MicroRNA 375 regulates proliferation and migration of colon cancer cells by suppressing the CTGF‐EGFR signaling pathway

Abstract

MicroRNA 375 (MIR375) is significantly down regulated in human colorectal cancer (CRC) tissues; we have previously identified MIR375 as a colon cancer associated microRNA (miRNA). We identified putative MIR375 target genes by comparing the mRNA microarray analysis data of MIR375‐overexpressing cells with the candidate MIR375 target genes predicted by public bioinformatic tools. We investigated that the connective tissue growth factor (CTGF) is a direct target gene of MIR375. Expression of CTGF, a ligand of epidermal growth factor receptor (EGFR), was markedly enhanced in human CRC tissues in comparison with the corresponding normal colon tissues. We demonstrated that the expression levels of molecules in EGFR signaling pathways were regulated by MIR375 in colorectal cells. Using immunohistochemistry and the xenograft of MIR375‐overexpressing colorectal cells in mice, we showed that MIR375 regulates cell growth and proliferation, angiogenesis, cell migration, cell cycle arrest, apoptosis, and necrosis in colon cells. Furthermore, results of MIR375 overexpression and cetuximab treatment indicated that the apoptosis and necrosis in colon cells were synergistically enhanced. Our results suggest that the down‐regulation of MIR375 modulates EGFR signaling pathways in human colorectal cells and tissues by increasing CTGF expression; therefore, MIR375 may have a therapeutic value in relation to human CRC.

Abbreviations

3′UTR

3′ untranslated region

CRC

colorectal cancer

CTGF

connective tissue growth factor

EGFR

epidermal growth factor receptor

IC₅₀

50% inhibitory concentration

miRNA

microRNA

PCR

polymerase chain reaction

PI

propidium iodide

qRT‐PCR

quantitative real‐time polymerase chain reaction

siRNA

small interfering RNA

WT

wild type

Colorectal cancer (CRC) is a common malignant tumor and is the third leading cause of cancer‐related mortality worldwide.1, 2 Overall survivals of patients with advanced CRC have not improved much in spite of significant advances in the management of CRC. Therefore, there is a need for further exploration of the molecular mechanisms underlying CRC tumorigenesis and for identification of the genes that are crucial for this deadly cancer.

MicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs that bind to the 3′ untranslated region (3′UTR) of their target mRNAs, leading to mRNA degradation or inhibiting translation.3 Accumulating evidence suggests that miRNAs act as tumor suppressors or oncogenes by targeting genes involved in cell proliferation, survival, apoptosis and metastasis.4-6 Actually, miRNAs have been shown to play important roles in many types of cancer.7

MIR375 is encoded by the chromosomal region 2q35 in humans. MIR375 has dual functions as a tumor suppressor8, 9 and an oncogene.10, 11 These dual functions of MIR375 depend on the targets. Recent expression profiling studies have shown that the expression of MIR375 is significantly reduced in various types of cancer including CRC.12 In our previous study, MIR375 was identified both as a CRC13 and a dextran sulfate sodium (DSS)‐induced mice colitis14 associated miRNA by miRNA expression profiling of CRC tissues versus healthy colorectal tissues and DSS‐induced colitis versus healthy colons, respectively. MIR375 was found to be significantly down‐regulated in both CRC and DSS‐induced colitis tissues.13, 14

The connective tissue growth factor (CTGF, also known as CCN2, NOV2, HCS24 or IGFBP8) was discovered during the screening of a cDNA expression library from human vein endothelial cells.15 CTGF is considered a multifunctional signaling modulator involved in a wide variety of biological and pathological processes, including cell proliferation, adhesion, migration, and extracellular‐matrix synthesis and has also been identified as a promitogenic and proangiogenic cofactor.16-20 CTGF is a predominant regulator activated during CRC tumorigenesis and metastasis.21

In this study, we identified the target genes of MIR375, and analyzed their function in colon cancer cells or human colorectal tissues. We identified CTGF as a MIR375 target gene in CRC and analyzed its functions in colon cancer cell lines. Overall, we demonstrated that MIR375 regulates cell proliferation and migration in colon cancer cells by suppressing the CTGF‐epidermal growth factor receptor (EGFR) signaling pathway.

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 (WKIRB‐201310‐BR‐00 l), we obtained colon cancer tissue samples from 11 patients with colon cancer (7 males and 4 females) and rectal cancer tissue samples from 2 patients with rectal cancer (2 males). The mean ages of the colon cancer patients and rectal cancer patients were 65.6 and 72 years, respectively. Four colon cancer tissue samples and matching healthy colon tissue samples (2 males and 2 females) were used to validate the endogenous MIR375 expression level. In addition, 1 separate colon cancer tissue sample with a matching healthy colon tissue sample and 2 rectal cancer tissue samples with matching healthy rectal tissue samples were used to assess CTGF mRNA expression. Six separate colon cancer tissue samples and matching healthy colon tissue samples (4 males and 2 females) were used to analyze CTGF protein expression levels.

