Emerging evidence indicates that the miR-17 family may have a causal role in human cancer tumorigenesis, but their specific effects on the occurrence of CRC (colorectal carcinoma) are still poorly understood. In the present study, we profiled CRC tissue samples by miRNA (microRNA) microarray and found that four members of the miR-17 family had higher expression in CRC tissues than in normal tissues. This finding was further validated by qRT-PCR (quantitative reverse transcription PCR). Transfecting CRC cells with an inhibitor of miR-17 lowered their ability to proliferate and induced G0/G1 arrest. We also confirmed that miR-17 exerted this function by directly targeting RND3 in vitro, and that the expression of miR-17 was negatively correlated with that of RND3 in CRC tissues and CRC cells. Moreover, miR-17 inhibition led to tumour growth suppression and up-regulation of RND3 expression in a nude mouse xenograft model. RND3 expression was found to be significantly lower in CRC tissues than in normal tissues and adenomas, indicating that RND3 may act as a tumour suppressor gene in CRC. In conclusion, the present study suggests that miR-17 plays an important role in CRC carcinogenesis by targeting RND3 and may be a therapeutic agent for CRC.

INTRODUCTION

CRC (colorectal carcinoma) is the third most common cancer in Western countries and one of the most common cancers in China [1,2]. Although its mortality rate has decreased slightly over the past three decades because of increased early diagnosis and advances in treatments [3], its incidence has increased in recent years in China [1]. The carcinogenesis of CRC involves multiple alterations of oncogenes and tumour suppressor genes, such as inactivating APC (adenomatous polyposis coli) and TP53 (tumour protein 53) and oncogenically mutating Ras and BRAF [4]. However, few of these genes are helpful for early diagnosis, and the molecular mechanisms underlying development and progression of CRC remain poorly understood. Therefore further understanding of its molecular mechanisms and identification of both early diagnostic markers and novel therapeutic targets are of great clinical value.

Current research suggests that differential expression of miRNAs (microRNAs) in CRC could be involved in the development and progression of CRC and may serve as biomarkers for CRC diagnosis and prognosis. miRNAs are snRNAs (small non-coding RNAs), 18–25 nucleotides long, which post-transcriptionally inhibit many genes by binding to the 3′-UTR (3′-untranslated region) of target mRNAs [5]. Many miRNAs aberrantly expressed in human tumours have been found to be involved in tumorigenesis [6]. For instance, overexpression of miR-17-92 in lung cancer may promote proliferation of lung cancer cells [7] and decreased expression of miR-143 in bladder cancer may suppress tumours by regulating Ras protein expression [8]. In CRC, several miRNAs are known to be differentially expressed and implicated in colorectal carcinogenesis. First, both miR-143 and miR-145 were found to be down-regulated in CRC [9], and miR-143 was found to target KRAS [10], ERK5 (extracellular-signal-regulated kinase 5) [11] and DNMT3A (DNA methyltransferase 3A) [12], the three genes known to be involved in colorectal carcinogenesis and cancer therapy. Subsequently, more miRNAs, when dysregulated in CRC, have been demonstrated to be associated with cell proliferation [13,14], apoptosis [15,16], invasiveness and metastasis [17,18]. Studies have shown that deregulated miR-106a could act as a marker of DFS (disease-free survival) and OS (overall survival) independent of tumour stage [19], and increased expression of miR-21 is associated with poor survival and poor therapeutic outcomes [20]. In the present study, we intended to find new miRNAs which may be potentially developed into novel biomarkers for CRC diagnosis and prognosis.

Gene expression profiling based on genome-wide microarrays has been widely used to characterize human cancers [2124], and microarray analysis of miRNA expression has revealed the consistent deregulation of some miRNAs in many cancers [20,2528]. We used microarray analysis to determine differential expression of miRNAs from samples of CRC tissue and adjacent normal tissue. A total of 44 miRNAs that were dysregulated in tumour tissues were screened. We further focused on the miR-17 family and confirmed their dysregulated expression levels by qRT-PCR (quantitative reverse transcription PCR) in clinical samples. Most importantly, we confirmed that miR-17 was up-regulated in CRC tissues and promoted cell proliferation, tumour growth and cell cycle progression by targeting the RND3 tumour suppressor gene.

MATERIALS AND METHODS

Patients and clinical samples

All fresh tumour tissue samples were obtained from patients diagnosed with CRC and undergoing radical colectomy at Nanfang Hospital, Southern Medical University in China. All cases were confirmed by pathological evaluation post-operatively. A total of 132 paraffin-embedded samples were obtained from the CRC patients diagnosed on the basis of histological and clinical findings at Nanfang Hospital between 2001 and 2007, including 41 CRC subjects, 42 adenoma subjects and 49 adjacent normal tissue subjects. The adjacent normal tissue samples were obtained from the normal colorectal tissue located 5 cm away from the tumour. The clinicopathological data of all the patients are listed in Supplementary Table S1 (at http://www.BiochemJ.org/bj/442/bj4420311add.htm). Collection of the samples and the present study were permitted by our institutional review board, and written informed consent was obtained from each patient. Patient samples were obtained following informed consent according to an established protocol approved by the Institute Research Ethics Committee of NanFang Hospital, which acts to meet the demands of the Declaration of Helsinki (2000) of the World Medical Association.

