In the present study, a novel signaling pathway of microRNA-141 (miR-141)/fused

In the present study, a novel signaling pathway of microRNA-141 (miR-141)/fused in sarcoma (FUS) was investigated in neuroblastoma (NB). had similar tumor-suppressive effects as miR-141 upregulation on NB cell proliferation, cell cycle progression, migration and cisplatin chemosensitivity. Our data indicate that miR-141 and the FUS gene, which are inversely correlated, play significant functional functions in regulating human NB. gene is usually aberrantly repeated at chromosome 2p24, producing in NB progression to advanced stages, aggressive metastasis and poor patient prognosis (3C5). Unfortunately, the underlying molecular mechanisms INK 128 contributing to NB pathogenesis, metastasis and apoptosis are large unknown. It, thus, presents a great challenge to identifying novel biomarkers or therapeutic targets INK 128 for early detection or treatment for young patients with NB. MicroRNAs (miRNAs) are families of highly conserved non-coding small RNAs that attach to the 3-untranslated region (3-UTR) of downstream target genes to post-transcriptionally suppress gene manifestation, thus regulating various cellular processes in both animals and human diseases (6). miRNAs have been shown to play crucial functions in NB pathogenesis, metastasis and apoptosis (7,8). Among many of the oncogenic or tumor-suppressor INK 128 miRNAs, miR-141 is usually highly expressed in the circulating plasma in patients with advanced stages of metastatic colon malignancy, suggesting an oncogenic INK 128 role as a colon malignancy biomarker (9). In contrast, miR-141 was shown to be downregulated in gastric and prostate cancer, presumably acting as a tumor-suppressor of cancer progression and metastasis (10,11). In NB, Rabbit polyclonal to XIAP.The baculovirus protein p35 inhibits virally induced apoptosis of invertebrate and mammaliancells and may function to impair the clearing of virally infected cells by the immune system of thehost. This is accomplished at least in part by its ability to block both TNF- and FAS-mediatedapoptosis through the inhibition of the ICE family of serine proteases. Two mammalian homologsof baculovirus p35, referred to as inhibitor of apoptosis protein (IAP) 1 and 2, share an aminoterminal baculovirus IAP repeat (BIR) motif and a carboxy-terminal RING finger. Although thec-IAPs do not directly associate with the TNF receptor (TNF-R), they efficiently blockTNF-mediated apoptosis through their interaction with the downstream TNF-R effectors, TRAF1and TRAF2. Additional IAP family members include XIAP and survivin. XIAP inhibits activatedcaspase-3, leading to the resistance of FAS-mediated apoptosis. Survivin (also designated TIAP) isexpressed during the G2/M phase of the cell cycle and associates with microtublules of the mitoticspindle. In-creased caspase-3 activity is detected when a disruption of survivin-microtubuleinteractions occurs although a previous study showed that miR-141 is usually differentially expressed among NB subtypes (12), the exact manifestation information or mechanisms of miR-141 in NB remain evasive. The fused in sarcoma (FUS) gene, which encodes an RNA binding protein, is usually associated with genetic disorders particularly neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) (13,14). In human prostate cancer, FUS is usually shown to be a tumor-suppressor gene as its overexpression inhibited cancer growth and promoted malignancy apoptosis (15). In INK 128 a recent study, FUS was reported to be highly expressed in NB SK-N-AS cells (16). However, comparable to miR-141, the exact manifestation and function of FUS in human NB are largely unknown. In the present study, we firstly evaluated the manifestation of miR-141 in both growth of NB tumors, 2-month-old female nude mice were subcutaneously inoculated in the right flank with either miR- 141-mimic- or C-miRNA-transduced IMR-132 cells (1106). The lengths and widths of the subcutaneous tumors were assessed weekly, and the tumor volume (V) was calculated using the formula: V = length width2/2. Five weeks later, the mice were sacrificed. Subcutaneous tumors were extracted and formalin-fixed and paraffin-embedded sections were prepared. Immunohistochemistry was then performed using an anti-Ki-67 antibody (Cell Signaling, USA). Luciferase reporter assay Wild-type (WT) human FUS 3-UTR and mutated (MU) FUS 3-UTR (with a MU sequence on the miR-141 binding site) were amplified from a human brain cDNA library and inserted between the luciferase reporter pmiR-REPORT (RiboBio, Guangzhou, China). Human HEK293T cells were co-transfected with 25 ng/ml of either the luciferase reporter with WT FUS 3-UTR (WT-3UTR), or the luciferase reporter with MU FUS 3-UTR (MU-3UTR), and 100 pmol of either miR-141-mimics or C-miRNA. Forty-eight hours after co-transfection, a Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) was carried out according to the manufacturer’s protocol. For each measurement, the comparative firefly luciferase activity was normalized to with transfection of C-miRNA. FUS downregulation assay A small-interfering RNA (siRNA) against the human FUS gene (FUS-siRNA), and its control siRNA (C-siRNA) were purchased from RiboBio. In the NB culture, IMR-32 and SH-SY5Y cells were transfected with 100 nM FUS-siRNA or C-siRNA. Forty-eight hours after transfection, healthy cells were subject to qRT-PCR examination to verify.

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