Long non-coding RNA NORAD/miR-224-3p/MTDH axis contributes to CDDP resistance of esophageal squamous cell carcinoma by promoting nuclear accumulation of β-catenin

Abstract

Background: Cis-diamminedichloro-platinum (CDDP)-based chemotherapy regimens are the most predominant treatment strategies for patients with esophageal squamous cell carcinoma (ESCC). Dysregulated long non-coding RNAs (lncRNAs) contribute to CDDP resistance, which results in treatment failure in ESCC patients. However, the majority of lncRNAs involved in CDDP resistance in ESCC remain to be elucidated.

Methods: The public Gene Expression Omnibus (GEO) dataset GSE45670 was analysed to reveal potential lncRNAs involved in CDDP resistance of ESCC. Candidate upregulated lncRNAs were detected in ESCC specimens by qRT-PCR to identify crucial lncRNAs. Non-coding RNA activated by DNA damage (NORAD) was selected for further study. Kaplan-Meier analysis and a COX proportional regression model were performed to analyse the potential of NORAD for predicting prognosis of ESCC patients. The role of NORAD in CDDP resistance were determined by conducting gain and loss-of-function experiments in vitro. Fluorescence in situ hybridization (FISH) was performed to determine the subcellular location of NORAD in ESCC cells. A public GEO dataset and bioinformatic algorithms were used to predict the microRNAs (miRNAs) that might be latently sponged by NORAD. qRT-PCR was conducted to verify the expression of candidate miRNAs. Luciferase reporter and Argonaute-2 (Ago2)-RNA immunoprecipitation (RIP) assays were conducted to evaluate the interaction between NORAD and candidate miRNAs. A miRNA rescue experiment was performed to authenticate the NORAD regulatory axis and its effects on CDDP resistance in ESCC cells. Western blotting was conducted to confirm the precise downstream signalling pathway of NORAD. A xenograft mouse model was established to reveal the effect of NORAD on CDDP resistance in vivo.

Results: The expression of NORAD was higher in CDDP-resistant ESCC tissues and cells than in CDDP-sensitive tissues and cells. NORAD expression was negatively correlated with the postoperative prognosis of ESCC patients who underwent CDDP-based chemotherapy. NORAD knockdown partially arrested CDDP resistance of ESCC cells. FISH showed that NORAD was located in the cytoplasm in ESCC cells. Furthermore, overlapping results from bioinformatic algorithms analyses and qRT-PCR showed that NORAD could sponge miR-224-3p in ESCC cells. Ago2-RIP demonstrated that NORAD and miR-224-3p occupied the same Ago2 to form an RNA-induced silencing complex (RISC) and subsequently regulated the expression of metadherin (MTDH) in ESCC cells. The NORAD/miR-224-3p/MTDH axis promoted CDDP resistance and progression in ESCC cells by promoting nuclear accumulation of β-catenin in vitro and in vivo.

Conclusions: NORAD upregulates MTDH to promote CDDP resistance and progression in ESCC by sponging miR-224-3p. Our results highlight the potential of NORAD as a therapeutic target in ESCC patients receiving CDDP-based chemotherapy.

Keywords: CDDP resistance; ESCC; MTDH; NORAD; miR-224-3p.

Fig. 1

NORAD expression is increased in CDDP-resistant ESCC tissues and correlated with poor prognosis of patients. a The differential lncRNAs between chemoradiotherapy-sensitive and chemoradiotherapy-resistant ESCC in public GSE45670 dataset. b Validated expression of 5 upregulated lncRNAs in the tissues from 93 ESCC patients by using qRT-PCR. Error bars indicate SD. *** P < 0.001. c The expression of NORAD in ESCC tissues and normal esophageal tissues in TCGA database. ** P < 0.01. d Representative images of RNA FISH of NORAD in ESCC tissues (× 400), which show that NORAD is predominantly located in the cytoplasm. Nuclei are stained with DAPI. FISH score of NORAD in CDDP-resistant ESCC group was higher, compared to CDDP-sensitive ESCC group. e Kaplan-Meier analysis curves of DFS and OS for ESCC patients with high (n = 48) or low (n = 45) NORAD expression. The cut-off value was calculated by using X-tile. f ROC curves of NORAD for predicting DFS and OS in ESCC patients. g Multivariate analyses of hazard ratios for DFS and OS in ESCC patients

