Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression

Abstract

Background: UCA1 is frequently upregulated in a variety of cancers, including CRC, and it can play an oncogenic role by various mechanisms. However, how UCA1 is regulated in cancer is largely unknown. In this study, we aimed to determine whether RNA methylation at N6-methyladenosine (m6A) can impact UCA1 expression in colorectal cancer (CRC).

Methods: qRT-PCR was performed to detect the level of UCA1 and IGF2BP2 in CRC samples. CRISPR/Cas9 was employed to knockout (KO) UCA1, METTL3 and WTAP in DLD-1 and HCT-116 cells, while rescue experiments were carried out to re-express METTL3 and WTAP in KO cells. Immunoprecipitation using m6A antibody was performed to determine the m6A modification of UCA1. In vivo pulldown assays using S1m tagging combined with site-direct mutagenesis was carried out to confirm the recognition of m6A-modified UCA1 by IGF2BP2. Cell viability was measured by MTT and colony formation assays. The expression of UCA1 and IGF2BP2 in TCGA CRC database was obtained from GEPIA ( http://gepia.cancer-pku.cn ).

Results: Our results revealed that IGF2BP2 serves as a reader for m6A modified UCA1 and that adenosine at 1038 of UCA1 is critical to the recognition by IGF2BP2. Importantly, we showed that m6A writers, METTL3 and WTAP positively regulate UCA1 expression. Mechanically, IGF2BP2 increases the stability of m6A-modified UCA1. Clinically, IGF2BP2 is upregulated in CRC tissues compared with normal tissues.

Conclusion: These results suggest that m6A modification is an important factor contributing to upregulation of UCA1 in CRC tissues.

Keywords: CRC; IGF2BP2; UCA1; m6A modification.

Fig. 1

Expression of UCA1 in CRC clinical specimens. A Analysis of TCGA CRC cohort revealed that the expression of UCA1 is significantly higher in CRC tissue than in normal tissue. B UCA1 expression in 443 colon cancer samples and 19 colon mucosa tissue from GEO database (GSE39582) C qRT-PCR analysis of UCA1 expression in our cohort containing 46 matched CRC tissues and adjacent normal tissues. D GEPIA database was used to analyze the differential UCA1 expression between CRC samples and normal controls (|Log2FC| cutoff = 1; p-value cutoff = 0.05). E UCA1 expression was associated with the pathological stage of CRC based on GEPIA database analysis. Error bars represent SD; *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 2

Knockdown of UCA1 suppresses cell proliferation in vitro and tumor growth in vivo. A The RNA level of UCA1 in HCT-116 UCA1 knockdown (KD) cells as determined by qRT-PCR. B UCA1 KD suppresses colony formation of HCT116 cells. Left, the representative picture of cell colonies; right, the statistical result. C UCA1 KD impairs cell proliferation ability of HCT116 cells as determined by CCK-8 assay. D and E UCA1 KD suppresses the tumor growth in vivo. Tumors were harvested at the end of experiment. D the picture of tumors; E the statistical analysis of tumor weight. Error bars represent SD; n = 3 for A ~ C; *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 3

Knockout of m6A writers downregulates UCA1 expression. A Me-RIP and qRT-PCR assays suggest the m6A modification of UCA1 in HCT-116 and DLD-1 cells. B and C Knockout of METTL3 impairs UCA1 expression in HCT-116 cells. B Representative western blot result showed knockout of METTL3 in HCT-116 cells. C downregulation of UCA1 in HCT-116 METTL3 KO cells was detected by qRT-PCR. D and E Knockout of METTL3 impairs UCA1 expression in DLD-1 cells. D, Representative western blot showing knockout of METTL3 in DLD-1 cells. E downregulation of UCA1 in DLD-1 METTL3 KO cells was detected by qRT-PCR. F and G Knockout of WTAP suppresses UCA1 expression in HCT-116 cells. F Representative western blot showing knockout of METTL3 in HCT-116 cells. G, downregulation of UCA1 in HCT-116 WTAP KO cells was detected by qRT-PCR. H and I Knockout of WTAP suppresses UCA1 expression in DLD-1 cells. H, Representative western blot showing knockout of WTAP in DLD-1 cells. I downregulation of UCA1 in DLD-1 WTAP KO cells was detected by qRT-PCR. Error bars represent SD, n = 3, **P < 0.01, ***P < 0.005

