Background
Gastric carcinoma is one of the most common malignancies and ranks second in terms of global carcinoma-related mortality [
1]. The clinical outcome of gastric cancer has gradually improved, but the prognosis of patients with advanced disease is still disappointing. Although alterations in a large number of oncogenic and tumor-suppressive genes are reportedly implicated in gastric carcinoma, the molecular mechanisms underlying the development of gastric carcinoma are still poorly understood. Identification of these mechanisms is necessary for the development of targeted clinical therapy.
MicroRNA (miRNA) is a small non-coding RNA molecule comprising about 22 nucleotides, which regulates the expression of target genes at the post-transcriptional level [
2]. It has been reported that miRNAs are frequently dysregulated in human cancers and play oncogenic or tumor-suppressive roles in carcinoma cells [
3‐
5]. In our previous study, using a miRNA microarray covering 470 human miRNAs (miRBase 9.1), we identified 39 miRNAs that were differentially expressed in gastric carcinoma relative to normal gastric epithelium; 6 of these were significantly downregulated and the other 33 were upregulated [
6]. We have also reported that miR-375 is the most down-regulated miRNA, and that its ectopic expression is able to induce apoptosis of gastric carcinoma cells through downregulation of PDK1 and 14-3-3zeta [
6], implying a tumor-suppressive role of miR-375 in gastric carcinoma cells. Another miRNA, miR-29c, has also been found to be downregulated next to miR-375 [
6]. MiR-29c is the member of the miR-29 family composed of miR-29a, -29b and -29c. Deep sequencing has revealed that this miRNA family is that most highly expressed in gastric tissues [
7], and that the read count of miR-29c is the highest among them [
7], suggesting that alteration of the miR-29 expression level has an impact on gastric cells. Therefore, we hypothesized that miR-29c could be a candidate tumor-suppressor miRNA in gastric carcinoma, as well as miR-375. In the present study, to test this hypothesis, we attempted to clarify the function of miR-29c in the tumorigenesis and/or progression of gastric cancer.
Discussion
MiR-29c has been reported to be downregulated in various types of tumors [
16‐
19], including gastric carcinoma [
6,
20]. In nasopharyngeal carcinomas, miR-29c reportedly regulates the expressions of extracellular matrix proteins [
17], implicating the involvement of miR-29c downregulation in tumor invasiveness. In esophageal squamous cell carcinoma, miR-29c has been shown to induce cell cycle arrest by regulating the expression of cyclin E [
16]. However, in gastric carcinoma, there have been no data to suggest the function and targets of miR-29c. In the present study, we showed that ectopic expression of miR-29c suppressed the proliferation of gastric carcinoma cells and their ability to form colonies on soft agar. Furthermore, the growth suppression was not accompanied by caspase activation, but occurred through a decrease in the rate of DNA synthesis, suggesting that miR-29c may suppress cell proliferation by regulating progression of the cell cycle. To our knowledge, this is the first report to describe the tumor-suppressive role of miR-29c in gastric carcinoma. The mechanism responsible for miR-29c downregulation in gastric carcinoma is still unknown. Since genomic loss at 1q32.2, where miR-29c is located, is rarely detected in gastric carcinoma, whereas 1q gain is frequently observed [
21], it is unlikely that genomic copy number alteration is responsible for the downregulation of miR-29c. In addition, we showed that miR-29c downregulation is probably not associated with DNA methylation or histone deacetylation. Other factors, such as interaction of transcriptional suppressors with the miR-29c promoter and post-transcriptional regulation may play a role. Further studies will be needed to clarify these issues.
In this study, we showed that miR-29c reduced the expression of RCC2 at both the mRNA and protein levels. The luciferase assay with a reporter containing the miR-29c binding sequence at the 3’UTR of RCC2 mRNA suggested that miR-29c directly targets the 3’UTR of RCC2 mRNA. Although RCC2 (also known as TD-60) is reportedly a component of the chromosomal passenger complex, which is a crucial regulator of chromosomes, the cytoskeleton, and membrane dynamics throughout mitosis [
22,
23], little has been known about the oncogenic relevance of RCC2. Mollinari and colleagues showed that RCC2 knockdown induced cell cycle arrest at prometaphase in HeLa cells due to failure of spindle assembly, suggesting that RCC2 regulates cell cycle progression by modulating chromosome segregation and cell cleavage [
24]. In this study, we showed for the first time that RCC2 is upregulated in gastric carcinoma tissues. Furthermore, knockdown of RCC2 induced growth suppression without caspase activation in gastric carcinoma cells. Together with the data reported previously by other groups, our results suggest that RCC2 contributes to the proliferation of gastric carcinoma cells by regulating progression of the cell cycle. Interestingly, growth suppression without caspase activation was also observed in miR-29c-transfected cells, suggesting that downregulation of RCC2 may be involved in the miR-29c-induced growth suppression. In addition, both miR-29c and RCC2 transfection decreased cell viability accompanied by a reduction of BrdU incorporation, suggesting that RCC2 may play a role in/around S phase of the cell cycle. Thus, our data suggest that miR-29c reduces the expression of RCC2 in gastric carcinoma cells, leading to suppression of their growth, not through induction of apoptosis but by a cell cycle regulation signal. Therefore, we can hypothesize that decreased expression of miR-29c results in enhanced expression of RCC2, and confers a growth advantage on gastric carcinoma cells. Because the contribution of RCC2 to cell cycle regulation signaling during S phase of the cell cycle is still poorly understood, further studies will be needed to clarify this issue.
On the other hand, PPIC siRNA-transfected cells did not exhibit growth suppression, although PPIC mRNA level was decreased. We demonstrated that PPIC is one of the target genes of miR-29c and was significantly upregulated in gastric cancer tissues. Therefore, PPIC may play some role as a downstream molecule of miR-29c, although it may not be involved in cell proliferation and apoptosis. It has been reported that PPIC, also known as cyclophilin C, is a cellular binding protein for the immunosuppressive drug cyclosporine A, which can suppress T-cell activation [
25‐
27]. Moreover, it has been reported that the natural cellular ligand for PPIC, cyclophilin C-associated protein (CyCAP, also kinown as MAC2BP) [
28], can act as a modulator of endotoxin signaling in vivo [
29]. These facts led us to speculate that aberrant expression of PPIC may affect some aspects of the immune response, such as inflammation, during gastric carcinogenesis. However, to assess the function of PPIC under miR-29c regulation, further studies will be needed.
In gastric carcinoma tissues, CDK6 upregulation was not observed, at least at the mRNA level, although CDK6 expression was suppressed at both the mRNA and protein levels by ectopic expression of miR-29c in MKN45 cells. These findings suggest that CDK6 expression in tissue is probably affected by other factors. Indeed, upregulation of CDK6 in gastric carcinoma has been reported by other research groups [
30,
31], and a portion of cases showing CDK6 overexpression harbored chromosomal amplification of 7q21.2, where the CDK6 gene is located [
31].
Conclusions
We have found that miR-29c was downregulated in a substantial proportion of gastric carcinomas and suppressed proliferation of gastric carcinoma cells, potentially by targeting RCC2. Furthermore, miR-29c reduced the ability of gastric carcinoma cells to form colonies on soft agar. Therefore, we propose that miR-29c may have a tumor-suppressive role in gastric carcinoma cells, and that its decreased expression may confer a growth advantage on tumor cells at least partly via aberrant expression of RCC2, one of the target genes of miR-29c. Future studies examining the mechanism of miR-29c downregulation and the function of RCC2 in cell cycle regulation will be required in order to clarify the significance of miR-29c-RCC2-dependent cell cycle regulation in gastric carcinoma cells.
Materials and methods
Tissues
Gastric carcinoma tissues were surgically resected from 12 patients at Oita University Hospital. Information on the patients is summarized in Additional file
1. The tumor samples were fixed in 10% buffered formalin, and then embedded in paraffin. Use of the tissue samples for all experiments was approved by all of the patients and by Oita University Ethics Committee.
Cell culture and transfection
The gastric carcinoma cell lines MKN45, MKN74 and MKN7 were purchased from JCRB, and maintained in RPMI supplemented with 10% FCS. The cells were transfected with 10 nM small RNAs (see below) using Lipofectamine RNAiMAX (Invitrogen) and with plasmids using X-tremeGENE 9 (Roche Diagnostics) in accordance with the manufacturer’s instructions.
Small RNAs: pre-miR-29c precursor miRNA and negative control precursor miRNA (Ambion), RCC2 and PPIC siRNA ON-TARGETplus SMARTpool and negative control pool (Dharmacon).
MTS assay, BrdU ELISA and caspase activity
Cells were transfected with RNA oligos, and cell viability was measured using the CellTiter96 aqueous one solution cell proliferation assay (Promega) after the indicated periods. The rate of DNA synthesis was determined 24 h after transfection, at a time point when significant differences in cell number by MTS assay were not observed between groups, using a Cell Proliferation ELISA BrdU kit (Roche) in accordance with the manufacturer’s instructions. Activities of caspases-3/7, -8 and −9 were measured using the Caspase-Glo assay (Promega) in accordance with the manufacturer’s instructions. Statistical comparisons were performed using unpaired Student’s t test.
