Long non-coding RNA UICLM promotes colorectal cancer liver metastasis by acting as a ceRNA for microRNA-215 to regulate ZEB2 expression

Abstract

Long non-coding RNAs (lncRNAs) are involved in the pathology of various tumors, including colorectal cancer (CRC). However, the role of lncRNA in CRC liver metastasis remains unclear. Methods: a microarray was performed to identify the differentially expressed lncRNAs between CRC tissues with and without liver metastasis. Survival analysis was evaluated using the Kaplan-Meier method and assessed using the log-rank test. In vitro and in vivo assays were preformed to explore the biological effects of the differentially expressed lncRNA in CRC cells. Results: the lncRNA UICLM (up-regulated in colorectal cancer liver metastasis) was significantly up-regulated in cases of CRC with liver metastasis. Moreover, UICLM expression was higher in CRC tissues than in normal tissues, and UICLM expression was associated with poor patient survival. Knockdown of UICLM inhibited CRC cell proliferation, invasion, epithelial-mesenchymal transition (EMT) and CRC stem cell formation in vitro as well as tumor growth and liver metastasis in vivo. Ectopic expression of UICLM promoted CRC cell proliferation and invasion. Mechanistic investigations revealed that UICLM induced its biological effects by regulating ZEB2, as the oncogenesis facilitated by UICLM was inhibited by ZEB2 depletion. Further study indicated that UICLM acted as a competing endogenous RNA (ceRNA) for miR-215 to regulate ZEB2 expression. Conclusions: taken together, our findings demonstrate how UICLM induces CRC liver metastasis and may offer a novel prognostic marker and therapeutic target for this disease.

Keywords: CRC; liver metastasis.; lncRNA UICLM; long non-coding RNA.

Figure 1

The lncRNA UICLM is up-regulated in CRC with liver metastasis. (A) Differentially expressed lncRNAs between CRC tissues with and without liver metastases. A lncRNA microarray was conducted in CRC samples with (n = 7) and without (n = 8) liver metastases. (B) The relative UICLM expression levels in CRC with liver metastases (n = 55) were significantly higher than in CRC tissues without liver metastases (n = 67; P < 0.01). (C) The relative UICLM expression levels in tumor tissues (n = 122) were significantly higher than in adjacent normal tissues (n = 122; P < 0.01). (D) Relative UICLM expression levels in different clinical stages (*P < 0.05, **P < 0.01). (E) Kaplan-Meier analysis of progression-free survival based on UICLM levels (determined by real-time PCR) in 122 CRC patients showed that patients with high UICLM levels (n = 61) had a worse clinical outcome than patients with low UICLM levels (n = 61) (P = 0.01). (F) Relative UICLM expression levels in CRC cell lines (SW620, SW480, LoVo, HT-29, HCT116, DLD-1, and RKO) and the immortalized colon epithelial cell line CCD-112CoN. (G) i) Analyses of UICLM expression in adjacent normal tissues, tumor tissues and liver metastasis by ISH, with U6 used as the internal control (blue, positive staining; red, negative staining). (ii) and (iii) The percent of ISH-positive samples in different groups. (H) Kaplan-Meier analysis of progression-free survival based on UICLM expression (determined by ISH) showed that patients who were UICLM-positive (n = 98) had significantly worse survival than patients who were UICLM-negative (n = 24) (P = 0.075).

Figure 2

Knockdown of UICLM suppresses CRC cell proliferation and colony formation in vitro as well as tumorigenesis in vivo. (A) Relative expression levels of UICLM in SW620 and DLD-1 cells after transfection with UICLM siRNAs (*P < 0.05). (B) Knockdown of UICLM expression significantly inhibited cell viability in SW620 and DLD-1 cells (*P < 0.05). (C) Knockdown of UICLM expression significantly inhibited colony formation in SW620 and DLD-1 cells (*P < 0.05) (Scale bar: 0.5 cm). (D) Knockdown of UICLM expression significantly inhibited CRC cell growth in nude mice, and the tumor weight and tumor volume were significantly reduced in the sh-UICLM group compared to that in the sh-NC group (*P < 0.05) (Scale bar: 5 cm). (E) and (F) Knockdown of UICLM significantly reduced UICLM and Ki-67 expression in vivo (*P < 0.05) (Scale bar: 100μm).

Figure 3

Knockdown of UICLM inhibits CRC cell migration and invasion in vitro as well as liver metastasis in vivo. (A) Knockdown of UICLM expression significantly inhibited cell migration and invasion in SW620 and DLD-1 cells as demonstrated by transwell assay (*P < 0.05) (Scale bar:100μm). (B) Knockdown of UICLM expression markedly suppressed cell motility in SW620 and DLD-1 cells as demonstrated by wound healing assay (*P < 0.05) (Scale bar: 100 μm). (C) Knockdown of UICLM significantly inhibited the expression of N-cadherin while increasing the expression of E-cadherin as demonstrated by Immunofluorescence analysis (Scale bar: 50 μm). (D) Real-time PCR analysis showed that knockdown of UICM significantly reduced the expression of mesenchymal markers (N-cadherin, Vimetin, Snail, Slug) while increasing the expression of epithelial markers (E-cadherin, α-catenin, β-catenin) (*P < 0.05). (E) All of the mice formed tumors in the spleen, 12 of 12 mice injected with SW620 sh-NC cells formed liver metastases, and 7 of 12 mice injected with SW620 sh-UICLM cells formed liver metastases (Scale bar, left: 5 mm; right: 100 μm). (F) The numbers of metastatic nodules in the livers were significantly reduced in mice injected with SW620 sh-UICLM cells (2.25 ± 0.75) compared with the numbers in those injected with SW620 sh-NC cells (9.41 ± 1.28) (**P < 0.01).