Fig. 4

Re-expression of m6A writer restores the expression of UCA1 in KO cells. A–D Re-expression of METTL3 in KO cells restores UCA1 RNA level. A and C the representative western blot showing re-expression of METTL3 in HCT-116 KO clone#13 and DLD-1 KO clone#11. B and D the RNA level of UCA1 was detected by qRT-PCR. E–H Re-expression of WTAP in KO cells rescues UCA1 RNA level. E and G the representative western blot showing re-expression of WTAP in HCT-116 KO clone#28 and DLD-1 KO clone#37. F and H the RNA level of UCA1 was detected by qRT-PCR. Error bars represent SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.005

Fig. 5

Knockout of METTL3 and WTAP impairs proliferation and survival of CRC cells. A METTL3 KO suppresses cell proliferation of HCT-116 cells, as determined by CCK-8 assay. B METTL3 KO inhibits colony formation of HCT-116 cells. Left, the representative picture of cell colonies; right, the statistical result. C and D Cell proliferation and colony formation assay in METTL3 KO DLD-1 cells. E KO of WTAP suppressed cell proliferation of HCT-116 cells. Cell proliferation was determined by CCK-8 assay. F WTAP KO inhibits colony formation of HCT-116 cells. Left, the representative picture of cell colonies; right, the statistical result. G and H Cell proliferation and colony formation assay in WTAP KO DLD-1 cells. Error bars represent SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.005

Fig. 6

IGF2BP2 serves as a reader for m6A modified UCA1. A The workflow of in vivo S1m-tagging RNA pulldown assay. B Detection of the YTHDF1, YTHDF2 and YTHDF3 by western blot, after in vivo RNA pulldown. C Detection of the IGF2BP1, IGF2BP2 and IGF2BP3 by western blot, after in vivo RNA pulldown. D The interaction between UCA1 and IGF2BP2 was confirmed by RIP assay. Error bars represent SD, n = 3, ***P < 0.005. E Mutation on m6A motif of UCA1 impairs the interaction of IGF2BP2 with UCA1. Left, the schematic of the A to C mutation on UCA1 m6A motif; right, the representative western blot showing that the mutant UCA1revealed little interaction with IGF2BP2 and UCA1, in contrast to WT UCA1

Fig. 7

The recognition by IGF2BP2 enhances UCA1 RNA stability. A silencing of IGF2BP2 suppressed IGF2BP2 RNA expression, as determined RNA expression, as determined by western blot. B IGF2BP2 siRNA2 suppresses UCA1 RNA level, as determined by qRT-PCR. C IGF2BP2 siRNA2 decreases UCA1 RNA stability. HCT-116 cells were transfected with IGF2BP2 siRNA2 or NC siRNA. Two days after transfection, the cells were treated with actinomycin D (2 μg/ml) for indicated times. UCA1 RNA stability was determined by qRT-PCR. D Analysis of TCGA database suggests that IGF2BP2 mRNA level is higher in CRC samples than in normal tissue. E Analysis of GEO database (GSE39582) indicates the differential expression of IGF2BP2 in CRC tissue and normal tissue. F qRT-PCR analysis of IGF2BP2 expression in our cohort of 46 matched CRC tumor tissues and adjacent normal tissues; Error bars represent SD, n = 3 (B&C), *P < 0.05, **P < 0.01, ***P < 0.005

Fig. 8

A schematic model of present work. IGF2BP2 stablizes m6A-modified UCA1 to promote CRC progression. See text for explanation