miR-196a-5p promotes metastasis of colorectal cancer via targeting IκBα

The human colorectal cancer (CRC) cell lines of SW480, SW620 and HCT116 were obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. and cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (BI, USA) at 37 °C in 5%CO₂. In order to evaluate the influence of NF-κB pathway on the migration, invasion and EMT of CRC cells, cells were treated with NF-κB pathway inhibitor BAY 11–7082 (10 μM, medchemexpress, USA) for 48 h before following experiments.

In order to construct the IκBα expression plasmid, the full-length cDNA of IκBα was amplified and inserted into pcDNA3.1 vector with HindIII and BamHI sites. Lipofectamine 2000 (Invitrogen, USA) was employed to transfect IκBα plasmid into SW480 cells. Further, miR-196a-5p mimics, NC mimics, miR-196a-5p inhibitor and NC inhibitor purchased from Shanghai GenePharma Co., Ltd. were transfected into CRC cells using the Lipofectamine® RNAiMAX (Invitrogen, USA) following the manufacturer’s protocol.

The lentiviral (LV) particles that over-expressing miR-196a-5p (LV-miR-196a-5p) and its negative control (LV-NC) or silencing miR-196a-5p (LV-anti miR-196a-5p) and its negative control (LV-anti NC) were purchased from Wanleibio Co., Ltd. CRC cells were seeded in 6-well plates at a density of 4 × 10⁵/well. 24 h latter, the lentivirus was added into the culture medium to infect the CRC cells. After screening with Purine toxin (Solarbio, China), CRC cells with stable miR-196a-5p over-expressed or knockdown were obtained.

The 3′-UTR region of wide-type of IκBα was amplified and inserted into pmirGLO plasmid at Nhe I and Sal I sites. Furthermore, miR-196a-5p mimics or the miR-196a-5p mutant was cotransfected with Luc-IκBα or the control into HEK 293 T cells using the Lipofectamine 2000 (Invitrogen, USA). Luciferase assays were carried out with the Dual-Luciferase Reporter Assay System (Promega, USA) referring to the instructions.

The total RNA from CRC cells in different conditions was extracted using the TRIpure kit (BioTeke, China). After quantization of concentration, RNA was reversely transcribed into cDNA by the Super M-MLV reverse transcriptase (BioTeke). Quantitative PCR was carried out to determine the expression levels of miR-196a-5p and IκBα using 2 × Power Taq PCR MasterMix (BioTeke) and SYBR Green (Solarbio, China). The procedure for IκBα was as follows: first cycle including 94 °C for 5 min, 94 °C for 10 s, 60 °C for 20 s, 72 °C for 30 s, and then 40 cycles of 72 °C for 2 min 30 s, 40 °C for 1 min 30 s, melting from 60 °C to 94 °C for 1 s every 1 °C. While the miR-196a-5p expression level was measured under following condition: 94 °C for 2 min, 94 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s followed with 40 cycles of 72 °C for 2 min 30 s, 40 °C for 5 min 30 s, melting from 60 °C to 94 °C for 1 s every 1 °C. The data were calculated by 2^-ΔΔCt or 2^-ΔCt method and sequences information was shown in Table 1.

CRC cells were seeded into 96-well plates at 3 × 10³/well. The cell viability was measured at 12 h, 24 h, 48 h and 72 h after transfection. CCK-8 regent (KeyGEN BioTECH, China) was added into culture medium 10 μl per well. After 1 h incubation, the optical density of solution was measured using a microplate reader at 450 nm.

The transfected or infected CRC cells were cultured in serum-free DMEM containing mitomycin C (Sigma, USA) for 1 h. Subsequently, a sterile 200 μl pipette tip was employed to cause scratches. Representative images of cells migrating into wounds were taken at 0 h, 24 h or 12 h after the scratch.

Cell invasion assay was performed on 24-well transwell chambers coated with Matrigel (BD, USA). In brief, the upper chamber was added into 200 μl DMEM containing 1 × 10⁴ CRC cells and the lower chambers were filled with 800 μl culture medium containing 30% FBS. After 24 h incubation, migrated cells were fixed with 4% paraformaldehyde for 20 min and stained with the crystal violet (Amresco, USA) for 5 min. Cells on lower surface were counted with an inverted microscope.

