This study assessed the clinical and functional relevance of the long noncoding RNA BBOX1-AS1 in non-small cell lung cancer (NSCLC). The authors aimed to determine whether differential expression of BBOX1-AS1 has diagnostic or prognostic value and to explore mechanisms by which BBOX1-AS1 may regulate tumor cell behavior.
BBOX1-AS1 expression was measured by reverse transcription-quantitative real-time PCR (RT-qPCR) in NSCLC tumor tissues, patient serum, and established cell lines. Clinical correlations used the chi-square test on a cohort of patients diagnosed with NSCLC at Pangang Group General Hospital between January 2018 and January 2021. Receiver operating characteristic (ROC) curves evaluated diagnostic performance of BBOX1-AS1 and miR-3940-3p. Kaplan-Meier survival curves and Cox regression analysis assessed prognostic value. Cell proliferation effects of BBOX1-AS1 knockdown were analyzed using the cell counting kit-8 (CCK-8) assay in the A549 cell line. Bioinformatics analyses and dual-luciferase reporter assays were employed to identify downstream miRNAs and potential target genes involved in BBOX1-AS1 function.
RT-qPCR results showed that BBOX1-AS1 expression was significantly upregulated in NSCLC tumor tissues, in patient serum, and in NSCLC cell lines (all P < 0.05). The consistent elevation across tissue, circulating, and in vitro samples supports the relevance of BBOX1-AS1 to NSCLC biology and its potential as a measurable biomarker.
High BBOX1-AS1 expression was significantly associated with larger tumor diameter, presence of lymph node metastasis, and more advanced TNM stage (all P < 0.05). These associations indicate that higher BBOX1-AS1 levels correlate with more aggressive or advanced disease features in the studied cohort.
The study evaluated diagnostic performance using ROC analysis and reported that serum BBOX1-AS1 achieved an area under the curve (AUC) of 0.858, while miR-3940-3p achieved an AUC of 0.852, indicating both markers had high diagnostic value for NSCLC in this dataset. miR-3940-3p expression was downregulated in NSCLC serum (P < 0.05) and showed a negative correlation with BBOX1-AS1 levels (r = -0.646, P < 0.001).
Prognostically, patients with high BBOX1-AS1 expression had a 5-year survival rate of 36.7%, compared with 61.1% for patients with low expression. Multivariate Cox regression identified high BBOX1-AS1 expression as an independent prognostic factor for NSCLC (hazard ratio [HR] = 0.496; 95% confidence interval [CI], 0.272–0.904; P = 0.022).
Functional experiments used siRNA to reduce BBOX1-AS1 expression in A549 cells and measured proliferation with the CCK-8 assay. At 72 hours, the BBOX1-AS1-siRNA group showed a lower absorbance value (0.67 ± 0.05) than the Mock group (1.17 ± 0.06) and the siRNA-NC group (1.06 ± 0.03), with P < 0.001. The authors report that knockdown of BBOX1-AS1 inhibited A549 cell proliferation, supporting a role for BBOX1-AS1 in promoting tumor cell growth.
Dual-luciferase reporter assays indicated that miR-3940-3p directly interacts with BBOX1-AS1, suggesting a potential regulatory axis by which BBOX1-AS1 may act as a competitive endogenous RNA or otherwise modulate miRNA activity. Bioinformatics analysis predicted ten potential target genes of miR-3940-3p relevant to the observed biology: MOV10, TP53, PABPC1, TIAL1, STAU1, IGF2BP1, FMR1, ELAVL1, CPEB1, and PABPN1. The study presents these genes as candidate downstream effectors mediating BBOX1-AS1/miR-3940-3p-associated functions; functional validation of these targets was not reported in the abstract.
The authors conclude that BBOX1-AS1 is highly expressed in NSCLC and that elevated expression is associated with more advanced disease and poorer long-term survival. Knockdown experiments indicate that reducing BBOX1-AS1 expression decreases A549 tumor cell proliferation. The negative correlation and direct interaction with miR-3940-3p, along with predicted miR-3940-3p target genes, provide a putative mechanistic framework for BBOX1-AS1's role in NSCLC. The study screens potential functional target genes and suggests BBOX1-AS1 as a candidate prognostic biomarker and a possible therapeutic target in NSCLC.
All authors declared no conflicts of interest.