Background
Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths, according to the GLOBOCAN series of the International Agency for Research on Cancer [
1]. Surgery is the only curative treatment, however, many patients have inoperable disease at diagnosis or have recurrent disease after resection [
2]. Therefore, it is crucial to elucidate the molecular mechanisms underlying the development of gastric cancer and to look for new molecular markers and therapeutic targets.
MicroRNAs (miRNAs) are a class of small, non-coding RNAs about 18–25 nucleotides in length. MiRNAs mainly function to negatively regulate gene expression by promoting mRNA degradation or inhibiting mRNA translation through interacting with perfect or imperfect complementary sequences between the miRNA seed and the 3′untranslated regions (3′UTR) of its target genes [
3]. MiR-29c belongs to the miR-29 family, which is composed of four species with identical seed sequences, namely miR-29a, miR-29b-1, miR-29b-2 and miR-29c [
4]. MiR-29c plays the role as tumor suppressor in several kinds of tumors. MiR-29c was shown to inhibit cell growth, cell migration and invasion in pancreatic cancer by targeting ITGB1 [
5]. In bladder cancer, miR-29c overexpression inhibited cell growth, suppressed cell migration and resulted in an accumulation of cells in the G1 phase during the cell cycle through the target gene CDK6 [
6]. MiR-29c was displayed to mediate the epithelial to mesenchymal transition (EMT) and negatively regulated Wnt/β-catenin signaling pathway via PTP4A and GNA13 in human colorectal carcinoma [
7]. MiR-29c down-regulation by CpG dinucleotide methylation of the promoter has been participated in cell invasion and increased sensitive to chemotherapy in basal-like breast tumors [
8]. Further studies on liver carcinoma that focused on the suppressive role of ionizing radiation-responsive miR-29c in the development of the disease via targeting WIP1 [
9]. In lung cancer, miR-29c was shown to suppress cell adhesion and metastasis by targeting integrin β1 and MMP2 [
10].
In the present study, we qualified the expression of miR-29c in gastric cancer tissues and evaluated its role in cell proliferation and induction of cell apoptosis. We found that miR-29c has a general decrease in gastric cancer tissues compared with the matched normal tissues. Overexpression of miR-29c reduced cell proliferation by promoting apoptosis and inducing cell cycle G1/G0 arrest in vitro, and inhibited the ability of tumorigenesis in gastric cancer cells in vivo. Furthermore, we demonstrated that miR-29c can decrease NASP expression and the effects observed following miR-29c overexpression are partially due to NASP depletion. Thus, all of these results suggest that miR-29c is a potential marker for diagnose and therapeutic target for treatment in gastric cancer.
Methods
Human samples
Sixty-seven pairs of tumor tissues and paired adjacent normal tissues were collected from patients with gastric cancer who underwent surgery at the Department of Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. All samples were diagnosed by pathological examination, clinicopathological data were reviewed and TNM staging classification was ranked base on criteria of American Joint Committee on Cancer (AJCC, 6th edition).
Cell lines
The human gastric cancer cell lines SNU-1 (ATCC No. CRL-5971), SNU-16 (ATCC No. CRL-5974), NCI-N87 (ATCC No. CRL-5822), AGS (ATCC No. CRL-1739) and KATOIII (ATCC No. HTB-103) were got from the American Type Culture Collection, MKN-45 (JCRB No. 0254) and MKN-28 (JCRB No. 0253) were obtained from the Japanese Cancer Research Resources Bank, and the others (BGC-823: CBP60477, SGC-7901: CBP60500) were obtained from Shanghai Institute for Biological Sciences, Chinese Academy of Science. The immortalized normal gastric mucosal epithelial cell line (GES-1) and the human embryonic kidney cell line 293 T (HEK 293 T) were preserved in our institute. Gastric cancer cell lines were cultured in RPMI-1640, while HEK 293 T cells were cultured in DEME, supplemented with 10% heat-inactivated fetal calf serum with 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Exponentially growing cells were used for experiments.
RNA extraction and quantitative PCR (qPCR)
Total RNA isolation from homogenized tissue samples and cell lines was performed using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using the miScript II RT Kit (Qiagen, Venlo, Limburg, Netherlands). MiR-29c qPCR was assayed by All-in-One qPCR Mix Kit (GeneCopoeia, Rockville, MD, USA) with specific primer on ABI 7900 system. Expression of miR-29c was normalized to U6 small nuclear RNA and analyzed by the 2-ΔΔCt method. NASP mRNA expression level was measured by SYBR Green real time PCR (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. GAPDH was used as an internal control. Following primers were used: NASP sense 5′- GCGTCCCAAATTGCCTGTTT -3′ antisense 5′- GCTTCACTATCCACATCCAGA-3′; GAPDH sense 5′-GGACCTGACCTGCCGTCTAG-3′ antisense 5′-GTAGCCCAGGATGCCCTTGA-3′.