Cells culture and reagents

The human CRC cell lines Caco2, SW480, HT29 and HCT116 were obtained from Korea Cell Line Bank (KCLB, Seoul, Korea) or American Type Culture Collection (ATCC, Manassas, VA). SW480, HCT116 and HT29 cells were cultured in RPMI 1640 (HyClone, Logan, UT) supplemented with 10% FBS in a humidified atmosphere containing 5% of CO₂ at 37°C. Caco2 cells were cultured in α‐MEM (HyClone) supplemented with 20% FBS in a humidified atmosphere containing 5% of CO₂ at 37°C.

RNA extraction and quantitative real‐time PCR

Total RNA was isolated with the TRIzol reagent (Invitrogen, Carlsbad, CA). After digestion with DNase and cleanup, RNA samples were quantified, aliquoted, and stored at −80°C. Total RNA samples that were isolated from the tissue samples and/or cultured cells were used as a template to synthesize cDNA for quantitative real‐time polymerase chain reaction (qRT‐PCR) by means of a StepOne Real‐time PCR system (Applied Biosystems, Foster City, CA).

The differential miRNA expression patterns were validated by 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). qRT‐PCR with the 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 served as endogenous controls for qRT‐PCR of miRNA and mRNA, respectively. Each sample was analyzed by qRT‐PCR in triplicate. Primers for both qRT‐PCR and TaqMan analysis are listed in Supporting Information Table S1.

Transfection of oligonucleotides

Molecules that mimic endogenous MIR375 (hsa‐miR‐375, Pre‐miRNA precursor AM17100), anti‐MIR375 (Anti‐hsa‐miR‐375, Pre‐miRNA precursor AM17000), and negative control were synthesized commercially (Ambion, Austin, TX) and transfected at 50 nM. Transfection of oligonucleotides was carried out according to our previously described methods.13, 14 Negative control small interfering RNA (siRNA; Ambion, Austin, TX) and CTGF siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected according the instructions of the supplier.

mRNA expression profiling to find MIR375 target genes

SW480 and Caco2 cells were transfected with MIR375 or MIR1 as a control. Total RNA was isolated 48 hr after the transfection, amplified, and purified using the Illumina TotalPrep RNA Amplification Kit (Ambion) to obtain biotinylated complementary RNA. Hybridization of the samples, signal detection, array scanning, data analysis, and filtering were carried out by our previously described methods.13, 14

A luciferase reporter assay

Wild‐type or mutant fragments of the 3′UTR of CTGF containing the predicted binding site for MIR375 were amplified by PCR using the primer set shown in Supporting Information Table S1. Analysis of the results of the luciferase assays was carried out according to our previously described method.13, 14

Bioinformatic analysis of miRNA

The TargetScan (http://www.targetscan.org) and miRWalk (http://www.umm.uni‐heidelberg. de/apps/zmf/mirwalk/index.html) algorithms were used to identify putative targets of MIR375.

Protein extraction and western blot analysis

Membranes with proteins were then incubated overnight at 4°C with primary antibodies to EGFR, p‐AKT1, KRAS and p‐ERK1/2, IGF1R and YAP1 (Cell Signaling Technology, Carlsbad, CA), CTGF, AKT, ERK1/2, PIK3CA, BRAF, TGFB1 and PKCA (Santa Cruz Biotechnology, Santa Cruz, CA). Protein extraction and western blot analysis were carried out according to our previously described methods.13, 14

The cell viability assay and migration assay

The cell viability assay and migration assay were carried out according to our previously described methods.13, 14

Flow cytometric analysis

After transfection with MIR375 or siCTGF or control in 6‐well plates for 72 hr, we analyzed the cell cycle in HCT116 and HT29 cells (10⁵/well) by flow cytometry as follows. Five hundred microliters of trypsin was added for detachment of the cells, which were washed twice with PBS before the cell pellets were resuspended in PBS and fixed in 70% ice‐cold ethanol (v/v) overnight at −20°C. The fixed cells were incubated in PBS containing 50 µg/mL RNase A, 0.25% Triton X‐100 and 0.1 mM EDTA for 30 min at 37°C. Propidium iodide (PI) was added to the cell suspension at the final concentration of 100 μg/mL, and the mixture was incubated for 15 min while covered with aluminum foil at room temperature. The cell cycle was analyzed by flow cytometry using the Cell Quest software and a FACSCalibur instrument (Becton Dickinson). Apoptosis and necrosis were analyzed by flow cytometry with the Annexin V‐fluorescein isothiocyanate Apoptosis Detection Kit according to our previously described method,13 using the HCT116 or HT29 cells transfected with MIR375, siCTGF, or control mock. Cetuximab (Erbitux; Merck, Darmstadt, Germany) was diluted with the medium to obtain the required final concentration before each experiment. Cetuximab (70 µg/mL) was added 24 hr after the transfection of MIR375 (50 nM), and the HCT116 or Caco2 cells were then incubated for 48 hr.

Transwell migration assays

The Cytoselect 24‐well Cell Migration Assay Kit (8 μm, CBA100, Cell Biolabs, San Diego, CA) was used to assess migration of the cells. The cells suspension was placed into the top chamber of each insert. The Transwell chambers with upper and lower culture compartments were separated by polycarbonate membranes with 8‐μm pores. Five hundred microliters of the RPMI 1640 medium containing 10% FBS was added into the bottom chamber to act as a chemoattractant, and the plate was incubated for 24 hr at 37°C in an incubator with a humidified atmosphere containing 5% of CO₂. After 24 hr, the cells that did not migrate were removed from the upper side of the Transwell membrane filter inserts using a cotton‐tipped swab. Migrating cells on the bottom side were stained with 400 μL of 0.1% crystal violet and washed several times in a beaker with water.

