Study On The Role Of MiR-200a In Epithelial-mesenchymal Transition And Biological Characteristics In Vitro Of Human Pancreatic Cancer Stem Cells

Objective To evaluate the role of miR-200a in the epithelial-mesenchymal transitioncharacteristics and biological characteristics in vitro of pancreatic cancer stem cells(PCSCs) isolated from human pancreatic cancer cell line PANC-1and to confer thepossibility of regulation of miR-200a in PCSCs as a new approach for prevention and/ortreatment of pancreatic cancer.Methods(1) Cell Culture and Flow cytometryHuman pancreatic adenocarcinoma cell line, PANC-1was cultured in DMEMsupplemented with10%fetal bovine serum (FBS),100U/ml penicillin G, and100Ug/mlstreptomycin. Dissociated cells were counted and transferred to a5ml tube, washed twicewith PBS, counted and resuspended in PBS at1×106cell/100μl. Then, the antibodies APCanti-human CD44, PE anti-human CD24and FITC anti-human ESA (each at a dilution of1:40) were added and incubated for20min on ice in dark. The respective isotype controlantibodies were used at the same concentrations according to the manufacturer’sinstructions. After washing twice with PBS, samples were resuspended in500μl PBS andanalyzed on a flow cytometer. Side-scatter and forward-scatter profiles were used toeliminate cell doublets. Cells were routinely sorted twice, and the cells were reanalyzed forpurity, which typically was>97%.Data were analyzed with BD FACS Diva software.(2) MiR-200a mimic Transfection The CD24+CD44+ESA+populations of PANC-1cells were plated in6well plates andincubated overnight. Cells were transfected with either control or miR-200a mimic at afinal concentration of25nM using EntransterTM-R transfection reagent. After6h oftransfection the medium was changed to avoid cell death during transfection.(3) Real-Time Reverse Transcriptase-PCRTotal RNA was extracted using Trizol according to the manufacturer’s instructions.E-cadherin, N-cadherin, vimentin and ZEB1, Oct4and Nanog mRNA were analysed byreal-time PCR with Power SYBR_green PCR master mix and datas were normalized toGAPDH expression.(4) miR expression analysisTotal RNA was reverse transcribed using the miScript Reverse Transcription KitMiR-200a, miR-200b, miR-200c were analysed by RT-qPCR using the miScript PCR Kit.Experiments were normalized to U6.(5) Western Blot AnalysisCells were lysed in RIPA lysis buffer and the protein concentration was determined.Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidenefluoride membrane. The membranes were blocked in5%skim milk in TBST buffercontaining0.1%Tween20and then incubated with indicated primary antibodies (Rabbitanti Oct4, l:2000; Rabbit anti Nanog,1:2000; Rabbit anti-N-cadherin,1:1000; Mouseanti-Vimentin,1:500; Rabbit anti-E-cadherin,1:1000; Rabbit Anti-ZEB1,1:1000) for2hat room temperature. HRP-conjugated secondary antibodies (HRP Goat anti-Rabbit IgGAntibody,1:5000; HRP Goat anti-Mouse IgG Antibody,1:5000) were incubated at roomtemperature for1h and detected using the enhanced chemiluminesence detectionsystem.Total protein was extracted from untreated and the cells treated by transfectingmiR-200a and subjected to western blot analysis as described to evaluate the expression ofE-cadherin, N-cadherin, ZEB1and vimentin. The data was adjusted against loading controlusing β-actin. (6) Transwell migration assay1×105PCSCs were plated in the top chamber onto the noncoated membrane andallowed to migrate toward serum-containing medium in the lower chamber. Cells werefixed after48hours of incubation with methanol and stained with0.1%crystal violet (2mg/ml). The number of cells invading through the membrane was counted under a lightmicroscope (three random fields per well)(7) Transwell invasion assay1×105cells were plated in the top chamber onto the Matrigel coated Membrane. Eachwell was coated freshly with Matrigel (60mg) before the invasion assay. Cells were platedin medium without serum or growth factors, and medium supplemented with serum wasused as a chemo-attractant in the lower chamber. The cells were incubated for48hours.Cells which did not invade through the pores were removed by a cotton swab. Cells on thelower surface of the membrane were fixed with methanol and stained with0.1%crystalviolet. The number of cells invading through the membrane was counted under a lightmicroscope (three random fields per well)(8) Cell Counting Kit-8（CCK-8）assay for cell proliferation and chemotherapysensitivity to gemcitabineThe cells of each group were planted at a density of1000-3000cells per well in96-well plates.10μl CCK-8solution was added into each well at a given time point (24h、48h、72h、96h) or at72h after different concentrations (50ng/ml、100ng/ml、200ng/ml、400ng/ml、800ng/ml、2000ng/ml) of gemcitabine added in. Then all groups were incubatedat37°C,5%CO2for3h. Absorbance was measured at450nm on a microplate reader.Results(1) In PANC-1,(1.442±0.532)%of cells were CD44+CD24+, and (0.492±0.334)%of cells were CD44+CD24+ESA+. The results showed that Oct4and Nanog expressionssignificantly increased in CD44+CD24+ESA+cells.(2) Real time RT-PCR result showed that miR-200a was significantly downregulated in PCSCs. The expressions of N-cadherin, vimentin and ZEB1were up-regulated, whilethe expression of E-cadherin exhibited down-regulation at mRNA levels in PCSCs.(3) It suggested that the overexpression of miR-200a in the PCSCs resulted in theup-regulation of epithelial marker E-cadherin while down-regulation of mesenchymalmarkers ZEB1, N-cadherin, Vimentin and human embryonic stem cell genes Oct4, Nanogat mRNA leve. By westernblot analysis, the results showed that the overexpression ofmiR-200a in the PCSCs had led to the up-regulation of E-cadherin and down-regulation ofZEB1, N-cadherin and Oct4, Nanog, but not Vimentin at protein level.(4) The Transwell inserts results showed that the number of PCSCs transfected withmiR-200a mimic which invaded and migrated to the lower side of the membrane wassignificantly decreased than PCSCs without transfection or transfected with NC mimic byabout0.33fold in invasion and0.22fold in migration, respectively.(5) The CCK-8assays demonstrated that the PCSCs treated by transfection ofmiR-200a mimic displayed decreasing cell proliferation and increasing sensitivity togemcitabine.ConclusionsIn this study, we found that miRNA played an important role in regulating thecharacteristics of cancer stem cells with EMT signatures. To up-regulate miR-200a maylead to the reversal of EMT phenotype and inhibit malignant biological characteristics ofPCSCs in vitro. It will provide theoretical and experimental evidence for designingmiRNA-based target therapies for pancreatic cancer in the future.