Enhancement Of RAAV Transduction Efficiency In Lung Ancer Cell Via MiRNA Library Screening

BackgroundIn recent years, gene therapy has made great progress. Thousands ofclinical trials have been conductetd so far. Its application has extended to avariety of diseases. However, limitation in vectors becomes a major hurdle forfurther development of gene therapy.rAAV，with the adavantages of a wide host range, non-pathogenicity,stable and long-term expression of exogenous gene,is deemed the mostpromising gene therapy vectors.A number of problems in clinical trials such as CD8T cell-mediatedimmune response and cytotoxic effect in liver cells caused by AAV2,invalidity in Parkinson’s disease and AAT gene defects, placed a higherrequirement on rAAV transduction efficiency.Current gene drug designing method view vectors and therapeutic genesseparately. Notably, some therapeutic genes can improve thee efficacy of thevehicle while play an anti-tumor role. In2009, Johnson et al. reported a amaximum of15fold and30fold change led by siRNA-mediated silencing. In2011, Wallen et al. identified50siRNAs that significantly promote rAAVgene expression.These results lend an insight for our study. microRNAs，about22nt long,are a class of small and non-coding RNAs that regulate gene expression atpost-transcriptional level. Aberrant expression of miRNA has been closelyrelated to cancer, especially lung cancer.ObjectiveThis paper will explore the function of miRNA in rAAV-based genetherapy via screening of a miRNA library containing nearly2000sequences insmall cell lung cancer cell NCI-H446. Further validation of these miRNAswill be conducted, analysis of its potential target genes and signalingpathways will be studied and the comprehensive mechanism of rAAV will befurther explored, thus opening the gate for gene therapy.Method （1） Cell culture, establish NCI-H446cell line in vitro;（2） Cotransfect EGFP siRNA and EGFP using lipoinfection to assess andestablish a proper transfection system;（3） Lipoinfection nearly2000miRNAs from a miRNAlibrary;（4） Transfect rAAV-GLUC, rAAV expression level is reflected by GLUCluminescence intensity;（5） Detect GLUC intensity with working solution made of coelenterazine;（6） Determine cell growth and viability using MTTassay;（7） Use statistical method including Robust Z score, Quartile based method,etc for normalization and hit identification;（8） Predict target genes of miRNAs identified as top hits and analyse thepossible mechanism through pathway analysis.Results（1） Completed the screening of a miRNAlibrary in2replicates and identifiedtop hits among the large data from the screening;（2） Efficacy of top hits were confirmed by biological validation;（3） Possible mechanism was discussed with analysis from various databaseand potential value is evaluated.