The xenograft model based on nude mice

Athymic female BALB/c nude mice (8 weeks old, 19–21 g) were purchased from Charles River Technology (Boston, MA) through Orient Bio Inc. (Sungnam, Gyeonggi, South Korea). The mock control or MIR375 or siCTGF were prepared by preincubating 100 nmol of miRNAs with 10 µL of Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) for 15 min and by mixing with HCT116 or HT29 (10⁷ cells) in a final volume of 200 µL in the RPMI 1640 medium. Then, the transfected cells were injected subcutaneously into both sides of the posterior flank of mice. Tumor progression was evaluated every 5 days by means of digital calipers and the formula for volume [(longest diameter) × (shortest diameter)2 × 0.525]. On Day 20, the tumor‐bearing mice were euthanized and subjected to necropsy where tumors were excised with a scalpel from the circumferential tissues and fixed in 10% neutrally buffered formalin, were embedded in paraffin, and 4‐µm slices were prepared and stained with H&E. All the mice were housed and maintained under specific pathogen‐free conditions in mesh cages under light illumination for 12 hr a day. All surgical and care procedures that were administered to the animals were in accordance with the Animal Care Committee of Wonkwang University (WKU14‐47).

Immunohistochemistry

Immunohistochemistry assay was carried out according to our previously described methods.14 The antibodies and dilutions were as follows: proliferating cell nuclear antigen (PCNA), a biotinylated monoclonal antibody clone PC10 (Zymed's PCNA Staining Kit, Invitrogen) 1:1500; the antigen identified by monoclonal antibody Ki 67 (Ki67), rabbit clone SP6 (Thermo Fisher Scientific, Fremont, CA) 1:150; and platelet endothelial cell adhesion molecule 1 (PECAM‐1, also known as CD31) Ab‐1, mouse clone JC/70 A (Thermo Fisher Scientific, Fremont, CA), 1:125. The number of PCNA or Ki67‐labeled nuclei was determined by counting the Ki67‐positive cells in at least 6 high‐magnification (40×) visual fields under a microscope in each slice. CD31‐positive areas were analyzed in at least 6 high‐magnification (40×) visual fields in each slice and processed in the ImageJ software (version 1.44; http://rsbweb.nih.gov/ij/index.html).

Statistical analysis

Each experiment was repeated at least 3 times, and consistent results were obtained. All of the data were expressed as mean ± standard deviation (SD). The differences between groups were analyzed by means of the GraphPad Prism 5.0 statistical software (GraphPad Software, San Diego) or Student's t test. Differences with a p values <0.05 were regarded as statistically significant.

Results

The MIR375 expression level in colon cancer tissues

We found that MIR375 is down‐regulated in human CRC tissue. To confirm this result, we compared MIR375 expression in 4 human healthy colon tissue samples and the matching colon cancer tissues by qRT‐PCR. MIR375 expression levels were significantly reduced in CRC tissue (p < 0.01; Fig. 1a).

Figure 1

Endogenous MIR375 expression in CRC tissues and CRC cell lines. (a) The expression of MIR375 was validated using four colon cancer tissue samples and matching healthy colon tissue samples. Expression levels were normalized to colon‐specific RNU48. The data are presented as the relative levels (ΔΔCT method) of the MIR375 in colon cancer tissues. P1, P2, P3 and P4 indicate patients with colon cancer. (b) The relative endogenous MIR375 expression levels in three CRC cell lines. The data are presented as a fold change in HT29 or SW480 cells relative to HCT116 cells. The data represent mean ± SD of three independent experiments, each carried out in duplicate. The p values were calculated by the t test (**p < 0.01).

mRNA expression profiling in MIR375‐overexpressing cells

To determine the levels of endogenous MIR375 in Caco2, HCT116, HT29 and SW480 cells, we carried out qRT‐PCR analysis using the total RNA isolated from the three different cell lines. As shown in Figure 1b, the MIR375 level was the highest in HT29 cells and lower in HCT116 and Caco2 cells (Fig. 1b). Hypermethylation of MIR375 has been demonstrated in CRC cell lines including HCT116 and SW480. Down regulation of MIR375 in HCT116 and SW480 cells compared with HT29 cells is due to hypermethylation of MIR375 in HCT116 and SW480 cells. However HT29 cells have no MIR375 methylation.22 To identify the genes down‐regulated by MIR375 overexpression, pre‐MIR375 was transfected into Caco2 or SW480 cells. Increased expression of MIR375 was observed 24 hr after transfection of HCT116, HT29, SW480 or Caco2 cells, thus confirming the transfection efficiency (Supporting Information Fig. S1A). The cells were harvested, and mRNAs were isolated 48 hr after the MIR375 transfection for mRNA expression profiling analysis by means of the Illumina HumanHT‐12 v4 Expression BeadChip. We identified 31 genes whose expression was down‐regulated 1.5‐fold in the cells overexpressing MIR375 (Supporting Information Table S2).

