MicroRNA-302a functions as a putative tumor suppressor in colon cancer by targeting Akt

Abstract

Micro RNAs (miRNAs) are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in colon cancer. MiRNA-302a expression was detected in 44 colon cancer tissues and 10 normal colon tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on colon cancer cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal colon tissues, miRNA-302a expression was downregulated in colon cancer tissues. Overexpression of miRNA-302a induced G1/S cell cycle arrest in colon cancer cells, and suppressed colon cancer cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibited AKT expression by directly binding to its 3' untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 pathway. These results reveal miRNA-302a as a tumor suppressor in colon cancer, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in colon cancer.

Figure 1. MiRNA-302a expression profiles in colon cancer tissue, normal prostate tissue, and colon cancer cells.

(A) MiRNA-302a expression was downregulated in colon cancer tissue compared with normal colon tissue. (B) Low levels of miRNA-302a expression were detected in four human colon cancer cell lines, HCT8, HCT116, HT29, and CaCO2. (C) High levels of miRNA-302a expression were detected in HCT8 and HCT116 colon cancer cells stably expressing miRNA-302a (*P<0.05).

Figure 2. Overexpression of miRNA-302a significantly inhibits cell proliferation in colon cancer cells in vitro.

CCK-8 assay was performed to measure proliferation in (A) HCT8 and (B) HCT116 cells. Data represent the mean ± standard deviation of the optical density (OD) value detected at 450 nm from three independent experiments. Cell proliferation was detected in (C) HCT8 and (D) HCT116 cells using EdU assay analyzed by flow cytometry. (E, F) Colony formation assays indicated fewer colonies in miRNA-302a overexpressing HCT8 cells. (*P<0.05).

Figure 3. Overexpression of miRNA-302a significantly inhibits cell proliferation in colon cancer cells in vivo.

HCT8-GFP cells and HCT8-302a cells were injected into the left and right posterior flank of nude mice, respectively (A, B). The tumor volume (C) and mass (D) in the HCT8-302a group were notably lower than in the HCT8-GFP group. (* P<0.05).

Figure 4. Overexpression of miRNA-302a induces G1/S phase cell cycle arrest in colon cancer cells.

The proportion of cells in G1-phase increased significantly in HCT8-302a cells (A, C) and HCT116-302a cells (B, D) compared with controls, while the proportion of cells in the S phase were notably less than the controls. (* P<0.05).

Figure 5. MiRNA-302a suppresses AKT expression by directly targeting its 3′ untranslated region.

AKT mRNA expression was remarkably suppressed in (A) HCT8-302a and HCT116-302a cells, and (B) HCT8-302a tumors. (C) Immunohistochemistry staining indicated lower expression of AKT in HCT8-302a tumors. (D) Relative luciferase activity was notably suppressed in wild-type AKT-3′ untranslated region (UTR) transfected cells. (E) Schematic representation of the luciferase reporter, which carried the wild-type or mutant AKT-3′ UTR. (* P<0.05).

Figure 6. Overexpression of miRNA-302a in colon cancer cells triggers alterations in the AKT-GSK3β-cyclin D1 signaling pathway.

Western blot analyses showed downregulated AKT and cyclin D1 levels, and upregulated GSK3β levels in miRNA-302a overexpressing HCT8 cells. PTEN and PI3K levels were not affected.