Targeting of Marcks By MiR-34a Overcomes Drug Resistance in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. Current therapies, such as the proteasome inhibitor bortezomib (BTZ), have improved the outcome of patients. Nevertheless MM remains an incurable disease with a high rate of relapse and development of drug resistance. So far, the pathogenic mechanisms underlying drug resistance of MM are not fully elucidated. Our previous study showed the overexpression of MARCKS (Myristoylated alanine-rich C-kinase substrate) protein in MM cells played an important role in drug resistance. However; the mechanism (s) underlying MARCKS overexpression and its association with drug resistance in MM have not been elucidated.

In the current study, to investigate whether miRNAs play a role in aberrant upregulation of MARCKS in drug resistance, several miRNA-target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Among 52 miRNA candidates that target MARCKS, the expression level of several miRNAs with higher score was assessed in drug resistant cell lines (8226-R5 and MM1.R) in comparison to the parental cells (8226 and MM1.S). We observed that miR-34a expression was most significantly downregulated in 8226-R5 and MM1.R cell lines relative to their parental lines, 8226 and MM1S. We also identified a negative correlation between MARCKS and miR-34a expression levels in 6 primary patient samples and two healthy donor samples. Next, to confirm direct targeting of MARCKS by miR-34a, two MM resistant cell lines (8226-R5 and MM1R) were transduced with lentiviral vectors harboring miR-34a precursor gene or scramble control and mRNA and protein level of MARCKS were assessed in miR-34a overexpressing cell lines in comparison to scramble control. Overexpression of miR-34a reduced MARCKS expression level in both mRNA and protein levels. In addition, the luciferase reporter assay was performed to validate MARCKS as a direct target of miR-34a. To this end, the 3' UTR sequence of human MARCKS was cloned into the luciferase-expressing vector pEZX-MT01 to the downstream of the firefly luciferase gene. Co-transfection of MARCKS-UTR or negative control luciferase reporter vectors together with miR-34a expressing or control vector showed that miR-34a overexpression significantly reduced the luciferase activity, indicating that miR-34a can directly target the MARCKS 3′ UTR.

To examine the functional effect of miR-34a on cell proliferation and apoptosis in MM cells, the miR-34a overexpressing or scramble control cell lines were treated with BTZ. Overexpression of miR-34a in drug resistant cell lines re-sensitized them to BTZ. Furthermore, FACS analysis revealed that overexpression of miR-34a could induce apoptosis in drug resistant MM cell lines as indicated by an increase in the percentage of annexin-V positive cells compared to the control, suggesting that downregulation of miR-34a is associated with acquired BTZ-resistance in MM.

To further elucidate the interaction of miR-34a and MARCKS, we knocked down MARCKS expression by transfecting shMARCKS lentiviral vector into 8226-R5 and MM1.R resistant cell lines and examined the miR-34a level in MARCKS-knockdown cells. We found that miR-34a is also regulated by MARCKS as silencing of MARCKS could restore the miR-34a expression level in MM resistant cell lines. In compatible with miR-34a overexpression experiments, following the knockdown of MARCKS, both cell lines had significantly lower viability after treatment with BTZ, in comparison to negative controls. We also showed that knockdown of MARCKS by shMARCKS silencing or suppression of MARCKS by miR-34a overexpression increased the mRNA and protein levels of some pro-apoptotic genes including PUMA which may be involved in induction of apoptosis in drug resistant MM cells.

In conclusion, our data indicate that miR-34a is downregulated in drug resistant MM cell lines and primary MM cells, which contributes to the drug resistance of MM through upregulation of MARCKS. Moreover, regulatory interaction between miR-34a and MARCKS provides the rationale that restoration of miR-34a may serve as a promising therapeutic approach by targeting MARCKS in patients with refractory/relapsed MM.