Identification of MIR375 target genes

For this purpose, we compared the 31 candidate target genes identified by the mRNA microarray analysis with the candidate MIR375 target genes predicted by the TargetScan and miRWalk algorithms. Of the 31 genes, 13 were finally identified as putative targets of MIR375 (Table 1). Among them, we focused on the CTGF gene in this study. The endogenous CTGF level was almost similar among the SW480, HT29 and HCT116 cell lines (Supporting Information Fig. S1B). Next, we tested whether MIR375 regulates CTGF mRNA and protein levels in HT29 cells. The CTGF mRNA level was lower in HT29 cells transfected with pre‐MIR375 than in untransfected control cells (p < 0.01; Fig. 2a). The cellular and extracellular (secreted into culture media) CTGF protein expression levels were also significantly reduced in MIR375‐overexpressing SW480 and HT29 cells (p < 0.01; Fig. 2b).

Figure 2

CTGF is a direct target of MIR375. (a) qRT‐PCR analysis of CTGF expression in HT29 cells. The data are presented as the fold change in MIR375‐overexpressing cells relative to untransfected cells. The experiment was performed in duplicate and repeated five times. (b) The cellular and extracellular CTGF protein levels in MIR375‐overexpressing SW480 and HT29 cells and untransfected cells. (c) CTGF mRNA and (d) protein expression in six pairs of human colon cancer tissue samples and adjacent healthy colon tissue samples. (e) Sequence alignment of the WT and mutated (MT) MIR375 target site in the 3′UTR of CTGF. (f) A luciferase reporter plasmid containing the WT or MT CTGF 3′UTR was cotransfected into HT29 cells with pre‐MIR1 as a negative control or pre‐MIR375. The results are shown as the relative firefly luciferase activity normalized to Renilla luciferase activity. Three independent experiments were conducted, and p values were calculated by the t test (**p < 0.01).

Table 1. The putative target genes of MIR375 identified and predicted by both the microarray analysis from the MIR375 overexpressed cells and the bioinformatics methods

Gene symbol	Accession	Gene name	Chromosome location	Functions
ACTRT3	NM_032487.3	Actin related protein T3	3q26.2	Cytoskeletal protein
C14orf159	NM_024952.5	Chromosome 14 open reading frame 159	14q32.11	–
CADM1	NM_014333.3	Cell adhesion molecule 1	11q23.2	Receptor
CEPT1	NM_006090.3	Choline/ethanolamine phosphotransferase 1	1p13.3	Transferase
CTGF	NM_001901.1	Connective tissue growth factor	6q23.1	Signaling molecule
CTPS1	NM_001905.1	CTP synthase 1	1p34.1	Enzyme (synthase)
GRM8	NM_000845.1	Glutamate receptor, metabotropic 8	7q31.3‐q32.1	Receptor
MOB1A	NM_018221.3	MOB kinase activator 1A	2p13.1	Select regulatory molecule
MTDH	NM_178812.2	Metadherin	8q22.1	Oncogenesis
PRDX1	NM_181696.1	Peroxiredoxin 1	1p34.1	Antioxidant enzymes
PTPMT1	NM_175732.1	Protein tyrosine phosphatase, mitochondrial 1	11p11.2	–
VASN	NM_138440.2	Vasorin	16p13.3	Receptor
WBP1L	NM_001083913.1	WW domain binding protein 1‐like	10q24.32	–

CTGF expression in human CRC tissues

On the basis of the findings described above, we evaluated CTGF expression in 6 human colon cancer tissues and the matching healthy colon tissues by qRT‐PCR and by western blotting. CTGF mRNA expression was increased in all colon cancer tissue samples when compared to the expression in healthy colon tissues (p < 0.01, Fig. 2c). CTGF protein expression was also increased (5 of the 6 pairs) in colon cancer tissues (Fig. 2d).

CTGF is a direct target of MIR375

To demonstrate a direct interaction between the CTGF 3′UTR and MIR375, we cloned the WT CTGF 3′UTR region (predicted to interact with MIR375) into a luciferase reporter vector (Fig. 2e). Luciferase activity was reduced by ∼28%, when the cells were co‐transfected with pre‐MIR375 (p < 0.01, Fig. 2f). As a control experiment, we cloned a mutated CTGF 3′UTR sequence lacking 8 of the complementary bases (Fig. 2e). As expected, repression of the luciferase activity was abrogated when the interaction between MIR375 and its target 3′UTR was disrupted (Fig. 2f). As additional control experiments, MIR1 instead of MIR375 was cotransfected with the WT and mutated CTGF 3′UTR constructs. Transfection of pre‐MIR1 did not affect the luciferase activity of either construct (Fig. 2f).

MIR375 regulates CTGF‐EGFR signaling pathways

We then determined which receptor (EGFR or IGF1R) is mainly regulated by means of MIR375‐transfected or CTGF siRNA (siCTGF)‐transfected colorectal cells. EGFR was more significantly affected (Fig. 3a) by MIR375 or siCTGF overexpression than IGF1R was (Supporting Information Fig. S3A). This result led us to focus on the CTGF‐EGFR signaling in colon cancer cells.

