Prohibitin 2 localizes in nucleolus to regulate ribosomal RNA transcription and facilitate cell proliferation in RD cells

Zilong Zhou,Lihua Zheng,Yongli Bao,Yin Wu,Kun Li,Zhenbo Song,Chunlei Yu,Luguo Sun,Yuxin Li,Wenbin Zhu,Yanxin Huang,Ying Sun,Guannan Wang,Huihan Ai,Lei Liu,Xinlei Yao,Kewei Ma

doi:10.1038/s41598-018-19917-7

Zilong Zhou, Lihua Zheng + Show 15 more

Open Access

https://doi.org/10.1038/s41598-018-19917-7

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Abstract

Prohibitin 2 (PHB2), as a conserved multifunctional protein, is traditionally localized in the mitochondrial inner membrane and essential for maintenance of mitochondrial function. Here, we investigated the role of PHB2 in human rhabdomyosarcoma (RMS) RD cells and found substantial localization of PHB2 in the nucleolus. We demonstrated that PHB2 knockdown inhibited RD cell proliferation through inducing cell cycle arrest and suppressing DNA synthesis. Meanwhile, down-regulation of PHB2 also induced apoptosis and promoted differentiation in fractions of RD cells. In addition, PHB2 silencing led to altered nucleolar morphology, as observed by transmission electron microscopy, and impaired nucleolar function, as evidenced by down-regulation of 45S and 18S ribosomal RNA synthesis. Consistently, upon PHB2 knockdown, occupancy of c-Myc at the ribosomal DNA (rDNA) promoter was attenuated, while more myoblast determination protein 1 (MyoD) molecules bound to the rDNA promoter. In conclusion, our findings suggest that nucleolar PHB2 is involved in maintaining nucleolar morphology and function in RD cells by regulating a variety of transcription factors, which is likely to be one of the underlying mechanisms by which PHB2 promotes tumor proliferation and represses differentiation. Our study provides new insight into the pathogenesis of RMS and novel characterizations of the highly conserved PHB2 protein.

Highlights

Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents, accounting for 5% of all pediatric tumors and over 40% of pediatric soft tissue sarcomas[1]
We further demonstrated that the expression of antigen Ki67, a nuclear protein associated with cell proliferation, substantially decreased in RD cells transfected with Prohibitin 2 (PHB2) siRNAs (Fig. 1c)
Using the RD cell line, we first showed the localization of PHB2, a repressor of skeletal muscle differentiation, in the nucleolus where it participated in regulation of Myc-mediated ribosomal RNA (rRNA) synthesis, which may serve as a mechanism to facilitate cell proliferation while inhibiting differentiation

Summary

Introduction

Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents, accounting for 5% of all pediatric tumors and over 40% of pediatric soft tissue sarcomas[1]. Accompanied by incomplete differentiation, cell proliferation is no longer arrested in these tumor cells[2]. MRFs, which include MyoD, myogenin, myogenic factor 5 (Myf5) and MRF4, synergistically cooperate with MEF2 to induce muscle-specific gene transcription and the onset of myogenesis. Among those factors, MyoD is considered to be muscle-determining, and the expression of myogenin is a typical early myogenic differentiation marker. We further showed that nucleolar PHB2 might be involved in maintaining the structure and function of the nucleolus in RD cells partly by modulating the transcription of rDNA through regulating the accessibility of c-Myc and MyoD to the rDNA promoter. Our results suggest a pro-tumorigenic role for PHB2 in human RMS at least partly through its specific localization in the nucleolus

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