Cellular differentiation assessments by measuring the degree of cellular internalization and membrane adsorption using designed peptides.

We demonstrate examples of cellular differentiation assessments, including cellular neurite outgrowth and fat cell maturation, by measuring the degree of membrane adsorption or cellular internalization using designed peptides. Because changes in the cellular membrane and cytosol during differentiation were shown to influence membrane adsorption and cellular internalization, we could successfully evaluate the extent of differentiation simply like stain indicators.

Placenta-specific miRNA (miR-512-3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells.

AIM: The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets. METHODS: A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay. RESULTS: By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system. CONCLUSION: These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.

SnoN/SKIL modulates proliferation through control of hsa-miR-720 transcription in esophageal cancer cells.

It is now evident that changes in microRNA are involved in cancer progression, but the mechanisms of transcriptional regulation of miRNAs remain unknown. Ski-related novel gene (SnoN/SKIL), a transcription co-factor, acts as a potential key regulator within a complex network of p53 transcriptional repressors. SnoN has pro- and anti-oncogenic functions in the regulation of cell proliferation, senescence, apoptosis, and differentiation. We characterized the roles of SnoN in miRNA transcriptional regulation and its effects on cell proliferation using esophageal squamous cell carcinoma (ESCC) cells. Silencing of SnoN altered a set of miRNA expression profiles in TE-1cells, and the expression levels of miR-720, miR-1274A, and miR-1274B were modulated by SnoN. The expression of these miRNAs resulted in changes to the target protein p63 and a disintegrin and metalloproteinase domain 9 (ADAM9). Furthermore, silencing of SnoN significantly upregulated cell proliferation in TE-1 cells, indicating a potential anti-oncogenic function. These results support our observation that cancer tissues have lower expression levels of SnoN, miR-720, and miR-1274A compared to adjacent normal tissues from ESCC patients. These data demonstrate a novel mechanism of miRNA regulation, leading to changes in cell proliferation.

Genes that integrate multiple adipogenic signaling pathways in human mesenchymal stem cells.

Adipogenesis is a well-characterized cell differentiation process. A large body of evidence has revealed the core transcription factors and signaling pathways that govern adipogenesis, but cross-talks between these cellular signals and its functional consequences have not been thoroughly investigated. We, therefore, sought to identify genes that are regulated by multiple signaling pathways during adipogenesis of human mesenchymal stem cells. Focusing on the early stage of adipogenesis, microarray analysis and quantitative RT-PCR identified 12 genes whose transcription levels were dramatically affected by the complete adipogenic induction cocktail but not by the cocktail's individual components. Expression kinetics of these genes indicate diverse mechanisms of transcriptional regulation during adipogenesis. Functional relationships between these genes and adipogenic differentiation were frequently unknown. This study thus provided novel adipogenic gene candidates that likely mediate communications among multiple signaling pathways within human mesenchymal stem cells.

Transcripts of unknown function in multiple-signaling pathways involved in human stem cell differentiation.

Mammalian transcriptome analysis has uncovered tens of thousands of novel transcripts of unknown function (TUFs). Classical and recent examples suggest that the majority of TUFs may underlie vital intracellular functions as non-coding RNAs because of their low coding potentials. However, only a portion of TUFs have been studied to date, and the functional significance of TUFs remains mostly uncharacterized. To increase the repertoire of functional TUFs, we screened for TUFs whose expression is controlled during differentiation of pluripotent human mesenchymal stem cells (hMSCs). The resulting six TUFs, named transcripts related to hMSC differentiation (TMDs), displayed distinct transcriptional kinetics during hMSC adipogenesis and/or osteogenesis. Structural and comparative genomic characterization suggested a wide variety of biologically active structures of these TMDs, including a long nuclear non-coding RNA, a microRNA host gene and a novel small protein gene. Moreover, the transcriptional response to established pathway activators indicated that most of these TMDs were transcriptionally regulated by each of the two key pathways for hMSC differentiation: the Wnt and protein kinase A (PKA) signaling pathways. The present study suggests that not only TMDs but also other human TUFs may in general participate in vital cellular functions with different molecular mechanisms.

Increased inhibitory ability of conjugated RNA aptamers against the HCV IRES.

Hepatitis C virus (HCV) translation begins within the internal ribosome entry site (IRES). We have previously isolated two RNA aptamers, 2-02 and 3-07, which specifically bind to domain II and domain III-IV of the HCV IRES, respectively, and inhibit IRES-dependent translation. To improve the function of these aptamers, we constructed two conjugated molecules of 2-02 and 3-07. These bound to the target RNA more efficiently than the two parental aptamers. Furthermore, they inhibited IRES-dependent translation about 10 times as efficiently as the 3-07 aptamer. This result indicates that combining aptamers for different target recognition sites potentiates the inhibition activity by enhancing the domain-binding efficiency.

A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III-IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId.

The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III-IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA-RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.

RNA aptamers targeted to domain II of hepatitis C virus IRES that bind to its apical loop region.

