A novel tumour suppressor protein encoded by circMAPK14 inhibits progression and metastasis of colorectal cancer by competitively binding to MKK6

Abstract

Background: The mitogen-activated protein kinase (MAPK) pathway is highly associated with the progression and metastasis of various solid tumours. MAPK14, a core molecule of the MAPK pathway, plays vital roles in the colorectal cancer (CRC). Recent studies have shown that circRNAs can affect tumour progression by encoding peptides. However, little is known regarding the potential protein translated from circMAPK14 and whether it plays a role in the carcinogenesis of colorectal cancer.

Methods: The RNA level and translatable potential of circMAPK14 in CRC was verified using qRT-PCR and public databases. RNase R digestion assay, qRT-PCR, sanger sequencing and FISH assays were utilised to verify the circular characteristics and subcellular localisation of circMAPK14. The suppressive role of circMAPK14 on the progression and metastasis of CRC was verified in vivo and in vitro. LC/MS analysis combined with western blotting demonstrated the presence and relative expression of circMAPK14-175aa. The underlying mechanism of circMAPK14-175aa action to inhibit CRC was identified by co-IP analysis. The binding of U2AF2 within the flanking introns of circMAPK14 was evaluated by RNA pull-down assay and RIP assay. Ultimately, luciferase reporter gene assays and ChIP assays confirmed that FOXC1 suppressed transcription of U2AF2 by binding to the U2AF2 promoter in the -400 bp to -100 bp region. RESULTS: We identified that hsa_circ_0131663 (termed circMAPK14) showed significantly decreased expression level in cells and tissue samples of CRC, and was primarily localised in the cytoplasm. A series of function experiments demonstrated that circMAPK14 influenced CRC progression and metastasis by encoding a peptide of 175 amino acids (termed circMAPK14-175aa). We also found that circMAPK14-175aa reduced nuclear translocation of MAPK14 by competitively binding to MKK6, thus facilitating ubiquitin-mediated degradation of FOXC1. Moreover, we described a positive feedback loop in CRC in which elevated FOXC1 expression was caused by reduced circMAPK14-175aa expression. This, in turn, decreased circMAPK14 biogenesis by suppressing U2AF2 transcription.

Conclusion: In summary, we reported for the first time that circMAPK14 functioned as a tumour-suppressor by encoding circMAPK14-175aa, which blocked the progression and metastasis of colorectal cancer.

Keywords: CircRNA; colorectal cancer; translation; ubiquitination.

FIGURE 1

Identification and characterisation of circMAPK14 in CRC tissues and cell lines. (A) The relative expression of circMAPK14 in 72 CRC tissues and matched normal samples. (B) Kaplan–Meier plot analysis of correlations between the circMAPK14 levels and OS of 72 CRC patients. (C) The relative expression of circMAPK14 in CRC cell lines (SW480, DLD‐1, LoVo, HT29, HCT116, Caco2) compared with control (NCM460). (D) Sanger sequencing was utilised to confirm that circMAPK14 is generated from 4 to 10 exon of MAPK14 by head‐to‐tail splicing. (E) CircMAPK14 was only amplified via divergent primers in cDNA rather than gDNA by northern blot assay, GAPDH was used as a control. The relative levels of circMAPK14 and MAPK14 mRNA were detected after the treatment of (F) RNase R and (G) actinomycin D in DLD‐1 and LoVo cells. (H) The analysis of FISH indicated that circMAPK14 was mainly located in the cytoplasm. (I), (J) The qRT‐PCR analysis of cell fractions to certificate the subcellular localisation of circMAPK14 in DLD‐1 and LoVo cells

FIGURE 2

CircMAPK14 inhibits the proliferation and migration of CRC cells. The effect of circMAPK14 on CRC proliferation was evaluated by (A) CCK8 assay, (B) colony formation assay and (C) EdU incorporation assay. (D) The flow cytometry assay was used to assess the apoptosis rate of CRC cells after altering the expression of circMAPK14. The effect of circMAPK14 on CRC invasion and migration was examined by (E) wound healing assay and (F) Transwell assay

