The microRNA pathway regulates differentiation and proliferation of human multipotent stromal cells from bone marrow

The microRNA (miRNA) pathway regulates differentiation and proliferation in a variety of organisms. Here we analyzed the function of the miRNA pathway in regulating differentiation and proliferation of human multipotent stromal cells from bone marrow (hMSCs).;Differentiation of hMSCs into osteoblasts and adipocytes was inhibited using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were upregulated during osteogenic differentiation, 20 during adipogenic differentiation of which 11 were commonly upregulated in both osteogenic and adipogenic differentiation. In silico models predicted that 5 of the upregulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.;We then assessed the role of the miRNA pathway in hMSC proliferation using Drosha and Dicer knockdown hMSCs. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSC proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing.