METTL14 suppresses proliferation and metastasis of colorectal cancer by down-regulating oncogenic long non-coding RNA XIST - PubMed (original) (raw)
METTL14 suppresses proliferation and metastasis of colorectal cancer by down-regulating oncogenic long non-coding RNA XIST
Xiao Yang et al. Mol Cancer. 2020.
Abstract
Background: N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulation in eukaryotic cells. However, understanding of m6A in colorectal cancer (CRC) is very limited. We designed this study to investigate the role of m6A in CRC.
Methods: Expression level of METTL14 was extracted from public database and tissue array to investigate the clinical relevance of METTL14 in CRC. Next, gain/loss of function experiment was used to define the role of METTL14 in the progression of CRC. Moreover, transcriptomic sequencing (RNA-seq) was applied to screen the potential targets of METTL14. The specific binding between METTL14 and presumed target was verified by RNA pull-down and RNA immunoprecipitation (RIP) assay. Furthermore, rescue experiment and methylated RNA immunoprecipitation (Me-RIP) were performed to uncover the mechanism.
Results: Clinically, loss of METTL14 correlated with unfavorable prognosis of CRC patients. Functionally, knockdown of METTL14 drastically enhanced proliferative and invasive ability of CRC cells in vitro and promoted tumorigenicity and metastasis in vivo. Mechanically, RNA-seq and Me-RIP identified lncRNA XIST as the downstream target of METTL14. Knockdown of METTL14 substantially abolished m6A level of XIST and augmented XIST expression. Moreover, we found that m6A-methylated XIST was recognized by YTHDF2, a m6A reader protein, to mediate the degradation of XIST. Consistently, XIST expression negatively correlated with METTL14 and YTHDF2 in CRC tissues.
Conclusion: Our findings highlight the function and prognostic value of METTL14 in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.
Keywords: Colorectal cancer; Long non-coding RNA; METTL14; RNA epigenetics; m6A.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Fig. 1
METTL14 was down-regulated in human colorectal cancer**_._** a Real-time PCR analysis of METTL14 and METTL3 expression levels in colorectal cancer and paired normal tissue. ns, not significant. **, p < 0.01. b Mega-sampler analysis of expression patterns of METTL14 and METTL3 in cancer and normal tissues from GEO dataset (GSE14333). P values were as indicated. c Representative images of immunohistochemical (IHC) staining of METTL14 in colorectal cancer and paired normal tissue. d Expression levels of METTL14 in different CRC cell lines and normal NCM460 cells. **, p < 0.01. e-f Kaplan-Meier analysis of RFS of CRC patients from our cohort (e) and GSE14333 dataset (f). g Multivariate analysis of clinical prognostic parameters for RFS of CRC patients in GSE14333 cohort. P values were as indicated. h Time-dependent ROC analysis showing the predictive ability of METTL14 of RFS of CRC patients. P values were as indicated. PCR was analyzed by paired student’s _t_-test (a) and nonpaired student’s _t_-test (d). Mega-sampler analysis of GEO data (b) was generated by R2 online tools (
). Difference of RFS (e and f) between two subgroups was analyzed with Log-rank test. Time-dependent ROC analysis was constructed with Cox model from ‘Survival’ R package
Fig. 2
Knockdown of METTL14 promoted proliferation and invasion of CRC cells in vitro**_._** a Knockdown of METTL14 were confirmed by real-time PCR (upper panel) and western blot (lower panel) in HCT116 and HT29 cells. si1, siMETTL14–1. si2, siMETTL14–2. **, p < 0.01. b Cell count assay was performed to measure the proliferation of CRC cells transfected with siMETTL14 compared with those transfected with siNC. OD450 values were compared at indicated time points. *, p < 0.05, **, p < 0.01. ****, p < 0.0001. c Representative images and quantification of colony formation assay of HCT116 and HT29 cells transfected with siMETTL14 or siNC. Magnification, 200×. **, p < 0.01, ****, p < 0.0001. d Transwell invasion assay was performed to determine the effects of METTL14 on invasive ability of CRC cells. Magnification, 200×. *, p < 0.05, **, p < 0.01. All data were representative of three independent experiments and shown as mean ± SD. P values were determined by Student’s _t_-test
Fig. 3