Microarrays of miRNA

Four pairs of CRC and adjacent normal samples were randomly selected for microarray analysis. RNAs from the fresh samples were isolated by the one-step TRIzol® method (CapitalBio) for miRNA microarray analysis, which was performed according to the manufacturer's instructions, as described previously [29,30]. Briefly, total RNA was extracted with TRIzol®. Next, low-molecular-mass RNA was isolated by precipitation with poly(ethylene glycol), labelled by the T4 RNA ligase method described by Thomson et al. [30], and then hybridized on to the miRNA microarray. The microarray contained 1320 probes in triplicate that corresponded to 988 human (including 122 predicted miRNAs), 627 mouse and 350 rat mature miRNAs, which were from the miRNA Registry (http://microrna.sanger.ac.uk/sequences/), miRbase Release 13.0 Version. Both tumour and its paired normal tissue were profiled at the same time. The microarrays were scanned with a LuxScan 10K-A laser confocal scanner, and the images were analysed with LuxScan 3.0 software (CapitalBio).

Microarray analysis

The data were preprocessed by subtracting the background from the average values of the replicate spots for each miRNA and by filtering out faint spots with expression signals less than 300. Then the expression data from different chips were normalized with global mean normalization. We identified those miRNAs that were differentially expressed in tumours compared with normal tissues with SAM (Significance Analysis of Microarrays), version 2.1 (http://www-stat.stanford.edu/~tibs/SAM/), where we set the FDR (false discovery rate) to less than 5% and the fold change to greater than 3. The results from the miRNA profiles were also analysed by PCA (principal component analysis) and further analysed by unsupervised hierarchical clustering with CLUSTER and TREEVIEW software (Stanford University, Stanford, CA, U.S.A.).

Cell culture and transfection

HCT116 and Lovo cell lines (wild-type p53) were obtained from the A.T.C.C. The HCT116 cells were cultured in RPMI 1640 (Gibco) and Lovo cells were cultured in DMEM (Dulbecco's modified Eagle's medium) (Gibco). Both media were supplemented with 10% FBS (fetal bovine serum) (Gibco). The miR-17 inhibitor, the siRNA (small interfering RNA) for RND3 and the NC (negative control) siRNA were synthesized and purified by GenePharma. The miR-17 inhibitor sequence was 5′-CUACCUGCACUGUAAGCACUUUG-3′ and the miRNA inhibitor NC siRNA sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. The NC siRNA had a sense sequence of 5′-UUCUCCGAACGUGUCACGUTT-3′ and an antisense sequence of 5′-ACGUGACACGUUCGGAGAATT-3′. The RND3 siRNA had a sense sequence of 5′-GCAGCUACUUAUAUCGAAUTT-3′ and an antisense sequence of 5′-AUUCGAUAUAAGUAGCUGCTT-3′. Both the single miRNAs and duplex siRNAs were transfected using Lipofectamine™ 2000 reagent (Invitrogen).

Real-time reverse transcription-PCR

The expression of mature miRNAs and RND3 in the fresh tissue samples or transfected cell lines was quantified by SYBR Green assays with primers and SYBR Green from TaKaRa Biotechnology. The mean cycle threshold was determined by triplicate PCR runs, and gene expression was calculated relative to U6 snRNA or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The primer for miR-17 was 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTACCTGC-3′, and the primer for U6 was 5′-AACGCTTCACGAATTTGCGT-3′. The specific RND3 PCRs used 5′-CTATGACCAGGGGGCAAATA-3′ as the sense primer and 5′-TCTTCGCTTTGTCCTTTCGT-3′ as the antisense primer. The miR-17 reactions used 5′-CTCAACTGGTGTCGTGGA-3′ as the universal primer and 5′-ACACTCCAGCTGGGCAAAGTGCTTACAGTGCA-3′ as the sense primer. The U6 reactions used 5′-CTCGCTTCGGCAGCACA-3′ as the sense primer and 5′-AACGCTTCACGAATTTGCGT-3′ as the antisense primer. The GAPDH reactions used 5′-ACCCAGAAGACTGTGGATGG-3′ as the sense primer and 5′-TGCTGTAGCCAAATTCGTTG-3′ as the antisense primer.

Bioinformatics

Potential miRNA targets were predicted and analysed with three publicly available algorithms: PicTar, TargetScan and miRanda [31]. The number of false-positive results was decreased by accepting only those putative target genes predicted by at least two programs.

Dual-luciferase reporter assay

The dual-luciferase reporter plasmid (psiCHECK-2 plasmid) that contained the 3′-UTR for both RND3 and mutRND3 was constructed by LAND (Landbiology Corporation). The RND3 3′-UTR cloned in the psiCHECK-2 plasmid was an approximately 814 bp sequence containing the conserved miR-17-binding site, which is located at position 905–1718 of the RND3 3′-UTR. HEK (human embryonic kidney)-293T cells (A.T.C.C.) were transfected with either the miR-17 mimic or the miR-17 inhibitor and also with the psiCHECK-2 plasmid in 24-well plates with Lipofectamine™ 2000 reagent. After 48 h, the cells were lysed in passive lysis buffer (Promega), and then luciferase activity was measured with a dual-luciferase reporter assay (Promega).

