| Summary: | 碩士 === 國立陽明大學 === 臨床醫學研究所 === 102 === let-7 is a family of microRNAs that express during stem cell differentiation and functions as a tumor suppressor. Our previous papers showed that let-7i, a member of the let-7 family microRNAs, is a metastasis repressor through targeting NEDD9 and AURKA. Furthermore, our unpublished study showed that let7i regulates the gene expression of ARID3A and ARID3B, which are the members of the AT-rich interaction domain (ARID) family proteins that play an essential role in development, tissue-specific gene expression and proliferation control. However, the mechanism of let-7-mediated repression of stem cell properties is unclear. Since the ARID3A shuttles between cytoplasm and nucleus and ARID3B constitutively locates in the nucleus, the transportation of ARID3A becomes an important issue for let-7 mediated stemness genes regulation. We therefore hypothesize that let-7 represses other targets which regulates the nucleo-cytoplasmic shuttling of ARID3A. Using softwere prediction, we found that the importin family proteins Importin 9, KPNA4 and KPNA1 are the candidate let-7 targets that regulate ARID3A transport. Manipulation of let7i only alters the expression of Importin 9 but not Kpna1 nor Kpna4. Coimmunoprecipitation experiment showed that Importin 9 interacts with ARID3A, and the association between IPO9 and ARID3A could be disrupted by the addition of Ran-Q69L, which is a mutant that traps Ran in its GTP bound form.We next analyzed the subcellular localization of ARID3A.ARID3B interacts with ARID3A to trap it in the nucleus. Disruption of ARID3A-ARID3B interaction via truncation of the interacting domain REKLES-β results in the accumulation of ARID3A in the cytoplasm. These results suggested that in head and neck cancer cells, let-7 orchestrates the assembly of ARID3A-ARID3B complex through Importin 9.The significance of ARID3A-ARID3B complex on regulating stemness genes needs to be further investigated.
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