Figure 3

MIR375 regulates CTGF/EGFR‐mediated signaling in colon cancer cell lines. (a) Western blot analysis of EGFR‐regulated molecules AKT and p‐AKT in HT29, SW480 or HCT116 cells. We conducted three independent experiments, and the p values were calculated by the t test (*p < 0.05; **p < 0.01). (b) Western blot analysis of KRAS or ERK1/2 or p‐ERK1/2 in HT29 and HCT116 cells. We conducted three independent experiments, and the p values were calculated by the t test (*p < 0.05; **p < 0.01). (c) Silencing of CTGF decreases EGFR, p‐EGFR, AKT and p‐AKT levels in HCT116 cells, (d) but not KRAS, ERK or p‐ERK expression in HT29 cells. We conducted three independent experiments, and the p values were calculated by the t test (ns: not significant, *p < 0.05; **p < 0.01). (e) The expression levels of KRAS, BRAF, ERK1/2 and p‐ERK1/2 are not changed in CaCO₂ cells by CTGF silencing. We performed three independent experiments, and the p values were calculated by the t test (ns: not significant, *p < 0.05).

To determine the functional interaction between MIR375 and CTGF in HT29 cells (KRAS WT, and BRAF and PIK3CA mutated), SW480 cells (BRAF and PIK3CA WT and KRAS mutated) and HCT116 cells (BRAF WT, and KRAS and PIK3CA mutated; Ahmed et al., 2013), we analyzed expression of the proteins involved in the CTGF‐EGFR cellular signaling by western blot analysis. EGFR, p‐EGFR, PIK3CA, AKT and p‐AKT protein expression levels were reduced by MIR375 transfection in HT29, SW480 and HCT116 cells, respectively (Fig. 3a). These results indicated that MIR375 regulates the CTGF‐EGFR‐PIK3CA‐AKT pathway in colon cancer cells. KRAS protein expression was almost unchanged by the MIR375 transfection in both HT29 and HCT116 cells, respectively (Fig. 3b).

ERK1/2 (p < 0.01) and p‐ERK1/2 (p < 0.01) protein expression was decreased significantly by MIR375 transfection of HT29 and HCT116 cells, respectively (Fig. 3b). These results indicated that there is another signaling pathway for EGFR and ERK1/2 that does not involve KRAS.

CTGF gene silencing downregulates EGFR‐AKT‐p‐AKT signaling

On the basis of the above results, we tested whether CTGF gene silencing by the siCTGF duplex affects the EGFR‐AKT or EGFR‐KRAS‐ERK signaling pathway. Western blotting results showed that CTGF (p < 0.01), EGFR (p < 0.05 and 0.01), p‐EGFR (p < 0.05 and 0.01), AKT (p < 0.05) and p‐AKT (p < 0.01 and 0.05, respectively) protein expression levels were markedly down‐regulated by siCTGF transfection in HCT116 and SW480 cells, respectively (Fig. 3c). KRAS and ERK1/2 expression levels were not decreased by CTGF gene silencing in HT29 cells (Fig. 3d). These findings suggested that CTGF regulates only the EGFR‐PIK3CA‐AKT signaling cascade not the EGFR‐KRAS‐ERK pathway.

MIR375 regulates both EGFR signaling pathways

To confirm the MIR375‐ or CTGF‐mediated pathways in CRC, we quantified expression of relevant proteins in Caco2 cells (KRAS, BRAF and PIK3CA WT) after MIR375 or siCTGF transfection. KRAS, BRAF, ERK1/2 and pERK1/2 expression levels were not changed by siCTGF transfection in Caco2 cells (Fig. 3e). This result clearly indicated that CTGF regulates only the EGFR‐PIK3CA‐AKT signaling pathway not the EGFR‐KRAS‐ERK signaling pathway. BRAF, ERK1/2 and p‐ERK1/2 expression levels were significantly reduced by MIR375 overexpression (p < 0.05; Fig. 3e). Although KRAS was also slightly down‐regulated in Caco2 cells by MIR375 overexpression, this change was not statistically significant (p = ns; Fig. 3e).

MIR375 inhibits CRC cell viability and cell cycle progression

We explored the biological functions of MIR375 in CRC cells. MTT assays showed that cell viability was stably reduced by MIR375 transfection in colon cancer cell lines SW480 (p < 0.05), HT29 (p < 0.01) and HCT116 (p < 0.05; Fig. 4a). In addition, forced overexpression of MIR375 resulted in significant accumulation of the G₁ population among HCT116 cells (p < 0.01) and HT29 cells (p < 0.05), respectively (Fig. 4b). This result indicated that MIR375 inhibited cell cycle progression in colon cancer cells.