To assess the effects of miR-196a-5p on tumor metastasis, we employed 4-week-old BALB/c nude mice to establish in vivo metastasis models. The thymic aplasia of the nude mice results in immunodeficiency and could avoid interference from host immune system. The nude mice used in present study were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China; License number: SCXK (Jing) 2014–0004) and housed in a standard laboratory environment (12 h day-night cycle; temperature: 25 ± 1 °C; humidity: 50 ± 5%). During the experiments, mice were free access to the water and food and no adverse events were observed. In present work, 24 mice were randomly divided into four groups: SW480-LV-NC group (n = 6); SW480-LV-miR-196a-5p group (n = 6); HCT116-LV-anti NC group (n = 6); HCT116-LV-anti miR-196a-5p group (n = 6). According to the experimental group dividing, 2 × 10⁶ transfected CRC cells were injected into spleens of the anesthetized nude mice and the spleens were resected 48 h latter. All mice were sacrificed 7 weeks after the injection and the hepatic metastases have been evaluated.

The fixed liver tissues were embedded in paraffin followed with slicing into 5 μm sections. After dewaxing, the sections were stained with hematoxylin (Solarbio, China) and eosin (Sangon, China). The metastatic foci were observed under the OLYMPUS DP73 microscope.

CRC cells or liver metastatic tissues were lysed with RIPA Lysis Buffer (Beyotime, China) containing 1% PMSF (Beyotime). Besides, the nuclear protein was isolated by the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein from different conditions were separated by 6–14% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk or 1% BSA, the membranes were incubated with following primary antibodies at 4 °C overnight: anti-E-cadherin (1:1000, 60,335–1-Ig, Proteintech, China), anti-N-cadherin (1:1000, 66,219–1-Ig, Proteintech, China), anti-fibronectin (1:500, 15,613–1-AP, Proteintech, China), anti-p-IκBα (1:500, bs-2513R, Bioss, China), anti-IκBα (1:500, bs-1287R, Bioss, China), anti-p65 (1:500, 10,745–1-AP, Proteintech, China), anti-β-actin (1:500, KGAA001, KeyGen, China) and Histone H3 (1:2000, AM8433, ABGENT, USA). After rinsed with TSBT, the membranes were incubated with their corresponding secondary antibodies at 37 °C for 45 min. The protein bands were visualized using the ECL reagent (Beyotime).

The CRC cells were seeded on slides and then fixed in 4% paraformaldehyde for 15 min. After permeabilized with 0.1% tritonX-100 (Beyotime, China) for 30 min at room temperature, the slides were blocked with goat serum (Solarbio, China). Subsequently, cells were incubated with antibodies of E-cadherin (1:100, 60,335–1-Ig, Proteintech, China), N-cadherin (1:100, 66,219–1-Ig, Proteintech, China), Fibronectin (1:50, 15,613–1-AP, Proteintech, China) overnight at 4 °C. Then CRC cells were incubated with the goat anti-rabbit Cy3-conjugated IgG (1:400, A0516, Beyotime) or goat anti-mouse Cy3-conjugated IgG (1:400, A0521, Beyotime). Lastly, cells were counterstained with DAPI (Sigma, USA) before capturing images using the fluorescence microscope (Olympus, Japan).

We firstly performed Real-time PCR to detect the expression level of miR-196a-5p in various CRC cell lines. As shown in Fig. 1a, the primary colon cell SW480 revealed a lowest expression of miR-196a-5p, while the metastatic colon cell SW620 depicted a 4.05-fold elevation of miR-196a-5p than SW480. We also observed that miR-196a-5p was highly expression in HCT116 cell while weak expression in Caco-2 and LoVo cell lines.

We then assessed whether the expression level of miR-196a-5p in CRC cells influence their proliferation. With this objective, we used miR-196a-5p mimic and inhibitor to induce or silence the miR-196a-5p expression in CRC cells (Fig. 1b, c and d). The results of CCK-8 demonstrated that over-expression of miR-196a-5p significantly accelerated the cell proliferation, while the cell viability was lower in miR-196a-5p-silenced cells than the control (Fig. 1e, f and g).