Transient transfection
Oligonucleotides hsa-miR-29c mimics (miR-29c), miR-control and siRNAs for NASP were purchased from GenePharma (Shanghai, China). Oligonucleotides and siRNAs were transfected into cells by carring out with Lipofectamine 2000 (Invitrogen) at a final concentration of 100 nM. The transfection efficiency was monitored by qPCR or Western blot.
Cell proliferation assay
Cell proliferation was accessed by colorimetric water-soluble tetrazolium salt (WST) method using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. After 24 h, cells transfected with miR-29c mimics or NASP siRNA were seeded into 96-well plates (2 × 103 cells/well), and the proliferation was monitored everyday for 5 days. The number of viable cells was determined by measurement of the absorbance at 450 and 600 nm using a Safire 2 microplate reader (TECAN, Switzerland).
Cells transfected with miR-29c mimics were resuspended with 0.3% soft agar in RPMI-1640 containing 10% FBS, then layered onto 0.6% solidified agar in RPMI-1640 containing 10% FBS in 6-well plates (1 × 103 cells/well) at 24 h post-transfection. These plates were incubated at 37 °C for 2 weeks. Colonies containing 50 cells or more were counted.
Apoptosis analysis
Cells transfected with miRNA or siRNA were harvested at 48 h after transfection cells and stained with Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen, CA, USA). Apoptotic cells were assessed in triplicates and repeated three times independently by flow cytometry (FACS Calibur, Becton Dickinson, NJ, USA).
Cell cycle analysis
At 48 h post-transfection with miRNA or siRNA, cells were fixed overnight using 70% ethanol at 4 °C, washed two times in cold phosphate-buffered saline, and incubated with 100 μg/ml RNase A and 50 μg/ml propidium iodide for 1 h at 37 °C. Analysis was performed on a FACS Calibur flow cytometry by measurement of the percentage of cells in various phases of the cell cycle.
Construction of plasmids and luciferase activity assay
Wild type NASP-3′ UTR or mutant NASP-3′ UTR containing the putative miR-29c binding sites were synthesized by Sangon, Shanghai, China. After digestion by MluI and SpeI, wild type and mutant NASP-3′ UTR were cloned into the MluI and SpeI precut pMIR-Report luciferase vector. In HEK 293 T cells pre-seeded 24-well, 100 ng pMIR/NASP-WT or MUT, together with 2 ng pRL-TK vector containing Renilla luciferase and 100 nM miR-29c mimics or miR-control were cotransfected by Lipofectamine 2000 (Invitrogen). After 48 h, relative luciferase activity was measured by dual-luciferase assay (Promega, Madison, WI, USA) according to the manufacturer’s instruction.
Western blot analysis
Cells in culture were lysed using M-PER reagents (Pierce, Rockford, IL, USA) in the presence of Cocktail protease inhibitor (Pierce). The concentration was measured by a BCA Protein Assay Kit (Pierce). Fifty micrograms protein samples were resolved with 5× Lane Marker Reducing Sample Buffer (Pierce), electrophoresed in 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, CA, USA). Labeled bands were detected using the ECL chemiluminescent kit (Pierce). Rabbit polyclonal anti-NASP (1:1000, Abcam, Cambridge, UK) and mouse monoclonal anti-GAPDH (1:10000, Kangchen, Shanghai, China) were used.
Retroviral transfection for stable cell lines
A genomic region including the primary transcript of miR-29c was cloned into the EcoRI-Xhol modified pMSCV-GW-RfA-PGK-EGFP retroviral vector, no insert vector as a control. HEK 293 T cells (1 × 106 cells/well) were seeded in 6-well plates 24 h prior to transfection, 2 μg of retroviral construct containing either miR-29c or miR-control, 2 μg of gag/pol and 2 μg of VSVG were cotransfected into HEK 293 T cells using 18 μl FuGENE6 HD (Roche, Indianapolis, IN, USA) in each well. At 48 and 72 h post-transfection, viruses were harvested and spin infected at 1500 rpm for 30 min at room temperaturewith 8 μg/ml of polybrene. GFP positive cells were sorted by flow cytometry and named RV-miR-29c and RV-miR-control, respectively.
Tumor xenograft model
SGC-7901 cells (100 μl, 1 × 106 cells) infected with RV-miR-29c or RV-miR-control were injected into the right flank region of 4-week-old male nude mice (Institute of Zoology, Chinese Academy of Sciences, Shanghai, China). Each group had five mice. Tumor volume was measured with caliper and calculated using the following formula: volume = (length × width2)/2. Mice were euthanized 4 weeks after injection and tumor nodules were removed and weighted. After tumor excision, the tumor nodules were fixed in 10% buffered formalin for further analysis. Animal study and experimental protocol was approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University.
