Interaction between miR‑206 and lncRNA MALAT1 in regulating viability and invasion in hepatocellular carcinoma

Abstract

MicroRNAs (miRNAs) are strongly associated to the progression of hepatocellular carcinoma (HCC), which presents a high potential for diagnosis and treatment; however, the role of miRNAs is still largely unknown. The aim of the present study was to examine the expression and the biological role of miRNA (miR)-206 in the development of HCC, and to identify the underlying molecular mechanism. Results from this study show that miR-206 was significantly downregulated in HCC tissues and cell lines. It was observed that low expression of miR-206 was linked to advanced TNM stage, tumor nodularity and venous infiltration in patients with HCC; low miR-206 expression was associated with shorter survival times. miR-206 overexpression using miR-206 mimics notably decreased the proliferative ability and increased apoptosis of MHCC97-H and HCCLM3 HCC cell lines. Overexpression of miR-206 suppressed invasiveness associated with reduced epithelial-mesenchymal transition. Moreover, the c-Met oncogene, which is upregulated in HCC tissues, was negatively associated with the expression of miR-206. Notably, it was shown that miR-206 may exert its antitumor effect through suppressing c-Met/Akt/mTOR signaling. Low expression of miR-206 was shown to be regulated by lncRNA MALAT1 in HCC. Collectively, this study presented evidence that miR-206 was controlled by lncRNA MALAT1 and partially suppressed the proliferation and invasion of HCC through the c-Met/Akt/mTOR signaling pathway. According to these results, understanding MALAT1/miR-206-dependent regulation may lead to potential approaches for diagnosis and prospective treatment of HCC.

Keywords: c-Met/Akt/mTOR signaling pathway; hepatocellular carcinoma; lncRNA MALAT1; microRNA-206.

Figure 1.

miR-206 is downregulated in HCC tissues and is associated with clinicopathologic features. (A) Heatmap of normalized expression levels of microRNAs in HCC tissues and matched tumor-adjacent tissues, based on the GSE10694 array dataset. Blue indicates low expression levels; red indicates high expression levels. (B) miR-206 expression was detected by reverse transcription-quantitative PCR in HCC and matched tumor-adjacent tissues (n=50). P<0.01 vs. Normal. The relationship between miR-206 and clinicopathological features of patients with HCC, including (C) TNM stage, (D) tumor nodule number and (E) venous infiltration. (F) Overall survival of patients with high or low miR-425-5p expression. HCC, hepatocellular carcinoma; miR, microRNA.

Figure 2.

Overexpression of miR-206 suppresses cell viability and induces apoptosis. (A) miR-206 expression of in the immortalized non-tumorigenic human hepatocyte cell line MIHA, which was used as the NC, and the HCC cell lines MHCC97-H, HCCLM3, Huh-7, and Hep3B. Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. MIHA. (B) Reverse transcription-quantitative PCR was used to assess the miR-206 transfection efficiency. (C) The MTT assay was used to examine cell viability. A commercial kit was used to measure the caspase-3 activity in (D) HCCLM3 and (E) MHCC97-H cells. (F) Flow cytometry was used to investigate apoptotic rates. Data are presented as the mean ± SD of three independent experiments. *P<0.05, **P<0.01 vs. Blank or mimics NC. HCC, hepatocellular carcinoma; miR, microRNA; NC, negative control.

Figure 3.

Overexpression of miR-206 suppresses cell migration and invasion. (A) Transwell Matrigel and (B and C) wound healing assays were performed to analyze the effects of miR-206 on MHCC97-H and HCCLM3 cell migration and invasion, respectively (magnification, ×200). Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. mimics NC group. (D) The protein expression levels of epithelial marker (E-cadherin and N-cadherin) and mesenchymal markers (Vimentin and Fibronectin) were assessed by western blotting; β-actin was used as a loading control. miR, microRNA; NC, negative control.

