Circular RNA protein tyrosine kinase 2 (circPTK2) promotes colorectal cancer proliferation, migration, invasion and chemoresistance

Abstract

The dysregulated circular RNAs (circRNAs) are linked to progression and chemoresistance in colorectal cancer (CRC). However, the role of circRNA protein tyrosine kinase 2 (circPTK2) in CRC progression and chemoresistance is uncertain. The circPTK2, microRNA (miR)-136-5p, m6A 'reader' protein YTH domain family protein 1 (YTHDF1), β-catenin and cyclin D1 abundances were examined via quantitative reverse transcription PCR or Western blotting. The progression was investigated by cell counting kit-8 (CCK-8), colony formation, transwell and xenograft analysis. The resistance to 5-fluorouracil (5-FU) and oxaliplatin was analyzed via detecting cell viability and apoptosis using CCK-8 analysis and flow cytometry. The binding relationship was examined through dual-luciferase reporter, RNA immunoprecipitation and pull-down analysis. In our study, circPTK2 abundance was enhanced in CRC and associated with liver metastasis, clinical stage and chemoresistance. CircPTK2 knockdown constrained cell proliferation, migration, invasion, resistance to 5-FU and oxaliplatin, and the Wnt/β-catenin signaling. MiR-136-5p was bound with circPTK2 and downregulated in CRC. MiR-136-5p knockdown attenuated the influence of circPTK2 silence on CRC progression and chemoresistance. YTHDF1 was targeted via miR-136-5p and upregulated in CRC samples and cells. MiR-136-5p targeted YTHDF1 to restrain CRC progression and chemoresistance. In addition, we confirmed that circPTK2 silence reduced xenograft tumor growth. In conclusion, circPTK2 interference suppressed CRC proliferation, migration, invasion and chemoresistance via regulating miR-136-5p and YTHDF1.Abbreviations: circRNAs: circular RNAs; CRC: colorectal cancer; circPTK2: circRNA protein tyrosine kinase 2; miR: microRNA; YTHDF1: YTH domain family protein 1; CCK-8: cell counting kit-8; 5-FU: 5-fluorouracil; RIP: RNA immunoprecipitation.

Keywords: Colorectal cancer; YTHDF1; circPTK2; miR-136-5p.

Figure 1.

circPTK2 abundance in CRC. (a) circPTK2 abundance was detected in CRC and normal tissues. (b) circPTK2 level was examined in CRC tissues with liver metastasis (LM)-positive or LM-negative. (c) circPTK2 abundance was measured in CRC samples at different stages. (d) circPTK2 expression was detected in CRC patients with chemo-sensitive or resistant. (e) circPTK2 abundance was determined in HT-29, LoVo, SW480, HCT116 and NCM460 cells. (f) The basic information of circPTK2 was shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

The function of circPTK2 knockdown on cell proliferation, migration, invasion and chemoresistance in CRC cells. HT-29 and LoVo cells were transfected with sh-NC, sh-circPTK2-1 or sh-circPTK2-2. (a) circPTK2 abundance was determined by qRT-PCR. (b and c) Cell proliferation was detected by CCK-8 assay. (d and e) Colony formation was analyzed by colony formation assay. (f-i) Cell migration and invasion were measured by transwell assay. (j-m) Cell viability was determined by CCK-8 assay. (n and o) β-catenin and cyclin D1 abundances were measured by WB analysis. ***P < 0.001.

Figure 3.

The interaction relationship of circPTK2 and miR-136-5p. (a and b) circPTK2 enrichment level was detected after Ago2 or IgG RIP assay in HT-29 and LoVo cells. (c) The targeted miRNAs of circPTK2 were predicted and analyzed via starBase and circinteractome. (d) The targeted miRNA levels in CRC were predicted via dbDEMC 2.0. (e and f) miRNA levels were measured after RNA pull-down analysis. (g) The binding site of circPTK2 and miR-136-5p. (h) miR-136-5p level was determined in HT-29 and LoVo cells with transfection of miR-NC or miR-136-5p mimic. (i and j) Luciferase activity was measured in HT-29 and LoVo cells with transfection of circPTK2-wt or circPTK2-mut and miR-NC or miR-136-5p mimic. (k) miR-136-5p expression was measured in CRC and normal samples. (l) miR-136-5p abundance was examined in HT-29, LoVo and NCM460 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

The influence of miR-136-5p knockdown on silence of circPTK2-mediated CRC progression and chemoresistance. (a) miR-136-5p abundance was determined in HT-29 and LoVo cells with transfection of anti-miR-NC or anti-miR-136-5p. (b-o) HT-29 and LoVo cells were transfected with sh-circPTK2-1 and anti-miR-136-5p. Cell proliferation (b and c), colony formation (d and e), migration (f and g), invasion (h and i), 5-FU resistance (j and k), oxaliplatin resistance (l and m), β-catenin and cyclin D1 protein levels (n and o) were measured by CCK-8 assay, colony formation assay, transwell assay and WB analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

The target association of miR-136-5p and YTHDF1. (a and b) YTHDF1 expression was predicted in COAD and READ via UALCAN. (c) The binding sequence of miR-136-5p and YTHDF1. (d and e) Luciferase activity was examined in HT-29 and LoVo cells with transfection of YTHDF1 3ʹ UTR-wt or YTHDF1 3ʹ UTR-mut and miR-NC or miR-136-5p mimic. (f and g) miR-136-5p and YTHDF1 abundances were detected after Ago2 RIP in HT-29 and LoVo cells. (h) YTHDF1 expression was examined after RNA pull-down analysis. (i and j) YTHDF1 protein level was examined in HT-29 and LoVo cells after transfection of miR-NC, miR-136-5p mimic, anti-miR-NC or anti-miR-136-5p. (k-m) YTHDF1 abundance was examined in CRC and normal samples. (n) YTHDF1 abundance was measured in HT-29, LoVo and NCM460 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

The influence of miR-136-5p and YTHDF1 on CRC progression and chemoresistance. (a) YTHDF1 protein level was examined in HT-29 and LoVo cells transfected with vector or oe-YTHDF1. (b-o) HT-29 and LoVo cells were transfected with miR-136-5p mimic and oe-YTHDF1. Cell proliferation (b and c), colony formation (d and e), migration (f and g), invasion (h and i), 5-FU resistance (j and k), oxaliplatin resistance (l and m), β-catenin and cyclin D1 protein expression (n and o) were examined by CCK-8 assay, colony formation assay, transwell assay and WB analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

The effect of circPTK2/miR-136-5p axis on YTHDF1 expression in CRC cells. (a and b) YTHDF1 protein expression was examined in HT-29 and LoVo cells transfected with sh-NC + anti-miR-NC, sh-circPTK2-1 + anti-miR-NC or anti-miR-136-5p. ***P < 0.001.

Figure 8.

The influence of circPTK2 knockdown on tumor growth. LoVo cells (5 × 10⁶ per mouse) were subcutaneously injected into the nude mice to form the xenograft tumors (n = 5). (a) Tumor volume was measured every 7 days. (b) The representative images of tumor in each group. (c) Tumor weight was examined in each group. (d) circPTK2 and miR-136-5p abundances were examined in tumors of each group. (e) IHC staining was used to assess the YTHDF1 and Ki-67 positive cells in tumors of each group. (f) YTHDF1, β-catenin and cyclin D1 abundances were measured in tumors of each group. **P < 0.01, ***P < 0.001.