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

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.

SUBMITTER: Xu Y

PROVIDER: S-EPMC8100523 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Objective: The present study aimed to explore the association between NFIX circular RNA (circNFIX) and miR-34a-5p in glioma. Furthermore, this study investigated the influence that circNFIX has on glioma progression through the upregulation of NOTCH1 via the Notch signaling pathway by sponging miR-34a-5p. Methods: We applied five methods, CIRCexplorer2, circRNA-finder, CIRI, find-circ and MapSplice2, to screen for circRNAs with differential expression between three glioma tissue samples and three paired normal tissue samples. The GSEA software was used to confirm whether significantly different pathways were activated or inactivated in glioma tissues. The binding sites between circNFIX and miR-34a-5p were confirmed by TargetScan. QRT-PCR and western blot were used to measure the relative expression levels of circNFIX, miR-34a-5p and NOTCH and identify their correlation in glioma. RNA immunoprecipitation (RIP) validated the binding relationship between circNFIX and miR-34a-5p, while the targeted relationship between NOTCH1 and miR-34a-5p was verified by a dual luciferase reporter assay. Cell viability and mobility were examined by a CCK-8 assay and wound healing assay, and a flow cytometry assay was employed to analyze cell apoptosis. The nude mouse transplantation tumor experiment verified that si-circNFIX exerted a suppressive effect on glioma progression in vivo. Results: Twelve circRNAs were differentially expressed between the tissue types. Of those, circNFIX was the sole circRNA to be overexpressed in glioma among the five methods of finding circRNAs. In addition, the Notch signaling pathway was considerably upregulated in tumor tissues compared with the paired normal brain tissues. It was determined that circNFIX acted as a sponge of miR-34a-5p, a miRNA that targeted NOTCH1. Downregulation of circNFIX and upregulation of miR-34a-5p both inhibited cell propagation and migration. Furthermore, a miR-34a-5p inhibitor neutralized the suppressive effect of si-circNFIX on glioma cells. Si-circNFIX and miR-34a-5p mimics promoted cell apoptosis. Moreover, it was demonstrated in vivo that si-circNFIX could suppress glioma growth by regulating miR-34a-5p and NOTCH1. Conclusion: CircNFIX was markedly upregulated in glioma cells. CircNFIX could regulate NOTCH1 and the Notch signaling pathway to promote glioma progression by sponging miR-34a-5p via the Notch signaling pathway. This finding provided a deeper insight into the function of circNFIX in human glioma cancer progression.

Project description:BackgroundCircular RNAs (circRNAs) are involved in diverse processes that drive cancer development. However, the expression landscape and mechanistic function of circRNAs in osteosarcoma (OS) remain to be studied.MethodsBioinformatic analysis and high-throughput RNA sequencing tools were employed to identify differentially expressed circRNAs between OS and adjacent noncancerous tissues. The expression level of circ_001422 in clinical specimens and cell lines was measured using qRT-PCR. The association of circ_001422 expression with the clinicopathologic features of 55 recruited patients with OS was analyzed. Loss- and gain-of-function experiments were conducted to explore the role of circ_001422 in OS cells. RNA immunoprecipitation, fluorescence in situ hybridization, bioinformatics database analysis, RNA pulldown assays, dual-luciferase reporter assays, mRNA sequencing, and rescue experiments were conducted to decipher the competitive endogenous RNA regulatory network controlled by circ_001422.ResultsWe characterized a novel and abundant circRNA, circ_001422, that promoted OS progression. Circ_001422 expression was dramatically increased in OS cell lines and tissues compared with noncancerous samples. Higher circ_001422 expression correlated with more advanced clinical stage, larger tumor size, higher incidence of distant metastases and poorer overall survival in OS patients. Circ_001422 knockdown markedly repressed the proliferation and metastasis and promoted the apoptosis of OS cells in vivo and in vitro, whereas circ_001422 overexpression exerted the opposite effects. Mechanistically, competitive interactions between circ_001422 and miR-195-5p elevated FGF2 expression while also initiating PI3K/Akt signaling. These events enhanced the malignant characteristics of OS cells.ConclusionsCirc_001422 accelerates OS tumorigenesis and metastasis by modulating the miR-195-5p/FGF2/PI3K/Akt axis, implying that circ_001422 can be therapeutically targeted to treat OS.

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

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