Figure 4

MIR375 regulates cell viability, cell cycle, cell apoptosis or necrosis, and cell migration in colon cancer cell lines. (a) Effects of MIR375 on viability of HT29, HCT116 and SW480 cells. Cell viability was determined by the MTT assay. (b) Flow cytometry was used to compare the cell cycle progression between mock‐ and MIR375‐transfected HCT116 and HT29 cells. The results are expressed as mean ± SD of three independent experiments. (c) Flow cytometric analysis of apoptosis or necrosis in MIR375‐transfected HT29 and HCT116 cells. The number in each box indicates the percentage of annexin V‐positive and/or PI‐positive cells. The experiments were repeated three times with duplicates. (d) The scratch wound assay was conducted in mock‐ and MIR375‐transfected HCT116 cells. The migration distance was measured at 0, 24, 48 and 72 hr after the cells were scratched. The results are expressed as mean ± SD of three independent experiments. (e) For the migration assay, we used 24‐well Transwell chambers with upper and lower culture compartments separated by polycarbonate membranes with 8 μm pores. Dye‐binding cells were quantified at by measuring absorbance at 560 nm (A₅₆₀). Compared with the mock control, the migration of MIR375‐transfected cells (upper panel) or siCTGF‐transfected cells (lower panel) was reduced. These experiments were performed three times. All p values were calculated by the t test (*p < 0.05; **p < 0.01). (f) The synergistic effects of MIR375 and cetuximab in terms of apoptosis and necrosis in colon cancer cell lines. Flow cytometric analysis of apoptosis and necrosis in MIR375‐transfected and/or cetuximab‐treated HCT116 cells. Cetuximab (70 µg/mL) with a fresh medium was added 24 hr after transfection of MIR375 (50 nM); then, HCT116 cells were incubated for 48 hr without a change of the medium. The number in each box indicates the percentage of annexin V‐positive and/or PI‐positive cells. Experiments were repeated three times with duplicates.

Effects of MIR375 transfection on apoptosis in colon cancer cells

We evaluated apoptosis rates in MIR375 or mock‐transfected colon cancer cell lines. As shown in Figure 4c and Supporting Information Figure S4A, apoptosis and necrosis in HT29 cells was stably enhanced after MIR375 transfection (Fig. 4c, p < 0.01) and siCTGF transfection (Supporting Information Fig. S4A, p < 0.01). The stably increased apoptotic and necrotic cell numbers were also observed in MIR375‐transfected HCT116 cells (Fig. 4c, p < 0.01) and siCTGF‐transfected HCT116 cells (Supporting Information Fig. S4A, p < 0.01).

The migratory ability of MIR375‐ or siCTGF‐transfected colon cancer cells

As shown in Figure 4d, in the scratch wound assay, the cell migratory ability was significantly inhibited in HCT116 cells transfected with MIR375 (Fig. 4d). The cell migration ability in MIR375‐transfected cells was significantly decreased 48 hr (p < 0.05) and 72 hr (p < 0.01) after the transfection (Fig. 4d). The ability of HCT116 cells to migrate through the insert membrane was also significantly inhibited by MIR375 (Fig. 4e, upper panel, p < 0.01) or by siCTGF (Fig. 4e, lower panel, p < 0.05).

Effects of MIR375 on tumor growth in mice with a xenograft of colon cancer cells

To study the effects of MIR375 on tumor growth in vivo, we used a xenograft tumor model consisting of athymic nude mice with subcutaneously implanted HCT116 or HT29 cells. After 20 days of transfection, mean tumor volume of MIR375‐transfected cells was 332.7 ± 26.2 mm³ (HCT116) and 255.45 ± 15.05 mm³ (HT29) and was significantly smaller than that in mock cells (532.9 ± 25.2 and 357.9 ± 20.1 mm³, respectively; p < 0.01; Figs. 5a and 5b). As compared to the mock control, the average tumor weight of MIR375‐transfected cancer cells was significantly reduced in both cell lines: HCT116 and HT29 (Fig. 5b, p < 0.01). We also compared with the mock control, the average tumor volume of siCTGF‐transfected cancer cells was significantly reduced in both cell lines: HCT116 and HT29 (Supporting Information Fig. S4B). This result indicated that the proliferative ability was reduced by MIR375 overexpression.

Figure 5

MIR375 inhibits colon cancer cell growth and angiogenesis in a xenograft mouse model. (a) MIR375 inhibits colon cancer cell growth in vivo. An image of xenograft tumors derived from MIR375‐ or mock‐transfected colon cancer cells: HCT116 (upper panel) or HT29 (lower panel) cells. We conducted three independent experiments using 2–3 mice (for HCT116 cells) and 3–4 mice (for HT29 cells), and the p values were calculated by the t test (p < 0.01). (b) Tumor volume and weight of MIR375‐transfected HCT116 and HT29 cells in nude mice. (n = 6, mean ± SD). The p values were calculated by the t test (*p < 0.05; **p < 0.01). (c) Expression of cell proliferation marker PCNA and Ki‐67 in tumors after subcutaneous transplantation of MIR375‐ or mock‐transfected HCT116 and HT29 cells (magnification ×400). (d) Expression of endothelial cell marker CD31 in tumors after subcutaneous transplantation of MIR375‐ or mock‐transfected HCT116 or HT29 cells (magnification ×400). The p values were calculated by the t test (*p < 0.05). (e) The EGFR signaling is regulated by MIR375 in human colon cancer cells. The diminished MIR375 expression leads to YAP1 up‐regulation and resulted in increased transcription for CTGF mRNA, and gave rise to the increasing translation of CTGF in the cytoplasm and extracellular regions. Up‐regulated CTGF binds to the extracellular domain of EGFR. Activated EGFR leads to activation of the oncogene PIK3CA, which is also directly down‐regulated by MIR375. Activated PIK3CA activates AKT and several downstream effectors. However, EGFR activates the oncogene KRAS, which in turn activates the oncogene BRAF, then MEK and ERK1/2, and leads to expression of growth‐promoting genes in the nucleus. While CTGF regulates only the EGFR‐PIK3CA‐AKT signaling pathway, MIR375 regulates both EGFR‐PIK3CA‐AKT and EGFR‐KRAS (or IGF1R‐PKC)‐BRAF‐MEK‐ERK signal pathways in colon cancer cells. Consequentially, the diminished MIR375 expression in colon cancer leads to cell survival, proliferation, migration, and angiogenesis. Solid lines and square dotted lines indicate MIR375‐mediated signaling and CTGF‐dependent signaling, respectively.

