MiR-130b plays an oncogenic role by repressing PTEN expression in esophageal squamous cell carcinoma cells

Abstract

Background: Esophageal carcinoma is one of the most common malignancies with high cancer-related morbidity and mortality worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate a wide variety of cellular processes, and also play an important role in the development and progression of cancers. In a previous microarray study, we demonstrated that miR-130b was upregulated in esophageal squamous cell carcinoma (ESCC) tissues. However, the biologic functions and the molecular mechanism of miR-130b in ESCC remain to be elucidated.

Methods: qRT-PCR assays were used to quantify miR-130b expression levels in ESCC samples. Novel targets of miR-130b were identified via a bioinformatics search and confirmed using a dual-luciferase reporter system. Western blotting and qRT-PCR assays were used to quantify the expression of the target gene PTEN (phosphatase and tensin homolog) and the downstream effector, Akt. ESCC cells over- or underexpressing miR-130b were analyzed for in vitro biologic functions.

Results: High levels of miR-130b were identified in 20 ESCC samples following comparison with adjacent non-neoplastic tissues. We confirmed that miR-130b interacted with the 3'-untranslated region of PTEN, and that an increase in the expression level of miR-130b negatively affected the protein level of PTEN. However, the dysregulation of miR-130b had no obvious impact on PTEN mRNA. As Akt is a downstream effector of PTEN, we explored if miR-130b affected Akt expression, and found that miR-130b indirectly regulated the level of phosphorylated Akt, while total Akt protein remained unchanged. Overexpression of miR-130b increased the proliferation of ESCC cells and enhanced their ability to migrate and invade. In contrast, the proliferation, migration, and invasion of ESCC cells were weakened when miR-130b expression was suppressed, which was reversed by PTEN-targeted siRNA.

Conclusion: The results indicate that miR-130b plays an oncogenic role in ESCC cells by repressing PTEN expression and Akt phosphorylation, which would be helpful in developing miRNA-based treatments for ESCC.

Figure 1

Expression of mature miR-130b in ESCC tissue specimens. The expression of miR-130b was detected by TaqMan qRT-PCR in 20 ESCC and matched normal esophageal tissue samples. The expression of miR-130b is presented on a 2^-ΔCT (×100) scale and normalized against the endogenous control U6. (A) Paired comparison of miR-130b expression in ESCC tissue and matched normal tissue. (B) Scatter plots with merged data.

Figure 2

Identification of PTEN as target of miR-130b. (A) Bioinformatic prediction by TargetScan indicated that PTEN mRNA contains a putative miR-130b binding region, located at 412–418 nt of 3′-UTR. (B) A dual-luciferase reporter assay was used to confirm the interaction of miR-130b with PTEN. A fragment of miR-130b that was predicted to bind to the PTEN 3′-UTR was cloned into firefly luciferase pGL3-control vector. The Renilla luciferase plasmid was co-transfected for normalizing luciferase activity. *P < 0.05, **P < 0.01 vs. corresponding controls.

Figure 3

MiR-130b regulates the expression of PTEN and phosphorylation of Akt. Eca109 and TE13 cells were transfected with miR-130b mimic (130bm group) or inhibitor (130bi group); siPTEN was also co-transfected with the miR-130b inhibitor to decrease PTEN expression (130bi + siPTEN group). Protein expression of PTEN, p-Akt and total Akt in Eca109 (A, C, E) and TE13 cells (B, D, F) was determined by Western blot analysis. PTEN mRNA expression was detected by SYBR Green real-time PCR at 24 h following transfection of Eca109 (G) and TE13 (H) cell lines. The values were normalized to the corresponding controls. GAPDH was the endogenous control. *P < 0.05, **P < 0.01 vs. corresponding controls; #P < 0.05 vs. 130bi group.

Figure 4

Effect of miR-130b on the proliferation of ESCC cellsin vitro. The viability of Eca109 and TE13 cells transfected with miR-130b mimic, negative control, miR-130b inhibitor, inhibitor negative control or siPTEN was detected by CCK-8 at 24, 48, 72 and 96 h. The absorbance was measured at 450 nm. (A, B) Proliferation curves of Eca109 cells. (C, D) Proliferation curves of TE13 cells. For colony formation (E, G) and soft-agar (F, H) assays in Eca109 cells, the following calculation was performed: colony-forming efficiency (%) = the number of colonies (>50 cells or larger than 50 μm)/the number of cells plated per well. The photographs were captured from digital camera or microscope at 40× magnification, in which the size bar was representative of 200 μm. The results are expressed as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 vs. corresponding controls; #P < 0.05, ##P < 0.01 vs. 130bi group.

Figure 5

MiR-130b regulates the migration and invasion of ESCC cells. 1 × 10⁵ Eca109 cells were added to the Transwell inserts. For the invasion assay, the wells were covered with 50 μL of 1 mg/mL Matrigel. Cells were transfected with miR-130b mimic, negative control, miR-130b inhibitor, inhibitor negative control or siPTEN. The migratory or invasive cells were counted from five random areas at 200× magnification. (A) Cell images captured by microscopy. (B) Histogram of cell migration results. (C) Histogram of cell invasion results. The results are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding controls; #P < 0.05, ##P < 0.01 vs. 130bi group.