Figure 4

UICLM regulates the stemness of CRC cells. (A) Knockdown of UICLM significantly reduced the SP cell proportion in SW620 (reduce from 9.52% to 2.91%) and DLD-1 (reduced from 5.34% to 2.44%) cells. (B) Knockdown of UICLM significantly reduced sphere formation in SW620 and DLD-1 cells (*P < 0.05) (Scale bar: 100 μm). (C) Knockdown of UICLM markedly reduced the expression of stemness-associated genes (Nanog, Oct-4, SOX2, Notch1), multiple drug-resistant transporter genes (ABCG2) and surface antigens associated with cancer stem cells (CD24, CD44, CD133, CD155 and CD166) in both SW620 and DLD-1 cells (*P < 0.05). (D)i-ii) 83.3% of the mice developed tumors when injected with 2.0 × 10⁵ SW620 sh-NC cells, whereas the tumor incidence was reduced to 33.3% when mice were injected with SW620 sh-UICLM cells (Group #1). The tumor incidence was 58.3% in the control group when the mice were injected with 1.0 × 10⁵ cells, and the tumor incidence was reduced to 16.6% in the sh-UICLM group (Group #2). The tumor incidence was 33.3% in the control group when the mice were injected with 1.0 × 10⁴ cells, and the tumor incidence was reduced to 8.3% in the sh-UICLM group (Group #3). iii) The tumor volume and tumor weight were significantly decreased in the sh-NC group compared with that in the sh-UICLM group (*P < 0.05) (Scale bar: 1.5 cm).

Figure 5

UICLM interacts with ZEB2 to promote cell proliferation and invasion in CRC cells. (A) Schematic flowchart showing the results of a transcriptome sequencing study on UICLM-associated pathways. CRC cells were treated with siRNAs for UICLM or control siRNAs, and the mRNA expression profiles were determined. The combination of GSEA and gene expression correlation analysis identified ZEB2 in the cell migration pathway as a potential regulatory target of UICLM. (B) Knockdown of GAPLINC caused alterations in multiple pathways, and the “cellular migration and invasion” pathway showed the highest significance. This result was based on the following principle: when most genes in a defined pathway (gene set) are affected, a higher enrichment score is assigned to that pathway. (C) i)ZEB2 was significantly overexpressed in CRC with liver metastasis compared to CRC without liver metastasis, ii) A positive correlation was found between the expression level of ZEB2 and UICLM (r = 0.75, P < 0.001) (Scale bar: 100 μm). (D) The relative ZEB2 mRNA level in CRC cell lines (SW620, SW480, LoVo, HT-29, HCT116, DLD-1, RKO) and the immortalized colon epithelial cell line CCD-112CoN (*P < 0.05). (E) i) Knockdown of UICLM significantly reduced the expression of ZEB2 mRNA in SW620 and DLD-1 cells (*P < 0.05), ii) Ectopic expression of UICLM significantly increased the expression of ZEB2 mRNA (*P < 0.05), iii) Knockdown of UICLM significantly reduced the expression of ZEB2 protein, and ectopic expression of UICLM increased the ZEB2 protein level in SW620 and DLD-1 cells. (F) i-ii) The cell invasion stimulated by ectopic expression of UICLM could be repressed by knockdown of ZEB2 (*P < 0.05) (Scale bar: 100 μm).

Figure 6

UICLM regulates ZEB2 expression by competing for miR-215. (A) i) Conservation of UICLM in the binding site of miR-215 predicted based on the human genome sequence in the UCSC Genome Browser, ii) Schematic representation of the predicted target site for miR-215 in UICLM, iii) The predicted miR-215 binding sites in ZEB2 mRNA 3'-UTR as predicted by the Targetscan algorithm.(B) i) Ectopic expression of miR-215 significantly reduced the level of UICLM in SW620 and DLD-1 cells (*P < 0.05), ii) Knockdown of UICLM significantly increased the expression of miR-215 in SW620 and DLD-1 cells (*P < 0.05). (C) Dual-luciferase assays indicated a significant reduction in luciferase activities after co-transfection of miR-215 and the wild-type UICLM reporter vector, but not the mutant-type UICLM (*P < 0.05). (D) A RIP assay was performed using input from cell lysates, anti-normal mouse IgG or anti-Ago2. The relative expression levels of UICLM and miR-215 were detected by quantitative real-time PCR (*P < 0.05). (E) i) Ectopic expression of miR-215 significantly reduced the expression of ZEB2 mRNA in SW620 and DLD-1 cells (*P < 0.05). ii) Ectopic expression of miR-215 significantly reduced ZEB2 protein levels in SW620 and DLD-1 cells. iii) The luciferase activity assay demonstrated that ectopic expression of miR-215 or knockdown of UICLM could significantly reduce the luciferase activity of wild-type but not mutant-type ZEB2 3'-UTR, and reduction of luciferase activity caused by UICLM suppression could be restored by miR-215 inhibition (*P < 0.05). (f) i) The relative expression of miR-215 in CRC tissues with and without liver metastasis (*P < 0.05). ii) The relative expression of miR-215 in different clinical stages; miR-215 was decreased in advanced stages compared with early stages (*P < 0.05, *P < 0.01). iii) An inverse correlation was found between the expression level of miR-215 and UICLM (r = -0.53, P < 0.001). iv)An inverse correlation was found between the expression level of miR-215 and ZEB2 (r = -0.72, P < 0.001).

Figure 7

Overexpression of UICLM reduces the level of miR-215, which increases the expression level of ZEB2, thus resulting in stimulated cell proliferation, migration, EMT, and CSC formation in CRC.