[Development and functional validation of a nano-delivery system of miR-16/polypeptide targeting ovarian cancer cells].

OBJECTIVE: To develop a nano-delivery system for targeted delivery of miR-16/polypeptide for enhancing cisplatin sensitivity of ovarian cancer. OBJECTIVE: R9-SS-R9 and cRGD-R9-SS-R9 peptides were synthesized and self-assembled with miR-16 molecules to form a nano-delivery system. The stability, particle size, potential and morphology of the nanoparticles were determined by agarose gel electrophoresis, particle size potentiometer and transmission electron microscopy. CCK-8 assay was used to assess the toxicity of the polypeptides in ovarian cancer cells. Stem loop qRT-PCR and living cell imaging were used to verify the uptake efficiency and intracellular distribution of the nanoparticles. Flow cytometry and Western blotting were performed to verify the effect of the nanoparticles for enhancing cisplatin sensitivity of ovarian cancer cells and explore the possible mechanism. OBJECTIVE: R9-SS-R9/miR-16 and cRGD-R9-SS-R9/miR-16 nanoparticles were successfully prepared. The nanoparticles, with a particle size below 150 nm, a dispersity index less than 0.1 and a potential of about 40 mV, showed a good serum stability. The polypeptide material had no obvious cytotoxicity. The miR-16/polypeptide nanoparticles could be efficiently absorbed by human ovarian cancer cells and were distributed in the cytoplasm. The nanoparticles significantly increased the intracellular expression level of miR-16 (P < 0.001) and decreased the expression of Bcl-2 and Chk-1 proteins in ovarian cancer cells, thus enabling miR-16 to promote apoptosis and enhance cisplatin sensitivity of the cells. OBJECTIVE: We successfully prepared a miR-16/polypeptide nano-delivery system for targeted delivery of miR-16 to ovarian cancer cells for enhancing cisplatin sensitivity of the cancer cells.

Long non-coding RNA C2dat1 regulates CaMKIIδ expression to promote neuronal survival through the NF-κB signaling pathway following cerebral ischemia.

Increasing evidence has demonstrated a significant role of long non-coding RNAs (lncRNAs) in diverse biological processes. However, their functions in cerebral ischemia remain largely unknown. Through an lncRNA array analysis in a rat model of focal cerebral ischemia/reperfusion (I/R), we have identified CAMK2D-associated transcript 1 (C2dat1) as a novel I/R-induced lncRNA that regulated the expression of CaMKIIδ in murine models of focal cerebral ischemia. C2dat1 mRNA was upregulated in a time-dependent manner in mouse cortical penumbra after focal ischemic brain injury, which was accompanied by increased expression of CaMKIIδ at transcript and protein levels. The expression patterns of C2dat1 and CAMK2D were confirmed in mouse Neuro-2a cells in response to in vitro ischemia (oxygen-glucose deprivation/reoxygenation, OGD/R). Knockdown of C2dat1 resulted in a significant blockade of CaMKIIδ expression, and potentiated OGD/R-induced cell death. Mechanistically, reduced CaMKIIδ expression upon silencing C2dat1 inhibited OGD/R-induced activation of the NF-κB signaling pathway. Further analysis showed that the downregulation of IKKα and IKKß expression and phosphorylation, and subsequent inhibition of IκBα degradation accounted for the inhibition of the NF-κB signaling activity caused by silencing C2dat1. In summary, we discovered a novel I/R-induced lncRNA C2dat1 that modulates the expression of CaMKIIδ to impact neuronal survival, and may be a potential target for therapeutic intervention of ischemic brain injury.

Direct binding of beta-arrestins to two distinct intracellular domains of the delta opioid receptor.

beta-Arrestins regulate opioid receptor-mediated signal transduction and play an important role in opiate-induced analgesia and tolerance/dependence. This study was carried out to measure the direct interaction between beta-arrestins and opioid receptor. Immunoprecipitation experiments demonstrated that beta-arrestin 1 physically interacts with delta opioid receptor (DOR) co-expressed in human embryonic kidney 293 cells in an agonist-enhanced manner and truncation of the carboxyl terminus of DOR partially impairs the interaction. In vitro data from glutathione-S-transferase pull-down assay showed that the carboxyl terminus (CT) and the third intracellular loop (I3L) of DOR are both capable of and either domain is sufficient for binding to beta-arrestin 1 and 2. Surface plasmon resonance determination further revealed that binding of CT and I3L of DOR to beta-arrestin is additive, suggesting these two domains bind at distinctly different sites on beta-arrestin without considerable spatial hindrance. This study demonstrated for the first time the direct binding of beta-arrestins to the two distinct domains, the carboxyl terminus and the third intracellular loop, of DOR.

