目的:B7同源物3蛋白(B7-H3)的异常表达已在包括结直肠癌(CRC)在内的各种癌症中检测到,并涉及调节CRC细胞的多种生物学功能。然而,其在CRC转移中的作用尚未确定。本研究旨在探索和解开B7-H3有助于迁移的潜在机制,CRC的侵袭和肌动蛋白细胞骨架。
方法:通过IHC染色测定CRC肿瘤样品中B7-H3和LIMK1的表达。进行了Transwell和F-actin免疫荧光染色测定,以探讨B7-H3在迁移中的作用,CRC细胞的侵袭和肌动蛋白丝积累。RNA-seq和Western印迹分析用于研究分子机制。
结果:B7-H3在CRC组织中高表达,免疫组织化学显示B7-H3与CRC患者的不良预后呈正相关。迁移和侵袭实验表明,B7-H3敲低能显著抑制CRC细胞的迁移和侵袭。B7-H3过表达具有相反的作用。此外,我们通过F-肌动蛋白免疫荧光染色和Westernblot确定B7-H3可以调节肌动蛋白细胞骨架和RhoA/ROCK1/LIMK1通路。重要的是,BDP5290,RhoA/ROCK1/(LIM结构域激酶1)LIMK1轴的抑制剂,逆转了B7-H3过表达对肌动蛋白丝积累的影响,迁移,和CRC细胞的侵袭。
结论:我们的研究得出结论,B7-H3促进CRC细胞肌动蛋白丝积累,迁移,并通过RhoA/ROCK1/LIMK1轴侵入。
OBJECTIVE: Aberrant expression of B7 homolog 3 protein (B7-H3) has been detected in various cancers including colorectal cancer (CRC) and implicated in modulating multiple biological functions of CRC cells. However, its role in CRC metastasis has not yet been determined. This study aims to explore and unravel the underlying mechanisms through which B7-H3 contributes to migration, invasion and actin cytoskeleton in CRC.
METHODS: The expression of B7-H3 and LIMK1 in CRC tumor samples was determined by IHC staining. Transwell and F-actin immunofluorescence staining assays were performed to explore the role of B7-H3 in migration, invasion and actin filament accumulating of CRC cells. RNA-seq and Western blot assays were used to investigate the molecular mechanisms.
RESULTS: B7-H3 was highly expressed in CRC tissues and positively associated with poor prognosis of CRC patients by immunohistochemistry. Migration and invasion assays showed that B7-H3 knockdown significantly inhibited the migration and invasion of CRC cells. B7-H3 overexpression had the opposite effect. Moreover, we determined that B7-H3 could regulate actin cytoskeleton and the RhoA/ROCK1/LIMK1 pathway by F-actin immunofluorescence staining and Western blot. Importantly, the BDP5290, an inhibitor of the RhoA/ROCK1/(LIM domain kinase 1) LIMK1 axis, reversed the effects of B7-H3 overexpression on actin filament accumulating, migration, and invasion of CRC cells.
CONCLUSIONS: Our study concluded that B7-H3 facilitated CRC cell actin filament accumulating, migration, and invasion through the RhoA/ROCK1/LIMK1 axis.