Introduction

MicroRNAs (miRNAs) are endogenously synthesized small noncoding RNA molecules of approximately 19–24 nucleotides. MiRNAs contribute to the regulation of crucial biological processes, such as cell proliferation, apoptosis, differentiation, and angiogenesis by controlling the stability and translation of target mRNAs [1, 2]. They are also implicated in the pathogenesis of various diseases as tumor suppressor genes or oncogenes [3, 4]. Accumulating evidence suggests that miRNAs act as tumor suppressors or oncogenes by targeting genes involved in cell proliferation, survival, apoptosis, and metastasis [5,6,7].

In our previous study, MIR196B was identified as a colorectal cancer (CRC) associated miRNA by miRNA expression profiling of CRC tissues versus healthy colorectal tissues, which showed that its expression was significantly up-regulated in CRC tissues [8]. MIR196B is a member of the MIR196 gene family (MIR196A1, MIR196A2, and MIR196B), which are transcribed from three different genes and are located in the homeobox (HOX) gene cluster regions in humans [9, 10]. The MIR196A1 gene is located on Chr. 17q21.32 between the HOXB9 and HOXB10 genes. The MIR196A2 gene is located between HOXC10 and HOXC9 on Chr. 12q13.13. The gene for MIR196B is located in an evolutionarily conserved region between HOXA9 and HOXA10 on Chr. 7p15.2. The mature nucleotide sequences of MIR196A1 and MIR196A2 are identical, whereas mature MIR196B differs from MIR196A by one nucleotide [10].

We previously detected 18 target genes of MIR196B by comparing the mRNA microarray analysis of MIR196B-overexpressed human colon cells with the bioinformatics-predicted candidate target genes, and suggested that MIR196B regulates FAS cell surface death receptor (FAS)-mediated apoptosis in colorectal cancer cells [8].

In this study, we validated the expression levels of HOXA5, HOXB6, and glycolipid transfer protein (GLTP) in MIR196B-transfected CRC cells, and showed that HOXA5, HOXB6 and GLTP are the direct target genes of MIR196B.

Material and Methods

Patients and Tissue Samples

The tissue samples used in this study were provided by the Biobank of Wonkwang University Hospital, a member of the National Biobank of Korea. With approval from the institutional review board and informed consent from the patients, we obtained colon cancer tissue from 2 colon cancer patients (1 male and 1 female) and rectal cancer tissue from 2 rectal cancer patients (2 males). The mean ages of the colon cancer and rectal cancer patients were 64.8 and 72 years, respectively. The colon cancer and matched normal colon tissue samples were used to analyze GLTP protein expression.

Cell Culture

The human CRC cell lines SW480 and Caco2 were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea) and American Type Culture Collection (ATCC, Manassas, VA, USA), respectively. SW480 cells were cultured in RPMI 1640 (HyClone, Logan, UT, USA) supplemented with 10% FBS in 5% CO2 at 37 °C in a humidified atmosphere. Caco2 cells were cultured in Alpha-MEM (HyClone) supplemented with 20% FBS in 5% CO2 at 37 °C in a humidified atmosphere.

RNA Extraction and Quantitative RT-PCR (qRT-PCR)

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to our previous reports [11, 12]. After digestion with DNase and cleanup, RNA samples were quantified, aliquoted, and stored at −80 °C. Total RNA isolated from tissue samples and/or cultured cells was used as a template to synthesize cDNA for qRT-PCR analysis in a StepOne Real-time PCR system (Applied Biosystems, Foster City, CA, USA).

The differential miRNA expression patterns were validated with the TaqMan qRT-PCR assay (Applied Biosystems) or the NCode VILO miRNA cDNA Synthesis kit for qRT-PCR and EXPRESS SYBR GreenER miRNA qRT-PCR kit (Invitrogen). Furthermore, qRT-PCR with SYBR Green dye (Applied Biosystems) was used to assess mRNA expression. RNU48 (for TaqMan qRT-PCR) or 5.8S (for SYBR qRT-PCR) and GAPDH were used as endogenous controls for the qRT-PCR of miRNA and mRNA, respectively. Each sample was run in triplicate.

MIR196B Transfection

SW480 cells (3 × 105) or Caco2 cells (1.5 × 105) were plated onto 6-well or 10-cm culture plates and cultured as described above. MIR196B (hsa-miR-196b, Pre-miR miRNA Precursor AM17100, Product ID: PM12946) was commercially synthesized (Ambion, Austin, TX, USA) and transfected at a concentration of 50 nmol/L using Lipofectamine RNAiMAX (Invitrogen) or siPORT™ NeoFX™ transfection agent (Ambion) according to the manufacturers’ recommendations. Cells were harvested 24–48 h (for miRNA and mRNA) or 48–72 h (for protein) after transfection for RNA or protein extraction.

