Table 1.
The sequence of PCR primers.
Fig 1.
Characterization of circTADA2A in melanoma cells.
(A) A schematic diagram showing that circTADA2A was formed by the back-splicing of linear TADA2A between the 5th and 6th exons. (B) The circTADA2A junction site was identified by Sanger sequencing. circTADA2A and its linear counterpart TADA2A in complementary DNA (cDNA) and genomic DNA (gDNA) was analyzed by qRT-PCR assays. (C, D) CircTADA2A and TADA2A mRNA were detected by qRT-PCR assays, RNA samples were treated with RNase R or mock treated without the enzyme. (E, F) qRT-PCR analysis for the abundance of circTADA2A and TADA2A in melanoma cells treated with Actinomycin D at the indicated time point. (G) qRT-PCR analysis for the abundance of circTADA2A in normal melanocytes cell line HeMa-Lp and melanoma cell line A375 and A2058. (H, I) Subcellular qRT-PCR analysis showing that circTADA2A was mainly localized in the nucleus. GAPDH and U1 were applied as positive controls in the cytoplasm and nucleus, respectively. (J) RNA fluorescence in situ hybridization analysis revealed the subcellular localization of circTADA2A.
Fig 2.
CircTADA2A suppresses proliferation, migration and invasion of melanoma cells.
(A) The expression of circTADA2A was determined by qRT-PCR in circTADA2A overexpression or knockdown melanoma cells. (B, C) The effect of circTADA2A on melanoma cell proliferation was determined by EdU assay (B), and colony formation (C). (D, E) The effect of circTADA2A on melanoma cell migration and invasion was determined by transwell assay (D), and wound healing assay (E). (F) The effect of circTADA2A on melanoma cell cycle was determined by flow cytometry.
Fig 3.
CircTADA2A interacts with CNBP protein in melanoma.
(A) qRT-PCR analysis of TADA2A mRNA expression in melanoma cells with circTADA2A overexpression. (B) RIP assay revealed that the enrichment of circTADA2A on Ago2 relative to IgG. (C) SDS-PAGE separation and silver staining of the immunoprecipitated proteins obtained from A375 cells (left) by sense and anti-sense probe of circTADA2A. The proteins pulled down by probes were overlapped with established RBPs and TFs (right). (D) RIP and qRT-PCR assays showing the relative interaction between circTADA2A and three proteins in melanoma cells stably transfected with empty vector (circ-Mock), circTADA2A, or linear circTADA2A (lin-TADA2A), with normalization to input of cells transfected with circ-Mock. (E) RNA pulldown assay indicating the direct interaction between circTADA2A and CNBP in melanoma cells. (F) MS assay depicting the identified CNBP peptides pulled down by circTADA2A. (G) Schematic diagram revealing the domains of CNBP truncations. (H) In vitro binding assay showing the enriched circTADA2A levels detected by qRT-PCR after incubation with full-length or truncations of Flag-tagged or GST-tagged recombinant CNBP protein validated by western blot. (I, J) Western blot assays (I) and qRT-PCT (J) showing the protein levels of CNBP in A375 and A2058 cells stably transfected with mock or circTADA2A.
Fig 4.
SLC38A1 was a direct downstream target of CNBP in melanoma.
(A) The differentially expressed genes from melanoma datasets (GSE31909 and GSE35388) were overlapped with predicted targets of CNBP from ENCORI database. (B) The abundance of five potential target mRNAs in melanoma cells with circTADA2A overexpression or knockdown was analyzed by qRT-PCR assay. (C) qRT-PCR assay showing the expression of SLC38A1 in melanoma cells stably transfected with mock or circTADA2A, and those cotransfected CNBP. (D) RIP assay was used to evaluate interaction between CNBP and SLC38A1 mRNA. (E) CHIP assay with primer sets indicating the interaction between CNBP and SLC38A1 promoter in melanoma cells. (F) CHIP assay indicating the interaction between CNBP and SLC38A1 promoter in melanoma cells stably transfected with circ-Mock, circTADA2A, and those cotransfected CNBP. (G) Dual-luciferase reporting experiment was applied to evaluate the binding strength between CNBP and SLC38A1 promoter with Mock, CNBP-OE or CNBP-KD vectors. (H) qRT-PCR assay showing the abundance of SLC38A1 in melanoma cells stably transfected with mock, CNBP, sh-NC or sh-CNBP. (I) Western blot assay showing the expression of SLC38A1 in melanoma cells stably transfected with mock, CNBP, sh-NC or sh-CNBP.
Fig 5.
CircTADA2A/CNBP suppresses the progression of melanoma cells via repressing SLC38A1 expression.
(A, B) EdU assay (A), and colony formation (B) assay indicating the cell proliferation of A375 and A2058 cells stably transfected with mock, circTADA2A, circTADA2A co-transfected with CNBP, or circTADA2A co-transfected with CNBP and sh-SLC38A1. (C, D) Transwell (C), and wound healing (D) assay indicating the cell migration and invasion of A375 and A2058 cells stably transfected with mock, circTADA2A, circTADA2A co-transfected with CNBP, or circTADA2A co-transfected with CNBP and sh-SLC38A1. (E) Western blot assay showing the expression of CNBP and SLC38A1 in melanoma cells stably transfected with circ-mock, circTADA2A, mock or CNBP. (F) Western blot assay showing the expression of PCNA and MMP-9 in melanoma cells stably transfected with mock, circTADA2A, circTADA2A co-transfected with CNBP, or circTADA2A co-transfected with CNBP and sh-SLC38A1.