m6A-induced lncRNA RP11 triggers the dissemination of colorectal cancer cells via upregulation of Zeb1

Abstract

Background: Long noncoding RNAs (lncRNAs) have emerged as critical players in cancer progression, but their functions in colorectal cancer (CRC) metastasis have not been systematically clarified.

Methods: lncRNA expression profiles in matched normal and CRC tissue were checked using microarray analysis. The biological roles of a novel lncRNA, namely RP11-138 J23.1 (RP11), in development of CRC were checked both in vitro and in vivo. Its association with clinical progression of CRC was further analyzed.

Results: RP11 was highly expressed in CRC tissues, and its expression increased with CRC stage in patients. RP11 positively regulated the migration, invasion and epithelial mesenchymal transition (EMT) of CRC cells in vitro and enhanced liver metastasis in vivo. Post-translational upregulation of Zeb1, an EMT-related transcription factor, was essential for RP11-induced cell dissemination. Mechanistically, the RP11/hnRNPA2B1/mRNA complex accelerated the mRNA degradation of two E3 ligases, Siah1 and Fbxo45, and subsequently prevented the proteasomal degradation of Zeb1. m⁶A methylation was involved in the upregulation of RP11 by increasing its nuclear accumulation. Clinical analysis showed that m⁶A can regulate the expression of RP11, further, RP11 regulated Siah1-Fbxo45/Zeb1 was involved in the development of CRC.

Conclusions: m⁶A-induced lncRNA RP11 can trigger the dissemination of CRC cells via post-translational upregulation of Zeb1. Considering the high and specific levels of RP11 in CRC tissues, our present study paves the way for further investigations of RP11 as a predictive biomarker or therapeutic target for CRC.

Keywords: CRC; Cell dissemination; LncRNA RP11; Zeb1; hnRNPA2B1; m6A.

Fig. 1

RP11 is increased in CRC cells and tissues. a Heat-maps of lncRNAs that were differentially expressed between stage I samples (a, cancer tissues) and matched adjacent normal samples (b, normal samples) (left) or between stage IV samples and matched adjacent normal samples (right). The colour scale shown on the left illustrates the relative RNA expression levels; red represents high expression, and green represents low expression. b Venn diagram showing the overlapping of 2-fold upregulated lncRNAs between stage I samples and normal samples, stage IV and normal, and stage IV and stage I. c Heat-maps of the 8 lncRNAs upregulated simultaneously between stage I samples and normal samples, between stage IV and normal, and between stage IV and stage I (Red probe targeted lncRNA RP11). d The relative fold of RP11 in 32 paired human colon cancer tissues versus its matched adjacent normal mucosa tissues. e The relative expression of RP11 in colon (left) and rectal (right) cancer tissues and their corresponding adjacent normal tissues based on data available from TCGA database. f The levels of RP11 in CRC cell lines and human colon mucosal epithelial NCM460 cells were measured by qRT-PCR. Data are presented as the mean ± SD from three independent experiments. *p < 0.05 compared with control

Fig. 2

RP11 triggers the dissemination of CRC cells both in vitro and in vivo. a CRC cells were transfected with the vector control or pcDNA/RP11 for 48 h, and proliferation was measured with a CCK-8 kit. b The wound healing of HCT-15 RP11 stable overexpression and control cells was recorded (left) and quantitatively analysed (right). c The in vitro invasion of HCT-15 RP11 stable overexpression and control cells was recorded (left) and quantitatively analysed (right). d The expression of EMT-related markers of HCT-15 or HCT-8 RP11 stable overexpression and control cells was verified by western blot analysis. e After transfection with si-NC or si-RP11 for 48 h, the expression of EMT markers in SW620 cells was verified by western blot analysis. f Tumour growth curves of HCT-15 RP11 stable overexpression and control cells in xenograft models at the indicated time intervals. g Weights of tumours derived from HCT-15 RP11 stable overexpression or control cells in xenograft models at the end of the experiment. h IHC analysis of Ki67-, vimentin- or fibronectin-stained paraffin-embedded sections obtained from xenografts. (I).HCT-15 RP11 stable overexpression and control cells were injected into nude mice via the tail vein. Representative images and H&E staining of metastatic liver tumours are shown. j The number of metastatic sites of tumours derived from HCT-15 RP11 stable overexpression or control cells was quantitatively analysed. Data are presented as the mean ± SD from three independent experiments. Bar = 200 μm. *p < 0.05, **p < 0.01 compared with control

Fig. 3

Upregulation of Zeb1 mediates the RP11-induced dissemination of CRC cells. a. The expression levels of EMT-TFs in HCT-15 or HCT-8 RP11 stable overexpression and control cells were verified by western blot analysis. After transfection with si-NC or si-RP11 for 48 h, the expression levels of EMT-TFs in SW620 cells were verified by western blot analysis. b Zeb1 expression in subcellular fractions of HCT-15 RP11 stable overexpression and control cells was verified by western blot analysis. c IHC analysis of Zeb1-stained paraffin-embedded sections obtained from xenografts. d The mRNA expression levels of EMT-TFs in HCT-15 RP11 stable overexpression and control cells were verified by qRT-PCR. e After transfection with si-NC or si-Zeb1 for 48 h, the wound healing of HCT-15 RP11 stable overexpression and control cells was quantitatively analysed. f After transfection with si-NC or si-Zeb1 for 48 h, the EMT markers of HCT-15 RP11 stable overexpression and control cells were detected by western blot analysis. g After treatment with 100 μg/ml CHX for the indicated times, Zeb1 expression in HCT-15 RP11 stable overexpression and control cells was detected by western blot analysis (left) and quantitatively analysed (right). h & i Zeb1 in HCT-15 or HCT-8 RP11 stable overexpression and control cells was immunoprecipitated for the detection of ubiquitylation. Data are presented as the mean ± SD from three independent experiments. Bar = 200 μm. **p < 0.01 compared with control