Fig. 2

NORAD contributes to CDDP resistance of ESCC. a The expression of NORAD in CDDP resistant and matched parental ESCC cells, normalized to GAPDH expression. b Representative images of RNA FISH of NORAD in KYSE30/CDDP-R and KYSE30 cells (× 400), which show that NORAD is predominantly located in the cytoplasm. Nuclei are stained with DAPI. c NORAD knockdown increases the sensitivity of KYSE30/CDDP-R cells to CDDP, detected by CCK-8 assay. d Overexpression of NORAD decreases the sensitivity of KYSE30 cells to CDDP, detected by CCK-8 assay. e NORAD knockdown decreases the colony formation ability in KYSE30/CDDP-R cells in the presence of CDDP. f Overexpression of NORAD increases the colony formation ability of KYSE30 in the presence of CDDP. g NORAD knockdown increases the CDDP-induced apoptosis rate in KYSE30/CDDP-R cells, and overexpression of NORAD decreases this rate in KYSE30 cells, detected by FCM. h NORAD knockdown facilitates CDDP to induce cell cycle arrest in KYSE30/CDDP-R cells. i Overexpression of NORAD suppresses CDDP-induced cell cycle arrest in KYSE30 cells. j NORAD knockdown increases the CDDP-induced γH2AX and cleaved caspase-3 in KYSE30/CDDP-R cells. k Overexpression of NORAD decreases the CDDP-induced γH2AX and cleaved caspase-3 in KYSE30 cells. In all cases, error bars denote SD of triplicates. **P < 0.01, ***P < 0.001

Fig. 3

NORAD sponges miR-224-3p in ESCC. a The differential miRNAs between CDDP-resistant and CDDP-sensitive ESCC cells in public GSE83362 dataset. b Predicted binding sites on NORAD to sponge miR-224-3p. c The effect of sh-NORAD on the expressions of miR-224-3p, miR-28-5p and miR-7-5p in ESCC cells, normalized to U6 expression. d The expression of NORAD and miR-224-3p in multiple clones of KYSE30/CDDP-R and TE1/CDDP-R cells. NORAD and miR-224-3p was negatively correlated. e NORAD is negatively correlated with miR-224-3p in ESCC specimens while not with miR-28-5p and miR-7-5p. f The expression of miR-224-3p in CDDP-resistant and matched parental ESCC cells, normalized to U6 expression. g The expression of NORAD and miR-224-3p in multiple clones of KYSE30 and TE1 cells. NORAD and miR-224-3p was negatively correlated. h Representative images of RNA FISH of NORAD and miR-224-3p in KYSE30/CDDP-R cells (× 1000), which show that NORAD and miR-224-3p are co-located in the cytoplasm. Nuclei are stained with DAPI. i The nuclear and cytoplasmic expression of NORAD and miR-224-3p in KYSE30/CDDP-R cells. j Luciferase reporter assay showing the luciferase activity of NORAD-WT, NORAD-Mut#1 and NORAD-Mut#2 in KYSE30/CDDP-R and TE1/CDDP-R cells which are co-transfected with miR-224-3p mimic. k Ago2-RIP assay shows that NORAD and miR-224-3p occupy the same Ago2 protein. In all cases, error bars denote SD of triplicates. **P < 0.01, ***P < 0.001

