Mir-125a Facilitates Cell Apoptosis Via Upregulating Wild Type P53

IntriductionmicroRNAs(miRNAs)are an extensive class of non-coding endogenous RNAs in animals and plants,appropriate 22 nucleotides in length,with the role of regulating gene expression by translational repression.Recent studies show that some miRNAs regulate cell proliferation and apoptosis processes that are important in cancer formation,thus,function as oncogenes and/or tumor suppressor genes,mir-125a was reported low-expressed in human breast cancer and lung cancer cell line A549 by microchip recently,and whether mir-125a is involved in the regulation of cell proliferation and apoptosis remains unknown.Therefore,we aimed to study the function of mir-125a in lung cancer cell.Materials and Methods1.Cell lineshuman bronchial epithelial(HBE),lung adenocarcinomal cell lines A549,SPC-A-1 and LTEP-a-2,lung squamous cell carcinomal cell lines SK-MES-1 and QG56,human giant cell lung carcinomal cell line BE1,large cell lung carcinomal cell lines NCL-H661and NCL-H460,small cell lung carcinomal cell line NCL-H446。2.miRNA sequencesmir-125a:5'-UCC CUG AGA CCC UUU AAC CUG UGA-3';anti-mir-125a:5'-CAC AGG UUA AAG GGU CUC AGG GAA;scrambled mir-125a:5'-GGA CGG CGA UCA GAU AAG AGU UC-3'Every sequence contains 2'-OMe modifications at every base without fluorescencent marker. 3.Cell cultureCells were propagated in DMEM or RPMI-1640 with 10%or 15%FBS 37℃,5% CO₂.4.Real time PCRTotal RNA was extracted using TRIZOL and reverse transcribed using TaqMan^? MicroRNA Reverse Transcription Kit.Real-time PCR were performed using TaqMan^? MicroRNA Assays HSA-MIR-125A and U18 as an internal standard according to a standard TaqMan^?PCR protocol on an Applied Biosystems 7900HT Fast Real-Time PCR System.All reations were run in triplicate.5.Transient transfectionA549 cells in subcontiguous condition were transfected with mature mir-125a (20μM),antisense inhibitor(20μM)and scrambled mir-125a with appropriate Lipofectamine^TM2000 according to the manufacturer's instruction.Cells were cultured in 37℃,5%CO₂ for 24 to 72 hours according the need of the assay.6.Flow cytometry(Annexin V-FITC)Every group was divided into four subgroups:blank control,only Annexin V,only FITC and Annexin V+FITC.Flow cytometry performed on BD FACSCalibur^TMFlow Cytometer.7.Western blotProteins were detected with coomassie brilliant blue method.And proteins were visualized by Super ECL Plus Reagent according to the manufacturer's protocol,β-actin as an internal control.8.Transwell invasional cabinet24h after transfection,100μl cells(2.5×10⁵/mL)in media without FBS were added to the up cabinet(the matrix were coverd 6 hours before),and 600μl media were added to the low cabinet,cultured in 37℃,5%CO₂.24h later,dyeing with hematoxylin, observing and counting.9.Thiazolyl blue assay(MTT)The OD value of each well was measured using a microplate readerwith a test wavelength of 490 nm.All the cells were run in 6 replicating wells and in continuous five days.The proliferation curve was plotted according the OD values.10.Statistical analysisSPSS 13.0 software was employed to analysis the data using t test.Results1.The expression levels of mir-125a in 9 cell lines we studied were lower than HBE except SK-MES-1,especially A549,NCL-H661,NCL-H460 and SPC-A-1 (p＜0.01).2.Sense mir-125a transfection can up-regulate mir-125a level obviously(P= 0.004)while antisense mir-125a transfection can down-regulate mir-125a level obviously(P=0.005).3.Sense mir-125a can facilitate cell apoptosis and inhibit cell invasional ability while antisense mir-125a can inhibit cell apoptosis and facilitate cells invasional ability. Sense and antisense mir-125a neither can affect cell proliferation.4.Sense mir-125a facilitates the apoptosis of A549 via upregulating wild type P53.Conclusionmir-125a is low-expressed in 8 lung cancer cell lines relative to HBE except SK-MES-1;mir-125a can inhibit cell invasion ability and facilitate cell apoptosis via upregulate the wild type P53.The mechanism of invasion inhibition is under further study.