Hu Qiande, Zhu Liang, Li Yuan, Zhou Jianjun, Xu Jun
Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai, 200120, China.
Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China.
Cell Biosci. 2021 Jan 28;11(1):25. doi: 10.1186/s13578-021-00534-3.
Alveolar Rhabdomyosarcoma (ARMS) is a pediatric malignant soft tissue tumor with skeletal muscle phenotype. Little work about skeletal muscle proteins in ARMS was reported. PAX3-FOXO1 is a specific fusion gene generated from the chromosomal translocation t (2;13) (q35; q14) in most ARMS. ACTA1 is the skeletal muscle alpha actin gene whose transcript was detected in ARMS. However, ACTA1 expression and regulation in ARMS have not been well investigated. This work aims to explore the expression, regulation and potential role of ACTA1 in ARMS.
ACTA1 protein was detected in the studied RH30, RH4 and RH41 ARMS cells. ACTA1 was found to be inhibited by PAX3-FOXO1 at transcription and protein levels by employing western blot, luciferase reporter, qRT-PCR and immunofluorescence assays. The activities of ACTA1 gene reporter induced by RhoA, MKL1, SRF, STARS or Cytochalasin D molecule were reduced in the presence of overexpressed PAX3-FOXO1 protein. CCG-1423 is an inhibitor of RhoA-MKL1-SRF signaling, we observed there was a synergistic effect between this inhibitor and PAX3-FOXO1 to suppress ACTA1 reporter activity. Furthermore, PAX3-FOXO1 overexpression decreased ACTA1 protein level and knockdown of PAX3-FOXO1 by siRNA enhanced ACTA1 expression. In addition, both MKL1 and SRF, but not RhoA were also found to be inhibited by PAX3-FOXO1 gene at protein levels and increased once knockdown of PAX3-FOXO1 expression. The association between MKL1 and SRF in cells was decreased accordingly with ectopic expression of PAX3-FOXO1. However, the distribution of MKL1 and SRF in nuclear or cytoplasm fraction was not changed by PAX3-FOXO1 expression. Finally, we showed that ACTA1 overexpression in RH30 cells could inhibit cell proliferation and migration in vitro and impair tumor growth in vivo compared with the control groups.
ACTA1 is inhibited by PAX3-FOXO1 at transcription and protein levels through RhoA-MKL1-SRF signaling pathway and this inhibition may partially contribute to the tumorigenesis and development of ARMS. Our findings improved the understanding of PAX3-FOXO1 in ARMS and provided a potential strategy for the treatment of ARMS in future.
肺泡横纹肌肉瘤（ARMS）是一种具有骨骼肌表型的儿童恶性软组织肿瘤。关于ARMS中骨骼肌蛋白的研究报道较少。PAX3-FOXO1是大多数ARMS中由染色体易位t(2;13)(q35;q14)产生的特异性融合基因。ACTA1是骨骼肌α肌动蛋白基因，其转录本在ARMS中被检测到。然而，ACTA1在ARMS中的表达和调控尚未得到充分研究。本研究旨在探讨ACTA1在ARMS中的表达、调控及潜在作用。
在所研究的RH30、RH4和RH41 ARMS细胞中检测到ACTA1蛋白。通过蛋白质免疫印迹、荧光素酶报告基因、qRT-PCR和免疫荧光分析发现，PAX3-FOXO1在转录和蛋白质水平上抑制ACTA1。在过表达PAX3-FOXO1蛋白的情况下，RhoA、MKL1、SRF、STARS或细胞松弛素D分子诱导的ACTA1基因报告基因活性降低。CCG-1423是RhoA-MKL1-SRF信号通路的抑制剂，我们观察到该抑制剂与PAX3-FOXO1之间存在协同作用以抑制ACTA1报告基因活性。此外，PAX3-FOXO1过表达降低了ACTA1蛋白水平，而通过小干扰RNA敲低PAX3-FOXO1则增强了ACTA1表达。另外，还发现MKL1和SRF在蛋白质水平上也受到PAX3-FOXO1基因的抑制，并且在敲低PAX3-FOXO1表达后二者表达增加。随着PAX3-FOXO1的异位表达，细胞中MKL1与SRF之间的关联相应降低。然而，PAX3-FOXO1的表达并未改变MKL1和SRF在细胞核或细胞质组分中的分布。最后，我们表明与对照组相比，RH30细胞中ACTA1的过表达可在体外抑制细胞增殖和迁移，并在体内损害肿瘤生长。
PAX3-FOXO1通过RhoA-MKL1-SRF信号通路在转录和蛋白质水平上抑制ACTA1，这种抑制可能部分促成了ARMS的发生和发展。我们的研究结果增进了对PAX3-FOXO1在ARMS中的理解，并为未来ARMS的治疗提供了潜在策略。