The internal ribosome entry site (IRES) is important for translation of hepatitis C virus (HCV) mRNA and has a unique RNA structure containing conserved domains I to IV. To investigate the function of domain II, we selected RNA aptamers that bind to domain II of HCV IRES by applying a simple and convenient selection method using a hybridized tag for fixing domain II RNA on magnetic beads instead of synthesizing long RNA. In addition, we employed surface plasmon resonance (SPR) technology to measure the binding affinity of each generation and to obtain detailed kinetic constants. The selected aptamers have a consensus sequence, 5'-UAUGGCU-3', which is complementary to the apical loop of domain II. The loop-loop interaction between the consensus sequence and domain II was confirmed by mutagenesis and nuclease mapping analyses. Binding affinities were dependent on the local structure containing the conserved sequence. The aptamers could inhibit IRES-dependent translation.

MiR-376c down-regulation accelerates EGF-dependent migration by targeting GRB2 in the HuCCT1 human intrahepatic cholangiocarcinoma cell line.

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3'-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.

The miR-221/222 cluster, miR-10b and miR-92a are highly upregulated in metastatic minimally invasive follicular thyroid carcinoma.

Minimally invasive follicular thyroid carcinoma (MI-FTC) is characterized by limited capsular and/or vascular invasion with good long-term outcomes. However, some cases of MI-FTC show a poor prognosis because of severe distant metastasis (i.e., metastatic MI-FTC). Nonetheless, no method has been established for predicting the prognosis of MI-FTC. This study was conducted to identify novel prognostic factors for metastatic MI-FTC by the use of microRNA (miRNA). Thirty-four patients with MI-FTC were categorized into two groups: the metastatic group, M(+) (n=12) and the non-metastatic group, M(-) (n=22). In the M(+) group, distant metastasis was recognized after the initial operation established the diagnosis of MI-FTC. In the M(-) group, no distant metastasis was recognized postoperatively for ≥ 10 years. Using laser microdissection followed by quantitative real-time PCR and PCR arrays, we performed a comprehensive expression profiling of 667 miRNAs in formalin-fixed, paraffin-embedded samples from the initial MI-FTC operation. Furthermore, we assessed the potential use of miRNAs as novel biomarkers for the metastatic potential of MI-FTC by logistic regression analysis. Comprehensive quantitative analysis of miRNA expression in MI-FTC samples revealed that the miR-221/222 cluster (i.e., miR-221, miR-222 and miR-222*), miR-10b and miR-92a were significantly upregulated in the M(+) group compared with the M(-) group. Interestingly, the expression levels of these miRNAs were also shown to be upregulated in widely invasive FTC (WI-FTC; n=13) that has distant metastasis and worse prognosis, indicating a close similarity in the miRNA expression between metastatic MI-FTC and WI-FTC. Logistic regression analysis revealed that miR-10b made a significant contribution to prognosis (OR 19.759, 95% CI 1.433-272.355, p=0.026). Our findings suggest that miR-10b is a potential prognostic factor for evaluating the metastatic potential of MI-FTC at an initial operation stage.

Isolation of RNA aptamers specific for the 3' X tail of HCV.

The 3' end of the HCV genome, designated as the 3' X tail, comprises an almost invariant 98-nucleotide sequence containing three highly conserved stem-loop structures (3' SL1, 3' SL2, and 3' SL3). Since these sequences are all critical for the initiation of negative-strand synthesis and essential for viral replication, they are attractive targets for novel anti-HCV drugs. To obtain effective RNA aptamers specific for the 3' X tail, and with the aim of developing novel inhibitors of HCV replication, we performed in vitro selection of aptamers with specificity for the 3' X tail. In vitro selection, namely SELEX (systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating nucleic acid sequences from a randomized oligonucleotide pool that have a high affinity for a target molecule. After four selection cycles, a pool of the 3' X tail-specific RNA aptamers were obtained. This RNA pool included 39 clones that could be divided into three main classes (cSL1, cSL2, and cSL3) which harbor complementary sequences to the apical loops of 3' SL1, 3' SL2, and 3' SL3, respectively. Biochemical analyses are in progress to evaluate whether these RNA aptamers have the potential to block HCV replication.

In vitro selection of RNA aptamers that bind to domain II of HCV IRES.

The translation of HCV starts at the internal ribosomal entry site (IRES) within the 5' untranslated region and domain II of IRES is essential for translation activity. However, the information of function is limited. We attempted to obtain RNA that bind HCV IRES domain II. Selected aptamers will give us much information, such as RNA-RNA interaction between IRES and other cellular RNAs as well as target sites for anti-HCV drugs. We developed a novel selection method using biotinylated DNA probe. This method can be applied to many RNAs and would identify the most suitable target position experimentally and simply. The binding kinetics of aptamers were analyzed by using Biacore. Furthermore, inhibition of translation by aptamers was analyzed.

Structure-inhibition analysis of RNA aptamers that bind to HCV IRES.

The translation of HCV starts at the internal ribosomal entry site (IRES) within the 5' untranslated region and IRES is well-conserved in HCV strains. We developed a novel selection strategy using biotinylated oligonucleotide probe and obtained RNA aptamers that bind HCV IRES domain II and domain III-IV, respectively. Selected aptamers specifically bound to target sequence via RNA-RNA interactions. These aptamers inhibited IRES-depend translation in vitro. Especially, 3-07 aptamer, which bound domain IIId, showed strong inhibition. Structure/function relationship of these aptamers was analyzed by mutagenesis, RNase mapping and binding kinetics.