MicroRNA‑190b expression predicts a good prognosis and attenuates the malignant progression of pancreatic cancer by targeting MEF2C and TCF4

Abstract

MicroRNAs (miRNAs/miRs) are key components of regulatory networks in cancer. Although miR‑190b is an important tumor‑related miRNA, its role in pancreatic cancer has not been extensively investigated. The aim of the present study was to examine the expression of miR‑190b in pancreatic cancer cell lines and tissues and evaluate its effects on cancer progression. Reverse transcription‑quantitative PCR (RT‑qPCR) analysis was used to measure miR‑190b expression levels in human pancreatic cancer cell lines and tissues, and the association between miR‑190b expression and clinicopathological characteristics was assessed. An in vitro Transwell invasion assay and an in vivo metastasis formation assay were performed using pancreatic cancer cells. The effect of miR‑190b on pancreatic cancer cell proliferation was evaluated using a Cell Counting Kit‑8 assay based on an in vivo xenograft mouse model. The direct targets of miR‑190b were predicted using bioinformatics tools and were validated through western blotting and luciferase reporter assays. Pancreatic cancer cell lines and tissues were found to express lower levels of miR‑190b compared with normal cells and adjacent non‑tumor tissues. Furthermore, high expression of miR‑190b was found to be positively correlated with low T, N and American Joint Committee on Cancer classifications, and predicted a good prognosis. miR‑190b was shown to exert suppressive effects on cancer cell proliferation, invasion and metastasis. In addition, it was also found that miR‑190b directly targeted myocyte enhancer factor 2C (MEF2C) and transcription factor 4 (TCF4) in pancreatic cancer, thus serving as a tumor suppressor and a predictor of good prognosis in pancreatic cancer. The immunohistochemistry and RT‑qPCR results indicated that the MEF2C and TCF4 expression levels were negatively correlated with the miR‑190b expression levels. The findings of the present study highlight the value of miR‑190b as a novel target candidate for pancreatic cancer diagnosis and therapy.

Keywords: microRNA‑190b; myocyte enhancer factor 2C; pancreatic cancer; prognosis; transcription factor 4; tumor suppressor.

Figure 1.

miR-190b expression in pancreatic cell lines and pancreatic tissues. Relative expression levels were determined by reverse transcription-quantitative PCR analysis. (A) miR-190b expression levels in pancreatic cancer cell lines relative to the normal human pancreatic ductal cell line HPDE. (B) Comparison of miR-190b levels between pancreatic cancer tissues and non-tumor tissues. (C) Non-parametric test demonstrated that patients with lower AJCC stage (I and II) had significantly higher miR-190b expression compared with those with higher AJCC stage (II and III) (P<0.01). (D) Survival curves according to miR-190b expression levels in patients with PDAC. The cut-off value of miR-190b expression used to generate the two groups of patients was a relative expression ratio of 0.5. Values are presented as mean ± SD. n=3. *P<0.05 and **P<0.01. miR, microRNA; AJCC, American Joint Committee on Cancer; PDAC, pancreatic ductal adenocarcinoma.

Figure 2.

miR-190b suppresses AsPC-1 cell invasion in vitro. (A) AsPC-1 cells were transfected with miR-190b mimics or NC. Following transfection with miR-190b mimics, miR-190b expression increased by 42.88±2.18-fold. (B) AsPC-1 cells were transfected with anti-miR-190b or anti-NC. Following transfection with anti-miR-190b, miR-190b expression decreased by 0.30±0.03-fold. (C) Representative photomicrographs of Transwell assay results for AsPC-1 cells (original magnification, ×100). (D) The numbers of miR-190b-transfected AsPC-1 cells invading through the Matrigel membrane were significantly lower compared with NC-transfected and parental AsPC-1 cells (blank control). Cells were counted in 16 independent symmetrical visual fields under an inverted microscope (original magnification, ×400) in three independent experiments. (E) The numbers of anti-miR-190b-transfected AsPC-1 cells invading through the Matrigel membrane were significantly higher compared with anti-NC-transfected and parental AsPC-1 cells (blank control). Cells were counted in 16 independent symmetrical visual fields under an inverted microscope (original magnification, ×400) in three independent experiments. (F) Representative photomicrographs of Transwell assay results for AsPC-1 cells (original magnification, ×100). Values are shown as mean ± SD; n=3. *P<0.05. miR, microRNA; NC, negative control.

