of FOG2. Subsequently, we transfected the recombinant vectors loaded FOG2 or shRRNA-FOG2 into P19 cells to further address the functional significance of FOG2 in uc.245-regulated cardiomyocyte-like differentiation. Interestingly, we found that overexpressing of FOG2 promoted cell proliferation, migration, and inhibited apoptosis both in uc.245 overexpressed and silenced P19 cells, especially in uc.245 silenced cell line. In addition, sh-FOG2 promoted cardiomyocyte-like differentiation and upregulated the expression of cardiomyogenic-specific markers at the gene and protein levels both in uc.245 overexpressed and silenced P19 cells. Similarly, this upregulation effect of sh-FOG2 was more obvious after uc.245 silencing. These findings suggest that FOG2 is a key mediator during uc.245-regulated differentiation of P19 cells into cardiomyocytes. It is expected that lnc-uc.245/FOG2 will become a promising therapeutic target for cardiac diseases.
每年，心脏病可能导致全球高发病率和死亡率。含有超保守元件 (UCEs) 的长非编码 RNA (lncRNA) 可能在心肌细胞分化中发挥重要作用。有必要进一步研究 lncRNA-UC 调节胚胎心脏发育的机制。在这项研究中，我们研究了 lnc-uc.245 在 DMSO 刺激下对 P19 细胞增殖、迁移、凋亡和心肌样分化的影响，并假设 lnc-uc.245 通过 FOG2 影响心肌细胞分化。分别将 pGPU6/GFP/Neo-uc.245 和 pGPU6/GFP/Neo-shRNA-uc.245 的慢病毒载体转染到 P19 细胞中以过表达或沉默 uc.245。进行 MTT 测定、Annexin V-FITC/PI 双重染色、划痕试验和 Transwell 试验，结果表明 uc.245 过表达可显著抑制 P19 细胞增殖、迁移和心肌样分化，但促进细胞凋亡。相反，sh-uc.245 处理则导致相反的变化。uc.245 过表达明显下调心肌生成特异性分子标志物（cTnI、ANP、α-MHC、Nkx2.5、GATA4、MEF2C）的表达，但显著上调 FOG2 的表达。随后，我们将携带 FOG2 的重组载体转染到 P19 细胞中，进一步研究 FOG2 在 uc.245 调节的心肌样分化中的功能意义。有趣的是，我们发现过表达 FOG2 可促进 uc.245 过表达和沉默 P19 细胞的增殖、迁移，并抑制凋亡，尤其是在 uc.245 沉默的细胞系中。此外，sh-FOG2 在 uc.245 过表达和沉默的 P19 细胞中均促进心肌样分化，并上调心肌生成特异性标志物的基因和蛋白水平的表达。同样，在 uc.245 沉默后，sh-FOG2 的这种上调作用更为明显。这些发现表明，FOG2 是 uc.245 调节 P19 细胞向心肌细胞分化过程中的关键介质。预计 lnc-uc.245/FOG2 将成为心脏疾病的有前途的治疗靶点。
