Aberrantly expressed microRNA (miRNA) are known to disrupt intracellular RNA networks in cancer cells. Exploring miRNA-dependent molecular networks is a major challenge in cancer research. In this study, we performed RNA-sequencing of breast cancer (BrCa) clinical specimens to identify tumor-suppressive miRNA in BrCa. In total, 64 miRNA were identified as candidate tumor-suppressive miRNA in BrCa cells. Analysis of our BrCa signature revealed that several miRNA duplexes (guide strand/passenger strand) derived from pre-miRNA were downregulated in BrCa tissues (e.g. miR-99a-5p/-3p, miR-101-5p/-3p, miR-126-5p/-3p, miR-143-5p/-3p, and miR-144-5p/-3p). Among these miRNA, we focused on miR-101-5p, the passenger strand of pre-miR-101, and investigated its tumor-suppressive roles and oncogenic targets in BrCa cells. Low expression of miR-101-5p predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0316). Ectopic expression of miR-101-5p attenuated aggressive phenotypes, e.g. proliferation, migration, and invasion, in BrCa cells. Finally, we identified seven putative oncogenic genes (i.e. High Mobility Group Box 3, Epithelial splicing regulatory protein 1, GINS complex subunit 1 (GINS1), Tumor Protein D52, Serine/Arginine-Rich Splicing Factor Kinase 1, Vang-like protein 1, and Mago Homolog B) regulated by miR-101-5p in BrCa cells. The expression of these target genes was associated with the molecular pathogenesis of BrCa. Furthermore, we explored the oncogenic roles of GINS1, whose function had not been previously elucidated, in BrCa cells. Aberrant expression of GINS1 mRNA and protein was observed in BrCa clinical specimens, and high GINS1 expression significantly predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0126). Knockdown of GINS1 inhibited the malignant features of BrCa cells. Thus, identification of tumor-suppressive miRNA and molecular networks controlled by these miRNA in Abbreviations BrCa, breast cancer; ESRP1, Epithelial splicing regulatory protein 1; GEO, Gene Expression Omnibus; GINS1, GINS complex subunit 1; HMGB3, High Mobility Group Box 3; MAGOHB, Mago Homolog B; miRNA, microRNA; RISC, RNA-induced silencing complex; SRPK1, Serine/Arginine-Rich Splicing Factor Kinase 1; TCGA, The Cancer Genome Atlas; TPD52, Tumor Protein D52; VANGL1, Vang-like protein 1.426 Molecular Oncology 14 (2020) 426-446 ª
The amino acid sequences of three variants of the Kunitz-type trypsin inhibitors, Tia, Tib, and Tic, obtained from some cultivars of soybean were determined by conventional methods. All three inhibitors consisted of 181 amino acid residues. The differences in the amino acid sequences are as follows: Tia E12 G55 Y62 H71 S74 M114 L120 P137 L176; Tib S F N R V I T V; Tic E. The amino acid sequences of Pro(60)-Ser(61) and Asp(154)-Asp(155)-Gly(156)-His(157) of Tia reported previously (Koide & Ikenaka (1973) Eur. J. Biochem. 32, 417-431) were amended to Ser(60)-Pro(61) and His(154)-Asp-Asp-Gly(157), respectively.
MicroRNAs (miRNAs) are unique in that a single miRNA molecule regulates a vast number of RNA transcripts. Thus, aberrantly expressed miRNAs disrupt tightly controlled RNA networks in cancer cells. Our functional screening showed that expression of miR-124-3p was downregulated in pancreatic ductal adenocarcinoma (PDAC) tissues. Here, we aimed to investigate the anti-tumor roles of miR-124-3p in PDAC cells and to identify miR-124-3p-mediated oncogenic signaling in this disease. Ectopic expression of miR-124-3p inhibited cancer cell migration and invasion in PDAC cells. Moreover, restoration of miR-124-3p suppressed oncogenic signaling, as demonstrated by reduced phosphorylation of focal adhesion kinase, AKT, and extracellular signal-regulated kinase, in PDAC cells. Our in silico database analyses and luciferase reporter assays showed that two cell-surface matrix receptors, integrin α3 (ITGA3) and integrin β1 (ITGB1), were directly regulated by miR-124-3p in PDAC cells. Overexpression of ITGA3 and ITGB1 was confirmed in PDAC clinical specimens. Interestingly, a large number of cohort analyses from TCGA database showed that high expressions of ITGA3 and ITGB1 were significantly associated with poor prognosis of patients with PDAC. Knockdown of ITGA3 and ITGB1 by siRNAs markedly suppressed the migration and invasion abilities of PDAC cells. Moreover, downstream oncogenic signaling was inhibited by ectopic expression of miR-124-3p or knockdown of the two integrins. The discovery of anti-tumor miRNAs and miRNA-mediated oncogenic signaling may provide novel therapeutic targets for the treatment of PDAC.