Fig. 4

Downregulation of Siah1 and Fbxo45 mediates the RP11-induced upregulation of Zeb1. a & b The mRNA expression levels of 7 reported target proteins related to Zeb1 stability in HCT-15 a or HCT-8 (b) RP11 stable overexpression and control cells were determined by qRT-PCR. c The protein expression of Siah1 and Fbxo45 in HCT-15 or HCT-8 RP11 stable overexpression and control cells was determined by western blot analysis. d IHC analysis of Siah1- or Fbxo45-stained paraffin-embedded sections obtained from HCT-15 RP11 stable overexpression and control xenografts. e HCT-15 cells were transfected with vector control, pcDNA/RP11, pcDNA/Siah1, or Fbxo45 alone or together for 48 h, protein expression was verified by western blot analysis. f RIP-PCR was performed to analyse the relative enrichment of RP11 by use of an antibody against Siah1 or Fbxo45 in HCT-15 cells. Data are presented as the means ± SD from three independent experiments. Bar = 200 μm. **p < 0.01 compared with control

Fig. 5

RP11 regulates Siah1 and Fbxo45 expression by forming the RP11-hnRNPA2B1-mRNA complex. a RNA pull-down analysis and MS identified hnRNPA2B1 as the specific protein interacting with RP11 in both HCT-15 and HCT-15 RP11 stable overexpression cells. The red arrow shows the position of hnRNPA2B1. b The secondary structure of RP11 was predicted (http://rna.tbi.univie.ac.at/). The red colour indicates strong confidence for the prediction of each base. c RNA pull-down detection of the interaction between hnRNPA2B1 and RP11, Siah1, or Fbxo45 in HCT-15 cells. d hnRNPA2B1 expression in the cytoplasmic and nuclear fractions of HCT-15 RP11 stable overexpression and control cells were analysed by western blot. e HCT-15 cells were transfected with pcDNA (vector) or pcDNA/hnRNPA2B1 for 24 h, and the expression of Siah1 and Fbxo45 was verified by western blot analysis. f &g The computational prediction of the interaction between RP11 and the Siah1 (f) or Fbxo45 (g) mRNA based on IntaRNA 2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp) [53]. h After in vitro transcription to generate biotin-labelled RP-11 and RP-11 AS, RIP-PCR was performed to analyse the relative enrichment of Siah1 or Fbxo45 mRNA on RP11 in HCT-15 cells. i & j After treatment with Act-D for the indicated times, the mRNA levels of Siah1 (i) or Fbxo45 (j) in HCT15 RP11 stable overexpression and control cells were measured by qRT-PCR. k Binding between hnRNPA2B1 and Siah1 mRNA or between hnRNPA2B1 and Fbxo45 mRNA in HCT-15 RP11 stable overexpression and control cells was analysed by RIP-PCR. Data are presented as the mean ± SD from three independent experiments. **p < 0.01 compared with control

Fig. 6

The m⁶A modification is involved in the upregulation of RP11 in CRC cells. a m⁶A RIP-qPCR analysis of RP11 in HCT-15, HCT-8 and NCM460 cells. b After transfection with vector control or ppB/Mettl3 for 24 h, RP11 expression was measured by qRT-PCR. c After transfection with vector control or pcDNA/Alkbh5 for 24 h, RP11 expression was measured by qRT-PCR. d After transfection with vector control or ppB/Mettl3 for 24 h, HCT-15 cells were further treated with Act-D for the indicated times, and RP11 expression was measured by qRT-PCR. e After transfection with vector control or ppB/Mettl3 for 24 h, the cytoplasmic, nuclear, and chromatin fractions of HCT-15 cells were separated for RNA extraction and qRT-PCR. f After transfection with vector control or ppB/Mettl3 for 24 h, binding between RP11 and hnRNPA2B1 in HCT-15 and HCT-8 cells was analysed by RIP-PCR using an antibody against hnRNPA2B1. Data are presented as the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 compared with control

Fig. 7

The m⁶A/RP11/Zeb1 axis and in vivo progression of CRC. a & b The relative mRNA expression of Zeb1 in two Oncomine datasets: Hong Colorectal (a), and Skrzypczak Colorectal 2 (b). c & d The relative mRNA expression of Zeb1 (c) and Fbxo45 (d) in patients with stage N0, N1, and N2 CRC based on data available from TCGA database. e DFS of CRC patients with high (n = 135) and low (n = 134) levels of RP11 was plotted according to the Kaplan-Meier method. f DFS of CRC patients with high (n = 135) and low (n = 135) levels of RP11/Siah1 was plotted according to the Kaplan-Meier method. g DFS of CRC patients with high (n = 135) and low (n = 135) levels of RP11/Fbxo45 was plotted according to the Kaplan-Meier method. (H) DFS of CRC patients with high (n = 135) and low (n = 134) levels of Zeb1 was plotted according to the Kaplan-Meier method. (I) DFS of CRC patients with high (n = 135) and low (n = 135) levels of Zeb1/Siah1 was plotted according to the Kaplan-Meier method. j DFS of CRC patients with high (n = 135) and low (n = 135) levels of Zeb1/Fbxo45 was plotted according to the Kaplan-Meier method. *p < 0.05, **p < 0.01 compared with control