Development and Screening of Subtractive RNAi Libraries from Ovarian Cancer Cell Lines

摘要

The Specific aims of this proposal are: (1) Construction of two subtractive RNAi libraries targeting mRNA transcripts predominantly present in an (a) advanced ovarian cancer cell line and (b) metastatic ovarian cancer cell line. (2) Screen for genes vitally important for survival and growth. The two malignant cell lines will be transfected with individual clones from the subtractive RNAi libraries and the effect on viability will be monitored. Clones from the subtractive RNAi libraries producing marked growth inhibition or causing apoptosis will be sequenced and the targeted transcripts identified by BLAST search. (3) Validation of the identified targets: a) confirmation of the growth inhibitory/apoptotic effect of the selected RNAi clones; b) verification of targeting the translation of the respective mRNAs by Western blot; c) verification of the increased transcription and translation of the target genes in the malignant cell lines by RT-PCR and Western blot. The proposed studies are expected to be the first, but very important, step towards identification of targets for therapy of advanced and metastatic ovarian cancer. Later the scope of the research will be widened by including samples from patients to confirm the data from the cell lines and eventually, to obtain additional information. The great advantage of our approach, compared to other modern methods (such as microarrays and total transcriptome RNAi libraries)is the relative simplicity and much lower costs.