Objective: MicroRNAs (miRNA) are single-stranded, small non-coding RNA molecules (∼21 nucleotides) that regulate protein-coding gene expression at the post-transcriptional level, mainly through interactions with the 3′-untranslated region of target mRNAs. Such interactions lead to mRNA degradation and/or translational repression, depending on the complementarity of the miRNA seed sequence with the mRNAs 3′-untranslated region. They can function as oncogenes or tumor suppressors, possessing a vital role in all stages of tumorigenesis and cancer progression. In the present study, we have investigated the clinical significance of a molecular signature consisting of 10 cancer-related miRNAs in multiple myeloma (MM): miR-15a, miR-16, miR-21, miR-221, miR-222, miR-25, miR-125, miR-155, miR-223, and miR-181a. These molecules were selected due to their well-documented role and clinical significance in numerous human malignancies.
Methodology: Bone marrow aspiration samples were collected from 94 patients with multiple myeloma (MM) and smoldering multiple myeloma (sMM) at the time of diagnosis and CD138+ plasma cells were positively selected using magnetic beads coated with an anti-CD138 antibody. Total RNA was isolated using TRIZol, 200ng RNA of each sample were polyadenylated at the 3′ end and reversely transcribed. An in-house developed real-time quantitative PCR assay was conducted and the results were biostatistically analyzed. For the normalization of the expression levels of each miRNA, the mean expression of two small nucleolar RNAs (RNU43 and RNU48) was used as reference.
Results: Seventy-six out of the 94 BM aspiration samples were derived from MM patients and 18 from sMM patients. The MM patients were classified, according to the R-ISS staging system, as follows: 15 patients with stage I disease, 42 patients with stage II, and 19 patients with stage III. Forty-nine myeloma patients presented with osteolytic lesions at diagnosis. The statistical analysis revealed significantly lower expression levels of miR-16 (p=0.036) and miR-155 (p=0.045) in CD138+ cells of MM patients, compared to those from sMM patients. Furthermore, miR-221 and miR-222 expression levels were negatively correlated with R-ISS; thus, miR-221 and miR-222 expression was significantly downregulated in MM patients with R-ISS stage III (p=0.004 and 0.034, respectively). Interestingly, the expression levels of miR-15a (p=0.048) and miR-16 (p=0.047) were decreased in MM patients with osteolytic lesions at the time of diagnosis, compared to those without osteolyses.
Conclusion: We conclude that miR-221/222 cluster correlates with more favorable R-ISS stage, revealing a potential favorable prognostic value in MM patients. MiR-15a and miR-16 correlate with the presence of osteolytic disease in MM. The observed decreased expression of these two miRNAs in symptomatic MM patients with osteolytic lesions could constitute a possible biomarker for the occurrence of bone disease. Moreover, decreased expression of miR-16 and miR-155 in MM patients, compared to sMM patients may indicate a putative predictive biomarker able to distinguish symptomatic patients from sMM patients. This ongoing study will further reveal the possible prognostic significance of this 10 miRNAs signature studied, when response to therapy, progression-free and overall survival is available.
