Extended Data Fig. 9: circFam53b RNA elicits anti-tumour immune response against mouse melanoma via encoding cryptic antigenic peptides.
From: Tumour circular RNAs elicit anti-tumour immunity by encoding cryptic peptides
(a) Heatmap of Z-score normalized log2(count+1) expression of the selected differentially expressed circRNAs between normal melanocyte cell line Melan-a and melanoma cell line B16F10 (n = 3). (b) Flowcharts indicating key steps involved in TSA discovery for details. Numbers in the charts indicate the number of circRNAs upregulated in B16F10 cells. (c) The expression of indicated circRNAs, normalized to Actb expression, in Melan-a and B16F10 cells, as evaluated by RT-qPCR. **P = 0.0079 (circAsh1l, circCbfb, circSlco3a1, circFam53b). (d) Relative quantitation of circFam53b and linFam53b levels evaluated by RT-qPCR is shown. *P = 0.0125, **P = 0.0015. (e) Relative abundance by RT-qPCR of circFam53b in different cell fractions of B16F10 cells. (f) FISH with junction-specific probes indicates the cellular localization of circFam53b in Melan-a and B16F10 cells. Scale bars, 5 μm. (g-k) B16F10 cells were transfected with siGFP, circFam53b siRNA-1 and siRNA-2 (sicirc-1 and sicirc-2, respectively). Relative quantitation of circFam53b (g) and linFam53b levels (h) by RT-qPCR is shown. (i) CCK-8 assays were used to detect cell viability. Abs, Absorbance. (j) Percentages of annexin V+ cells are shown, evaluated by flow cytometry. (k) Migrated cell counts per field were shown, determined by Transwell migration assays. (l) The efficiency of in vitro circularization and purification of circFam53b and linFam53b were examined by RNase R digestion and denaturing PAGE gel. (m) Cytotoxic effect on B16F10 cells induced by splenic T cells from immunized mice was assessed by LDH assay. (n) The putative IRES activity in circFam53b was determined by the relative luciferase activity of Luc/ Rluc. (o, p) Immunoblotting for Flag expression (o) and circFam53b-221 (p) in HEK293T cells with indicated transfection. (q) MS identified circFam53b-encoded unique peptide sequences in HEK293T cells transfected with circFam53b (n = 3). (r) Immunoblotting for circFam53b-221 in Melan-a and B16F10 cells. (s) MHC peptide binding predictions for circFam53b-encoded peptides to H-2-Db or H-2-Kb using IEDB algorithm (Rank, Score1) and SYFPEITHI (Score2). The circFam53b-encoded unique amino acid sequences are shown in red. (t) MS analysis identified circFam53b(187-196) as the MHC-I binding peptide from B16F10 cells pulled down by H-2-Db. (u, v) C57BL/6 mice were immunized with peptides encoded by circFam53b and linFam53b in combination with adjuvant, poly(I:C). (u) Percentages of PI+ B16F10 cells induced by the splenic T cells of immunized mice are shown, evaluated by flow cytometry. (v) Cytotoxic effect on B16F10 cells was assessed by LDH assay. Representative image of n = 3 independent experiments (f, l, o, p, r, t). For gel source data of Extended Data Fig. 9l, o, p, r and t, see Supplementary Fig. 7. Results are mean ± s.d. of n = 5 (c), n = 3 (d, e, g-k, m, n, u, v) independent experiments producing similar results. ****P < 0.0001. P values, compared with Melan-a cells (c), cells with indicated treatment (d), untreated B16F10 cells (-) (g-k, m, u, v), HEK293T cells transfected with IRES-WT (n), were determined by two-tailed Wilcoxon rank-sum tests (c), two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test (g-k, m, n, u, v) or with Tukey’s multiple-comparisons test (d).