Plasmid Construct and Luciferase Assays

Wild-type or mutant fragments of the 3′ untranslated region (UTR) of HOXA5, HOXB6 and GLTP containing the predicted binding site for MIR196B, were amplified by PCR using the primer set shown in Table 1. The PCR product was cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA). Analysis of the luciferase assay results was carried out according to our previously described method (Mo et al., 2015) [8]. Briefly, cells (5 × 104/well) were seeded onto 24-well plates and co-transfected with wild-type or mutant HOXA5, HOXB6 and GLTP constructs (500 ng/well) or with 50 nM MIR196B or MIR-1 (negative control) using Lipofectamine 2000 (Invitrogen Life Technologies) or siPORT™ NeoFX™ Transfection Agent (Ambion). Firefly and Renilla luminescence was measured 24 h after transfection using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. Non-transfected cells were used for background subtraction, and the ratio of firefly reporter luminescence to Renilla reporter luminescence (control) was calculated. All experiments were performed in triplicate and repeated at least three times.

Table 1 Primer sequences used for qRT-PCR analysis and luciferase assays in this study
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Antibodies and Western Blot Analysis

SW480 or Caco2 cells (2 × 105 cells/well) were seeded onto 6-well plates and incubated for 72 h. Whole cell lysates were prepared by incubation in RIPA buffer supplemented with a protease inhibitor mixture for 30 min at 4 °C. Protein was collected by centrifugation at 12,000 rpm for 30 min at 4 °C. Equal amounts of protein (50 μg; determined by the Bradford assay) were boiled in Laemmli buffer, subjected to 12.0% or 15.0% SDS-PAGE, and then transferred onto PVDF membranes. The membranes were incubated with blocking buffer (5% BSA in TBS containing 0.1% Tween-20 [TBS-T]) for 2 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies to HOXA5 (Pierce Biotechnology) and HOXB6 and GLTP (Santa Cruz Biotechnology), and washed three times for 10 min per wash with TBS-T. Membranes were incubated with HRP-conjugated rabbit or mouse IgG secondary antibodies for 1 h at room temperature. After washing three times for 10 min in TBS-T, protein was detected with ECL solution (Millipore Corporation, Billerica, MA, USA) and the FluorChem E System (Cell Biosciences, Santa Clara, CA, USA). After protein detection, some membranes were stripped with stripping buffer for 1 h at room temperature and re-probed with an antibody to GAPDH (0411; Santa Cruz Biotechnology), which was used as a loading control. Protein expression was evaluated using the ImageJ software (version 1.44; http://rsbweb.nih.gov/ij/index.html).

Statistical Analysis

Each experiment was repeated at least three times with consistent results. All of the data were represented as mean ± standard deviation(SD). Statistical differences were analyzed by the Student′s t-test, and p-values of less than 0.05 (p < 0.05) were regarded as statistically significant.

Results

Validation and Expression of HOXA5 and HOXB6 Genes

The expression levels of HOXA5 and HOXB6 transcripts in un-transfected and transfected cells were compared by qRT-PCR. As shown in Fig. 1a, HOXA5 and HOXB6 mRNA levels were downregulated in SW480 and Caco2 cells transfected with pre-MIR196B, when compared to levels in the un-transfected control cells (Fig. 1a).

Fig. 1
figure 1

MIR196B directly down-regulates HOXA5 and HOXB6 expression. (a) Validation of MIR196B target genes. The expression of HOXA5 and HOXB6 was downregulated by MIR196B overexpression in SW480 or Caco2 cells. CRC cells were transfected with precursor MIR196B. Total RNA extracts were prepared 48 h after transfection, and HOXA5 and HOXB6 mRNA levels were examined by qRT-PCR. Values were normalized to levels in un-transfected Caco2 cells in three independent experiments. (b) Sequence alignment of the MIR196B seed region and the putative binding sites in the HOXA5 and HOXB6 3′ UTRs. The wild-type (WT) or mutant (MT) 3′ UTR of the HOXA5 or HOXB6 gene, containing the MIR196B target sequence, was cloned into a pmirGLO Dual-Luciferase miRNA Target Expression Vector. Each luciferase reporter construct was co-transfected into SW480 cells with either pre-MIR1 as a negative control or pre-MIR196B. After 24 h, luciferase activity was measured in the cell extracts. In the presence of the WT HOXA5 (c) or HOXB6 (d) 3′ UTR, transfection with pre-MIR196B inhibited luciferase activity (P < 0.01; two-sided t-test). Inhibition was not observed with the reporter constructs containing the MT 3′ UTR target sequences or with MIR1 transfection. (e) Western blot analysis in MIR196B- or mock-transfected SW480 cells. The HOXA5 and HOXB6 bands were quantified relative to the appropriate loading controls using the ImageJ software and are shown relative to the protein level in mock-transfected cells. The data are representative of three independent experiments

