Let-7 Microrna Family Members Regulate Cell Proliferation in Multiple Myeloma

MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. In particular, the Let-7 miRNA family members have been described to act as tumor suppressors, as demonstrated both in solid cancer and hematologic malignancies. However, the role of Let-7 in multiple myeloma (MM) has not been studied.

Circulating miRNA profiling has been performed in MM patients compared to healthy individuals using TaqMan human miRNA profiling, and validated by qRT-PCR. Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. Exosomes were then evaluated for their miRNA content, by qRT-PCR. Gain- and loss-of functions studies were performed on MM cell lines (MM.1S; U266), using Let-7-mimic and Lin28B siRNA, respectively. Scramble probe-transfected cells were used as control. Cell proliferation and cell survival have been evaluated by using BrdU assay and MTT assay, respectively. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot.

We identified a MM specific signature characterized by down-regulation of miRNA-15a, −19b, −21, let-7b, let-7c and over-expression of miR-720 (P < 0.001). Circulating exosomes were studied at ultrastructural level showing positivity for CD81 and CD63, as demonstrated by immunogold labeling and electron microscopy. The same miRNA signature was found in the circulating exosomes, suggesting that circulating miRNAs may be transported by exosomes. The Let-7 family members were significantly decreased in peripheral blood of MM patient compared to healthy individuals, suggesting that Let-7 family could be down-regulated in MM cells. We then performed qRT-PCR in MM primary cells; and found that the Let-7 family is significantly down-regulated in MM primary cells, especially for Let-7b and c (5 fold change, P< 0.05). Over-expression of Let-7b and Let-7c in MM cells (U266; MM1S) transfected decreased cell proliferation. The RNA binding protein Lin28B is known to regulate the Let-7 family: we therefore performed Lin28-loss of function studies which led to up-regulation of the Let-7 family members, in MM transfected cells; and found that Lin28B-knockdown cells presented with reduced cell proliferation, supported by down-regulation of c-Myc and K-Ras, known to be Let-7-related targets.

This data indicate that Let-7 miRNA family members play an important role in modulating MM biology, thus providing the basis for the development of new miRNA-based target therapies and biomarker in this disease.

Ghobrial:Novartis: advisory board, advisory board Other; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: advisory board, advisory board Other.