Circular RNA circSIPA1L1 Contributes to Osteosarcoma Progression Through the miR-411-5p/RAB9A Signaling Pathway.

Yining Xu,Yizhen Huang,Jianjun Ma,Jun Gao,Shuying Shen,Rujie Zheng,Di Qiao,Kangmao Huang,Teng Yao,Haonan Ni

doi:10.3389/fcell.2021.642605

Abstract

Recently, various studies have identified circular RNAs (circRNAs) to play a significant role in tumorigenesis, thereby showing potential as novel tumor biomarkers. circSIPA1L1 is a newly discoveredcircular RNA, which is formed by back-splicing of SIPA1L1 and is found increased in osteosarcoma (OS). Nevertheless, the specific functions of circSIPA1L1 in OS remain unknown. In the present study, circSIPA1L1 was obtained from a previously reported circRNA microarray in the GEO database (GSE96964). Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the mRNA level of circSIPA1L1 in OS cell lines and tissue samples. Bioinformatics analysis, luciferase reporter assays, real-time PCR, RNA pull-down assays and RNA immunoprecipitation (RIP) were employed to verify the binding of circSIPA1L1 with miR-411-5p. Xenograft tumor models were established to identify the role of circSIPA1L1 in vivo. A series of in vitro experiments, such as western blotting, colony formation, transwell assays and anoikis assay were employed to confirm the relationship across circSIPA1L1, miR-411-5p, and RAB9A. Our study confirmed circSIPA1L1 to be upregulated in both human OS samples and OS cell lines. Mechanistically, circSIPA1L1 could serve as a miR-411-5p molecular sponge to increase RAB9A expression, which was confirmed to be a tumor promoter mediating carcinogenesis. Silencing of circSIPA1L1 attenuated the vitality, invasion, migration and proliferation of OS cell lines both in vivo and in vitro. miR-411-5p inhibition or RAB9A overexpression reversed the anti-tumor effects caused by circSIPA1L1 knockdown. Briefly, circSIPA1L1 could function as a driver gene in OS and initiate OS tumorigenesis through the miR-411-5p/RAB9A signaling pathway, which might become a potential therapeutic biomarker for OS treatment.

Highlights

Osteosarcoma (OS), a neoplasm with high aggressiveness and formidable distant metastatic characteristics, is the third most prevalent and lethal primary bone malignancy across children, youth, and adolescents (Ritter and Bielack, 2010)
We validated using Quantitative real-time polymerase chain reaction (qRT-PCR) that the abundance of circSIPA1L1 was notably increased in OS cell lines (U20S, 143B, HOS, and MG-63) in comparison with hFOB1.19 cells
To verify the stability of circSIPA1L1, RNase R was used in the assay. circSIPA1L1 exhibited high tolerance to RNase R treatment while the levels of linear form of signal induced proliferation associated 1 like 1 (SIPA1L1) were significantly decreased upon exposure to RNase R (Figure 1F)

Summary

Introduction

Osteosarcoma (OS), a neoplasm with high aggressiveness and formidable distant metastatic characteristics, is the third most prevalent and lethal primary bone malignancy across children, youth, and adolescents (Ritter and Bielack, 2010). While the long-term overall survival of patients suffered from OS with no metastasis is greater than 60%, the 5-year survival rate of patients in an advanced disease stage could be as low as 20%, owing to distant metastasis and recurrence (Gianferante et al, 2017; Kumar et al, 2018). With advancement in high-throughput sequencing and bioinformatics approaches, a growing number of circRNAs have been demonstrated to be highly expressed in multiple human cancers, and to play crucial roles in a wide variety of biological processes in cancer (Han et al, 2018; Cheng et al, 2019). The specific function and mechanisms of circSIPA1L1 in OS have not been adequately elucidated

Methods

Results

Conclusion