Figure 3.

miR-190b suppresses MIA PaCa-2 cell invasion in vitro. (A) MIA PaCa2 cells were transfected with miR-190b mimics or NC. Following transfection with miR-190b mimics, miR-190b expression increased by 354.62±69.44-fold. (B) MIA PaCa-2 cells were transfected with anti-miR-190b or anti-NC. Following transfection with anti-miR-190b, miR-190b expression decreased by 0.34±0.05-fold. (C) Representative photomicrographs of Transwell assay results for MIA PaCa-2 cells (original magnification, ×100). (D) The numbers of miR-190b-transfected MIA PaCa-2 cells invading through the Matrigel membrane were significantly lower compared with NC-transfected and parental MIA PaCa-2 cells (blank control). Cells were counted in 16 independent symmetrical visual fields under a light microscope (original magnification, ×400) in three independent experiments. (E) The numbers of anti-miR-190b-transfected MIA PaCa-2 cells invading through the Matrigel membrane were significantly higher compared with anti-NC-transfected and parental MIA PaCa-2 cells (blank control). Cells were counted in 16 independent symmetrical visual fields under a light microscope (original magnification, ×400) in three independent experiments. (F) Representative photomicrographs of Transwell assay results for MIA PaCa-2 cells (original magnification, ×100). Values are shown as mean ± SD; n=3. *P<0.05. miR, microRNA; NC, negative control.

Figure 4.

miR-190b suppresses pancreatic cancer cell proliferation in vitro. CCK-8 assay demonstrated that (A) miR-190b suppressed AsPC-1 cell proliferation and (B) anti-miR-190b significantly promoted AsPC-1 cell proliferation at 72 and 96 h. CCK-8 assay demonstrated that (C) miR-190b suppressed MIA PaCa-2 cell proliferation and (D) anti-miR-190b significantly promoted MIA PaCa-2 cell proliferation at 72 and 96 h. miR, microRNA; CCK-8, Cell Counting Kit-8; NC, negative control. *P<0.05.

Figure 5.

miR-190b suppresses pancreatic cancer growth and metastasis in vivo. (A) Xenograft mouse models were used to detect the proliferative ability of AsPC-1 cells in vivo. Images of tumors in each group up to day 16 after injection. (B) Mean tumor weight in each group. (C) Tumor growth curves based on in vivo tumor volume. (D) Representative microscopic images showing histological morphology (H&E staining; magnification, ×100) and Ki67 expression [magnification, ×100 and ×400 (inset)]. Scale bar, 100 µm. (E) Representative images of mouse lung tissues (upper panels) and corresponding histological sections (lower panels). Cell aggregates with dark-stained nuclei represent lung metastases (arrows). (F) Data are shown graphically by the number of lung metastases at 4 weeks in each mouse injected with 1×10⁶ AsPC-1 cells. Values are shown as mean ± SD; n=8. *P<0.05. miR, microRNA; NC, negative control.

Figure 6.

MEF2C and TCF4 are direct targets of miR-190b in pancreatic cancer. (A) Potential target genes of miR-190b were predicted by using bioinformatic analyses. (B) Luciferase activities of MEF2C, TCF4, MEF2C-MUT and TCF4-MUT in AsPC-1 cells transfected with miR-190b mimics or NC. (C) Western blot analyses of MEF2C and TCF4 in transfected and parental AsPC-1 cells. Levels were normalized to those of GAPDH, and endogenous MEF2C and TCF4 levels were notably reduced in miR-190b-transfected AsPC-1 cells. Values are shown as mean ± SD. n=3. *P<0.05. miR, microRNA; MEF2C, myocyte enhancer factor 2C; TCF4, transcription factor 4; NC, negative control; WT, wild-type; MUT, mutated.

Figure 7.

High expression of MEF2C and TCF4 is associated with reduced miR-190b expression. (A and B) MEF2C and TCF4 expression in PDAC tissues was determined using immunohistochemistry. Positive expression is shown as brown-yellow particles distributed in the cell nucleus and cytoplasm. The cellular staining was classified using a scale of 0-2 as follows: 0, negative; 1, weakly positive; and 2, moderately positive. Scale bar, 200 µm. Magnification, ×100 (main panels) and ×200 (insets). (C) The non-parametric test demonstrated that miR-190b levels were significantly lower in samples with high expression of MEF2C and TCF4. (D) Pearson's correlation analysis demonstrated that miR-190b levels were significantly negatively correlated with MEF2C and TCF4 levels in pancreatic cell lines. Values are shown as mean ± SD. n=3. *P<0.05. miR, microRNA; MEF2C, myocyte enhancer factor 2C; TCF4, transcription factor 4; PDAC, pancreatic ductal adenocarcinoma.