Fig. 4

MTDH is a direct target of miR-224-3p. a Venn diagram showing the putative target genes of miR-224-3p computationally predicted by 4 algorithms (miRmap, TargetScan, miRWalk and DIANA microT). b The TOP20 upregulated genes in chemoradiotherapy-resistant ESCC, compared to chemoradiotherapy-sensitive ESCC, in public GSE45670 dataset. c Representative images of positive and negative IHC staining of MTDH in ESCC tissues (× 200). MTDH predominantly locates in cytoplasm. d The expression of MTDH is higher in CDDP-resistant ESCC cells than in matched parental cells. e Predicted binding sites on 3′-UTR of MTDH to sponge miR-224-3p and matched established mutant sequences. f Luciferase reporter assay shows the luciferase activity of MTDH-3′-UTR-WT, MTDH-3′-UTR-Mut#1, MTDH-3′-UTR-Mut#2, MTDH-3′-UTR-Mut#3 and MTDH-3′-UTR-Mut#4 in KYSE30/CDDP-R cells co-transfected with miR-224-3p mimic. g The effects of miR-224-3p mimic on the expression of MTDH in KYSE30/CDDP-R cells transfected with MTDH-3′-UTR-WT, MTDH-3′-UTR-Mut#1, MTDH-3′-UTR-Mut#2, MTDH-3′-UTR-Mut#3 or MTDH-3′-UTR-Mut#4. h NORAD knockdown downregulates MTDH expression in KYSE30/CDDP-R cells and miR-224-3p mimic rescues this downregulation. i Overexpression of NORAD upregulates MTDH expression in KYSE30 cells and miR-224-3p inhibitor rescues this downregulation. j Three-dimensional scatter plot of NORAD, miR-224-3p and NORAD expressions in 11 CDDP-resistant and 82 CDDP-sensitive ESCC tissues from patients. g-i Error bars denote SD of triplicates. ***P < 0.001

Fig. 5

The NORAD/miR-224-3p/MTDH axis promotes CDDP resistance in ESCC cells by promoting nuclear accumulation of β-catenin. a The effects of NORAD/miR-224-3p/MTDH axis on the colony formation ability of KYSE30/CDDP-R and KYSE30 cells in the presence of CDDP. b The effects of NORAD/miR-224-3p/MTDH axis on CDDP induced apoptosis of KYSE30/CDDP-R and KYSE30 cells. c NORAD knockdown facilitates CDDP to induce cell cycle arrest in KYSE30/CDDP-R cells while miR-224-3p inhibitor rescued the arrest. d Overexpression of NORAD suppresses CDDP-induced cell cycle arrest in KYSE30 cells while miR-224-3p mimic neutralizes the suppression. e The effects of NORAD/miR-224-3p/MTDH axis on the expression and phosphorylation of MAPK, Akt, NF-κB and β-catenin in KYSE30/CDDP-R cells. f NORAD knockdown decreases the nuclear expression of MTDH in KYSE30/CDDP-R cells, detected by western blotting. g NORAD knockdown decreases the nuclear expression of MTDH in KYSE30/CDDP-R cells, detected by immunofluorescence. In all cases, error bars denote SD of triplicates. **P < 0.01, ***P < 0.001

Fig. 6

The NORAD/miR-224-3p/MTDH axis promotes progression of ESCC cells. a The effects of the NORAD/miR-224-3p/MTDH axis on migratory ability of KYSE30/CDDP-R and KYSE30 cells. b The effects of the NORAD/miR-224-3p/MTDH axis on invasive ability of KYSE30/CDDP-R and KYSE30 cells. c The effects of the NORAD/miR-224-3p/MTDH axis on the expression of E-cadherin, N-cadherin and MMP9 in KYSE30/CDDP-R and KYSE30 cells. In all cases, error bars denote SD of triplicates. **P < 0.01, ***P < 0.001

Fig. 7

The NORAD/miR-224-3p/MTDH axis contributes to CDDP resistance of ESCC in vivo. a KYSE30/CDDP-R cell-formed xenograft tumors of sacrificed mice with or without CDDP treatment (3 mg/kg, three times a week) at the end of the experiment and their growth curves. b KYSE30 cell-formed xenograft tumors of sacrificed mice with or without CDDP treatment (3 mg/kg, three times a week) at the end of the experiment and their growth curves. c Representative images of IHC staining of MTDH in xenograft tumors (× 100). Scale bars, 200 μm. d Representative images of nuclear and cytoplasmic expression of MTDH in xenograft tumors, detected by IHC (× 200). Scale bar, 20 μm. e Representative images of CDDP-induced γH2AX and cleaved caspase-3 in KYSE30/CDDP-R cell-formed xenograft tumors, detected by IHC (× 200). Scale bar, 20 μm. f IHC scores of γH2AX and cleaved caspase-3 in KYSE30/CDDP-R cell-formed xenograft tumors. g The summary figure showing the effect of the NORAD/miR-224-3p/MTDH axis in ESCC. Error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001