Establishment of Multiple Myeloma Cell lines with Hepatocyte growth factor (HGF) overexpression and knockdown

Abstract

Multiple myeloma is the malignancy of plasma cells which causes 0.9 % of all cancer related deaths. These malignant plasma cells acquire chromosomal abnormalities and complex genetic instability. Hepatocyte growth factor (HGF) is a multifunctional cytokine promoting cell proliferation, survival, motility, scattering, differentiation and morphogenesis. HGF/c-MET pathway plays an important role in multiple myeloma pathogenesis and in extravasation and homing of myeloma cells to bone marrow microenvironment. The analysis of gene expression of HGF is important for better understanding the pathology of myeloma. The role of a gene involved in a disease can be better understood by gene knock down and overexpression analysis. RNAi is generally used in biomedical research to study the outcome of knock down of gene expression. RNAi can be exploited in experimental settings by exogenous introduction of dsRNAs or constucts which express shRNAs (short hairpin RNA).

In this work we have established a method for making retroviral-shRNA expression constructs based on micro RNA-adapted short hairpin RNA (sh RNA mir) design. By using this method we have generated five HGF-shRNA expression constructs. Additionally, 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 knock down 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) transduced with lentiviral HGF–shRNA transduction particles were showing 50-60 % knock down in their HGF mRNA and protein expression. Besides this, non HGF producing human myeloma cell lines INA-6, transduced with HGF expression plasmids, were tested for overexpression of HGF gene. Both HGF overexpressing cell lines (INA-6-HGF and INA-6-HGF-2) showed expression of HGF at the mRNA and at the protein level. However the overexpression of the HGF protein was not as high as the expression of HGF protein in JJN-3 cell line which is a high HGF producing cell line. These HGF knock down and over expressing cell lines can be used as tissue culture models to investigate the role of HGF protein in pathology of multiple myeloma. Similarly the retroviral-HGF-shRNA expression constructs can contribute to understanding HGF response in multiple myeloma via gene knock down studies.