Western blot analysis

Protein was extracted from the cells and the tumour tissues from mice were injected with miR-17 inhibitor-transfected cells and NC siRNA-transfected cells using the Whole Protein Extraction Kit (KeyGEN KGP250), and the concentrations were determined using the BCA (bicinchonic acid) protein assay kit (Beyotime). Equal amounts of protein were separated by electrophoresis on SDS/PAGE (12% gel) and then transferred on to PVDF membranes (Immobilon P; Millipore). The membranes were blocked with 5% (w/v) non-fat dried skimmed milk powder solution for 1 h, and then incubated overnight at 4°C with a mouse monoclonal antibody specific for RND3 (1:500; Abcam), a monoclonal antibody against CCND1 (cyclin D1) (1:200; Santa Cruz Biotechnology), a monoclonal antibody against c-Myc (1:500; Bioworld Technology) or a mouse monoclonal antibody against β-actin (1:1000; Santa Cruz Biotechnology). After washing with TBST (Tris-buffered saline with Tween; 20 mM Tris/HCl, pH 7.6, 150 mM NaCl and 0.05% Tween 20), the membranes were incubated with a secondary antibody against mouse IgG. Then the membranes were washed, and protein was detected with an ECL (enhanced chemiluminescence) kit (Pierce).

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] cell proliferation assays

Tumour cells were transiently transfected either with 80 nM of the miR-17 inhibitor (GenePharma) or with 50 nM of the RND3 siRNA (GenePharma). After 24 h, the cells were plated at 2×103 cells per well in 96-well plates. The viability of the cells was assessed using the MTT assay (Sigma) daily for 1 week.

Cell cycle assay using flow cytometry

Cells were transfected with miR-17 inhibitor or with siRNA against RND3 for 48 h, then starved for 16 h in 0.5% FBS-containing medium, and then stimulated for 8 h in medium containing 10% FBS. The cells were then trypsinized, collected by centrifugation, washed in PBS, and fixed overnight at 4°C in 70% ethanol. After the cells were washed twice with PBS, their DNA was stained with the Cell Cycle Detection Kit (KeyGEN). The cells were then analysed with a FACScalibur flow cytometer (Becton Dickinson).

Tumorigenicity assays in nude mice

All experimental procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication nos. 80-23, revised 1996) and were performed according to the institutional ethical guidelines for animal experiments. Cells were transfected with the miR-17 inhibitor or NC siRNA for 72 h. Then transfected cells (1×106 cells) were suspended in 100 μl of PBS and injected subcutaneously into either side of the posterior flank of the same female BALB/c athymic nude mouse at 4–6 weeks of age. Before injection, a Trypan Blue assay was performed to assess cell viability after transfection of the miR-17 inhibitor to eliminate transfection interference. Tumour growth was examined every 2 days for at least 2 weeks. Tumour volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L×W2)×0.5. At 2 weeks later, the tumour was removed and weighed. Analyses of tumorigenesis and IHC (immunohistochemistry) staining were performed.

IHC staining analysis

IHC staining was performed using a Dako Envision System (Dako) following the manufacturer's recommended protocol. Briefly, all paraffin sections, 4 μm in thickness, were baked for 2 h at 65°C. Sections were de-paraffinized with xylene and rehydrated with graded ethanol to distilled water. Sections were submerged in EDTA antigenic retrieval buffer (pH 8.0) and subjected to high-pressure treatment. After being treated with 0.3% H2O2 for 15 min to block the endogenous peroxidase, the sections were treated with 1% BSA for 30 min to reduce non-specific binding and then mouse monoclonal anti-RND3 antibody (1:400; Abcam), monoclonal anti-PCNA (proliferating-cell nuclear antigen) antibody (1:400; Santa Cruz Biotechnology), monoclonal anti-CCND1 antibody (1:400; Santa Cruz Biotechnology) or monoclonal anti-c-Myc antibody (1:400; Bioworld Technology) was incubated with the sections overnight at 4°C. After washing, the sections were incubated with peroxidase-labelled polymer conjugated to a secondary antibody for 30 min. For colour reactions, diaminobenzidine was used. For negative controls, the antibody was replaced by normal goat serum.

Staining evaluation

The IHC-stained tissue sections were scored separately by two pathologists blinded to the clinical parameters. The RND3 staining was assessed by scanning the entire tissue section to assign score. The staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The percentage of positive staining areas relative to the entire carcinoma-involved area for CRC samples or relative to the entire section for the normal samples scored the extent of staining as 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–80%) or 4 (81–100%). The final staining score of 0–12 for RND3 was the product of multiplying the intensity and extent scores. This relatively simple and reproducible scoring method gives highly concordant results between independent evaluators and has been used in a previous study [32]. Tissues with a final staining score of 6 or higher were considered positive for statistical evaluation.

Statistical analysis

Statistics were analysed with the SPSS statistical software package, version 13.0 (SPSS). The Student's t test and the one-way ANOVA test were performed for qRT-PCR, MTT analyses and tumour growth curve, and the Mann–Whitney U test was performed for IHC staining. The correlation between miR-17 and RND3 was analysed by Spearman's rank correlation. A value of P<0.05 indicated a significant difference.

