Department of Respiratory the First Affiliated Hospital of Soochow University Suzhou China.
Department of Respiratory the Second Affiliated Hospital of Nantong University China.
FEBS Open Bio. 2019 Mar 19;9(4):605-617. doi: 10.1002/2211-5463.12606. eCollection 2019 Apr.
We previously reported that the expression of G protein-coupled receptor kinase 6 (GRK6) is significantly downregulated in lung adenocarcinoma (LADC) tissues, and low expression levels of GRK6 are correlated with poor survival prognosis. However, the specific regulatory mechanisms and functions of GRK6 in LADC remain unknown. Here, we report that GRK6 mRNA expression levels are downregulated in LADC tissues compared to those in matched adjacent non-tumor tissues ( < 0.001). The promoter of the gene was found to be hypermethylated in LADC tissues, and its methylation was correlated with both GRK6 expression and pathology grade. promoter hypermethylation may predict shorter overall survival. Treatment with 5-aza-2'-deoxycytidine significantly enhanced gene expression. Four binding sites of CCAAT/enhancer-binding protein-α (C/EBPα) in the CpG island of the gene promoter were predicted , of which three sites were further confirmed by ChIP. Decreased binding of C/EBPα to binding sites 1, 3 and 4 of the gene promoter was observed in LADC tissues. Inhibition of C/EBPα significantly inhibited GRK6 expression, while overexpression of C/EBPα significantly promoted GRK6 expression. In addition, overexpression of significantly suppressed, while knockdown promoted cell migration and invasion. Overexpression of enhanced E-cadherin expression and suppressed vimentin expression, and silencing of had the opposite effects. Furthermore, ectopic expression of significantly decreased matrix metalloproteinase (MMP) 2 and MMP7 protein expression levels. Our findings suggest that hypermethylation of the gene promoter suppressed binding of C/EBPα, thereby contributing to the promotion of cell migration and invasion. The methylation status of the promoter might be suitable for use as an epigenetic biomarker, and the C/EBPα-GRK6 signaling pathway may be a potential target for LADC.
我们之前报道过，G 蛋白偶联受体激酶 6（GRK6）在肺腺癌（LADC）组织中的表达显著下调，GRK6 表达水平低与不良生存预后相关。然而，GRK6 在 LADC 中的具体调控机制和功能仍不清楚。在这里，我们报告在 LADC 组织中，与匹配的相邻非肿瘤组织相比，GRK6 mRNA 表达水平下调（<0.001）。发现基因启动子在 LADC 组织中发生超甲基化，其甲基化与 GRK6 表达和病理分级均相关。基因启动子的异常高甲基化可能预示着总生存期更短。用 5-氮杂-2'-脱氧胞苷处理可显著增强基因表达。基因启动子 CpG 岛上预测到 CCAAT/增强子结合蛋白-α（C/EBPα）的四个结合位点，其中三个位点通过 ChIP 进一步得到证实。在 LADC 组织中观察到 C/EBPα与基因启动子结合位点 1、3 和 4 的结合减少。C/EBPα 抑制显著抑制 GRK6 表达，而 C/EBPα 的过表达则显著促进 GRK6 表达。此外，过表达显著抑制，而敲低则促进细胞迁移和侵袭。过表达增强了 E-钙黏蛋白的表达并抑制了波形蛋白的表达，而沉默则产生相反的效果。此外，过表达显著降低了基质金属蛋白酶（MMP）2 和 MMP7 蛋白表达水平。我们的研究结果表明，基因启动子的异常高甲基化抑制了 C/EBPα 的结合，从而促进了细胞迁移和侵袭。基因启动子的甲基化状态可能适合作为表观遗传生物标志物，而 C/EBPα-GRK6 信号通路可能是 LADC 的一个潜在靶点。
