LncRNA CHRF-induced miR-489 loss promotes metastasis of colorectal cancer via TWIST1/EMT signaling pathway

Abstract

microRNA-489 (miR-489) is a novel cancer-related miRNAs and functions as a tumor suppressor in human cancers. While, the clinical significance of miR-489 and its role in colorectal cancer (CRC) remain rarely known. Here, we found that the levels of miR-489 in CRC tissues were significantly lower than those in matched tumor-adjacent tissues. Furthermore, decreased levels of miR-489 also observed in CRC cell lines compared to HIEC cells. Clinicopathological analysis revealed that miR-489 underexpression was positively correlated with advanced pT stage, pN stage and AJCC stage. Moreover, miR-489 low expressing CRC patients showed a obvious shorter survival. Functionally, miR-489 restoration inhibited cell migration and invasion as well as epithelial-mesenchymal transition (EMT) in HCT116 cells, while miR-489 loss facilitated these cellular processes in SW480 cells. In vivo experiments revealed that miR-489 overexpression reduced the number of metastatic nodules in nude mice liver. Notably, TWIST1 was recognized as a direct downstream target of miR-489 in CRC cells. Interestingly, TWIST1 restoration abrogated the effects of miR-489 on CRC cells with enhanced cell migration, invasion and EMT process. Furthermore, overexpression of long noncoding RNA cardiac hypertrophy-related factor (lncRNA CHRF) was inversely correlated with miR-489 expression in CRC tissues. CHRF knockdown increased the expression of miR-489 and suppressed EMT events of HCT116 cells, while CHRF overexpression showed opposite effects on miR-489 expression and EMT in SW480 cells. Taken together, this work support the first evidence that lncRNA CHRF-induced miR-489 loss facilitates metastasis and EMT process of CRC cells probably via TWIST1/EMT signaling pathway.

Keywords: CRC; LncRNA CHRF; TWIST1/EMT; metastasis; miR-489.

Figure 1. The expression and prognostic significance of miR-489 in CRC

(A) miR-489 expression differences between CRC tissues and matched tumor-adjacent tissues. n = 80, *P < 0.05 by t test. (B) Relative expression of miR-489 in five CRC cell lines (HCT116, Caco2, HT29, SW620 and SW480) and a normal human intestinal epithelial cell line (HIEC) detected by qRT-PCR. n = three independent experiments with similar results, *P < 0.05 by ANOVA. (C) and (D) CRC patients were divided into miR-489 high expression group (n = 40) and miR-489 low expression group (n = 40) based on the median level of miR-489 expression. miR-489 low expressing CRC patients showed a significant decreased overall survival and disease-free survival compared to miR-489 high expressing cases. P < 0.05 by Log-rank test.

Figure 2. miR-489 regulates migration and invasion of CRC cells

(A) HCT116 cells that were transfected with precursor miR-489 and scrambled control, respectively, were subjected to qRT-PCR for miR-489 expression. n = three independent experiments with similar results, *P < 0.05 by t test. (B) Wound healing assays indicated that miR-489 overexpression inhibited migration of HCT116 cells. (C) The number of invaded cells was reduced after miR-489 restoration in HCT116 cells. n = three independent experiments with similar results, *P < 0.05 by t test. (D) SW480 cells that were transfected with miR-489 inhibitors and scrambled control, respectively, were detected by qRT-PCR for miR-489 expression. n = three independent experiments with similar results, *P < 0.05 by t test. (E) miR-489 loss facilitated the migratory ability of SW480 cells. (F) miR-489 knockdown promoted invasion of SW480 cells in vitro. n = three independent experiments with similar results, *P < 0.05 by t test.

Figure 3. miR-489 overexpression inhibits liver metastasis of CRC in nude mice

HCT116 cells that were transfected with precursor miR-489 or scrambled control were injected to the spleen subcapsular. HE staining revealed that miR-489 overexpression significantly reduced liver metastases of HCT116 cells. n = 5, *P < 0.05 by t test. Scale bar: 2 mm.