RESULTS

Expression of the miR-17 family increased in CRC

Microarray analysis of miRNAs from the four pairs of CRC and adjacent normal tissues identified 44 miRNAs that were differentially expressed in CRC. Of them, 19 miRNAs had increased expression and 25 miRNAs had lowered expression in the CRC tissues compared with the normal tissues (see Supplementary Table S2 at http://www.BiochemJ.org/bj/442/bj4420311add.htm, fold-change >3 or <0.333, and FDR=0%). The list included six members of the miR-17 family: miR-17, miR-18a, miR-18b, miR-20a, miR-19a and miR-106a. They all had increased expression, as revealed by clustering by unsupervised hierarchy (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/442/bj4420311add.htm). Furthermore, PCA indicated 19 differentially expressed miRNAs that contributed to the differences between cancer and normal tissue, including five members of the miR-17 family (see Supplementary Table S3 at http://www.BiochemJ.org/bj/442/bj4420311add.htm). qRT-PCR was performed to test whether the members of the miR-17 family could be overexpressed in CRC. The overexpression of the four miR-17 members, miR-17, miR-106a, miR-18a and miR-18b, were further confirmed by qRT-PCR in 21 pairs of CRC and normal clinical tissue samples. As shown in Figure 1, expression of the four miRNAs tended to be up-regulated in cancer tissue samples. Further statistical analysis showed a significant up-regulation of all of the four miRNAs in CRC tissues (Figure 1, P=0.009 for miR-17, P=0.007 for miR-106a, P=0.001 for miR-18a and P=0.001 for miR-18b).

The miR-17 family is overexpressed in CRC

Figure 1
The miR-17 family is overexpressed in CRC

The expression of miR-17 (A), miR-18a (B), miR-18b (C) and miR-106a (D) relative to that of U6 were compared between CRC tissues and adjacent normal tissues by qRT-PCR. The histogram indicates the differential expression of miR-17 between cancer and normal tissues. Box plots illustrate the median, 25th and 75th percentiles and the range of expression levels of miR-17. *or ° indicates the extreme outlying values, and “19” signifies the identity of the sample whose miRNA expression had this value. **P<0.01.

Figure 1
The miR-17 family is overexpressed in CRC

The expression of miR-17 (A), miR-18a (B), miR-18b (C) and miR-106a (D) relative to that of U6 were compared between CRC tissues and adjacent normal tissues by qRT-PCR. The histogram indicates the differential expression of miR-17 between cancer and normal tissues. Box plots illustrate the median, 25th and 75th percentiles and the range of expression levels of miR-17. *or ° indicates the extreme outlying values, and “19” signifies the identity of the sample whose miRNA expression had this value. **P<0.01.

Inhibiting miR-17 reduced the proliferation of Lovo cells and induced G0/G1 arrest

Although several studies have reported that the miR-17 family may be a potential oncogene in many human cancers [3335], there has been no research on the role of miR-17 in CRC tumorigenesis. Therefore we focused on the role of miR-17, the most important component of the miR-17 family, in CRC by transfecting Lovo cells with an inhibitor of miR-17. The transfection efficiency was determined by qRT-PCR (Figure 2A, P<0.001). Cells transfected with the miR-17 inhibitor had lower levels of cell proliferation than the NC siRNA or mock control cells (Figure 2B, F=964.521, P<0.001). The Lovo cells transfected with the miR-17 inhibitor had a higher percentage of cells in the G1-phase than those transfected with NC siRNA (Figure 2C, P<0.01). These results indicated that miR-17 could enhance proliferation and promote the progression of the cell cycle.

Inhibiting miR-17 reduced Lovo cell proliferation and induced G0/G1 arrest

Figure 2
Inhibiting miR-17 reduced Lovo cell proliferation and induced G0/G1 arrest

(A) The level of miR-17 was measured by qRT-PCR in Lovo cells transfected with the miR-17 inhibitor and NC siRNA. The values indicate the means±S.E.M., n=3, **P<0.01. (B) Lovo cells were transfected with the miR-17 inhibitor and NC siRNA for 24 h and then grown in normal medium for 7 days and assessed using a daily MTT assay. The values indicate the means±S.E.M., n=3, F=964.521 and P<0.001 using one-way ANOVA. (C) Lovo cells were transfected with the miR-17 inhibitor for 72 h and then collected for FACS analysis. Cells transfected with the miR-17 inhibitor showed an altered cell cycle profile, with significantly more cells with G1/G0 DNA content. The values indicate the means±S.E.M., **P<0.01.

Figure 2
Inhibiting miR-17 reduced Lovo cell proliferation and induced G0/G1 arrest

(A) The level of miR-17 was measured by qRT-PCR in Lovo cells transfected with the miR-17 inhibitor and NC siRNA. The values indicate the means±S.E.M., n=3, **P<0.01. (B) Lovo cells were transfected with the miR-17 inhibitor and NC siRNA for 24 h and then grown in normal medium for 7 days and assessed using a daily MTT assay. The values indicate the means±S.E.M., n=3, F=964.521 and P<0.001 using one-way ANOVA. (C) Lovo cells were transfected with the miR-17 inhibitor for 72 h and then collected for FACS analysis. Cells transfected with the miR-17 inhibitor showed an altered cell cycle profile, with significantly more cells with G1/G0 DNA content. The values indicate the means±S.E.M., **P<0.01.

RND3 was a target of miR-17

Since miRNAs exert their biological functions by suppressing their target genes, we searched further for genes targeted by miR-17 that may be involved in cell proliferation and cell cycle progression. The gene RND3 was predicted to be a potential target of miR-17 by computer-based sequence analysis by both TargetScan Human 5.1 and miRanda (Figure 3A). As the binding site of RND3 is conserved between human and mouse species, we sought to validate the regulation of RND3 by miR-17 through a luciferase reporter assay that could verify the direct interaction between miR-17 and the RND3 3′-UTR. We co-transfected HEK-293T cells with a psiCHECK-2 vector containing either the 3′-UTR for RND3 or mutated 3′-UTR for RND3 (Figure 3B), and a mimic of miR-17 or an inhibitor of miR-17. Overexpression of miR-17 reduced luciferase activity from the reporter vector containing the 3′-UTR of RND3 (Figure 3C, P=0.003). Moreover, mutation of the putative binding site on the RND3 3′-UTR abrogated this repression, supporting the direct interaction of miR-17 with RND3. However, cells co-transfected with the miR-17 inhibitor and a psiCHECK-2 vector containing the 3′-UTR of RND3 showed no alteration in luciferase activity (Figure 3C). The reason may be that the exogenetic 3′-UTR of RND3 was not attached to the endogenous miR-17 and decreased miR-17 expression, owing to the miR-17 inhibitor, would not increase luciferase activity in the reporter vector containing the 3′-UTR of RND3.

Validating predicted binding sites between miR-17 and RND3

Figure 3
Validating predicted binding sites between miR-17 and RND3

(A) Both TargetScan and miRanda predicted that miR-17 would target the 3′-UTR of RND3. (B) A schematic diagram illustrates the construct of Luc (luciferase)–RND3 3′-UTR and Luc–RND3 mutated (mut) 3′-UTR. Either RND3 3′-UTR or RND3 mut 3′-UTR was cloned into a psiCHECK-2 control plasmid downstream of the firefly luciferase coding region between the XhoI and NotI sites. (C) HEK-293T cells were co-transfected with the psiCHECK-2 constructs containing either the RND3 3′-UTR or RND3 mut 3′-UTR and either the miR-17 inhibitor or the miR-17 mimic for 48 h. The dual-luciferase reporter assay measured the activity of firefly luciferase 48 h after transfection. The relative luciferase activity was first normalized with Renilla luciferase activity and then compared with the psiCHECK control. The values are means±S.E.M., n=3, **P<0.01.

Figure 3
Validating predicted binding sites between miR-17 and RND3

(A) Both TargetScan and miRanda predicted that miR-17 would target the 3′-UTR of RND3. (B) A schematic diagram illustrates the construct of Luc (luciferase)–RND3 3′-UTR and Luc–RND3 mutated (mut) 3′-UTR. Either RND3 3′-UTR or RND3 mut 3′-UTR was cloned into a psiCHECK-2 control plasmid downstream of the firefly luciferase coding region between the XhoI and NotI sites. (C) HEK-293T cells were co-transfected with the psiCHECK-2 constructs containing either the RND3 3′-UTR or RND3 mut 3′-UTR and either the miR-17 inhibitor or the miR-17 mimic for 48 h. The dual-luciferase reporter assay measured the activity of firefly luciferase 48 h after transfection. The relative luciferase activity was first normalized with Renilla luciferase activity and then compared with the psiCHECK control. The values are means±S.E.M., n=3, **P<0.01.

miR-17 expression negatively correlated with RND3 expression

The interaction of miR-17 and RND3 was further validated by analysing whether miR-17 regulated the expression of endogenous RND3 in fresh CRC tissue and cells. The mRNA expression levels of both miR-17 and RND3 were analysed by qRT-PCR in 19 samples from patients with CRC. Unfortunately, the correlation between miR-17 and RND3 mRNA expression was only analysed in 19 samples, because two samples had been used up before we detected RND3 expression. The mRNA level of RND3 was negatively correlated with that of miR-17 (Figures 4A and 4B, R=−0.537, P=0.018). On the other hand, cells transfected with miR-17 had increased expression of RND3 at the mRNA and protein levels compared with mock control and NC siRNA-transfected control cells (Figure 4C). This result suggests that miR-17 may directly regulate the expression of endogenous RND3.

Expression of miR-17 negatively correlates with that of RND3 in CRC and Lovo cells

Figure 4
Expression of miR-17 negatively correlates with that of RND3 in CRC and Lovo cells

The endogenous expression of miR-17 and RND3 were examined by qRT-PCR in 19 patients with CRC. A trend towards a negative correlation between the expression levels was shown in the histogram (A) or scatter plots (B), with RND3 mRNA expression on the y axis and miR-17 mRNA expression on the x axis (R=−0.537, P=0.018). Lovo cells transfected with miR-17 inhibitor increased RND3 expression after transfection, as evaluated by qRT-PCR and Western blots (C). The values are means±S.E.M., n=3, **P<0.001.

Figure 4
Expression of miR-17 negatively correlates with that of RND3 in CRC and Lovo cells

The endogenous expression of miR-17 and RND3 were examined by qRT-PCR in 19 patients with CRC. A trend towards a negative correlation between the expression levels was shown in the histogram (A) or scatter plots (B), with RND3 mRNA expression on the y axis and miR-17 mRNA expression on the x axis (R=−0.537, P=0.018). Lovo cells transfected with miR-17 inhibitor increased RND3 expression after transfection, as evaluated by qRT-PCR and Western blots (C). The values are means±S.E.M., n=3, **P<0.001.

