Splicing factor derived circular RNA circUHRF1 accelerates oral squamous cell carcinoma tumorigenesis via feedback loop

Abstract

Emerging evidences have suggested the vital roles of circular RNA (circRNA) in the human cancers. However, the underlying biological functions and biogenesis of circRNA in the oral squamous cell carcinoma (OSCC) is still ambiguous. Here, we investigate the oncogenic roles and biogenesis of the novel identified circRNA, circUHRF1 (hsa_circ_0002185), in the OSCC tumorigenesis. Results showed that circUHRF1 was markedly upregulated in the OSCC cells and tissue, besides, the overexpression was closely correlated with the poor prognosis of OSCC patients. Functionally, circUHRF1 promoted the proliferation, migration, invasion, and epithelial mesenchymal transformation (EMT) in vitro and the tumor growth in vivo. Mechanically, circUHRF1 acted as the sponge of miR-526b-5p, thereby positively regulating c-Myc. Transcription factor c-Myc could accelerate the transcription of TGF-β1 and ESRP1. Moreover, splicing factor ESRP1 promoted the circularization and biogenesis of circUHRF1 by targeting the flanking introns, forming the circUHRF1/miR-526b-5p/c-Myc/TGF-β1/ESRP1 feedback loop. In conclusion, our research identified the oncogenic roles of circUHRF1 in the OSCC tumorigenesis and EMT via circUHRF1/miR-526b-5p/c-Myc/TGF-β1/ESRP1 feedback loop, shedding light on the pathogenic mechanism of circUHRF1 for OSCC and providing the potential therapeutic target.

Fig. 1

circUHRF1 is highly expressed in the OSCC tissue and cells. a RT-PCR showed the significant overregulation of circUHRF1 in the OSCC cells. b Schematic diagram presented the formation of circUHRF1 from the 12, 13 exon of UHRF1 gene. c Sanger sequencing identified the existence of circUHRF1. The red arrow indicates the conjunction sites of 12, 13 exon of UHRF1 gene. d The agarose gel electrophoresis was performed using the divergent primers and convergent primers to verify the existence of circUHRF1 using SCC25 cells. e The subcellular distribution of circUHRF1 was identified using RNA-FISH using SCC25 and CAL27 cells. The red (Cy3-labeled probe) indicated the circUHRF1. DAPI indicated the nucleus. f RT-PCR showed the transcript half-life of circUHRF1 in SCC25 cells treated with transcription inhibitor Actinomycin D. g RNase R was administrated to the extracted RNA to measure the relative expression of circular form (circUHRF1) and the linear form (UHRF1 mRNA). h RT-PCR showed the enrichment of circUHRF1 in the enrolled OSSCC individuals and normal controls. i The survival rate of OSCC patients who with high level of circUHRF1 and lower level. **p-value < 0.01

Fig. 2

Knockdown of circUHRF1 represses the proliferation, migration, invasion, and EMT of OSCC cells in vitro. a shRNA targeting circUHRF1 junction sites (Exon 13–Exon 12) were constructed (left). Stable silencing shRNA were transfected into OSCC cells (SCC25, CAL27) to silence circUHRF1 expression (middle). The linear UHRF1 mRNA levels were detected using RT-qPCR (right). b Colony formation assay was performed in the SCC25 and CAL27 cells after stable transfection. c EdU assay was performed for the DNA synthesis in the SCC25 and CAL27 cells after stable transfection. d Wound healing assay illustrated migrative ability after the knockdown of circUHRF1 or negative control. e, f Transwell assay for migration and invasion was carried out to detect the invasive ability and migrative ability of OSCC cells (SCC25, CAL27). g The protein markers of EMT were detected using western blot analysis, including mesenchymal markers (N-cadherin, Vimentin) and the epithelial marker (E-cadherin). **p-value < 0.01

Fig. 3

circUHRF1 acts as the sponge of miR-526b-5p in OSCC cells. a Online bioinformatics tools CircInteractome (https://circinteractome.nia.nih.gov/) indicated the miR-526b-5p target for circUHRF1. b The wild-type and mutant sequences were constructed corresponding with the miR-526b-5p for the luciferase reporter assay. c The luciferase activity in the cotransfection of miR-526b-5p and circUHRF1 wild type or mutant. d Biotinylated-circUHRF1 probe was performed for RNA pull-down assay. RT-PCR revealed the miR-526b-5p expression in the pulled down SCC25 cells. e RT-PCR indicated the miR-526b-5p level in the SCC25 cells with transfection of circUHRF1 shRNA. f RT-PCR illustrated the miR-526b-5p level in the OSCC (SCC25, CAL27) cells. g RNA-FISH indicated the circUHRF1 and miR-526b-5p subcellular location. h The Pearson correlation analysis indicated the negative correlation within miR-526b-5p and circUHRF1. **p-value < 0.01