FIGURE 3

CircMAPK14 encodes a novel protein of 175 amino acids, termed as circMAPK14‐175aa. (A) The translation potential of circMAPK14 was showed in the schematic diagram. (B) The wild‐type or two mutant of IRES sequence were cloned between Rluc and Luc reporter genes. (C) The activity of IRES sequence was tested by dual luciferase reporter assay. (D) The description of circMAPK14‐174aa sequence and MAPK14 sequence and the antibody (ab170099) recognised the same residues of them. (E) The activity of IRES sequence was verified by western blotting assay. (F) The total protein of HCT116 cell transfected with circMAPK14‐ov and IRES‐mut#1 was separated by electrophoresis and sliver staining. (G) Cut the corresponding gel band of prediction to perform LC‐MS/MS analysis. (H) The existence of circMAPK14‐175aa was revealed by western blotting assay. The expression of MAPK14 and circMAPK14‐175aa was determined in CRC (I) tissues and (J) cell lines

FIGURE 4

CircMAPK14 suppresses the CRC malignant phenotype via 175aa in vitro. After transfection of circMAPK14‐ov, circMAPK14 ATG mut, circMAPK14 IRES mut#1 and linearised 175aa‐ov in DLD‐1 and LoVo cells, the abilities of proliferation were evaluated by (A) and (B) CCK8 assay, (C) and (D) colony formation assay, (E) and (F) EdU incorporation assay. (G), (H) The apoptosis rates were evaluated by flow cytometry assay. The abilities of invasion and migration were evaluated by (I) and (J) wound healing assay, (K) and (L) Transwell assay

FIGURE 5

CircMAPK14 attenuated tumourigenicity and metastasis of CRC via 175aa in vivo. (A), (B) The above cells were injected subcutaneously into nude mice to assess the suppressive function of circMAPK14‐175aa. In vivo tumourigenicity was monitored, and mice were sacrificed after 30 days. (C), (D) The xenograft tumours were stained with ki67. (E) The above cells were injected into the tail vein of nude mice to assess the suppressive effect on lung metastasis of circMAPK14‐175aa. Mice lung were subjected to H&E staining. (F) The above cells were injected into the spleen of nude mice to assess the suppressive effect on liver metastasis of circMAPK14‐175aa. Mice liver were subjected to H&E staining

FIGURE 6

CircMAPK14‐175aa represses the phosphorylation of MAPK14 by competing with MKK6 and subsequently promotes the degradation of FOXC1 via ubiquitination. (A) LEFT panel, total protein from Flag‐circMAPK14‐175aa transfected HEK293T cells were separated via SDS‐PAGE. Right panel, MKK6 was identified by LC‐MS analysis. (B) Mutual interaction of Flag‐circMAPK14‐175aa and MKK6 were confirmed by Co‐IP assay. (C) Immunofluorescence colocalisation between Flag‐circMAPK14‐175aa and MKK6. Blue represent DAPI, green represent Flag‐circMAPK14‐175aa, red represent MKK6. (D) When the level of 175aa was rose, the amount of MAPK14 but not 175aa binding with MKK6 was apparently reduced by Co‐IP assay against MKK6 antibody. (E) The relative expression of FOXC1 mRNA was detected by qRT‐PCR after alteration of 175aa level. (F) Upper panel, the relative level of FOXC1 protein was detected by western blotting assay after alteration of 175aa level. Lower panel, the relative level of p‐MAPK14 in the nucleus was detected by WB assay after alteration of 175aa level. (G) The time‐course experiment with WB assay was conducted after the treatment with cycloheximide (CHX, an inhibitor of protein synthesis). The results of WB assay showed that the ubiquitin‐proteasome system (UPS) played major roles in the degradation of FOXC1 after the treatment with (H) MG132 (a proteasome inhibitor). (I) Overexpression of circMAPK14 facilitated the FOXC1 degradation via ubiquitination. (J) The downstream target genes (SOX4, SOX13 and MMP10) levels were correspondingly changed along with the degradation of FOXC1

FIGURE 7

The mechanism diagram of circMAPK14‐175aa encoded by circMAPK14 on inhibiting tumourigenicity and metastasis of CRC by competing with MKK6