Long noncoding RNA NORAD, a novel competing endogenous RNA, enhances the hypoxia-induced epithelial-mesenchymal transition to promote metastasis in pancreatic cancer

Abstract

Background: Pancreatic cancer, one of the top two most fatal cancers, is characterized by a desmoplastic reaction that creates a dense microenvironment, promoting hypoxia and inducing the epithelial-to-mesenchymal transition (EMT) to facilitate invasion and metastasis. Recent evidence indicates that the long noncoding RNA NORAD may be a potential oncogenic gene and that this lncRNA is significantly upregulated during hypoxia. However, the overall biological role and clinical significance of NORAD remains largely unknown.

Methods: NORAD expression was measured in 33 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of NORAD on pancreatic cancer cells were studied by overexpression and knockdown in vitro. Insights into the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatics analyses and luciferase assays. In vivo, metastatic potential was identified using an orthotopic model of PDAC and quantified using bioluminescent signals. Alterations in RhoA expression and EMT levels were identified and verified by immunohistochemistry and Western blotting.

Results: NORAD is highly expressed in pancreatic cancer tissues and upregulated in hypoxic conditions. NORAD upregulation is correlated with shorter overall survival in pancreatic cancer patients. Furthermore, NORAD overexpression promoted the migration and invasion of pancreatic carcinoma cells, while NORAD depletion inhibited EMT and metastasis in vitro and in vivo. In particular, NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p, thereby promoting EMT.

Conclusions: Elevated expression of NORAD in pancreatic cancer tissues is linked to poor prognosis and may confer a malignant phenotype upon tumor cells. NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p. This finding may contribute to a better understanding of the role played by lncRNAs in hypoxia-induced EMT and provide a potential novel diagnostic and therapeutic target for pancreatic cancer.

Fig. 1

Analysis of lncRNA expression profiles in PDAC databases indicates that NORAD increases during hypoxia. a Venn analysis of non-coding RNAs that are significantly upregulated or downregulated in GSE15471 and GSE16515; b Heatmap of hypoxia-related lncRNA expression profiles; c Relative expression of hypoxia-related lncRNAs in SW1990 after 48 h of hypoxic or normoxic treatment. *, statistical significance, P < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

NORAD promotes cell migration and invasion in vitro and in vivo. a The migration and invasion abilities of tumor cells were assessed using wound healing assays as well as transwell migration and invasion assays. Left panel: wound healing assays (photographed after 24 h), middle panel: transwell migration assays (photographed after 12 h), right panel: transwell invasion assays (photographed after 12 h); b The distances between wound edges after 0 h and 24 h; c-d Histograms showing the numbers of cells that have migrated or invaded after 0 h and 12 h; e SW1990-sh-NORAD and SW1990-sh-NC were labeled with firefly luciferase and injected into the abdominal cavity of nude mice (n = 5). The bioluminescent signal was assessed 3 weeks after injection; f Histogram showing the bioluminescent signal intensity detected using a noninvasive In Vivo Imaging System; g Representative H & E staining images of liver and gastric wall from the different groups. Yellow star: micrometastatic folic, red triangle: gastric mucosa. *p < 0.05 **p < 0.01, ***p < 0.001

Fig. 3

NORAD can directly bind to miR-125a-3p and inhibit its expression. a NORAD contains a sequence that is complementary to miR-125a-3p; b the expression of NORAD was negatively associated with the expression of miR-125a-3p in 33 PDAC tissues; c miR-125a-3p was upregulated when NORAD was knocked down in SW1990 and PANC-1; d Relative expression of miR-125a-3p was significantly decreased when NORAD was overexpressed in BxPC-3 and Canpan-1, whereas this phenomenon disappeared when the NORAD MRE was mutated. *, statistical significance, P < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

NORAD increases the expression of RhoA and acts as a ceRNA for miR-125a-3p. a RhoA expression is negatively correlated with miR-125a-3p in GSE32688. The results were expressed as RMA Values; b NORAD and RhoA mRNA levels are positively associated in 33 PDAC tissues; c miR-125a-3p can complement the seed region of RhoA, which is similar to the NORAD MRE. d NORAD increases RhoA protein level. Upper panel: western blot of RhoA was performed. Relative density was shown below each lane. Bottom panel: IHC staining of orthotopic implantation tumor tissues; e RhoA protein level is increased by NORAD-wt but not by NORAD-mut; f The 3′-UTR of RhoA (wt or mut) was fused to the luciferase coding region and transfected into SW1990 with along with a miR-125a-3p mimic to confirm that RhoA is a target of miR-125a-3p. A miR-239b-5p mimic was used as a negative control; g RhoA-3′-UTR-wt-luc was transfected into SW1990-sh-NC or SW1990-sh-NORAD. Inhibitors of miR-125a-3p or miR-239b-5p were added. Rno-miR-239b-5p was used as a negative control. The histogram indicates the luciferase values measured 48 h after transfection. *, statistical significance, P < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

NORAD promotes EMT via regulating RhoA expression. a Western blot analysis of E-cadherin, N-cadherin, Vimentin and ZEB1. GAPDH was used as a loading control. Relative density was shown below each lane; b Western blot analysis of E-cadherin, RhoA and RhoA downstream protein Rock1. Relative density was shown below each lane; c transwell migration and invasion assays, NC: negative control, NORAD: cell lines with NORAD overexpressed, NORAD + CCG-1423: NORAD overexpressed cell lines incubated with 300 nM CCG-1423 72 h; d Histograms of numbers of migration and invasion cells*, statistical significance, P < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

NORAD is upregulated in pancreatic cancer and is correlated with the prognosis of PDAC patients. a NORAD expression profiles in GSE15471 and GSE16515. The results are expressed as log2 RMA Values; b Relative NORAD expression in 33 paired PDAC as quantified by q-PCR. Results were expressed as log2 (2^-△△Ct);c NORAD expression was investigated in the cancer tissue compared with its adjacent normal tissues. The results were presented as log 2-fold change of tumor tissues relative to normal tissues; d-e Kaplan–Meier estimator of overall survival (OS) and recurrence-free survival (RFS). P values were calculated by the log-rank test. *, statistical significance, P < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Schematic diagram showing that NORAD increases the expression of RhoA proteins via sponging of its inhibitor miR-125a-3p, thus bridging hypoxia and metastasis