RND3 mediated the effect of miR-17 on promoting cell proliferation and cell cycle progression

The ability of miR-17 to promote cell survival and cell cycle progression by targeting RND3 was confirmed with an RND3 siRNA whose transfection efficiency was verified by qRT-PCR and Western blot analysis (Figures 5A and 5B). The MTT assay found that cell proliferation was inhibited by the miR-17 inhibitor and enhanced by RND3 siRNA. Moreover, the reduced proliferation due to inhibiting miR-17 was attenuated after cells were co-transfected with RND3 siRNA (Figure 5C, F=1374.362, P<0.001 for Lovo cells; F=1172.702, P<0.001 for HCT116 cells). This result indicated that RND3 reversed the promoting effect of miR-17 on proliferation of the CRC cells. On the other hand, inhibiting RND3 promoted progression of the cell cycle (Figure 5D) and inhibiting miR-17 induced arrest of the cell cycle in the G0/G1 phase compared with the NC (Figure 5C, P<0.01). The Lovo cells co-transfected with both the miR-17 inhibitor and RND3 siRNA had no significant change (Figure 5C), suggesting that inhibiting RND3 reversed cell cycle arrest in G0/G1 caused by inhibiting miR-17. This result confirmed that RND3 mediated the effect of miR-17 on promoting cell proliferation and cell cycle progression.

RND3 mediates the miR-17 effect on promoting cell proliferation and cell cycle progression

Figure 5
RND3 mediates the miR-17 effect on promoting cell proliferation and cell cycle progression

(A) The level of RND3 was validated by qRT-PCR and Western blot analysis in Lovo and HCT116 (wtp53) cells after they were transfected for 48 h with siRNA for RND3, the miR-17 inhibitor or both. The values are means±S.E.M., n=3, **P<0.01. (B) Lovo cells and HCT116 cells were transfected for 24 h with siRNA for RND3, the miR-17 inhibitor or both, and then grown in normal medium for 1 week, with the MTT assay being performed daily. The values are means±S.E.M., n=3. The Lovo cells had values of F=1374.362 and P<0.001. The HCT116 cells had values of F=1172.702 and P<0.001. (C) The effect of the miR-17 inhibitor on the progress of the cell cycle could be reversed by inhibiting RND3. Lovo cells were transfected with miR-17 inhibitor for 72 h and then collected for FACS analysis. The values are means±S.E.M., **P<0.01.

Figure 5
RND3 mediates the miR-17 effect on promoting cell proliferation and cell cycle progression

(A) The level of RND3 was validated by qRT-PCR and Western blot analysis in Lovo and HCT116 (wtp53) cells after they were transfected for 48 h with siRNA for RND3, the miR-17 inhibitor or both. The values are means±S.E.M., n=3, **P<0.01. (B) Lovo cells and HCT116 cells were transfected for 24 h with siRNA for RND3, the miR-17 inhibitor or both, and then grown in normal medium for 1 week, with the MTT assay being performed daily. The values are means±S.E.M., n=3. The Lovo cells had values of F=1374.362 and P<0.001. The HCT116 cells had values of F=1172.702 and P<0.001. (C) The effect of the miR-17 inhibitor on the progress of the cell cycle could be reversed by inhibiting RND3. Lovo cells were transfected with miR-17 inhibitor for 72 h and then collected for FACS analysis. The values are means±S.E.M., **P<0.01.

miR-17 inhibition suppressed CRC tumorigenesis in vivo

As described above, we observed a dramatic change in cell proliferation in Lovo cells transfected with the miR-17 inhibitor. Next, the Lovo cells transfected with the miR-17 inhibitor or NC siRNA were injected subcutaneously into nude mice. Before the transfected cells were injected into nude mice, a Trypan Blue assay was performed to assess cell viability after transfection of the miR-17 inhibitor. It showed that the transfection of the miRNA inhibitor did not affect the viability of the CRC cells. We detected a clear difference in tumour size 17 days after the injection. As shown in Figure 6(A), by the 17th day, mice injected with the miR-17 inhibitor-transfected cells had significantly smaller tumours, by weight, than mice injected with the NC siRNA-transfected cells (P<0.01). The tumour growth curves indicated that the difference was significant 10 days after cell injection (Figure 6B, F=41.644, P<0.01). Then the tumours were removed and sectioned for H/E (haematoxylin/eosin) staining and IHC, using an anti-PCNA antibody, an anti-RND3 antibody, an anti-CCND1 antibody and an anti-c-Myc antibody. The tumour tissues from mice injected with the miR-17 inhibitor-transfected cells showed more strongly positive RND3 expression in cytoplasm, and more weakly positive PCNA and CCND1 expression in nuclei than those from mice injected with the NC siRNA-transfected cells (Figure 6C). As a positive control, c-Myc was expressed in both tumour tissues from mice injected with miR-17 inhibitor-transfected cells and NC siRNA-transfected cells, but a little weakly positive in tumour tissues from mice injected with miR-17 inhibitor-transfected cells (Figure 6C). Further Western blot analysis of CCND1 and c-Myc showed that tumours from mice injected with NC siRNA-transfected cells had a higher expression of CCND1 and c-Myc than those from mice injected with miR-17 inhibitor-transfected cells (Figure 6D). Taken together, these results suggested that a CRC formed by the injection of Lovo cells was suppressed by miR-17 inhibition.

