miR-338-3p inhibits epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma cells

Down-regulation of the miRNA miR-338-3p correlates with the invasive ability of hepatocellular carcinoma (HCC) cells. However, it is currently unclear whether down-regulation of miR-338-3p induces epithelial-mesenchymal transition (EMT), which may be the underlying mechanism governing HCC invasion. Here, we demonstrate that restoration of miR-338-3p expression via transfection of a miR-338-3p mimic reversed EMT and inhibited the motility and invasiveness of HCC cells. Conversely, silencing of endogenous miR-338-3p expression with a miR-338-3p-specific inhibitor induced EMT and enhanced HCC cell motility. Additionally, Snail1 (an upstream regulatory protein of EMT) and Gli1 (a key transcription factor in the sonic hedgehog (SHH) signaling pathway) expression was up-regulated in cells treated with the miR-338-3p inhibitor and down-regulated by the miR-338-3p mimic. Further analyses demonstrated that miR-338-3p inhibitor-induced EMT in HCC cells was blocked by treatment with a small interfering RNA (siRNA) targeting Snail1, that the SHH signaling pathway was required for both miR-338-3p inhibitor-induced EMT and up-regulation of Snail1, and that miR-338-3p targeted a sequence within the 3′-untranslated region of N-cadherin mRNA. Notably, miR-338-3p expression was significantly down-regulated in HCC samples from patients with metastases and was associated with poor metastasis-free survival rates. Lastly, correlations between the expression levels of miR-338-3p and E-cadherin, Smoothened (SMO), Gli1, Snail1, N-cadherin, and vimentin were confirmed in HCC xenograft tumors and HCC patient specimens. Our findings suggest that miR-338-3p suppresses EMT and metastasis via both inhibition of the SHH/Gli1 pathway and direct binding of N-cadherin. miR-338-3p is a potential therapeutic target for metastatic HCC.


Cell lines and transfection
MHCC-97H and SMMC-7721 cells were transfected with 50 nM of the miR-338-3p mimic or negative control miRNA (RiboBio, Co., Ltd., Guangzhou, China), and 50 nM of the miR-338-3p inhibitor or the negative control miRNA (RiboBio), respectively, using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Meanwhile, the small interfering RNA (siRNA) specific to SMO and the corresponding control siRNA (RiboBio) were transfected into SMMC-7721 cells, as described previously [1]. The three siRNA duplex oligonucleotides specific to human Snail1 and N-cadherin mRNA utilized in this study were also synthesized by RiboBio. For our analyses, we chose to utilize Snail1 siRNA1 and N-cadherin siRNA1 as they effectively inhibited endogenous Snail1 and N-cadherin expression, respectively. For these experiments, SMMC-7721 cells were transfected with 100 nM of the Snail1 siRNA1, the N-cadherin siRNA1, or the control siRNA using Lipofectamine 2000 reagent. After the aforementioned treatments, morphological changes in HCC cells were monitored by inverted phasecontrast microscopy, and siRNA knockdown efficiencies were confirmed by quantitative reverse transcription PCR (qRT-PCR) and western blotting analyses. All RNA oligoribonucleotides used in this study are listed in Supplementary Table 2.

RNA extraction and qRT-PCR
Total RNA, including small RNAs, was extracted from tissues or cells using TRIzol reagent (Invitrogen), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized and qRT-PCR was performed as previously described [2]. The primers used for detection of human SMO, Gli1, Snail1, N-cadherin, E-cadherin, vimentin, and β-actin expression were reported previously [3]. A TaqMan miRNA assay kit (Applied Biosystems, Waltham, MA, USA) was used for the detection of mature miR-338-3p expression, as described previously [4]. The expression levels of target genes were normalized to that of U6 and β-actin for miRNAs and mRNAs assays, respectively. All primers used in this study are listed in Supplementary Table 2.

Cell migration and invasion assays
The cell migration and invasion assays were performed as previously described [5]. Briefly, for the migration assay, at 24 h after transfection, cells (5×10 4 / well) were seeded into the upper chamber of a 24-well Transwell insert (Corning, New York, NY) in serum-free medium. After 24-h incubation, the cells on the upper surface of the filter were wiped off with a cotton swab, and the cells that had invaded into the lower surface of the filter were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope at 200× magnification. The cellular invasion potential was determined by these same procedures but with a Matrigel coating on the filter.

Wound healing assay
The transfected cells were seeded on to 24-well plates and grown in Dulbecco's modified Eagle's medium (DMEM). After 48-h incubation and once the culture reached approximately 90% confluence, an artificial homogenous wound was created on the monolayer with a sterile 200-μL plastic micropipette tip. After scratching, the cells were washed twice with PBS and incubated for additional 48 h. The images of cells migrating into the wound were observed at indicated times and captured with an inverted microscope under 100× power (BX50; Olympus, Tokyo, Japan).

Western blotting analysis
Western blotting was conducted according to a standard method described previously [1]. In brief, the total protein was extracted from tissues or cells, and protein concentration was measured with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA,). The samples with equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA,). The membranes were incubated with primary antibodies against E-cadherin (Cell Signaling Technology, West Grove, PA) at 1:800, N-cadherin (Cell Signaling Technology) at 1:800, Gli1 (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500, SMO (Santa Cruz Biotechnology) at 1:500, Snail1 (Cell Signaling Technology) at 1:800, vimentin (Santa Cruz Biotechnology) at 1:500, and GAPDH (Santa Cruz Biotechnology at 1:1000 overnight at 4°C, followed by washes with TBS and incubation with the corresponding secondary HRP-conjugated antibodies (Santa Cruz Biotechnology). Protein bands were detected with an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL) and exposure to X-ray film.

Immunohistochemistry
Immunohistochemistry was performed as described previously [5]. The degree of immunohistochemical staining was examined and scored independently by two observers. The staining intensity was scored according to the following criteria: no staining (0), weak staining (1), moderate staining (2), and strong staining (3), as described previously [2]. The cut-off values for high and low expression of indicated proteins were chosen based on a measurement of heterogeneity using the log-rank test. Images were captured at 200× magnification using the AxioVision Rel. 4.6 computerized image analysis system (Carl Zeiss).

Luciferase reporter assay
Cadherin gene CDH2 3´-untranslated region (CDH2 3´-UTR) luciferase reporter constructs were generated by cloning either the wild type or mutant 3´-UTR sequence of the CDH2 coding sequence into the pEZX-MT01-reporter construct (GeneCopoeia) downstream of the luciferase gene using the primers listed in Supplementary Table S2. PCR products were cloned into PEZX-MT01 at the same site, and the identities of the resulting clones (pEZX-MT01-CDH2 3´-UTR-WT and pEZX-MT01-CDH2-3´-UTR-m) were confirmed by sequencing analysis. To confirm binding between miR-338-3p and the 3´-UTR of CDH2, the mutant CDH2 3´-UTR-m reporter was transfected into MHCC-97H cells together with pre-miR-338-3p. At 48 h after transfection, cells were harvested and analyzed using a Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI, USA). For these analyses, firefly luciferase activity was normalized to Renilla luciferase activity. The cDNA of human SMO (lacking the 3´-UTR) was PCR amplified and inserted into pEZ-Lv201 expression vector using the primers listed in Supplementary