We subsequently investigated the influence of miR-196a-5p on the cell mobility. Data from wound healing assay indicated that over-expression of miR-196a-5p in SW480 cells enhanced the migration rate to 26.30 ± 2.70% and 50.83 ± 4.11% at 12 and 24 h, respectively (Fig. 2a). We also observed that knockdown of miR-196a-5p in SW620 and HCT116 cells decreased the migration rates at 24 h (Fig. 2a). Interestingly, The SW480 cell migration at 24 h was higher than that of SW620 cells [14, 15]. We further found that over-expression of miR-196a-5p in SW480 cells increased the number of invading cells by 31%, while knockdown of miR-196a-5p reduced the invading cells by 35–55% in SW620 and HCT116 cells (Fig. 2b).

The effects of miR-196a-5p on liver metastasis were also been evaluated in our present study. We firstly established SW480 cells stably over-expressed miR-196a-5p, and HCT-116 cells with miR-196a-5p knockdown (Fig. 3a). After injection these CRC cells into spleens for seven weeks, laparotomy was conducted. We observed that SW480 cells which over-expressed miR-196a-5p induced more metastatic nodules formation in liver, while knockdown of miR-196a-5p in HCT-116 cells attenuated the tumor metastasis (Fig. 3b). As shown in Fig. 3c, histologic analyses further verified that injection with miR-196a-5p-expressed CRC cells increased the number of micrometastatic lesions in liver, whereas injection of miR-196a-5p knockdown cells had opposite effects. The western blot results showed that the expression of IκBα and E-cadherin was decreased, and N-cadherin was increased in liver metastatic nodules induced by miR-196a-5p overexpressing cells (Additional file 1a). Conversely, we found that IκBα and E-cadherin protein levels were increased, and N-cadherin protein levels were decreased in liver metastatic nodules induced by miR-196a-5p downregulated cells (Additional file 1b).

Western blot assay and immunofluorescence staining were performed to evaluate the effects of miR-196a-5p on the expression of epithelial markers E-cadherin and mesenchymal markers N-cadherin and fibronectin. As shown in Fig. 4 and Fig. 5, over-expression of miR-196a-5p down-regulated the E-cadherin, whereas up-regulated the N-cadherin and fibronectin. Contrastingly, E-cadherin was increased while N-cadherin and fibronectin were decreased in miR-196a-5p knockdown CRC cells.

We employed BAY 11–7082, the inhibitor of NF-κB signaling pathway, to investigate the role of NF-κB pathway in EMT. As shown in Fig. 6a, BAY 11–7082 decreased the phosphorylation level of IκBα and nuclear expression of p65 in SW620 and HCT116 cells. BAY 11–7082 up-regulated the total expression level of IκBα in CRC cells. Furthermore, BAY 11–7082 increased the expression of E-cadherin whereas decreased the expression of N-cadherin and fibronectin in both SW620 and HCT116 cells (Fig. 6b).

By analyzing the database of microrna.​org, we found that IκBα may be a downstream target of the miR-196a-5p. The expression of IκBα and its correlation with miR-196a-5p were evaluated in CRC cells (Additional file 2). The result showed a negative correlation tendency between miR196 and IκBα expression in CRC cells (r = − 0.372), but no significance (p = 0.54). We performed dual luciferase reporter assay to determine whether there was an interaction between IκBα mRNA and miR-196a-5p. As shown in Fig. 7a, miR-196a-5p mimic decreased the luciferase activity of reporter containing 3′UTR of IκBα, while miR-196a-5p mutant did not have similar effects. We found that induced the expression of miR-196a-5p down-regulated the IκBα both in mRNA and protein level, whereas miR-196a-5p knockdown had opposite effects (Fig. 7b and c). We further observed that over-expression of miR-196a-5p increased the nuclear p65 level, and knockdown of miR-196a-5p decreased the expression of nuclear p65 (Fig. 7c). Moreover, we employed IκBα plasmid to induce the expression of IκBα in SW480 cells (Additional file 3). As shown in Fig. 8a and b, inducing the IκBα expression by transfection of IκBα plasmid significantly decreased the migration rate and number of invading cells in miR-196a-5p overexpressing SW480 cells. Further, over-expression of IκBα in miR-196a-5p overexpressing CRC cells increased E-cadherin levels and inhibited N-cadherin and fibronectin levels (Fig. 8c).