MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4

Abstract

Background: Tumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known.

Results: miR-20a-5p negatively regulated Smad4 by directly targeting its 3'UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo, but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients' clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients.

Methods: Five miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo. The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan-Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data.

Conclusions: miR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer.

Keywords: Smad4; colorectal cancer; metastasis; miR-20a-5p; prognosis.

Figure 1. miR-20a-5p negatively regulated Smad4 in human colorectal cancer

(A) 293T cells were co-transfected with the negative control (NC) or miRNA mimics and the luciferase reporter construct containing the wild-type Smad4 3′-UTR. The luciferase activity of the NC was set to 1, the others were normalized to the NC. *P < 0.05 was considered to be statistically significant. (B and C) The mRNA expression of miR-20a-5p (P = 0.0002) and Smad4 (P < 0.001) in 10 CRC patients’ tissues with and without metastasis was evaluated by qRT-PCR. (D) The correlation between miR-20a-5p and Smad4 mRNA expressions in 10 colorectal cancer patients was evaluated using Spearman's correlation analysis (P = 0.031, R² = 0.234). (E) The expression of miR-20a-5p in different colorectal cancer cells was evaluated by qRT-PCR. FHC was used as a control. (F) HT29 cells were infected with miR-20a-5p-expressing lentiviral vector and HCT116 cells were infected with miR-20a-5p-knockingdown lentiviral vector. The luciferase reporter construct containing the wild-type Smad4 3′-UTR. Empty vector was used as a control, *P < 0.05. (G) The expression levels of Smad4 mRNA and protein were evaluated using qRT–PCR and western blot analysis in empty vector-infected or miR-20a-5p vector infected HT29 cells and negative control or anti-miR-20a-5p vector infected HCT116 cells, respectively, *P < 0.05.

Figure 2. Up-regulated miR-20a-5p promoted the invasion and metastasis of colorectal cancer in vitro and in vivo

(A) Wound healing assay (B) Migration and (C) invasion assays were performed both in empty vector-infected or miR-20a-5p vector infected HT29 cells and negative control or anti-miR-20a-5p vector infected HCT116 cells, respectively. The results were presented as the mean ± SD of values.(A) The “wound” was created in a cell monolayer, and captured the images at the beginning and at 48 h. Compared the images to quantify the closed rate of the cells,**P < 0.01.(B and C) The number of cells that passed through the membrane was counted in 10 fields (**P < 0.01, magnification ×200). (D) Impact of miR-20a-5p on tumor metastasis in vivo. The number of metastatic liver colonies was counted. The images of normal liver tissue (1) and metastatic colony tissue (2) stained by HE were presented (***P < 0.001, magnification × 200).

Figure 3. miR-20a-5p promoted the epithelial-to-mesenchymal transition of colorectal cancer cells

(A) The alteration of phenotypic observed under microscopy (magnification × 200) in miR-20a-5p overexpressing HT29 cells and miR-20a-5p knocking-down HCT116 cells. (B) Expression of E-cadherin and N-cadherin were evaluated at mRNA and protein levels (*P < 0.05, **P < 0.01).

Figure 4. miR-20a-5p mediated Smad4 expression by directly binding to its 3′-UTR in CRC cell lines

(A) The complementary sequences between the position 1357–1363 of wild-type or mutant human Smad4 3-UTRs mRNA and human miR-20a-5p. (B) HT29 cells were co-transfected with the control vector, miR-20a-5p or mutant miR-20a-5p mimics and the luciferase reporter construct containing the wild-type or mutant Smad4 3′-UTR. (C) HCT-116 cells were co-transfected with the negative control, anti-miR-20a-5p or mutant-miR-20a-5p mimics and the luciferase reporter construct containing the wild-type or mutant Smad4 3′-UTR. For each experiment, the results were normalized to the luciferase activity detected in the cells transfected with the control vector. (*P < 0.05 **P < 0.01 ***P < 0.001).

Figure 5. miR-20a-5p promoted the onco-process of CRC cells via Smad4 repression

miR-20a-5p overexpressing and negative control HT29 cells were transfected with empty vectors or Lv-Smad4. miR-20a-5p knocking down and negative control HCT-116 cells were transfected with empty vectors or shSmad4. (A) Wound healing assay, migration and invasion assays were performed in HT29 cells. Smad4 overexpression partially abrogated the increased wound healing, invasion and metastasis capacity induced by upregulation of miR-20a-5p. (B) Wound healing assay, migration and invasion assays were performed in HCT116 cells. Smad4 knockdown partially restored the decreased wound healing, invasion and metastasis capacity induced by downregulation of miR-20a-5p. (C) The expression level of E-cadherin and N-cadherin mRNA was evaluated by qRT-PCR. Overexpression or knockdown of Smad4 partially reversed the promoted or inhibited EMT due to miR-20a-5p upregulation or downregulation. (D) The expression level of E-cadherin and N-cadherin protein was evaluated by western blot analysis. (E) The number of tumor colonies formed in animal models. Overexpression or knockdown of Smad4 partially reversed the number of liver metastatic colonies resulting from miR-20a-5p upregulation or downregulation. (*P < 0.05; **P < 0.01, ***P < 0.001).

Figure 6. High level of miR-20a-5p was correlated with the metastasis and predicted poor prognosis in colorectal cancer patients

(A) The expression of miR-20a-5p mRNA in 544 patients normal and paired tumor tissues through qRT-PCR (P < 0.001). (B) The expression of miR-20a-5p mRNA in metastasis and non-metastasis tumor tissues. (C) The expression of Smad4 protein in tissues through IHC staining method: C1 normal tissue with low miR-20a-5p and Smad4 strong staining; C2 non-metastasis tumor tissue with high miR-20a-5p and Smad4 moderated staining, C3 metastatic tumor tissue with high miR-20a-5p and Smad4 weak staining. (D) Disease free survival (DFS) and (E) overall survival (OS) rates in 544 patients by Kaplan–Meier analysis with log-rank test (**P < 0.001). (F and G) overall survival (OS) rates in patients with tumor relapse (P = 0.003) and without tumor relapse patients (P = 0.547) by Kaplan–Meier analysis with log-rank test.