Fig. 4

c-Myc functions as the target of circUHRF1/miR-526b-5p. a The c-Myc wild type and mutant sequence at 3′-UTR and miR-526b-5p were constructed. b Luciferase reporter assay indicated the luciferase activity after the cotransfection with c-Myc wild type or mutant and miR-526b-5p mimics or controls. c RT-PCR indicated the c-Myc mRNA after miR-526b-5p mimics transfection or miR-526b-5p inhibitor transfection. d, e Western blot analysis illustrated the c-Myc protein in OSCC cells (SCC25, CAL27) with miR-526b-5p mimics or inhibitor transfection. f, g Western blot analysis indicated the c-Myc protein in the transfection of circUHRF1 shRNA with miR-526b-5p inhibitor. **p-value  < 0.01

Fig. 5

c-Myc promotes the transcription of TGF-β1 and ESRP1. a JASPAR (http://jaspar.genereg.net/) and RegRNA (http://regrna2.mbc.nctu.edu.tw/detection.html) indicated that c-Myc shared with the binding sites with the promoter region of TGF-β1. b ChIP-PCR was performed in SCC25 cells to detect the enrichment of potential binding sequences using the c-Myc antibody. IgG acts as the negative control. c RT-PCR showed the TGF-β1 mRNA level in SCC25 cells transfected with c-Myc overexpressed plasmid orc-Myc silencing siRNA. d The luciferase activities were tested in the luciferase reporter assay in SCC25 cells after the vector (wild type and mutant) and c-Myc. e Bioinformatics tools indicated that c-Myc shared with the binding sites with the promoter region of ESRP1. f ChIP-PCR showed the binding of c-Myc with the promoter region of ESRP1. g RT-PCR showed the ESRP1 mRNA level with c-Myc overexpressed plasmid orc-Myc silencing siRNA transfection. h The luciferase reporter assay indicated the binding of the wild type or mutant with c-Myc. i Western blotting analysis for the TGF-β1 and ESRP1 protein level after the transfection of c-Myc overexpression plasmid and siRNA. j The positive correlation within c-Myc and TGF-β1 and ESRP1 based on the GEPIA dataset based on the TCGA (http://gepia.cancer-pku.cn/index.html). k Schematic diagram suggested the activation of c-Myc for the transcription of TGF-β1 and ESRP1 in OSCC cells. **p-value < 0.05

Fig. 6

Splicing factor ESRP1 accelerates the biogenesis of circUHRF1. a Multiple ESRP1 motifs (GGT-rich) were found in the flanking of circUHRF1. The minigenes of these motifs were constructed, including wild type and mutant. b RNA-binding protein immunoprecipitation (RIP) presented the binding of ESRP1 with the motifs minigenes, including wild type and mutant. c RT-PCR showed the circUHRF1 expression in SCC25 cells transfected with ESRP1 silencing shRNA or controls with these mutant sequences (one motif mutant). d RT-PCR showed the circUHRF1 expression when the two or three motifs were mutated. e RT-PCR showed the linear transcript expression levels (exon 12–13, exon 11–12, and exon 13–14) after the ESRP1 knockdown. f Schematic diagram illustrates that Splicing factor ESRP1 accelerates the biogenesis of circUHRF1 via targeting the flanking intron. **p-value < 0.05

Fig. 7

circUHRF1/miR-526b-5p/c-Myc/TGF-β1/ESRP1 feedback loop regulates the OSCC metastasis. a, b The invasive and migrative ability of SCC25 cells was tested using the transwell assay with the transfection of miR-526b-5p inhibitor, circUHRF1 shRNA (sh-circUHRF1), c-Myc siRNA (si-c-Myc), and ESRP1 pcDNA plasmid (pcDNA–ESRP1). c The DNA synthesis measured by EdU assay. d The protein markers of EMT were detected using western blot analysis, including mesenchymal markers (N-cadherin, Vimentin) and the epithelial marker (E-cadherin). **p-value < 0.01

Fig. 8

Knockdown of circUHRF1 inhibits the OSCC tumor growth in vivo. a The tumor volume of mice injected with circUHRF1 knockdown and control group. b The tumor weight in circUHRF1 knockdown and control group. c The luciferase was monitored using a bioluminescence in vivo imaging system. Bioluminescence image showed the tumor metastasis. d IHC staining and HE staining of the tumor showed that circUHRF1 knockdown reduced the tumor phenotypes. **p-value < 0.01

Fig. 9

circUHRF1/miR-526b-5p/c-Myc/TGF-β1/ESRP1 feedback loop regulates the OSCC cell’s EMT