Human tissues
A total of 92 pairs of gastric cancer and matched adjacent noncancerous tissue samples used in this study were collected from 2006 to 2015 at Zhejiang provincial people’s hospital, the Affiliated Hospital of Hangzhou medical college. Written informed consents were obtained from all patients. The collection and use of tissues were performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the Medical Ethics Committee from Hangzhou medical college and Zhejiang provincial people’s hospital. The clinical data of these tissues are listed in Table 1.
Immunohistochemistry
The gastric cancer and matched adjacent noncancerous tissues were fixed in 4% paraformaldehyde at 4°C overnight, dehydrated and embedded in paraffin. Next, the paraffin-embedded tissue samples were cut into serial 3μm section, dewaxed and hydrated, and then 3% hydrogen peroxide was utilized to block endogenous peroxidase. Subsequently, immunohistochemical staining was carried out using primary antibody Cyclin D3 (1:150) and CDK6 (1:100) overnight at 4°C. After incubation with the biotinylated secondary antibody for 30 min at room temperature, the sections were developed by diaminobenzidine (DAB), which were followed by counterstaining with hematoxylin solution and sealing. All information of antibodies used are provided in Supplementary Table 1. A positive result was regarded as the presence of yellow-brown particles in the nucleus and cytoplasm. The staining was divided by color intensity into not colored, light yellow, brown yellow and brown, and graded with 0, 1, 2 and 3 points, respectively. According to the percentage of positive cells in visual field cells, the score was 0 point in 0-5%, 1 point in 6-25%, 2 points in 26-50%, 3 points in 51-75% and 4 points in > 76%. To calculate the final score, the two items were multiplied. Two observers who are blinded read the results separately.
Cell culture
The human gastric cancer cell lines (MKN-45, SGC-7901, AGS and MGC-803) and the human gastric epithelial cell line (GES-1) were provided from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin and streptomycin (Invitrogen, San Diego,CA) in a humidified incubator at 37 °C with 5% CO2.
RNA extraction and quantitative RT-PCR
Total RNA was extracted from cell lines or human tissues using TRIzol Reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The concentration and quality of RNA were determined using NANODROP 2000 (Thermo Scientific, Rockford, IL, USA). TaqMan miRNA probes (Applied Biosystems, Foster City, CA) were used to quantify mature miRNAs expression level according to the manufacturer’s instructions. The relative amount of miRNA expression was normalized to U6 snRNA expression in this study by the equation 2−ΔΔCT, where ΔΔCT=(CTmiRNA−CTU6)target−(CTmiRNA−CTU6)control. SYBR Green method was used to quantify mRNAs (Cyclin D3, CDK6, CDK4, Rb, c-Myc, Cyclin A2, Cyclin E1, Cyclin E2 and GAPDH) expression level according to the manufacturer’s instructions. The relative amount of each mRNA was normalized to GAPDH by the equation 2−ΔΔCT, where ΔΔCT=(CTmRNA−CTGAPDH)target− (CTmRNA−CTGAPDH)control. All sequences of the primers used are provided in Supplementary Table 2.
Protein isolation and western blot
Cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with freshly added proteinase inhibitors PMSF (Beyotime, Shanghai, China) and PI (Thermo Scientific, Rockford, IL, USA) for 30 min on ice. Tissue samples were frozen solid with liquid nitrogen, ground into a powder and then lysed in RIPA lysis buffer supplemented with PMSF and PI on ice for 30 min. After centrifugation at 12,500 r/m, 4°C for 10 min, the supernatants were collected and the concentration of proteins were quantified using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were separated by SDS-PAGE gel and transferred to PVDF membrane (Millipore Corporation, Billerica, MA, USA). After blocking in 5% skim milk, the PVDF membranes were incubated with primary antibodies overnight at 4°C. After washing and incubating with secondary antibodies, the bands were detected with the SuperSignal West Pico chemiluminescence substrate (BIO-RAD, USA). All information of antibodies used are provided in Supplementary Table 1.
Plasmid construction and siRNA interference assay
A mammalian expression plasmid pcDNA3.1+ and pCMV-3xflag were purchased from Genescript (Nanjing, China). The full-length open reading frame (ORF) of Cyclin D3 was amplified from cDNA that reverse transcription of MGC-803 cells mRNA, the ORFs of CDK6 and c-Myc were amplified from cDNA that reverse transcription of SGC-7901 cells mRNA, and subcloned into pcDNA3.1+or pCMV-3xflag plasmid digested with Hind III and EcoR I, respectively, generating pcDNA3.1+-CyclinD3, pcDNA3.1+-CDK6, pcDNA3.1+-c-Myc, pCMV-3xflag-Cyclin D3 and pCMV-3xflag-CDK6 plasmid. To construct pLKO.1-TRC-shRNA plasmid, we used annealing oligos of Cyclin D3 or CDK6 subcloned into pLKO.1-TRC vector digested with Age I and EcoR I, generating pLKO.1-TRC-Cyclin D3-shRNA and pLKO.1-TRC-CDK6-shRNA. All sequences of the clone primers used are provided in Supplementary Table 2. Two siRNAs (siRNA-I and siRNA-II) targeting Cyclin D3, CDK6 and c-Myc were designed and synthesized by Ribobio (Guangzhou, China). A scrambled siRNA served as a negative control (siRNA-NC). The siRNA sequences are provided in Supplementary Table 3.
