ESTABLISHMENT OF MULTIPLE MYELOMA CELL LINES WITH REDUCED EXPRESSION OF HEPATOCYTE GROWTH FACTOR USING RNAI APPROACH

Abstract

Objective: To establish the gene knockdown system (RNAi) using different Multiple Myeloma Cell lines (MMCL). Material and Methods: RNAi is generally used in biomedical research to study the outcome of knockdown of gene expression. In this research, retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) were designed and constructed. The sequence of the cloned fragments in the vector constructs was determined, and the effect of constructs in MMCLs was examined using qRT-PCR and Western blot analysis. Results: In this work, we established a method for making retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) design. The HGF producing human myeloma cell line JJN-3 transduced with lentiviral HGF-shRNA transduction particles were analyzed for their effect on the HGF gene expression. HGF knockdown MMCL was analyzed both at mRNA level and protein levels. Two out of five clones of myeloma cell line JJN-3 (JJN3-sh3308 and JJN3-sh47137) were transduced with lentiviral HGF–shRNA transduction particles were showing 50-60 % knockdown in their HGF mRNA and protein expression. Conclusion: HGF knockdown cell lines can be used as tissue culture models to investigate the role of HGF protein in the pathology of multiple myeloma. Further, the retroviral-HGF-shRNA expression constructs can study HGF response in various multiple myeloma via gene knockdown studies.