Establishment Of A Lung Adenocarcinoma Cell Line Stably Transfected By MiRNA-145Gene And Study Of The Proliferation And Invasion On The Cell

Objective: Establish a new lung adenocarcinoma cell lines A549of stable expressover-expression、 silence-expression and empty carrier miRNA-145, and verify theeffects of miRNA-145on the A549cell proliferation and invasion.Methods:1.Establishment of A549cell line of stably transfected by miRNA-145gene.The initial concentration of screening by G418the its determine Screening ofconcentration.Using to obtein miRNA-145gene recombinant plasmid ofover-expression、 silence-expression and empty carrier-expression,TransfectmiRNA-145into human lung adenocarcinoma cell line A549by cationic lipofectinmedium,Then,to observe the green fiuorescent protein by Fluorescence microscopeand flow cytometry instrument to detection transfection efficiency,after screenedwith G418, we establish a new lung adenocarcinoma cell lines stableover-expression miRNA-145(Named for A549/H) and silence-expressionmiRNA-145(Named for A549/L) and empty carrier-expressionmiRNA-145(Named for A549/E). according to kit for extraction and reversetranscription of miRNA, The primer of has-miR-145and U6weredesigned and synthesized by Guangzhou BioLink(BioLink,China). Therelative expression of mRNA was analyzed using software.After made sure thestandard curve and melting curve,we compare miRNA-145gene expressionlevel in the experimental group.2.Validation of Effects of the Proliferation and invasion in stable transfectionA549/H cell group.The groups of the experiment: A549/H cell; A549/E cell;A549cell.Using CCk8methods to detect liver and growth,observe growth curvein three groups cell,Study the proliferation ability of A549/H. Using Transwell cellinvison experiment methods to detect the invison of Effects in three groups cell. Results:1.Through the initial concentration of screening by G418,we ultimatelydetermine200μg/ml of Screening of concentration.2. Through detecting green fiuorescent protein and flow cytometry instrument,the stably transfected efficiency of over-expression group、silence-expression groupand mpty carrier-expression group is80%.3. Result of qRT-PCRshow that: Compared to the normal group, miRNA-145expression level of A549/H cell are significantly increased (P <0.05), miRNA-145expression level of A549/L cell are significantly dropped (P<0.05),but emptycarrier group compared to the A549cell group with no significant difference (P>0.05).4.Results of CCK8show that: This cell of A549/H group,A549/E group andA549group growth24hours later, OD respectively is0.624、0.709、0.690;48hourslater, OD respectively is0.703、0.777、0.763；72hours later, OD respectively is0.724、0.794、0.779；96hours later, OD respectively is0.759、0.834、0.825.Compared to the A549cell group, OD of A549/H cell group significantlydropped in the24hours,48hours,72hours,96hours. Compared to the A549cellgroup, proliferation ability of A549/H cell group significantly dropped (P<0.05),butcompared to A549/e group and A549cell group with no significant difference (P>0.05).5.Results of Transwell tumor cell invasion experimental show that:TheA549/H group, A549/e group and A549cell group Cells by Throughing thematrigel respectively is:41,73,81. Compared to the A549cell group, invasionability of A549/H cell group significantly dropped (P<0.05),but compared toA549/e group and A549cell group with no significant difference (P>0.05).Conclusions:1. Successfully established of three types of lung adenocarcinoma cell linename as A549/H whose miRNA-145gene is stably excessived and A549/L whosemiRNA-145gene is stably silenced and A549/e whose miRNA-145gene is stablyempty,offer a cell model in subsequently of gene regulation. 2. Lung adenocarcinoma cell A549/H proliferation and invasion is inhibitedeffectively after stably transfected by miRNA-145,discuss the function andmechanism of miRNA-145furtherly in lung adenocarcinoma, and also to providethe evidence for lung adenocarcinoma which are targetly treated by miRNA-145.