Long noncoding RNA CRNDE stabilized by hnRNPUL2 accelerates cell proliferation and migration in colorectal carcinoma via activating Ras/MAPK signaling pathways

Abstract

Recent studies have furthered our understanding of the function of long noncoding RNAs (lncRNAs) in numerous biological processes, including cancer. This study investigated the expression of a novel lncRNA, colorectal neoplasia differentially expressed (CRNDE), in colorectal carcinoma (CRC) tissues and cells by real-time RT-PCR and in situ hybridization, and its biological function using a series of in vitro and in vivo experiments to determine its potential as a prognostic marker and therapeutic target. CRNDE was found to be upregulated in primary CRC tissues and cells (P<0.05), and the upregulation of CRNDE expression is a powerful predictor of advanced TNM stage (P<0.05) and poor prognosis for CRC patients (P=0.002). The promoting effects of CRNDE on the cell proliferation, cell cycling and metastasis of CRC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Mechanistically, it was demonstrated that CRNDE could form a functional complex with heterogeneous nuclear ribonucleoprotein U-like 2 protein (hnRNPUL2) and direct the transport of hnRNPUL2 between the nucleus and cytoplasm. hnRNPUL2 that was accumulated in the cytoplasm could interact with CRNDE both physically and functionally, increasing the stability of CRNDE RNA. Moreover, gene expression profile data showed that CRNDE depletion in cells downregulated a series of genes involved in the Ras/mitogen-activated protein kinase signaling pathways. Collectively, these findings provide novel insights into the function and mechanism of lncRNA CRNDE in the pathogenesis of CRC and highlight its potential as a therapeutic target for CRC intervention.

Figure 1

CRNDE expression is upregulated in CRC and could be an independent prognostic factor for the prediction of the overall survival of CRC patients. (A) Expression levels of CRNDE in paired CRC and adjacent non-cancerous tissues. (B) Expression levels of CRNDE in CRC and colon mucosa epithelial (NCM460) cell lines. (C) Expression analysis of CRNDE in normal colorectal mucosa and CRC tissues by ISH. (a) Negative expression of CRNDE in normal colorectal mucosa. (b) High expression of CRNDE in a tumor tissue sample and weak expression of CRNDE in its normal mucosal counterpart were observed in one filed of a tissue sample from a single patient. (c) High expression of CRNDE in CRC tissue. Scale bars are shown in the lower right corner of each picture. (d) Graphical illustration of statistical CRNDE distribution in CRC patients. (E) Kaplan–Meier analysis of overall survival in all patients with CRC according to CRNDE expression. *P<0.05

Figure 2

CRNDE depletion inhibits cell proliferation, cell cycling, invasion and migration in CRC cells in vitro. (a) DLD1 and HCT116 cells were infected with Lv-siCRNDE to establish two cell lines with stable knockdown of CRNDE expression. CRNDE levels in DLD1 and HCT116 cells after shRNA-mediated knockdown of CRNDE were detected by real-time RT-PCR. (b) CCK-8 assays were performed to determine the proliferation of CRNDE-depleted CRC cells. (c) Colony-forming assays were performed to determine the effects of CRNDE depletion on the growth of CRC cells. (d) Cell cycle progression was analyzed by flow cytometry. (e) Apoptosis assays were performed to determine the effects of CRNDE depletion on CRC cells by flow cytometry (upper) and by western blot analysis (lower). (f) Matrigel invasion assays were used to determine the effects of CRNDE depletion on the invasion ability of CRC cells. (g) Scratch-wound-healing assays were performed to determine the effects of CRNDE depletion on the migration ability of CRC cells. The experiments were performed in triplicate, and the data are expressed as the mean±S.D. *P<0.05, **P<0.01 and ***P<0.001

Figure 3

The CRNDE depletion inhibits the growth and metastasis of human CRC cells in mice in vivo. (a) DLD1 cells with stable CRNDE downregulation and control cells were inoculated into nude mice. These graphs show the tumor xenografts 4 weeks after ectopic-subcutaneous implantation in nude mice. (b) DLD1 cells with downregulated CRNDE expression exhibited attenuated tumor growth in nude mice. The effect of CRNDE on CRC tumor growth was evaluated based on tumor volume in the two groups. (c) Representative pictures of lung metastasis by hematoxylin and eosin (H&E) staining in nude mice 4 weeks after tail vein injection with CRNDE downregulated DLD1 cells or control cells. (d) Statistical comparisons of lung metastasis and pulmonary tumor colonies per view in the two groups of mice after tail vein injection

Figure 4

Overexpression of CRNDE promotes cell proliferation, cell cycling, invasion and migration in CRC cells in vitro. (a) Overexpression of CRNDE induced significantly higher growth rates in CRC cells. (b) Overexpression of CRNDE increased the capacity to form colonies in CRC cells. (c) Overexpression of CRNDE induced a significant increase in cells at G2-phase relative to mock cells. (d and e) Invasion/migration assays using Matrigel Transwell and wound-healing assays for CRC cells. CRNDE overexpression promoted the invasion and migration of CRC cells. The experiments were performed in triplicate; the data are expressed as the mean±S.D. *P<0.05, **P<0.01 and *** P<0.001

Figure 5

CRNDE interacts with hnRNPUL2 protein and directs its localization. (a) RNA pull-down assays were used to identify proteins associated with CRNDE. Biotinylated CRNDE and antisense RNA were incubated with cell extracts, and the associated proteins were resolved by SDS-PAGE. Specific bands were excised and submitted for mass spectrometry, and hnRNPUL2 was identified. (b) Western blotting analysis of the specific interaction of hnRNPUL2 with CRNDE. (c) RIP experiments were performed in DLD1 cells using an hnRNPUL2 antibody or nonspecific IgG, and specific primers were used to detect CRNDE. RIP enrichment was determined as the amount of RNA associated with hnRNPUL2 or IgG relative to the input control. (d) CRNDE did not induce the expression level of hnRNPUL2 in SW480 and DLD1 CRC cells. CRNDE-induced subcellular relocalization of hnRNPUL2 protein was identified by western blot analysis (e) and immunofluorescence microscopy analysis (f). Scale bars=10 μm. Increased hnRNPUL2 was seen in the cytoplasm following CRNDE overexpression in SW480 and LS174T cells. Conversely, the downregulation of CRNDE could reduce the cytoplasmic levels of hnRNPUL2 expression in DLD1 and HCT116 cells. (g) Cytoplasmic hnRNPUL2-stabilized CRNDE RNA. Cells were transfected with hnRNPUL2-siRNA or control siRNA for 48 h and were then exposed to actinomycin D (1 μg/ml), and total RNA was isolated at the indicated times and subjected to real-time RT-PCR to assess the half-life of CRNDE RNA. (h) Cytoplasmic hnRNPUL2 induced CRNDE RNA levels in CRC cells

Figure 6

CRNDE activates Ras/MAPK signaling pathways. mRNA expression profiles were detected in CRNDE knockdown DLD1 cells and control cells. (a) KEGG pathway analysis of the genes downregulated in CRNDE-depleted cells. (b) Hierarchical clustering analysis of Ras/MAPK signaling genes which were downregulated in the CRNDE-depleted DLD1 cells. (c) A subset of genes was detected by real-time RT-PCR. The experiments were performed in triplicate, and the data are expressed as the mean±S.D.