CircBIRC6 Contributes to the Development of Non-Small Cell Lung Cancer via Regulating MicroRNA-217/Amyloid Beta Precursor Protein Binding Protein 2 Axis
Non-small cell lung cancer (NSCLC) is a prevalent and aggressive form of lung cancer, accounting for 80% to 85% of all lung cancer cases. Despite advancements in early detection and treatment, the prognosis for NSCLC patients remains poor, particularly for those diagnosed at advanced stages. This underscores the urgent need to explore the molecular mechanisms underlying NSCLC pathogenesis to identify novel therapeutic targets.
Circular RNAs (circRNAs) have emerged as significant regulators in cancer biology. These single-stranded, loop-closed non-coding RNAs play crucial roles in various cancers, including NSCLC. One such circRNA, circBIRC6, has been implicated in NSCLC progression. This study delves into the role of circBIRC6 in NSCLC, focusing on its interaction with microRNA-217 (miR-217) and amyloid beta precursor protein binding protein 2 (APPBP2).
The study began by collecting NSCLC tissues and adjacent non-tumor tissues from patients at Shanghai Ninth People’s Hospital. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression levels of circBIRC6, APPBP2 messenger RNA (mRNA), BIRC6 mRNA, and miR-217. Western blot assays were used to measure protein levels of APPBP2, E-cadherin, N-cadherin, and vimentin. Functional assays, including colony formation, transwell migration and invasion, and flow cytometry analysis, were conducted to evaluate the effects of circBIRC6 on NSCLC cell behaviors. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to determine the interactions among circBIRC6, miR-217, and APPBP2. Additionally, a murine xenograft model was used to investigate the role of circBIRC6 in tumor formation in vivo.
The results revealed that circBIRC6 was significantly overexpressed in NSCLC tissues and cell lines compared to normal tissues and cells. Specifically, circBIRC6 levels were 11.070 ± 4.388 in NSCLC tissues versus 0.979 ± 0.343 in normal tissues (P < 0.001). Similarly, APPBP2 mRNA and protein levels were elevated in NSCLC tissues and cells. These findings suggested that both circBIRC6 and APPBP2 might play roles in NSCLC progression.
To explore the functional significance of circBIRC6, the researchers performed RNase R treatment and subcellular fraction assays. CircBIRC6 was found to be resistant to RNase R, indicating its stability, and was predominantly localized in the cytoplasm of NSCLC cells. Knockdown of circBIRC6 using small interfering RNAs (siRNAs) significantly inhibited colony formation, migration, and invasion of NSCLC cells while promoting apoptosis. Specifically, colony numbers decreased, migration and invasion abilities were repressed, and apoptosis rates increased in circBIRC6-deficient cells. Additionally, circBIRC6 knockdown led to increased E-cadherin levels and decreased N-cadherin and vimentin levels, indicating inhibition of the epithelial-mesenchymal transition (EMT) process.
The study then investigated the underlying mechanisms by which circBIRC6 regulates NSCLC progression. Bioinformatics analysis predicted that miR-217 could bind to circBIRC6. Dual-luciferase reporter assays confirmed this interaction, showing that miR-217 transfection reduced the luciferase activity of wild-type circBIRC6 but not mutant circBIRC6. RIP assays further validated the interaction, demonstrating increased enrichment of circBIRC6 and miR-217 in anti-argonaute 2 (Ago2) immunoprecipitates compared to control groups.
MiR-217 was found to be downregulated in NSCLC tissues and cells. Overexpression of circBIRC6 reduced miR-217 levels, while circBIRC6 knockdown increased miR-217 levels. Functional rescue experiments showed that inhibition of miR-217 reversed the effects of circBIRC6 knockdown on NSCLC cell behaviors, including colony formation, migration, invasion, and apoptosis. These results indicated that circBIRC6 exerts its oncogenic effects in NSCLC by sponging miR-217.
Next, the study explored the relationship between miR-217 and APPBP2. Bioinformatics analysis predicted that miR-217 could target APPBP2. Dual-luciferase reporter assays confirmed this interaction, showing that miR-217 transfection reduced the luciferase activity of wild-type APPBP2 3’UTR but not mutant APPBP2 3’UTR. RIP assays further validated the interaction, demonstrating increased enrichment of miR-217 and APPBP2 in anti-Ago2 immunoprecipitates compared to control groups.
MiR-217 overexpression significantly decreased APPBP2 protein levels in NSCLC cells, while miR-217 inhibition increased APPBP2 levels. Functional rescue experiments showed that overexpression of APPBP2 reversed the effects of miR-217 overexpression on NSCLC cell behaviors, including colony formation, migration, invasion, and apoptosis. These results suggested that miR-217 regulates NSCLC progression by targeting APPBP2.
The study also examined the correlations among circBIRC6, miR-217, and APPBP2 in NSCLC tissues. Pearson’s correlation coefficient analysis revealed a negative correlation between miR-217 levels and both circBIRC6 and APPBP2 mRNA levels, and a positive correlation between circBIRC6 and APPBP2 mRNA levels. These findings suggested that circBIRC6 positively regulates APPBP2 expression by sponging miR-217.
Finally, the role of circBIRC6 in tumor growth was investigated using a murine xenograft model. CircBIRC6 knockdown significantly reduced tumor volume and weight in nude mice. Analysis of the harvested tumors showed decreased circBIRC6 and APPBP2 levels and increased miR-217 levels in the circBIRC6-deficient groups compared to control groups. These results demonstrated that circBIRC6 knockdown inhibits tumor growth in vivo.
In conclusion, this study revealed that circBIRC6 is overexpressed in NSCLC and promotes NSCLC progression by sponging miR-217 and upregulating APPBP2. The findings provide new insights into the molecular mechanisms of NSCLC and suggest that circBIRC6 could serve as a potential therapeutic target for NSCLC treatment.
doi.org/10.1097/CM9.0000000000001940
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