Reagents
The oligonucleotides of miR-29b-3p mimics and negative control miRNA were synthesized by Thermo Fisher Scientific (Waltham, MA). The sequence of the oligonucleotides used for miR-29b-3pmimics
5’-UAGCACCAUUUGAAAUCAGUGUU-3.
Lipofectamine RNAiMAX was purchased from Invitrogen (Carlsbad, CA). Rabbit mAbs to E-cadherin and calretinin, were purchased from Abcam (Cambridge, UK), Cell Signaling Technology (Danvers, MA). Recombinant- TGF-b1 was purchased from R&D systems (Minneapolis, MN). Rabbit anti-vimentin mAb and anti-rabbit Ig conjugated with AlexaFluor 488 or AlexaFluor 595® were from Invitrogen. DAPI was obtained from Dojindo (Kumamoto, Japan). Anti-integrin b1 mAb and RGD peptide were purchased from Cayman Chemical Co. (Ann Arbor, MI).
Cell culture
Human gastric cancer cells, NUGC-4, MKN45 were obtained from Riken (Tsukuba JAPAN), and a bone marrow derived mesenchymal stem cell line, UE6E7T-12, was obtained from the Japan Health Science Foundation (Tokyo, Japan). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO), 100 U/mL penicillin, and 100 mg/mL streptomycin (Life Technologies, Grand Island, NY) at 37℃ in a 5% CO2 cell culture incubator.
Isolation and culture of human peritoneal mesothelial cells (HPMC)
HPMC were isolated from 2-4 cm3 samples of omentum collected from consenting patients undergoing sleeve gastrectomy. Omental samples were momentarily placed in a half TrypLE Express (Thermo Fisher Scientific, Waltham, MA) with pure PBS, and incubated in a thermostatic tank at 37℃ for 2h. The supernatants were collected after filtration by 100µm-pore nylon mesh, were centrifuged at 1,500 rpm and 4℃ for 5minites. Explants were seeded into collagen coated 10 cm2 tissue culture dishes (Falcon, Becton Dickinson, Oxford, UK) and cultured in DMEM with 20% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37℃ in 5% CO2 cell culture incubator (Riera, McCulloch et al. 2006).
Sample collection and miRNA purification and digital polymerase reaction (PCR)
Cell line miRNAs were isolated from cell pellets using the MiRNeasy kit (Qiagen, Hilden, Germany), according to the manufactures’ instructions. cDNA was synthesized starting from 100 μg of extracted RNA using the TaqMan miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Applied BioSystems, Foster City, CA) following manufacturer’s instructions. Total miR-29b-3p cell levels were then quantified using the digital (dd) PCR system (Bio-Rad Laboratories, Hercules, CA). Briefly, 10 μg of synthesized cDNA were added to a 20 μl PCR reaction mixture containing 10 μl of digital PCR™ supermix (Bio-Rad Laboratories), 1 μl of TaqMan primer/probe mix (Applied BioSystems) and RNase-free H2O. Droplets were generated by loading the mixture onto a plastic cartridge with 70 μl of QX100 Droplet Generation oil (Bio-Rad Laboratories). Cartridges were then placed into the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets generated from each sample were transferred to a 96-well PCR plate (Eppendorf, Hamburg, Germany), and PCR amplification was carried out on the C1000 Touch Thermal Cycler (Bio-Rad Laboratories), according to the manufacturer’s protocol. The plate was then loaded on the QX200 Droplet Reader (Bio-Rad Laboratories) and read automatically. The fraction of PCR-positive droplets was quantified assuming a Poisson distribution. QuantaSoft software was used to obtain the concentration results in number of copies per microliter for each sample.
Introduction of MMT and transfection of miR-29b-3p
HPMCs were seeded in 6-well plates at 50%–60% confluence and induced with 10 ng/ml of TGF-β1. Then, the cells were transfected with miR-29b-3p mimic or negative controls with lipofectamine RNAiMAX in HPMC at a final concentration of 50nM, and cultured for 48 hr at 37℃, according to the manufacturer’s instructions and then used for the following experiments
Immunofluorescence
HPMC (5×104) were plated in 24-well collagen coated plates, incubated with 10ng/ml of TGF-b1 and then transfected miR-29b or negative controls for 48h. Cells were washed with PBS, fixed in 4% paraformaldehyde for 10min at 37℃ and then permeabilized with 0.5% Tween-20 in PBS for 20min. Subsequently, cells were blocked for 1h with 3% BSA in PBS at room temperature. Cells were then incubated for 1 hr at room temperature with mAbs to E-cadherin (1:200), calretinin (1:500), vimentin (1:1000), and fibronectin (FN) (1:150). Then, cells were washed 3 times with PBS and incubated for 30min at room temperature with the appropriate fluorescence conjugated secondary anti-rabbit antibodies conjugated with AlexaFluor 488 or AlexaFluor 595® (1:2000). Lastly, the nucleus was counterstained with DAPI (1:1000) for 5 min. Glass coverslips were placed on slides and the preparations were visualized under a fluorescence microscope (Keyense, Osaka, JAPAN).
Cell proliferation
HPMCs (1.0x104 cells) transfected with miR-29b or negative controls were cultured with or without TGF-b1 in 96-well culture plate for 24 hr and incubated with MTS (DOJINDO, Kumamoto, Japan) diluted in normal culture media at 37℃ for an additional 3hr. Proliferation rates were determined and quantification was performed on a microtiter plate reader (Spectra Rainbow; Tecan) according to the manufacturer’s protocol.
Cell migration
HPMCs (5.0x105 cells) transfected with miR-29b or negative controls were cultured with or without TGF-b1 for 48 hours. Cells were re-suspended in DMEM and seeded on an 8-µm pore membrane in 24-well plate and 10% FBS was added to the lower chamber as a chemoattractant. After 24 h, non-migrated cells were gently removed with a cotton swab. Migrated cells on the lower surface of the culture insets were stained with Dif-Quick (Sysmex, Kobe, Japan) and counted.
Cell adhesion assay
HPMCs (5.0x104 cells) transfected with miR-29b or negative controls were cultured with or without TGF-b1 in 24-well culture plates. Fluorescein labelled NUGC-4 (1.0 x104 cells/well) were then added, incubated for 15min for all cells to attach to the MC monolayer. After gentle washing with warmed media 3 times, the number of NUGC cells remaining attached were counted under a fluorescent microscope.
Statistical analysis
Data were represented as mean ± standard deviation. The significance of the differences between groups was assessed with a one-way ANOVA using GraphPad Prism8. Differences were considered significant when P<0.05.