Nanos3, a cancer-germline gene, promotes cell proliferation, migration, chemoresistance, and invasion of human glioblastoma

Fengyu Zhang; Ruilai Liu; Cheng Liu; Haishi Zhang; Yuan Lu

doi:10.1186/s12935-020-01272-1

Nanos3, a cancer-germline gene, promotes cell proliferation, migration, chemoresistance, and invasion of human glioblastoma

Cancer Cell Int. 2020 May 26:20:197. doi: 10.1186/s12935-020-01272-1. eCollection 2020.

Authors

Fengyu Zhang^#¹, Ruilai Liu^#¹, Cheng Liu¹, Haishi Zhang², Yuan Lu¹

Affiliations

¹ Department of Laboratory Medicine, Huashan Hospital, Fudan University, 12 Wulumuqi Road, Jing-an District, Shanghai, 200040 China.
² Department of Neurosurgery, Huashan Hospital, Fudan University, 12 Wulumuqi Road, Jing-an District, Shanghai, 200040 China.

^# Contributed equally.

Abstract

Background: Radiotherapy, chemotherapy, and surgery have made crucial strides in glioblastoma treatment, yet they often fail; thus, new treatment and new detection methods are needed. Aberrant expression of Nanos3 has been functionally associated with various cancers. Here, we sought to identify the clinical significance and potential mechanisms of Nanos3 in human glioblastoma.

Methods: Nanos3 expression was studied in nude mouse glioblastoma tissues and glioblastoma cell lines by immunohistochemistry, Western blot, and RT-PCR. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing assay was performed to generate the Nanos3 knockdown glioblastoma cell lines. The effects of Nanos3 on glioblastoma cells proliferation, migration, invasion, chemoresistance, germ cell characteristics, and tumor formation were analyzed by CCK8, transwell, cell survival experiments and alkaline phosphatase staining in vitro and in nude mouse models in vivo. Correlation between the expression of stemness proteins and the expression of Nanos3 was evaluated by Western blot.

Results: We found that Nanos3 was strongly expressed in both glioblastoma cell lines and tissues. Western blot and sequencing assays showed that the Nanos3 knockdown glioblastoma cell lines were established successfully, and we discovered that Nanos3 deletion reduced the proliferation, migration, and invasion of glioblastoma cells in vitro (P < 0.05). Nanos3 knockdown enhanced the sensitivity of glioblastoma cells to doxorubicin (DOX) and temozolomide (TMZ) (P < 0.05), and Nanos3^+/- glioblastoma cell lines did not show the characteristics of the germline cells. In addition, Nanos3 deletion inhibited subcutaneous xenograft tumor growth in vivo (P < 0.001). Moreover, the oncogenesis germline protein levels of CD133, Oct4, Ki67, and Dazl decreased significantly in glioblastoma cells following Nanos3 knockdown.

Conclusions: Both in vitro and in vivo assays suggest that Nanos3, which is a cancer-germline gene, initiates the tumorigenesis of glioblastoma via acquiring the oncogenesis germline traits. These data demonstrate that ectopic germline traits are necessary for glioblastoma growth.

Keywords: CRISPR/Cas9; Cancer-germline; Glioblastoma; Nanos3; Tumorigenicity.