Construction of Stably Transfected Cell Lines
The recombinant plasmids were verified by sequencing and co-transfected with pMD2G, pSPAX2 into 293T cells to produce recombinant lenti-virus. SGC-7901 cells were infected with lenti-shRNA-NC (control) or lenti-shRNA-Cyclin D3 and lenti-shRNA-CDK6. Forty-eight hours later, the virus-infected cells were cultured in the growth medium with 2.5 μg/mL puromycin for selection. The knockdown efficiency of Cyclin D3 and CDK6 in surviving cells were confirmed by Western blot.
Xenograft assays in nude mice
Four-week-old athymic BALB/c nude (nu/nu) mice were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China). All mice were housed in SPF animal facility. The animal studies were approved by the Animal Care and Use Committee at Hangzhou Medical College. The methods were performed in accordance with the approved guidelines by Hangzhou Medical College. They were equally divided into 3 groups (6 mice/group) and injected subcutaneously with untreated 2×106 SGC-7901 cells (Mock) or SGC-7901 cells infected with the control lentiviral vector (lenti-shRNA-NC) or Cyclin D3/CDK6 knockdown lentiviral vector (lenti-shRNA-Cyclin D3/CDK6). After subcutaneous implantation of cells, animals were observed daily for tumor growth, and measured the tumor volume weekly from 14 days post-implantation. Tumors were harvested at 28 days post-implantation, photographed and the volumes and weights of the tumors were recorded. Parts of tumors were used for protein and RNA extraction, and remainder were fixed in 4% paraformaldehyde and were subjected to immunohistochemical analysis using Cyclin D3, CDK6, PCNA and Ki-67 staining. All information of antibodies used are provided in Supplementary Table 1.
miRNA target prediction
The miR-15a/16 can simultaneously target Cyclin D3 and CDK6, which were determined using algorithms from TargetScan (http://genes.mit.edu/targetscan/), and RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/).
Luciferase assay
To construct a luciferase reporter carrying the mRNA 3′UTR of Cyclin D3 or CDK6 with a putative miR-15a and miR-16 binding sites, we amplified a 674 bp Cyclin D3 3′UTR region including each two binding sites of miR-15a and miR-16 from MGC-803 cDNA, and amplified a 1471 bp CDK6 3′UTR region including each two binding sites from SGC-7901 cDNA. All sequences of the clone primers used are provided in Supplementary Table 2. The PCR amplified product was cloned into the pMIR-Report plasmid (Ambion, Austin, TX, USA) at the Spe I & Mlu I site. The pMIR-Report plasmid that carried the mutant Cyclin D3 or CDK6 3′UTR region was using the Site-Directed Gene Mutagenesis Kit and specific primers containing mutated nucleotides, the sequences of these primers used are provided in Supplementary Table 2. These insertions were verified by DNA sequencing. For the luciferase reporter assays, 293 T cells were cultured in 24-well plates, and each well was transfected with 0.2 μg firefly luciferase reporter plasmid, 0.15μg β-galactosidase expression plasmid (Ambion, Austin, TX, USA), and equal amounts (25 pmol) of miR-15a or miR-16 or mixture using Lipofectamine 3000 (Invitrogen). The β-galactosidase plasmid was used as transfection control. After 24 hours transfection, the luciferase activity was analyzed using luciferase assay kits (Promega, Madison, WI, USA).
Cell cycle assay
To assess the cell cycle, collected cells were washed with PBS buffer in twice, and fixed in 75% ethanol overnight. Then, the fixed cells were washed with PBS buffer and incubated with 50μg/ml RNase A for 30 min at 37 °C, followed by staining for DNA content was performed using 50 mg/ml propidium iodide (BD Biosciences, San Jose, CA). Analysis was performed on a fluorescence-activated cell-sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA) with Cell Quest Pro software.
Cell proliferation assay
Cells were seed into 96-well plates and incubated in RPMI 1640 medium supplemented with 5% FBS. The cell proliferation rate was measured using the Cell Counting Kit-8 (CK04-500, Dojindo, Japan) after 12, 24, 36, 48, 60h transfection according to the manufacturer’s instruction. Absorbance was measured at a wavelength of 450 nm.
EdU proliferation assay
To assess cell proliferation, SGC-7901 cells and MGC-803 cells were seeded into 6-well plates, after 24 hours transfection, equal amounts were seeded in triplicate into 96-well plates allowed to attach overnight. The cell proliferation was measured using EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Cells were incubated in 50μM Edu for 5 hours and fixed in 4% paraformaldehyde for 30 min at room temperature. Next, these cells were permeabilization in PBS with 0.5% Triton X-100 for 10 min. Subsequently, the cells were incubated in Apollo staining solution for 30 min and then incubated in Hoechst 33342 for 30 min. The proportion of nucleated cells incorporating EdU was determined by fluorescence microscopy.
Statistical analysis
All data was analyzed using Student’s t-test in SPSS statistical software and presented as the mean value ± SD, with p value <0.05 was considered statistically significant. p<0.05 (indicated by *), <0.01 (indicated by **) or <0.001 (indicated by ***).