Direct and differential interaction of beta-arrestins with the intracellular domains of different opioid receptors.

beta-arrestins have been shown to play important roles in regulation of signaling and desensitization of opioid receptors in many in vivo studies. The current study was carried out to measure the direct interaction of beta-arrestins with two functional intracellular domains, the third intracellular loop (I3L) and the carboxyl terminus (CT), of delta-, mu-, and kappa-opioid receptors (DOR, MOR, and KOR, respectively). Results from the pull-down assay using glutathione S-transferase fusion proteins demonstrated that beta-arrestins (1 and 2) were able to bind to the I3L of DOR and to the CT of DOR and KOR. Surface plasmon resonance measurement gave similar results with typical dissociation equilibrium constant (K(D)) values in the micromolar range. The site-directed mutagenesis experiment further revealed that certain specific serine/threonine residues in these receptor domains play a critical role in their interaction with beta-arrestins. Taken together, our data clearly indicated that beta-arrestins interact differentially with the functional domains of different opioid receptors; this may provide a possible molecular basis for differential regulation of opioid receptors by beta-arrestins.

beta-arrestin differentially regulates the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this implicates multiple interaction sites between beta-arrestin and CXCR4.

The chemokine receptor CXCR4 has recently been shown to be a co-receptor involved in the entry of human immunodeficiency virus type 1 into target cells. This study shows that coexpression of beta-arrestin with CXCR4 in human embryonic kidney 293 cells attenuated chemokine-stimulated G protein activation and inhibition of cAMP production. Truncation of the C-terminal 34 amino acids of CXCR4 (CXCR4-T) abolished the effects of beta-arrestin on CXCR4/G protein signaling, indicating the functional interaction of the receptor C terminus with beta-arrestin. On the other hand, receptor internalization and the subsequent activation of extracellular signal-regulated kinases were significantly promoted by coexpression of beta-arrestin with CXCR4, whereas the C-terminal truncation of CXCR4 did not affect this regulation of beta-arrestin, suggesting that beta-arrestin can functionally interact with CXCR4 with or without the C terminus. Moreover, beta(2)V54D, the dominant inhibitory mutant of beta-arrestin 2, exerted no effects on CXCR4/G protein signaling, but strongly influenced receptor internalization and extracellular signal-regulated kinase activation. Further cross-linking experiments demonstrated that beta-arrestin as well as beta(2)V54D could physically contact both CXCR4 and CXCR4-T. Glutathione S-transferase pull-down assay showed that beta-arrestin was able to bind efficiently in vitro to both the third intracellular loop and the 34-amino acid C terminus of CXCR4. Taken together, our data clearly establish that beta-arrestin can effectively regulate different functions of CXCR4 and that this is mediated through its distinct interactions with the C terminus and other regions including the third loop of CXCR4.

Glycine68 to histidine73 has an important role in the function of human tumor necrosis factor alpha.

Mutant human tumor necrosis factor alpha(hTNF alpha) genes have been constructed by in vitro mutagenesis and expressed in Escherichia coli. A deletion involving Gly68 to His73 in hTNF alpha remarkably decreased the solubility and biological activity of hTNF alpha. From the above and results of a molecular dynamics simulation it is proposed that the region of Gly68 to His73 in hTNF alpha has an important role in the maintenance of the 3-D structure and modulation of the biological activity of hTNF alpha.

Expression of soluble, active human macrophage colony stimulating factor in Escherichia coli.

Human macrophage colony stimulating factor (M-CSF) has been successfully overexpressed in Escherichia coli AD494 (DE3) with an expression level of approximate 26% of the total cellular proteins. The truncated human M-CSF gene encoding the amino-terminal 149 amino acids was subcloned into the prokaryotic expression vector pET11d under the control of the inducible T7 promoter. Nearly 40% of the recombinant protein was in the soluble fraction which showed obvious stimulating effects on mouse macrophage colony formation and had an M-CSF specific activity of approximately 1 x 10(6) units/mg soluble protein.