Figure 4. miR-489 restrains EMT events in CRC cells

(A) HCT116 cells were transfected with precursor miR-489 and scrambled control, respectively. Western blotting and IF analysis indicated the miR-489 overexpression up-regulated E-cadherin while down-regulated Vimentin. miR-489 overexpressing HCT116 cells showed the morphology of epithelial-like cells. (B) SW480 cells that were transfected with miR-489 inhibitors and scrambled control, respectively. miR-489 loss facilitated EMT process and represented the morphology of mesenchymal-like cells.

Figure 5. miR-489 regulates TWIST1 abundance in CRC cells

(A) The potential miR-489 binding site in wild type (wt) 3′-UTR sequence of TWIST1. The underlined part is the mutant site designed for mutant (mt) 3′-UTR sequence of TWIST1. (B) Overexpression of miR-489 decreased the luciferase activity of wt 3′-UTR of TWIST1, while miR-489 overexpression showed no effect on the luciferase activity of mt 3′-UTR of TWIST1 in HCT116 cells. n = three independent experiments with similar results, *P < 0.05 by t test. (C) and (D) HCT116 and HT29 cells that were transfected with correspongding miRNA vectors were confirmed by qRT-PCR and immunoblotting for TWIST1 expression. n = three independent experiments with similar results, *P < 0.05 by t test. (E) An inverse correlation between the levels of miR-489 and TWIST1 mRNA expression was observed in CRC tissues. n = 80, P < 0.01 by Spearman's rank correlation test.

Figure 6. Representative immunohistochemical staining of TWIST1, E-cadherin and Vimentin in CRC tissues

CRC tissues were subjected to immunohistochemical staining for TWIST1, E-cadherin and Vimentin expression. miR-489 low-expressing CRC tissue showed strong staining of TWIST1 and Vimentin (A and E), and weak staining of E-cadherin (C). While, weak signal of TWIST1 and Vimentin (B and F), and strong signal of E-cadherin (D) were observed in miR-489 high-expressing tumor. Scale bar: 100 μm.

Figure 7. TWIST1 restoration abolishes the effects of miR-489 in CRC cells

(A) miR-489 overexpressing HCT116 cells were transfected with TWIST1 expression vector. TWIST1 overexpression abrogated the inhibitory effects of miR-489 on EMT process. (B) TWIST1 re-expression promoted migration of miR-489 overexpressing HCT116 cells. (C) TWIST restoration facilitated invasion of miR-489 overexpressing HCT116 cells. n = three independent experiments with similar results, *P < 0.05 by ANOVA.

Figure 8. LncRNA CHRF negatively regulates miR-489 expression in CRC

(A) CHRF expression differences between CRC tissues and matched tumor-adjacent tissues. n = 80, *P < 0.05 by t test. (B) An inverse correlation between the levels of miR-489 and CHRF expression was observed in CRC tissues. n = 80, P < 0.01 by Spearman's rank correlation test. (C) HCT116 that were transfected with CHRF siRNA or scrambled siRNA were detected by qRT-PCR. n = three independent experiments with similar results, *P < 0.05 by t test. (D) CHRF knockdown significantly increased the expression of miR-489 in HCT116 cells. n = three independent experiments with similar results, *P < 0.05 by t test. (E) SW480 cells that were transfected with CHRF vector or empty vector were confirmed by qRT-PCR. n = three independent experiments with similar results, *P < 0.05 by t test. (F) CHRF overexpression notably reduced the expression of miR-489 in SW480 cells. n = three independent experiments with similar results, *P < 0.05 by t test.

Figure 9. LncRNA CHRF induced miR-489 loss promotes EMT process of CRC cells

(A) CHRF knockdown reduced the expressions of TWIST1 and Vimentin, while increased E-cadherin expression in HCT116 cells. (B) CHRF knockdown contributed to the morphology of epithelial-like cells in HCT116 cells. (C) CHRF overexpression increased the levels of TWIST1 and Vimentin, while reduced E-cadherin expression in SW480 cells. (D) CHRF overexpression facilitated the EMT process of SW480 cells.