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Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer-functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that inhibit gene expression post-transcriptionally. By regulating their target genes, miRNAs play important roles in tumor generation and development. Recently, the mir-200 family was revealed to inhibit the epithelial-mesenchymal transition, which is viewed as an essential step in early tumor metastasis. Here, we used luciferase assays to demonstrate that mir-200b interacts with predicted target sites in the 3′ untranslated region of RND3. In HeLa cells, mir-200b directly reduced the expression of RND3 at the mRNA and protein levels, which thereby promoted expression of the downstream protein cyclin D1 and increased S-phase entry. In conclusion, our study demonstrates a novel role for mir-200b in cell cycle progression and identifies RND3 as a novel mir-200b target. [ABSTRACT FROM AUTHOR]

Recently, the identification of miRNA targets has received much attention. The strategies to determine miRNA targets include bioinformatic prediction and experimental assays. The bioinformatic prediction methods are mainly based on the confirmed rules of interaction between miRNAs and their targets, and are carried out by programs, such as miRanda, TargetScan, TargetScanS, RNAhybrid, DIANA-microT, PicTar, RNA22 and FindTar, which follow well-known principles. The experimental assays to find miRNA targets employ immunoprecipitation of AGO proteins to identify interacting mRNAs, or the analysis of mRNA or protein levels to identify genes which can be regulated by miRNAs. The improvement of current bioinformatic and experimental assays and the development of novel assays will enable greater efficiency in the identification of miRNA targets and thus facilitate miRNA research. This paper describes progress in the prediction and identification of miRNA targets. [ABSTRACT FROM AUTHOR]

Aptamers which specifically recognize targets are selected from random oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX). In this paper, capillary electrophoresis (CE) as a separation approach has been introduced to SELEX procedure. The high efficiency of CE gives rise to greatly shorten the selection procedure. The results from enzyme-linked assay and dot blot experiment show that an enrichment pool has been obtained after four rounds selection, which can specifically recognize ricin. [ABSTRACT FROM AUTHOR]

Inherited mutations in the breast and ovarian cancer susceptibility gene BRCA1 are associated with high risk for developing breast and ovarian cancers. Several studies link BRCA1 to transcriptional regulation, DNA repair, apoptosis and growth/tumor suppression. BRCA1 associates with p53 and stimulates transcription in both p53 dependent and p53-independent manners. BRCA1 splice variants BRCA1a (p110) and BRCA1b (p100) associates with CBP/p300 co-activators. Here we show that BRCA1a and BRCA1b proteins stimulate p53-dependent transcription from the p21WAF1/CIP1 promoter. In addition, the C-terminal second BRCA1 (BRCT) domain is sufficient for p53 mediated transactivation of the p21 promoter. Previous studies emphasized the importance of the BRCT domain, which shows homology with p53 binding protein (53BP1), in transcriptional activation, growth inhibition and tumor suppression. Our findings demonstrate an additional function for this domain in protein – protein interaction and co-activation of p53. We also found that BRCA1a and BRCA1b proteins interact with p53 in vitro and in vivo. The p53 interaction domain of BRCA1a/1b maps, in vitro, to the second BRCT domain (aa 1760 – 1863). The BRCT domain binds to the central domain of p53 which is required for sequence specific DNA binding. These results demonstrate for the first time the presence of a second p53 interaction domain in BRCA1 proteins and suggests that BRCA1a and BRCA1b proteins, like BRCA1, function as p53 co-activators. This BRCT domain also binds in vitro to CBP. These results suggest that one of the mechanisms by which BRCA1 proteins function is through recruitment of CBP/p300 associated HAT/FAT activity for acetylation of p53 to specific promoters resulting in transcriptional activation. [ABSTRACT FROM AUTHOR]

Elk-1, an ets related gene codes for at least two splice variants Elk-1, which regulates c-fos transcription and ΔElk-1, both of which function as transcriptional activators. To investigate the role of Elk-1 and ΔElk-1 proteins in apoptosis; we have developed rat fibroblast cell lines and human breast cancer cell lines expressing Elk-1 and ΔElk-1. The expression of Elk-1 and ΔElk-1 proteins in the Elk-1/ΔElk-1 transfectants were analysed by immunofluorescence, immunohistochemistry, and Western blot analysis. The Elk-1 unlike ΔElk-1 transfectants showed a shortened and flattened morphology compared to the parental cells. We have found that calcium ionophore treatment of Rat-1 Elk-1, MCF-7 Elk-1, Rat-1 ΔElk-1 and MCF-7 ΔElk-1 transfectants resulted in programmed cell death. These results indicate that constitutive expression of Elk-1 and ΔElk-1 proteins triggers apoptosis in Rat-1 fibroblasts and breast cancer cells when treated with calcium ionophore. [ABSTRACT FROM AUTHOR]

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes nuclear phosphoproteins that function as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using different BRCA1 antibodies by immunofluorescence and immunohistochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/p100) which are phosphoproteins containing phosphotyrosine. Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc finger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that specifically interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that... [ABSTRACT FROM AUTHOR]

This corrects the article DOI: 10.1038/srep45235 [ABSTRACT FROM AUTHOR]

Breast cancer is a disease with high heterogeneity. Many issues on tumorigenesis and progression are still elusive. It is critical to identify genes that play important roles in the progression of tumors, especially for tumors with poor prognosis such as basal-like breast cancer and tumors in very young women. To facilitate the identification of potential regulatory or driver genes, we present the Breast Cancer Integrative Platform (BCIP, http://omics.bmi.ac.cn/bcancer/). BCIP maintains multi-omics data selected with strict quality control and processed with uniform normalization methods, including gene expression profiles from 9,005 tumor and 376 normal tissue samples, copy number variation information from 3,035 tumor samples, microRNA-target interactions, co-expressed genes, KEGG pathways, and mammary tissue-specific gene functional networks. This platform provides a user-friendly interface integrating comprehensive and flexible analysis tools on differential gene expression, copy number variation, and survival analysis. The prominent characteristic of BCIP is that users can perform analysis by customizing subgroups with single or combined clinical features, including subtypes, histological grades, pathologic stages, metastasis status, lymph node status, ER/PR/HER2 status, TP53 mutation status, menopause status, age, tumor size, therapy responses, and prognosis. BCIP will help to identify regulatory or driver genes and candidate biomarkers for further research in breast cancer. [ABSTRACT FROM AUTHOR]