The effect of miR-17 on tumour growth and RND3 expression in a nude mouse xenograft model

Figure 6
The effect of miR-17 on tumour growth and RND3 expression in a nude mouse xenograft model

(A) Left-hand panel, NC siRNA- and miR-17 inhibitor-transfected Lovo cells (1×106 cells) were injected subcutaneously into either posterior flank of the same nude mice respectively. Photographs illustrate the representative features of tumour growth 17 days after inoculation. Right-hand panel, 17 days after the injection, the tumour was weighed. The values are means±S.E.M., n=5, **P<0.01. (B) Tumour sizes were measured and tumour growth curves were obtained. The values are means±S.E.M, n=5, *P<0.05 and **P<0.01. (C) Tumours formed by cells transfected with the miR-17 inhibitor or NC siRNA were subjected to H/E staining and IHC probed with an anti-PCNA antibody, an anti-RND3 antibody, an anti-CCND1 antibody and an anti-c-Myc antibody. The tumour tissues from mice injected with miR-17 inhibitor-transfected cells had higher RND3 expression and weaker PCNA, CCND1 and c-Myc expression than those from mice injected with NC siRNA-transfected cells. The red line indicates 10 μm. (D) Tumours formed by cells transfected with the miR-17 inhibitor or NC siRNA were subjected to Western blot analysis for CCND1 and c-Myc. CCND1 and c-Myc were more highly expressed in tumours formed by cells transfected with the miR-17 inhibitor than in those formed by cells transfected with NC siRNA. The five mice were named as ID 1, ID 2, ID 3, ID 4 and ID 5.

Figure 6
The effect of miR-17 on tumour growth and RND3 expression in a nude mouse xenograft model

(A) Left-hand panel, NC siRNA- and miR-17 inhibitor-transfected Lovo cells (1×106 cells) were injected subcutaneously into either posterior flank of the same nude mice respectively. Photographs illustrate the representative features of tumour growth 17 days after inoculation. Right-hand panel, 17 days after the injection, the tumour was weighed. The values are means±S.E.M., n=5, **P<0.01. (B) Tumour sizes were measured and tumour growth curves were obtained. The values are means±S.E.M, n=5, *P<0.05 and **P<0.01. (C) Tumours formed by cells transfected with the miR-17 inhibitor or NC siRNA were subjected to H/E staining and IHC probed with an anti-PCNA antibody, an anti-RND3 antibody, an anti-CCND1 antibody and an anti-c-Myc antibody. The tumour tissues from mice injected with miR-17 inhibitor-transfected cells had higher RND3 expression and weaker PCNA, CCND1 and c-Myc expression than those from mice injected with NC siRNA-transfected cells. The red line indicates 10 μm. (D) Tumours formed by cells transfected with the miR-17 inhibitor or NC siRNA were subjected to Western blot analysis for CCND1 and c-Myc. CCND1 and c-Myc were more highly expressed in tumours formed by cells transfected with the miR-17 inhibitor than in those formed by cells transfected with NC siRNA. The five mice were named as ID 1, ID 2, ID 3, ID 4 and ID 5.

RND3 may act as a tumour suppressor gene in colorectal carcinogenesis

After we found that RND3 reversed the effect of miR-17 on promoting cell proliferation and cell cycle progression, we explored whether RND3 functioned as a tumour suppressor in CRC. We evaluated the expression of RND3 in the 132 paraffin-embedded samples. The CRC samples had significantly lower expression of RND3 than the adjacent normal tissue samples and the adenoma samples as analysed by IHC (Figure 7, P<0.01), suggesting that RND3 might act as a tumour suppressor gene involved in the malignant transformation process from adenoma to carcinoma in colorectal carcinogenesis.

Expression of RND3 detected immunohistochemically

Figure 7
Expression of RND3 detected immunohistochemically

Staining intensity by IHC was moderate in the cytoplasm of normal colorectal epithelium cells (A), moderate for the colorectal adenoma cells (B) and weak for colorectal cancer cells (C). The original magnification was 400×, and the red line indicates 10 μm. (D) The histogram indicates that the positive rate of RND3 expression was lower in CRC than in normal colorectal epithelium and adenoma cells. **P<0.01.

Figure 7
Expression of RND3 detected immunohistochemically

Staining intensity by IHC was moderate in the cytoplasm of normal colorectal epithelium cells (A), moderate for the colorectal adenoma cells (B) and weak for colorectal cancer cells (C). The original magnification was 400×, and the red line indicates 10 μm. (D) The histogram indicates that the positive rate of RND3 expression was lower in CRC than in normal colorectal epithelium and adenoma cells. **P<0.01.

DISCUSSION

Previous reports have strongly suggested a direct role of miRNAs in tumorigenesis [26,27,36,37]. Our results from the present study confirmed the overexpression of four members of the miR-17 family (miR-106a, miR-17, miR-18a and miR-18b) in CRC compared with adjacent normal tissues. Furthermore, we demonstrated that miR-17 promoted proliferation of cells and progression of the cell cycle through targeting RND3 in CRC cells, as well as promoting tumour growth in a nude mouse xenograft model. The expression level of RND3 decreased as the miR-17 expression level increased, and their expression were negatively correlated in CRC tissues. Together, the findings suggested that miR-17 may contribute to the development and progression of CRC by suppressing RND3.

