Department of Biochemistry and Molecular Biology, Central University of Kerala, Tejaswini Hills, Periye, Kasaragod, Kerala, 671316, India.
CRP-10, Cancer Research, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram, 695014, India.
Mol Biol Rep. 2023 Oct;50(10):8623-8637. doi: 10.1007/s11033-023-08682-0. Epub 2023 Sep 1.
The process of transdifferentiating epithelial cells to mesenchymal-like cells (EMT) involves cells gradually taking on an invasive and migratory phenotype. Many cell adhesion molecules are crucial for the management of EMT, integrin β4 (ITGB4) being one among them. Although signaling downstream of ITGB4 has been reported to cause changes in the expression of several miRNAs, little is known about the role of such miRNAs in the process of EMT.
The cytoplasmic domain of ITGB4 (ITGB4CD) was ectopically expressed in HeLa cells to induce ITGB4 signaling, and expression analysis of mesenchymal markers indicated the induction of EMT. β-catenin and AKT signaling pathways were found to be activated downstream of ITGB4 signaling, as evidenced by the TOPFlash assay and the levels of phosphorylated AKT, respectively. Based on in silico and qRT-PCR analysis, miR-383 was selected for functional validation studies. miR-383 and Sponge were ectopically expressed in HeLa, thereafter, western blot and qRT-PCR analysis revealed that miR-383 regulates GATA binding protein 6 (GATA6) post-transcriptionally. The ectopic expression of shRNA targeting GATA6 caused the reversal of EMT and β catenin activation downstream of ITGB4 signaling. Cell migration assays revealed significantly high cell migration upon ectopic expression ITGB4CD, which was reversed upon ectopic co-expression of miR-383 or GATA6 shRNA. Besides, ITGB4CD promoted EMT in in ovo xenograft model, which was reversed by ectopic expression of miR-383 or GATA6 shRNA.
The induction of EMT downstream of ITGB4 involves a signaling axis encompassing AKT/miR-383/GATA6/β-catenin.
上皮细胞向间充质样细胞（EMT）的转化过程涉及细胞逐渐呈现出侵袭和迁移表型。许多细胞粘附分子对于 EMT 的管理至关重要，整合素β4（ITGB4）就是其中之一。尽管已经报道 ITGB4 下游的信号转导会导致几种 miRNA 的表达发生变化，但对于这些 miRNA 在 EMT 过程中的作用知之甚少。
将 ITGB4 的细胞质结构域（ITGB4CD）异位表达于 HeLa 细胞中，以诱导 ITGB4 信号转导，间充质标志物的表达分析表明 EMT 的诱导。通过 TOPFlash 测定和磷酸化 AKT 的水平，分别证实了 ITGB4 信号转导下游的 β-连环蛋白和 AKT 信号通路的激活。基于计算机模拟和 qRT-PCR 分析，选择 miR-383 进行功能验证研究。miR-383 和 Sponge 被异位表达于 HeLa 细胞中，然后通过 Western blot 和 qRT-PCR 分析表明，miR-383 在后转录水平上调节 GATA 结合蛋白 6（GATA6）。针对 GATA6 的 shRNA 的异位表达导致 EMT 的逆转和 ITGB4 信号转导下游的 β-catenin 激活。细胞迁移实验表明，ITGB4CD 的异位表达显著促进了细胞迁移，而 miR-383 或 GATA6 shRNA 的异位共表达则逆转了这一过程。此外，ITGB4CD 在鸡胚异种移植模型中促进 EMT，而 miR-383 或 GATA6 shRNA 的异位表达则逆转了这一过程。
ITGB4 下游 EMT 的诱导涉及一个包含 AKT/miR-383/GATA6/β-catenin 的信号轴。