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HOXA5 and HOXB6 Are Direct Targets of MIR196B

To determine whether MIR196B directly regulates HOXA5 and HOXB6 mRNA, we used a luciferase reporter system. The predicted MIR196B recognition sites in the HOXA5 and HOXB6 3′ UTRs were cloned into a pmirGLO Dual-Luciferase miRNA Target Expression Vector (pmirGLO-HOXA5 WT and pmirGLO-HOXB6 WT). We also generated mutated (MT) MIR196B binding sites by site-directed mutagenesis (pmirGLO-HOXA5 MT and pmirGLO-HOXB6 MT) (Fig. 1b). The base pairing between the MIR196B seed sites and the HOXA5 and HOXB6 mRNA targets is depicted in Fig. 1b. Subsequently, the vectors were co-transfected into SW480 cells with pre-MIR196B or pre-MIR1. MIR196B inhibited luciferase activity in cells transfected with pmirGLO-HOXA5 WT and pmirGLO-HOXB6 WT (~27% and ~30%, respectively, vs. controls) (Fig. 1c and d). In contrast, transfection of MIR196B did not reduce luciferase activity in cells transfected with pmirGLO-HOXA5 MT or pmirGLO-HOXB6 MT vectors, and transfection of pre-MIR1 did not reduce the luciferase activity in cells transfected with the WT or MT constructs (Fig. 1c and d).

MIR196B Regulates HOXA5 and HOXB6 Expression in CRC Cells

We investigated whether MIR196B regulated HOXA5 and HOXB6 protein expression. MIR196B was transfected into SW480 cells, and western blot analyses were performed using cells isolated 48 h after transfection. HOXA5 and HOXB6 expression levels were significantly downregulated in cells transfected with MIR196B (Fig. 1e). These results suggest that the expression of HOXA5 and HOXB6 is directly regulated by MIR196B in CRC cells.

GLTP Is a Direct Target of MIR196B

To demonstrate a direct interaction between the GLTP 3′ UTR region and MIR196B, we cloned the WT GLTP 3′ UTR region, which is predicted to interact with MIR196B into a luciferase vector (Fig. 2a). Luciferase activity was reduced by approximately 25%, when cells were co-transfected with pre-MIR196B (P < 0.01, Fig. 2b). As a control experiment, we cloned a mutated GLTP 3′ UTR sequence lacking ten of the complementary bases. As expected, repression of luciferase activity was abolished when the interaction between MIR196B and its target 3′ UTR was disrupted (Fig. 2b). As additional control experiments, MIR1 instead of MIR196B was co-transfected with the WT and MT GLTP 3′ UTR constructs. Transfection of pre-MIR1 did not affect the luciferase activity of either construct (Fig. 2b). These results suggest that MIR196B directly regulates GLTP expression in CRC cells.

Fig. 2
figure 2

GLTP is a direct target gene of MIR196B. (a) Sequence alignment of the MIR196B seed region and the putative binding sites in the GLTP 3′ UTRs. The wild-type (WT) or mutant (MT) 3′ UTR of the GLTP gene containing the MIR196B target sequence was cloned and transfected in CRC cells. (b) After 24 h, luciferase activity was measured in the cell extracts. In the presence of the WT GLTP 3′ UTR, transfection with pre-MIR196B inhibited luciferase activity (P < 0.01; two-sided t-test). Inhibition was not observed with the reporter constructs containing the MT 3′ UTR target sequences or with MIR1 transfection. Luciferase activity was determined using the dual luciferase assay. Results are shown as the relative firefly luciferase activity normalized to Renilla luciferase activity. (c) GLTP mRNA and protein levels in MIR196B- or mock-transfected Caco2 or SW480 cells. Total RNA or protein extracts were prepared 48 h or 72 h after transfection, and GLTP mRNA or protein levels were examined by qRT-PCR or western blot analysis, respectively (P < 0.01; two-sided t-test). (d) GLTP expression in 4 pairs of human CRC tissue samples and adjacent normal colon tissue samples. The data are representative of three independent experiments

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We also investigated whether MIR196B regulated GLTP mRNA and protein levels in SW480 cells. The GLTP mRNA level was lower in SW480 cells transfected with pre-MIR196B than in un-transfected control cells (P < 0.01, Fig. 2c). GLTP protein expression was also downregulated in MIR196B-overexpressing cells (P < 0.01, Fig. 2c).