Triple-negative breast cancer (TNBC) is an aggressive type of cancer associated with a poor prognosis. Identification of novel therapeutic targets in TNBC is urgently needed. Here, we investigated the microRNA (miRNA) expression signature of TNBC using clinical specimens. In total, 104 miRNAs (56 upregulated and 48 downregulated) were significantly dysregulated in TNBC tissues; miR-204-5p showed the most dramatic downregulation. We then examined the antitumor roles of miR-204-5p in breast cancer (BC) cells. Notably, cancer cell migration and invasion were significantly reduced by ectopic expression of miR-204-5p in BC cells. Genome-wide gene expression analysis and in silico database search revealed that 32 genes were putative miR-204-5p targets. High expression of AP1S3, RACGAP1, ELOVL6, and LRRC59 was significantly associated with poor prognosis in patients with BC, and adaptor-related protein complex 1 sigma 3 subunit (AP1S3) was directly regulated by miR-204-5p, as demonstrated by luciferase reporter assays. AP1S3 overexpression was detected in TNBC clinical specimens and enhanced cancer cell aggressiveness. We further analyzed downstream RNA networks regulated by AP1S3 in BC cells. Overall, this miRNA signature is expected to be an effective tool for identification of miRNA-mediated molecular mechanisms of TNBC pathogenesis.
Introduction. Taka-amylase A (TAA) [EC 3.2.1.1 a-1,4-glucan 4-glucanohydrolase, Aspergillus oryzae] which was crystallized first by Akabori et al. in 19511) is a glycoprotein consisting of a single polypeptide chain of 478 amino acid residues with an amino (N)terminal alanine and a carboxy (C)-terminal serine.2~'3> The partial amino acid sequences of the N-and C-terminal regions,4~ '5> the carbohydrate chain structure° 7) and X-ray crystallographic analysis8> of the enzyme have been reported. This paper describes the complete amino acid sequence of TAA. Materials and methods. Crystalline TAA prepared from Takadiastase Sankyo9> was further purified by DEAE-cellulose column chromatography.10~ The homogeneous enzyme judged by polyacrylamide gel electrophoresis was reduced and carboxymethylated.'1 The cyanogen bromide cleavage at methionine residues of the reducedcarboxymethylated TAA (RCM-TAA) was performed in 70% formic acid for 24 h at room temperature. After removal of excess reagents by repeated lyophilization, the CNBr fragments were fractionated by gel filtration on a Sephadex G-75 column, by affinity chromatography on a Concanavalin A-Sepharose column, by ion-exchange chromatography on a SP-Sephadex C-25 column or an AG50W x 2 column and by paper electrophoresis. Methionine-containing peptides were isolated from tryptic and chymotryptic digests of maleylated RCM-TAA by a combination of gel filtration on Sephadex G-75 and G-50 columns, paper electrophoresis and high performance liquid chromatography. Amino acid compositions of peptides were determined with a Hitachi 8355 automated amino acid analyzer after hydrolysis of the samples with twice-distilled HCl containing 1 (v/v) phenol in evacuated sealed tubes at 110°C for 24, 48 and 72 h. Sequence analyses of the fragments were mostly performed by automated Edman degradation using a Beckman model 890C liquidphase sequencer in the presence of Polybrenel2) or an LKB 4020 College,
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