Mir-137 and Mir-197 within Chromosome 1p Minimal Deletion Region Regulate Apoptotic Activity in Multiple Myeloma by Targeting MCL 1

Abstract 322

Chromosome 1p deletion is detected in nearly 20 % of patients diagnosed with multiple myeloma (MM) and confers a poor prognosis. To pursue our discovery for potential tumor suppressor gene(s) in the 1p21 deletion region, we searched for all miRNA genes that are mapped to the minimal deletion region (1p12-1p21). Defining these nucleotides as start and end locations in miRBase search lists identifies five miRNAs located this deletion region. In the present study, we focused on the functional role of miR-137and miR-197 in MM since they have been shown to be deregulated in the solid tumors.

Low expressions of miR-137 and miR-197 were validated in MM1S, MM1R, and My5 cell lines harboring del (1p12-1p21) compared with H929 and U266 cell lines without such deletion by qRT-PCR analysis. In order to evaluate the involvement of miRNA-137 and -197 in MM cells, we transfected pre-miR-137 and pre-miR-197 in above cell lines. A decrease in cell proliferation of MM cells was observed in a dose dependent manner in MM1S, MM1R, and MY5 cells but not in H929 cells. Overexpression of miR137 or miRNA-197 induces cell apoptosis by increased amount of Annexin V–positive cells and increased percentage of sub-G1 population in MM1S cells.

In an attempt to identify the differentially expressed apoptosis genes between miRNAs-treated and untreated MM cells, we conducted a customized human apoptosis RT2 Profiler PCR Array assay to monitor the expression of 84 key genes, which were further validated by qRT-PCR analysis. We observed regulation of apoptosis-linked genes in a proapoptotic manner, indicating that these two miRNAs could sensitize cells for apoptotic events. In cells treated with miR-137 or -197, the expression of the anti-apoptotic gene-MCL1 showed no noticeable changes, whereas the other genes coding for proapoptosis-inducing and -supporting products such as BAD, BAX, BID, and BIM were markedly increased. In addition, transfection with miR-137 or -197 in MM1S cells resulted in an increase of caspase-9, caspase-3, and PARP, but not caspase-8, at 48 hours post-transfection. Importantly, MCL1 protein in MM1S cells at 72 hours of treatment with miR-137 or -197 was significantly decreased compared to scrambled treatment by western blotting assay. These results suggest these 2 miRNAs induced apoptosis in MM cells may be mediated by extrinsic apoptotic pathways through targeting MCL1 gene.

To further identify miR-137 and -197 targets, we firstly used bioinformatics analysis. Comparing the results obtained from the different searches, we found that the MCL1 protein was predicted as a target of miR-137 and -197 by the miRanda algorithm (www.microrna.org/microrna/home.do and http://www.targetscan.org/vert_52/). RNAhybrid also predicted a possible binding region of miR-137 and -197 in the 3' untranslated region (UTR) of MCL1. To validate MCL1 as miR-137 and -197 target, we cloned the 3' UTR sequence of human MCL1 into the luciferase-expressing vector pEZX-MT01 to control downstream of the luciferase stop codon. MM1S cells were transiently transfected with this construct in the presence of pre-miR-137 or pre-miR197, or a scrambled oligonucleotide acting as a negative control. As reported in luciferase plate reader, miR-137 or miR-197 significantly reduced luciferase activity compared with the scrambled control miRNA. This indicates that miR-137 and -197 bind to the 3'UTR of MCL1 and impair its mRNA translation. In order to further confirm that the region was specific for binding with miR-137 and -197, we generated the deletion mutants of 3'UTR of MCL1 lacking the binding site for miR137 and miR-197, respectively. The mutants were subsequently cloned into the luciferase gene following Renilla gene, and then cotransfected with pre-miR-137 or -197 in MM1S cells. miR-137 and -197 added singularly or at the same time, did not significantly reduce luciferase activity in the presence of the 3'UTR of MCL1 mutated sequence.

In conclusion, our data suggest that miR-137 and -197 induce apoptosis in myeloma cells at least in part through the control of MCL1 protein expression. Deregulated miR-137 or -197 may play an important role in the subset of high-risk MM with 1p deletions.