Immunohistochemistry (IHC)
Blocks of formalin-fixed, paraffin-embedded mouse subcutaneous tumors were used. Tissue sections (5 μm) were deparaffinized with xylene, rehydrated in ethanol, antigen retrieval was performed by boiling in 10 mM citrate buffer (pH 6.0) for 30 min. After inhibition endogenous peroxidase activity with 0.3% H2O2 for 10 min, sections were blocked in 2% serum in PBS for 30 min, incubated with Ki-67 (dilution 1:50, Dako, Carpinteria, CA, USA) or NASP (dilution 1:100) at 4 °C overnight, followed by secondary antibody incubation and visualized with Envision System (Dako). Sections were counterstained with hematoxylin.
Statistics
Experimental data were expressed as the mean ± SD. Pearson χ
2 test was applied to examine the relationship between the miR-29c expression level and clinicopathologic parameters, unpaired t test was used to analyzed the differences between two groups. All statistical analyses were performed using the SPSS 15.0 software, and a P value less than 0.05 was considered statistically significant.
Discussion
Accumulating studies have focused on the role of miRNAs play in regulating cell proliferation process in gastric cancer. MiR-29c has been shown to be down-regulated in gastric cancer, but the downstream targets differ and it is not clear how miR-29c mediates cell responses in varying cell contexts. Han et al. reported that miR-29c involved in the initiation of gastric carcinogenesis by directly targeting ITGB1 [
12]. They found restoration of miR-29c inhibits cell proliferation, adhesion, invasion and migration in gastric cancer. Another research group verified the downregulation of miR-29c in gastric cancer patients and assessed proliferation and colony formation ability of miR-29c by targeting RCC2 [
13]. It was showed that all the members of miR-29 family were down-regulated in gastric cancer and miR-29c was more significant as a signature miRNA than miR-29a or 29b for gastric cancer. Furthermore, they demonstrated that miR-29 family directly targeted CCND2 and MMP2 to influence gastric cancer progression [
14]. It also has been reported that miR-29c regulates the expression of many oncogenes, such as CDK6, CDC42, p85α, DNMT3a and DNMT3b in other types of cancers [
6,
15,
16].
In present study, we indicate that miR-29c acts as a tumor suppressor by suppressing cell growth through CCK-8 and colony formation assays, promotes apoptosis and arrests cell cycle at G1/G0 stage by targeting NASP. Furthermore, the down-regulation of NASP can elite the phenotypes caused by miR-29c. As a histone chaperone, NASP binds both core and linker histones that is proved to present in all dividing cells [
17]. Two splice variants of NASP have been reported: testicular NASP (tNASP), which is mainly expressed in testis, stem cells, embryonic tissues and malignant tumors; somatic NASP (sNASP), which exists in all somatic mitosis cells. Both types of NASP specifically bind to histone H1, H3 and H4 and affect chromatin assembly, lead to the association with DNA replication, cell proliferation and cell cycle progression [
18]. Previous studies have investigated the role of NASP in renal cell carcinoma [
19]. Fang et al. showed that tNASP has a relative high level in human renal cell carcinoma and tNASP knockdown effectively suppresses cell proliferation and induces G1 phase arrest through ERK/MAPK signaling pathway. Additional studies indicated that depletion of tNASP inhibited cell proliferation and promoted apoptosis in prostate cancer cells [
20]. However, the mechanisms that result in elevated NASP expression level are still unclear. Our study suggests one mechanism that contributes to the elevated NASP levels in tumors is a deregulation of miR-29c and further supports targeting NASP as a therapeutic strategy in gastric cancer.
Our study also demonstrates that the expression level of miR-29c is lower in 67 cases of gastric cancer compared with matched normal tissues, and the expression also decreases in nine gastric cancer cell lines versus GES-1. The relationship between the miR-29c expression level and the clinicopathological factors in human gastric cancer samples was further analyzed. However, miR-29c expression level did not show any correlation with the clinicopathological parameters. It is consistent with the result of our previous study [
21]. Among 15 candidate miRNAs selected from microRNA array, miR-29c showed no correlation with the clinicopathological features assessed by qPCR in 40 pairs of gastric cancer samples. Liu et al. evaluated the role of miR-29c, miR-124, miR-135a and miR-148a in predicting lymph node metastasis and tumor stage in gastric cancer, they showed a week relationship between miR-29c expression level and gastric cancer stage on the basis of
P = 0.049 in 60 gastric cancer tissues [
22]. More samples and further studies are needed to disclosure the relationship between miR-29c and the clinicopathological features in gastric cancer.
Acknowledgements
We would like to thank Jun Ji and Qu Cai for their assistance in this study.