Abstract
Constitutive hedgehog (Hh) signaling is associated with the genesis of medulloblastomas (MB). The objective of this study is to identify special microRNAs (miRNAs) regulated by the Hh pathway, and to clarify the role of miRNAs during the genesis of MB induced by sustained Hh activation. In the primary screening, we used stem-loop RT-PCR to test the expression of 90 different miRNAs in the wildtype (WT) and Ptc−/− MEF cell lines. In the secondary screening, the miRNAs screened from the first screening were validated in the Sufu−/− MEF cell lines. We then verified the expression of miRNAs both in the normal cerebellar tissues and the MB induced by activated Hh pathway, and examined the expression of the other 21 miRNA members of the miR-154 cluster in the MB and normal cerebellum. In the first screening, 13 miRNAs showed significant differential expression in WT and Ptc−/− MEF cell lines, while 10 of them had significant difference in the Sufu−/− MEF cell line. Compared to the normal mouse cerebellum, only 2 miRNAs in 15 miRNAs were differentially expressed between the MB and normal cerebellar tissues. Among 21 members of the miR-154 cluster, 6 miRNAs were downregulated in the MB. Our study demonstrated that miR-154 may be regulated by the Hh pathway, and the activation of the Hh pathway led to the downregulation of the miR-154 cluster, resulting in the genesis of MB.
Highlights
Hedgehog (Hh) is one of the primary morphogenic signals that specify the patterns of cell growth and differentiation during vertebrate development
"stem cell" tumor caused by the abnormal proliferation of granular cells in the internal granule layer (IGL) of the cerebellum, which come from the differentiation of granular cell precursors (GCPs) in the external germinal layer (EGL)[6,7]
Screening of miRNAs regulated by the Hh signaling pathway in WT mouse embryonic fibroblast (MEF), Ptc-/- MEF, and Sufu-/- MEF cell lines
Summary
Hedgehog (Hh) is one of the primary morphogenic signals that specify the patterns of cell growth and differentiation during vertebrate development. Sustained Hh signaling pathway activity can lead to congenital anomalies and development of various tumors[1]. MB is a "stem cell" tumor caused by the abnormal proliferation of granular cells in the internal granule layer (IGL) of the cerebellum, which come from the differentiation of granular cell precursors (GCPs) in the external germinal layer (EGL)[6,7]. When the Hh pathway is abnormally activated, GCP cells cannot exit the cell cycle; they continue to proliferate and form a large number of granule cells accumulating in the EGL layer, and develop MB. The mechanism of this pathological process remains elusive[8,9]
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