Histopathology in mice with a xenograft of MIR375‐transfected colon cancer cells

The tumors were analyzed histologically using H&E and immunohistochemically using antibodies against PCNA, Ki67, and CD31. PCNA‐ or Ki67‐immunoreactive cells were found in the xenografted tumors, and the PCNA‐ or Ki67‐positive cells were quantified by histological analysis. MIR375‐transfected tumors contained significantly decreased numbers of PCNA‐ or Ki67‐positive cells compared with mock control tumors for both HCT116 and HT29 cells (p < 0.01; Fig. 5c).

Effects of MIR375 on tumor angiogenesis in mice with a xenograft of colon cancer cells

We checked the effect of MIR375 on angiogenesis in mice bearing HCT116 or HT29 cells xenografts. As for angiogenesis, CD31 immunohistochemical analysis revealed that the blood vessel network was well developed in the tumors from the mock control, whereas the development of the blood vessel network appeared to be inhibited by MIR375 for both HCT116‐ and HT29‐derived tumors (p < 0.05; Fig. 5d). These results suggested that the decreased cell density may be due to impairment of angiogenesis by MIR375. We also checked the effect of CTGF on proliferation ability and angiogenesis in mice bearing HT29 cells xenografts. siCTGF‐transfected tumors contained significantly decreased numbers of Ki67‐positive cells (p < 0.05) and CD31‐positive blood vessel networks (p < 0.01) compared with mock control tumors for both HT29 cells (Supporting Information Fig. S4C).

The synergistic effects of MIR375 and cetuximab on apoptosis or necrosis in colon cancer cells

We examined the IC₅₀ values of cetuximab using the MTT assay. The IC₅₀ of cetuximab for HCT116 cells was 350 ± 0.02 µg/mL for 48 hr incubation. HCT116 cells were treated with 70 µg/mL cetuximab, at a 1/5 concentration of IC₅₀. We tested whether MIR375 plays a functional role in cetuximab‐induced apoptosis or necrosis. The apoptosis and necrosis rates were significantly increased (p < 0.05) in HCT116 cells after cetuximab treatment as compared with untreated control cells (Fig. 4f). MIR375 overexpression increased (p < 0.05) the cetuximab‐induced apoptosis and necrosis in HCT116 cells compared with the cells treated with cetuximab alone (Fig. 4f). These results showed that overexpression of MIR375 upregulated the cetuximab‐induced apoptosis and necrosis in colon cancer cells (Fig. 4f).

Discussion

In this study, we showed that MIR375 expression was down‐regulated in human colon cancer tissues compared with matching healthy colon tissues (Fig. 1a). In addition to our results, down‐regulation of MIR375 has been demonstrated in several types of cancer, such as HCC, gastric cancer, melanoma and glioma.23-26 MIR375 is up‐regulated in other tumors such as prostate cancer11 and breast cancer.27 In our previous study, MIR375 was identified both as a CRC13 and a DSS‐induced mice colitis14 associated miRNA by miRNA expression profiling of CRC tissues versus healthy colorectal tissues and DSS‐induced colitis versus healthy colons, respectively. In present study, we found that MIR375 regulates CTGF and the CTGF‐EGFR signaling pathway by directly down‐regulating CTGF expression in colorectal cells (Figs. 2 and 3). These data suggest that the CTGF‐EGFR signaling cascade is up‐regulated in CRC as a result of reduced MIR375 expression.

In CRC, high expression of CTGF correlates with an advanced clinical stage and lymph node metastasis.28 Another study also showed that CTGF mRNA is up‐regulated in CRC compared to healthy colon tissues but is down‐regulated in Dukes stage C colon cancer in comparison with Dukes stages A and B.29 Other researchers demonstrated that TAZ‐AXL‐CTGF co‐overexpression is associated with increased expression of genes that are related to colon cancer progression.30 We showed here that CTGF mRNA and protein are overexpressed in CRC tissues compared to adjacent non‐tumorous tissues (Figs. 2c and 2d), and that MIR375 directly reduces the expression of CTGF in colon cancer cells (Figs. 2e and 2f). These results suggest that MIR375 targets CTGF as a potential tumor suppressor in CRC.

EGFR is phosphorylated by the binding of CTGF and activates the downstream signaling cascades.31 The EGFR pathway is involved in several cellular responses, such as cell proliferation, migration, and differentiation.32-34 We analyzed the CTGF‐EGFR signaling cascades mediated by MIR375 in several colon cancer cells that have the most common mutations such as those in genes KRAS, PIK3CA, BRAF and TP53.35 CTGF‐EGFR signals are affected by MIR375 overexpression or by CTGF gene silencing by means of siCTGF (Fig. 3). EGFR‐PIK3CA‐AKT signaling is down‐regulated by MIR375 overexpression in HT29, SW480 and HCT116 cells (Fig. 3a) and by siCTGF in HCT116 and SW480 cells (Fig. 3c). ERK1/2 and p‐ERK1/2 expression levels are also down‐regulated by MIR375 overexpression in HT29 and HCT116 cells (Fig. 3b). Nevertheless, ERK1/2 and p‐ERK1/2 expression levels are not changed by siCTGF in HT29 and Caco2 cells (Figs. 3d and 3e). These results indicate that MIR375 regulates both the EGFR‐PIK3CA‐AKT pathway and EGFR‐BRAF‐ERK1/2 pathway, but CTGF that is affected by MIR375 is involved only in the EGFR‐PIK3CA‐AKT signaling pathway in colon cancer cells. These results led us to study the biological function of MIR375 in colon cancer cells, and we found that MIR375 can regulate cell proliferation (Fig. 4a), cell cycle progression (Fig. 4b), apoptosis and necrosis (Fig. 4c) and cell migration (Figs. 4d and 4e) via control over EGFR signaling pathways in colon cancer cells.

KRAS expression was not changed by MIR375 overexpression in HT29 and HCT116 cells (Fig. 3b). This finding prompted us to do an additional experiment involving Caco2 cells. BRAF and p‐ERK1/2 expression levels were found to be significantly reduced by MIR375 overexpression (Fig. 3e). KRAS expression was also slightly decreased by MIR375 overexpression, without statistical significance (Fig. 3e).

We also tested whether MIR375 directly regulates ERK expression. This is because ERK has a putative binding site for MIR375. Luciferase activity was not different between WT and mutated sequences of ERK 3′UTR (Supporting Information Fig. S2). These results suggest that MIR375 does not interact directly with ERK in colon cancer cells, and that unknown signals such as insulin‐like growth factor 1 receptor (IGF1R) may exist in CRC that are controlled by MIR375. Actually, IGF1R is down‐regulated by MIR375 overexpression in colon cancer cells (Supporting Information Fig. S3A). Cells with an altered IGF1R pathway seem to escape EGFR inhibitor (cetuximab)‐mediated cell death by activation of the PIK3CA pathway.36 YAP1, TGFβ1 and PKC expression levels are also down‐regulated by MIR375 overexpression in colon cancer cells (Supporting Information Fig. S3B). PIK3CA and YAP1 are 2 other target genes in colon cancer cells that have been identified as a direct target of MIR375.22, 37 PIK3CA is a key molecule in AKT signaling, a well‐recognized pathway that regulates cancer cell survival and proliferation.33 YAP1 is mainly known as an effector of the Hippo signaling pathway involved in cell growth, division and apoptosis.38, 39

Overexpression of MIR375 revealed significant inhibition of cell proliferation in the MTT assay (Fig. 4a). In nude mice, colon cancer cells overexpressing MIR375 showed a significantly lower growth rate as compared with control cancer cells (Fig. 5). The volumes, weight and cellular proliferation of a tumor xenograft of HCT116 cells or HT29 cells were significantly reduced by MIR375 overexpression (Figs. 5a–5c). Angiogenesis was also significantly reduced in xenografts of HCT116 cells and HT29 cells by MIR375 overexpression (Fig. 5d). In addition, flow cytometric analysis revealed significant enhancement of apoptosis in MIR375‐transfected cells compared to the control cells (Fig. 4c). Moreover, overexpression of MIR375 arrested the cell cycle in the G1 phase (Fig. 4b). Collectively, our results indicate that MIR375 targets CTGF and functions as a tumor suppressor in colon cancer.

Cetuximab, a chimeric monoclonal antibody, is an EGFR inhibitor used for the treatment of metastatic CRC. As shown in Figure 4c, MIR375 overexpression increased the apoptotic and necrotic cell ratio among HCT116 cells. Therefore, our results suggest that MIR375 could be used to develop an anticancer therapy for human CRC. We also observed synergistic effects of MIR375 and cetuximab in HCT116 cells (Fig. 4f). These findings indicated that combined treatment with cetuximab and MIR375 may be effective against CRC.

In summary, we showed that MIR375 expression is suppressed in the tissues of CRC patients. We identified CTGF as a putative MIR375 target gene using mRNA microarray analysis and bioinformatic tools and showed that CTGF is a direct target of MIR375. CTGF expression is increased in the tumor tissues of CRC. Our results suggest that MIR375 regulates both EGFR signaling pathways (CTGF‐EGFR‐PIK3CA‐AKT and EGFR‐KRAS‐BRAF‐ERK1/2) with or without CTGF‐mediated expression, and as a result, MIR375 regulates cell growth and proliferation, migration, cell cycle arrest in the G₁ phase, apoptosis and/or necrosis and angiogenesis via the EGFR pathway in colon cancer cells (Fig. 5e). We also showed that MIR375 has a synergistic anticancer effect with cetuximab on colon cancer cells.

Acknowledgements

The biospecimens for this study were provided by the Biobank of Wonkwang University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health and Welfare.