Fig. 1: Screen and characterization of circRFWD3 in HNSCC.

A Clustered heatmap showing the 32 differentially expressed circRNAs in three groups of human HNSCC tissues and adjacent normal tissues (red indicates upregulated circRNAs, and blue indicates downregulated circRNAs). The rows represent groups of HNSCC tissue samples (C, normal tissue; T, tumor tissue), and columns represent circRNAs’ numerical ID obtained from circBase. B The genomic loci of the RFWD3 gene and circRFWD3 were primarily transcribed from exons 7 and 8 of RFWD3, which were detected by qRT-PCR and validated by Sanger sequencing amplified with divergent primers. C, D The expression level of circRFWD3 in 43 normal tissues and 17 HNSCC tissues (data from TCGA) and in normal HOK cells and three HNSCC cell lines. E Agarose gel electrophoresis with RT-PCR products using divergent and convergent primers in both gDNA and cDNA. Red arrows showed that divergent primers could amplify circRFWD3 from cDNA. F, G qRT-PCR was used to determine the abundance of circRFWD3 and linear RFWD3 mRNA in UM1 and HN31 cells treated with RNase R. Results demonstrated circRFWD3 was tolerant to RNase R. H CirRFWD3 resistance to actinomycin D (ActD) was detected by the qRT-PCR assay. I RNA FISH showed that circRFWD3 located in the cytoplasm in UM1 and HN31 cells, scale bars, 10 μm. Data were shown as the mean value ± SD of three independent experiments. The asterisks indicate significant differences (Student’s t-tests, *P < 0.05, **P < 0.01, ***P < 0.001).