Actin filament‐associated protein 1‐antisense RNA1 promotes the development and invasion of tongue squamous cell carcinoma via the AFAP1‐AS1/miR‐133a‐5p/ZIC2 axis

Abstract

AbstractBackgroundThe present study aimed to explore the biological role and underlying mechanism of the long non‐coding RNA actin filament‐associated protein 1‐antisense RNA1 (lncRNA AFAP1‐AS1) in the progression of tongue squamous cell carcinoma (TSCC).MethodsA quantitative reverse transcriptase‐PCR (RT‐qPCR) was conducted to assess relative levels of the miR‐133a‐5p, lncRNAs AFAP1‐AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1‐AS1, miR‐133a‐5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial–mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual‐luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR‐133a‐5p, as well as between miR‐133a‐5p and lncRNA AFAP1‐AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1‐AS1. The potential biological pathway for AFP1‐AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1‐AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1‐AS1.ResultsIn TSCC cell lines and specimens, lncRNA AFAP1‐AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1‐AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1‐AS1 could upregulate ZIC2 by competitively inhibiting miR‐133a‐5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1‐AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1‐AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1‐AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator‐activated receptor signaling pathway was significantly correlated with low expression of AFP1‐AS1. Finally, the results of drug prediction indicated that the group with high AFAP1‐AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib.ConclusionsThe upregulation of lncRNA AFAP1‐AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1‐AS1/miR‐133a‐5p/ZIC2 axis, aids in the progression of TSCC.