Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a subpopulation present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein.Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.In eukaryotic cells, the nuclear envelope, which separates the nucleoplasm from the cytoplasm, is composed of the inner and outer nuclear membranes (INM 1 and ONM, respectively), the latter membrane being continuous with the endoplasmic reticulum. The two membranes are separated by a thin lumen and are joined at nuclear pores (1-3). Underlying the INM is a meshwork of various lamin isoforms (4) that are in close contact with the INM and its resident proteins (5). Characterized INM proteins in mammalian cells include the lamin B receptor (6), lamin-associated polypeptides 1 and 2 (Lap1 and Lap2) (7), emerin (8), and Man1 (9). These proteins possess a hydrophilic N-terminal region that protrudes into the nucleoplasm as well as one or more hydrophobic regions leading to predicted single or multispanning transmembrane domains. INM proteins are immobilized in the nuclear envelope through their interaction with lamins and/or heterochromatin (10, 11).Beside their structural role, lamina components also exert additional functions through their ability to interact with effector proteins involved in various regulatory processes. For example, the lamin B receptor directly binds to HP1 (12), a heterochromatin protein involved in transcription repression. Lap2 (13,14), emerin (15), and Man1 (16) interact with the barrier-to-autointegration factor, a 10-kDa DNA-binding protein (17). The range of regulatory functions pe...
The Androgen Receptor (AR) plays a key role in prostate biology and in the progression of prostate cancer (PCa) to castration resistance. The role of microRNAs (miRNAs) in aberrant AR signaling have not been fully characterized. Here we screened a library of 810 miRNA mimics to identify miRNAs that alter AR activity in complementary functional assays including protein lysate microarray (LMA) quantification of AR and PSA protein levels, AR transcriptional reporter activity, and AR-positive PCa cell viability. Candidate AR-regulating miRNAs were verified through AR transcriptional reporter and cell viability assays. MiRNA binding sites were found within the AR 3′-untranslated region (UTR) and within the AR and AR-V7 coding regions. MiRNA activity was characterized by western blotting, 3′-UTR reporter assay, and AR-GFP and AR-V7-GFP reporter assays. Results uncovered miR-30 family members as direct AR inhibitors. Inhibition of endogenous miR-30b-3p and miR-30d-5p enhanced AR expression and androgen-independent cell growth. Droplet digital RT-PCR quantification of miR-30c-5p and miR-30d-5p revealed significantly reduced levels in metastatic castration resistant PCa (CRPC), when compared to healthy prostate tissues. MiR-30d-5p levels were inversely correlated with AR activity, as measured by PSA mRNA, in metastatic CRPC. Collectively, these studies provide a comprehensive evaluation of AR-regulating miRNAs in PCa.
Further dissection of the role of PURalpha in cell growth regulation may reveal a novel target for HRPC.
We demonstrate a novel and robust microfluidic chip with combined functions of continuous culture and output of PC-3 prostate cancer cells. With digital controls, polydimethylsiloxane (PDMS) flexible diaphragms are able to apply hydrodynamic shear forces on cultures, detaching a fraction of attached cancer cells from the surface for output while leaving others for reuse in subsequent cultures. The fractions of detached cells and remaining cells can be precisely controlled. The system has not only the advantages of small size, high cell culture efficiency, and digital control, but also of simple fabrication at low cost, easy operation and robust performance. The chip performs 9 passages during 30 days of continuous culture and shows promise as a durable design suitable for long-term cell output.
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