Figure 4.

c-Met is a direct target of miR-206. (A) The putative target site of miR-206 and c-Met. The red box shows the mutated target site of miR-206 and c-Met. (B) Relative luciferase activity of c-Met wt or mut 3′-UTR in 293T cells following transfection with the miR-206 mimics, inhibitor or corresponding NC, as indicated). Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. mimics NC; ^##P<0.01 vs. inhibitor NC. (C) c-Met protein expression after transfection with miR-206 mimic or miR-206 inhibitor was measured by western blotting; β-actin was used as a loading control. (D) Reverse transcription-quantitative PCR was used to determine the mRNA expression levels of c-Met in HCC and matched tumor-adjacent tissues (n=50). Data are presented as the mean ± SD of three independent experiments. (E) Correlation between miR-206 and c-Met in HCC tissues was determined by Pearson's correlation coefficient. HCC, hepatocellular carcinoma; miR, microRNA; mut, mutant; NC, negative control; UTR, untranslated region; wt, wild-type.

Figure 5.

c-Met overexpression reverses the inhibitory effects of miR-206 mimics in HCC cells. (A) c-Met protein expression was determined by western blotting; β-actin was used as a loading control. Cell viability was measured by MTT assay in transfected (B) HCCLM3 and (C) MHCC97-H cells. (D) Apoptotic rates were determined by flow cytometry. (E) Transwell Matrigel invasion and (F) wound healing migration assays were used to examine how miR-206 affected HCC cell migration and invasion, respectively (200× magnification). Data are presented as the mean ± SD of three independent experiments. ^#P<0.05, ^##P<0.01 vs. miR-206 mimics. HCC, hepatocellular carcinoma; miR, microRNA.

Figure 6.

Overexpression of miR-206 blocks activation of AKT/mTOR pathway. (A and B) western blot was used to identify the protein expression levels of c-Met, p-c-Met, AKT, p-AKT, p-mTOR and mTOR in HCCLM3 cells after miR-206 mimics transfection. (C and D) western blot was used to identify the protein expression levels of c-Met, p-c-Met, AKT, p-AKT, p-mTOR and mTOR in MHCC97-H cells after miR-206 mimics transfection. Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. mimics NC. HCC, hepatocellular carcinoma; miR, microRNA; NC, negative control; p-, phosphorylated.

Figure 7.

LncRNA MALAT1 negatively regulates miR-206 expression in HCC. (A) Expression of MALAT1 in HCC and matched tumor-adjacent tissues (n=50). (B) A negative correlation was identified between MALAT1 and miR-206 in a cohort with 50 patients with HCC. (C) The expression of MALAT1 in HCC cell lines MHCC97-H, HCCLM3, Huh-7 and Hep3B; MIHA used as NC. Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. MIHA. (D) Schematic representation of the predicted binding site for miR-206 and lncRNA MALAT1. (E) Relative luciferase activity of MALAT1 wt or mut in 293T cells following transfection with the miR-206 mimics, inhibitor or corresponding NC. Data are presented as the mean ± SD of three independent experiments. **P<0.01; ^##P<0.01. (F) lncRNA MALAT1 expression levels following transfection with miR-206 mimic or miR-206 inhibitor were measured by reverse transcription-quantitative PCR. **P<0.01; ^##P<0.01. (G) Verification of successful downregulation of MALAT1 by siRNA and overexpression of MALAT1 by pcDNA-MALAT1 vector transfections in MHCC97-H and Huh-7 cells. **P<0.01; ^##P<0.01. (H) MALAT1 knockdown increased, whereas overexpression of MALAT1 decreased miR-206 expression in HCCLM3 and Huh-7 cells. **P<0.01; ^##P<0.01. (I) lncRNA MALAT1 increases c-Met expression via sequestering miR-206 at post-transcription level, leading to the activation of AKT/mTOR signaling pathway, thus promotes HCC cell growth, migration and invasion. HCC, hepatocellular carcinoma; hsa, Homo sapiens; lncRNA, long non-coding RNA; miR, microRNA; NC, negative control.