5-aza-dC and TSA treatment
For treatment with 5-aza-2’-deoxycytidine (5-aza-dC; Sigma-Aldrich), MKN45 cells (8 x10
4) were seeded into 35-mm dishes on day 0 and exposed to 5 μmol/L 5-aza-dC from day 1 to day 4. Trichostatin A (TSA; Sigma-Aldrich) was added into the cells on day 3 at a concentration of 100 nmol/L. The cells treated with either 5-aza-dC, TSA, or both were harvested on day 4. Two independent experiments were performed (Additional file
2).
Tissue sections (10 μm thick) from formalin-fixed, paraffin-embedded samples were stained with toluidine blue (Wako, Japan). Tumor cells and normal cells were collected separately under microscopic observation. Extraction of total RNA containing miRNA was performed using a RNeasy FFPE kit (QIAGEN) in accordance with the manufacturer’s instructions for miRNA containing total RNA.
Total RNA from cell lines was extracted with a miRNeasy Mini kit (QIAGEN) for quantitive RT-PCR, whereas total RNA for gene-expression microarray was extracted with a RNeasy Mini kit (QIAGEN) in accordance with the manufacturer’s instructions.
Quantitative RT-PCR
Quantitative RT-PCR for miRNA and mRNA was performed in the same way as for our previous work (standard curve, Taqman method) [
6,
32]. Levels of miRNA expression were determined relative to RNU44 and those of mRNA expression were determined relative to KPNA6.
Two or three days after transfection, cells were subjected to soft agar assays using 96-well plates containing semi-solid medium (10% FBS-DMEM, 0.4% agar) with 2.3 × 103 (MKN45) and 2 × 103 (MKN74) cells/well on a base layer of medium (10% FBS-DMEM, 0.6% agar). The cells were grown for 8–9 days, and the colonies were observed using a light microscope. To quantify the efficiency of colony formation, the CytoSelect™ 96-Well Cell Transformation Assay (Cell Biolabs) was used in accordance with the manufacturer’s instructions. Three independent experiments were performed.
Antibodies
Anti-CDK6 (Cell Signaling Technology) and anti-GAPDH mouse monoclonal antibodies (Ambion), and anti-RCC2 (Cell Signaling Technology) rabbit polyclonal antibody were used as primary antibodies. GAPDH was detected as an internal control by immunoblotting.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde for 10 min at RT. After washing with PBS, the cells were permeabilized in 0.3% Triton X-100/PBS for 10 min. The cells were then preincubated in 2% BSA/PBS for 30 min, followed by incubation for 2 h with anti-phospho-histone H3 (S10) (6 G3) antibody (Cell Signaling Technology). After washing with 0.1% Triton X-100/PBS, the cells were incubated with Alexa568-conjugated secondary antibody for 1 h. After a further wash, the cells were counterstained with Alexa488-conjugated phalloidin. The mounted cells were observed with a fluorescence microscope (Leica).
Gene-expression microarray
Three hundred nanograms of total RNA was subjected to microarray analysis in a similar way to that described in our previous study [
6,
21]. Briefly, Cy3-labeled cRNA was generated using a Quick Amp labeling kit (Agilent Technologies) and then hybridized to a human 44 K oligoarray (G4112F#014850, Agilent Technologies). Microarray images were obtained using a laser confocal scanner G2565BA (Agilent Technologies) and analyzed using Feature Extraction v.9.5.3.1 (Agilent Technologies) with the manufacturer’s recommended settings. The resulting data were subsequently imported into GeneSpring GX10 software (Agilent Technologies). For comparisons among multiple arrays, probe set data were median-normalized per chip. Then, the data were centered across the genes, followed by filtering based on signal intensity and flagged values. Differentially expressed genes were identified using a filter based on a fold-change of 2.0. All data are available at GEO via NCBI under Accession No.GSE38581 (
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38581).
Luciferase reporter assay
Reporter plasmids were constructed as described previously, with modifications [
6]. Double-stranded oligonucleotides corresponding to the wild-type (wt-3’UTR) or mutant (mut-3’UTR) miR-29c binding site in the RCC2 3’UTR or PPIC 3’UTR were synthesized and ligated between the SpeI and HindIII restriction sites of the reporter plasmid pMIR-Report (Ambion). The oligonucleotides used are described in Additional file
3. At 24 h after transfection with pre-29c or pre-Neg, a reporter plasmid containing the wt-3’UTR or mut-3’UTR and a plasmid expressing
Renilla luciferase (pRL-CMV) (Promega) were co-transfected into MKN45 cells. Firefly luciferase activity derived from pMIR-Report was normalized to
Renilla luciferase activity from pRL-CMV. Normalized luciferase activity in cells transfected with wt-3’UTR was compared with that in cells transfected with mut-3’UTR.
Competing interests
The authors have no conflict of interest.
Authors’ contribution
MiM, CN, YT and MaM conceived experiment and wrote the paper. MiM, CN and YT carried out experiments. All authors analyzed data. All authors had final approval of the submitted and published versions.