Knockdown of METTL14 promoted proliferation and invasion of CRC in vivo. a Xenograft tumors formed by HCT116-shMETTL14 or -shNC cells in nude mice. b-c Quantitative analysis of tumor volume (b) and tumor weight (c) of xenografts. Tumor volume was compared at indicated time points and tumor weight was measured at the end point. *, p < 0.05, **, p < 0.01. d Xenograft tumor sections stained with hematoxylin and eosin (HE), METTL14 and Ki-67 were examined by immunohistochemistry. Magnification, 200×. e Analysis of METTL14 IHC score and Ki-67 positive rate in xenograft tumor tissue from different groups. **, p < 0.01. f Analysis of metastatic liver nodules in different group of mice. Liver tissue of mice were counterstained with HE and typical metastatic lesions were indicated with black arrow. **, p < 0.01. P values were determined by Student’s _t_-test
Fig. 4
METTL14 suppressed the growth and invasion of CRC by targeting lncRNA XIST. a Volcano plot showed DEGs between shMETTL14 and control cells. METTL14 and interested genes which were reported to possess multiple m6A residues were indicated with black circles. b The levels of XIST were examined by real-time PCR in HCT116 and HT29 cells transfected with siNC or siMETTL14, respectively. **, p < 0.01. c Real-time PCR showing expression levels of XIST in CRC tissue and paired normal tissue. ****, p < 0.0001. d and e Negative correlation of METTL14 and XIST expression in CRC tissue from our center (d) or from TCGA public dataset (e). R and p values were as indicated. f and g Cell count assay and transwell assay showed the effects of METTL14 and XIST on the proliferative and invasive ability of CRC cells. Inhibition of XIST suppressed the increased cell growth (f) and invasive ability (g) resulted from knockdown of METTL14. *, p < 0.05. **, p < 0.01. All data were representative of three independent experiments. Means ± SD were shown. Statistical analysis was conducted using Student’s _t_-test. Correlation of METTL14 and XIST was evaluated by Pearson’s correlation analysis
Fig. 5
METTL14 down-regulated XIST through m6A methylation activity. a Two CRC cell lines were transfected with METTL14-overexpression (METTL14-OE) plasmids and siWTAP as indicated. Protein band intensity of METTL14 and WTAP were quantified by western blot assay and shown as fold change. Statistical histograms were not shown. b The alteration of XIST expression in METTL14-OE and METTL14-OE + siWTAP cells was analyzed by real-time PCR. ns, not significant, **, p < 0.01. c-d The effects of WTAP and METTL14 on proliferative ability of CRC cells were measured by cell count assay (c) and colony formation assay (d). ns, not significant, *, p < 0.05, **, p < 0.01. e Quantitative analysis of transwell invasion assay showing the effects of METTL14 and WTAP on invasive ability of CRC cells. ns, not significant, *, p < 0.05, **, p < 0.01. f Quantitative analysis of m6A-methylated XIST in control, siMETTL14–1 and siWTAP cells. Methylated XIST was immunoprecipitated with m6A antibody and then measured by real-time PCR. Equal amount of total RNA was used as input. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001. All data were representative of three independent experiments. Means ± SD were shown. P values were determined by Student’s _t_-test
Fig. 6
YTHDF2 mediated the recognition and degradation of m6A-methylated XIST. a RIP assay confirmed the association between XIST and m6A reader protein YTHDF2 in CRC cells. GAPDH mRNA was used as a non-target control. ns, not significant, **, p < 0.01, ***, p < 0.001. b RNA pull-down assay confirmed XIST was specifically recognized by YTHDF2. EGFP RNA was used as RNA control. GAPDH was used as protein control. c Real-time PCR showed the level of XIST in control and shYTHDF2 CRC cells. *, p < 0.05. d Negative correlation of METTL14 and XIST expression in CRC tissue from our center (right panel) or from TCGA public dataset (left panel). R and p values were as indicated. e The expression levels of XIST in shYTHDF2 and control cells were quantified by real-time PCR at indicated time points after actinomycin D treatment and the decay rate of XIST was evaluated with a linear regression model. *, p < 0.05. f Summary of the mechanism by which METTL14 regulates the malignant phenotype of CRC. Data were presented as the mean ± SD of at least three independent experiments. P values were determined by Student’s _t_-test
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