GLTP Expression Levels in Human CRC Tissues

On the basis of the findings described above, we evaluated GLTP expression in 4 human CRC tissues and the matching normal CRC tissues by western blotting. GLTP protein expression was decreased (3 of the 4 pairs) in CRC tissues compared to adjacent non-tumorous tissues (Fig. 2d).

Discussion

MiRNAs have been implicated as important regulators of gene expression in a variety of biological processes, as well as in various diseases as tumor suppressor genes or oncogenes [2,3,4]. In our previous study, we showed that MIR196B expression was up-regulated in human CRC tissues compared to matching healthy CRC tissues, and also suggested that MIR196B regulates FAS expression levels in CRC cells [8]. We used mRNA microarray analysis and bioinformatics tools to identify MIR196B target genes in CRC. Of the 18 genes identified in our previous study, five genes (HOXA5, HOXA9, HOXB6, HOXB7, and HOXC8) were members of the HOX gene family. This is not surprising because MIR196B is located between HOXA9 and HOXA10 on chromosome 7. Other studies have also previously identified HOXA9 [9, 13, 14], HOXB7 [15], and HOXC8 [16] as MIR196B target genes.

In this study, we showed that HOXA5 and HOXB6 are also MIR196B target genes in CRC cells (Fig. 1). HOXA5 and HOXB6 (Fig. 1a) expression was down-regulated by MIR196B overexpression in SW480 cells. Furthermore, HOXA5 and HOXB6 protein levels were also downregulated (Fig. 1e). These results suggest that the expression of HOXA5 and HOXB6 was directly regulated by MIR196B in CRC cells.

HOXA5 binds directly to a putative HOX-binding motif in the p53 promoter region. A reduction in the HOXA5 expression level correlates with the loss of p53 expression in breast cancer (Raman et al., 2000) [17]. The loss of HOXA5 gene expression in human breast cancer also correlates with the progression to higher-grade lesions [18], mammary gland homeostasis, and carcinogenesis [19]. HOXA5 participates in the developmental regulation of the lung. HOXA5−/− mice exhibited impaired postnatal lung development, indicating that HOXA5 has a critical role in lung ontogeny [20]. HOXA5 was also involved in the development and patterning of the mouse lung [21]. Furthermore, abnormal expression of HOXA5 was associated with lung tumorigenesis [22]. Recently, MIR196A has been shown to be significantly upregulated in non-small cell lung cancer (NSCLC) tissues, and that MIR196A regulates NSCLC cell proliferation, migration, and invasion, partially via the downregulation of HOXA5 [23]. HOXA5 and HOXB6 were verified as MIR196B targets in this study. Thus, MIR196B may correlate with the downregulation of HOXA5 expression in early colorectal carcinogenesis.

GLTP is a small cytosolic single-polypeptide protein that is able to catalyze the intermembrane transfer of glycosphingolipids between two membranes [24, 25]. The overexpression of human GLTP was found to dramatically alter the cell phenotype [26]. Human GLTP is located on chromosome 12 (12q24.11) and its expression is regulated by specific protein-1 (Sp1) and Sp3 [27, 28]. These results suggest that GLTP could be a significant player in cellular sphingolipid and overall lipid metabolism [29, 30]. The accumulating results suggested that glycolipids play an important role in biological functions such as recognition and cell signaling events as well as oncogenesis [31,32,33]. Although there have been many reports on glycolipid function, the exact functions and regulation of GLTP still remain unclear.

In this study, we showed that GLTP is a direct target of MIR196B in CRC cells (Fig. 2b). Our result also showed that the up-regulated MIR196B modulates the mRNA and protein levels of GLTP in CRC cells (Fig. 2c). As expected, the expression of GLTP protein is down-regulated in CRC tissues compared to adjacent non-tumorous tissues (Fig. 2d). Thus, these results suggest that the up-regulated MIR196B levels in CRC cells may regulates GLTP expression levels during colorectal carcinogenesis and therefore affect cell–cell communication or functions in CRC tissues.

Although we did not study in more detail the signaling mechanisms of HOXA5, HOXB6 and GLTP by the regulation of MIR196B, our results suggest that HOXA5, HOXB6 and GLTP were direct target genes of MIR196B in CRC cells. As the result, the up-regulated MIR196B in the CRC tissue regulate the expression levels of HOXA5, HOXB6 and GLTP during colorectal carcinogenesis. Our results could provide a valuable resource for further functional studies of HOXA5, HOXB6 and GLTP genes in the oncogenesis of various organs.