Role of MicroRNAs in Resistance to Dexamethasone in Multiple Myeloma Cell Lines

Abstract

Multiple Myeloma is a B-cells malignant disease of unknown etiology. It accounts for 1% of all cancers and 14% of hematologic malignancies. Despite the advances in treatment it remains an incurable disease. One of the commonly used drugs in the treatment of multiple myeloma is Dexamethasone. Most patients respond to this drug, but eventually most then become refractory to the treatment with steroids. MicroRNAs (miRNA) are a newly identified class of small RNAs that play important regulatory roles at the post transcriptional level. They usually act by binding to the 3′untranslated region (3′UTR) of messenger RNA (mRNA) and either block their translation or lead to degradation. MM1 cell line is a well known myeloma cell line. By continued exposure to Dexamethasone two variants of MM1 cell line i.e.; MM1.S and MM1.R were developed. MM1.S remains sensitive while MM1.R is resistant to the action of Dexamethasone. It has been reported that MM1.R cell line expresses very minimal levels of glucocorticoid receptor (GR) mRNA and protein, hence rendering it resistant to the action of steroids. We hypothesized that altered regulation of microRNAs between the two cell lines leads to the down regulation of glucocorticoid receptor hence contributing to resistance to the action of steroids. QRT-PCR and Western blot were conducted to demonstrate decreased expression of GR mRNA and protein in MM1.R cell line. We identified certain microRNAs based on bioinformatics with putative docking sites in 3′UTR of GR mRNA. DNA sequencing was used to rule out any mutation in binding sites of these microRNAs. No such mutation was found in MM1.S or MM1.R cell line. qRT-PCR was than used to analyze the differential expression of predicted microRNAs. Several microRNAs were found to be differentially expressed between the two cell lines, including microRNA-183 (p < 0.05). We then transfected inhibitors and mimics of pertinent microRNAs and were able to up or down regulate the GR mRNA. We were also able to show some evidence of apoptosis in MM1.R cell line with Dexamethasone exposure. These studies suggest that microRNAs regulate expression of GR and play an important role in conferring resistance to the action of steroids. These findings shed further light on the underlying mechanisms of steroid resistance and may have important therapeutic implications in the treatment of multiple myeloma.