Generally, miRNAs function as post-transcriptional repressors that exert their biological functions by suppressing their target genes. A number of mRNAs involved in distinct pathways are targeted by miR-17, an important component of the miR-17 family. In B-cell lymphomas, miR-17 may promote tumour growth by targeting E2F1 and may increase angiogenesis by targeting thrombospondin-1 to function as an oncogene [36,38]. However, miR-17 has a role as a tumour suppressor in breast cancer cells, where overexpressed miR-17 may suppress the proliferation of breast cancer cells by repressing the expression of AIB1 (amplified in breast cancer-1) and CCND1 [39,40]. More than 20 genes involved in the transition between the G1 and S-phases have been found to be targeted by miR-17 through ingenuity pathways analysis [41]. Both oncogenic and tumour suppressive functions have been attributed to miR-17 in this genetic network, since miR-17 has been observed to suppress genes both for and against proliferation. These findings suggest that it may depend on the type of cell and the function of the target mRNAs whether miR-17 functions as an oncogene or tumour suppressor gene [42]. However, functions of miR-17 and its targets have not been reported in CRC. We found that, consistent with the oncogene role of miR-17, down-regulating miR-17 in CRC cells could reduce cell proliferation, induce arrest of cells in the G1/S stage and suppress tumour growth. Furthermore, we validated the targeting of RND3, a negative regulator of cell proliferation and the progression of the cell cycle [43], by miR-17. Thus miR-17, overexpressed in CRC, could function as an oncogene.

The RND3 gene is an atypical member of the Rho GTPase family, which are crucial regulators of cytoskeletal dynamics, the progression of the cell cycle and cell proliferation [43]. Our studies suggest that RND3 could act as a tumour suppressor by inhibiting the progression of cell cycle and the proliferation of cells. Increased expression of RND3 inhibits the progression of the cell cycle, predominantly in the G1 phase [44,45]. Consistent with its role in inhibiting the progression of cell cycle, RND3 has also been shown to inhibit cell proliferation. Non-steroidal anti-inflammatory drugs have been found to reduce cancer growth by increasing the expression of RND3 in CRC cells [46]. Also, RND3 expression levels were found to be decreased in prostate cancer specimens compared with normal specimens, and a reduced level of RND3 was predicted to enhance proliferation of prostatic stromal cells [47,48]. The down-regulation of RND3 induced the hyper-proliferation of keratinocytes [49]. The present study also found that down-regulating RND3 expression with siRNA promoted cell proliferation, CRC tissues had decreased RND3 expression levels and RND3 expression levels negatively correlated with miR-17 expression levels. This suggests RND3 may act as a tumour suppressor gene in CRC.

The RND3 gene is a downstream target of miR-17, mediating the role of miR-17 in promoting cell cycle progression and proliferation. As shown by the present study, the miR-17 inhibitor decreased cell proliferation and the decrease was reversed when the tumour cells were co-transfected with RND3 siRNA. This indicates possible mechanisms where expression of RND3 in colon cells may inhibit proliferation of tumour cells and that inhibiting RND3 by miR-17 could contribute to CRC development and progression.

In conclusion, the findings of the present study indicate that miR-17, as an oncogene in CRC carcinogenesis, may play an important role in suppressing RND3 so that the tumour cells proliferate, the cell cycle progresses and the tumour grows. Our results also demonstrate that RND3 may act as a tumour suppressor gene in CRC carcinogenesis. This assertion dovetails nicely with previous reports [50] that an individual miRNA can act as an oncogene by regulating tumour suppressor genes in a series of events related to generation and development of a tumour. Potentially, miR-17 may target many genes, including those that directly and indirectly affect tumorigenesis. Therefore further research will need to investigate other miR-17 target genes more, which could also play an important role in CRC carcinogenesis, so as to dissect the complex network of miR-17 and its target genes in CRC carcinogenesis.

Abbreviations

     
  • CCND1

    cyclin D1

  •  
  • CRC

    colorectal carcinoma

  •  
  • FBS

    fetal bovine serum

  •  
  • FDR

    false discovery rate

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • H/E

    haematoxylin/eosin

  •  
  • HEK

    human embryonic kidney

  •  
  • IHC

    immunohistochemistry

  •  
  • miRNA

    microRNA

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

  •  
  • NC

    negative control

  •  
  • PCA

    principal component analysis

  •  
  • PCNA

    proliferating-cell nuclear antigen

  •  
  • qRT-PCR

    quantitative reverse transcription PCR

  •  
  • siRNA

    small interfering RNA

  •  
  • snRNA

    small non-coding RNA

  •  
  • UTR

    untranslated region

AUTHOR CONTRIBUTION

The concept of this study was from Dehua Wu and Li Liu. All authors contributed to the study design. Hesan Luo was primarily responsible for the research implementation of the study and contributed to miRNA microarray, qRT-PCR, IHC, cell cycle assay, MTT assay and data analysis. Jinjin Zou was responsible for the collection of clinical samples and the dual-luciferase reporter assay. Zhongyi Dong was responsible for the tumorigenicity assay and Western blotting analysis. Qin Zeng was responsible for IHC and Western blot analysis. Dehua Wu and Li Liu contributed to the research implementation of the study. Hesan Luo was primarily responsible for paper preparation. Dehua Wu and Li Liu helped draft the paper or critically revised the paper. All authors reviewed and approved the final version of the paper.

FUNDING

This work was supported by the Natural Science Foundation of Guangdong Province, China [grant number 10151051501000062] and the National Nature Science Foundation of China [grant numbers 81172056 and 81172586].

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Supplementary data