LncRNA RP11-89 facilitates tumorigenesis and ferroptosis resistance through PROM2-activated iron export by sponging miR-129-5p in bladder cancer

Long non-coding RNAs (lncRNAs) act as important regulators of tumorigenesis and development in bladder cancer. However, the underlying molecular mechanisms remain elusive. We previously identified a novel lncRNA signature related to immunity and progression in bladder cancer. Here we further explored the function of RP11-89, a lncRNA discovered in the previous signature. Loss- and gain-of function experiments were performed using CCK-8 assay, flow cytometry, Transwell assays, scratch tests and subcutaneous nude mouse models. High-throughput RNA sequencing was conducted to identify dysregulated genes in bladder cancer cells with RP11-89 knockdown or overexpression. Regulation of RP11-89 on miR-129-5p and PROM2 was explored through luciferase reporter assay, RIP assay and RNA pull-down assay. RP11-89 promoted cell proliferation, migration and tumorigenesis and inhibited cell cycle arrest via the miR-129-5p/PROM2 axis. We found that RP11-89 “sponges” miR-129-5p and upregulates PROM2. Elevated PROM2 in cells was associated with attenuated ferroptosis through iron export, formation of multivesicular bodies and less mitochondrial abnormalities. We demonstrated that RP11-89 is a novel tumorigenic regulator that inhibits ferroptosis via PROM2-activated iron export. RP11-89 may serve as a potential biomarker for targeted therapy in bladder cancer.Subject terms: Tumour biomarkers, Tumour biomarkers     

Studies on the interaction mechanism between hexakis(imidazole) manganese(II) terephthalate and DNA and preparation of DNA electrochemical sensor

The interaction between hexakis(imidazole) manganese(II) terephthalate ([Mn(Im)(6)](teph).4H(2)O) and salmon sperm DNA in 0.2M pH 2.30 Britton-Robinson buffer solution was studied by fluorescence spectroscopy and cyclic voltammetry. Increasing fluorescence was observed for [Mn(Im)(6)](2+) with DNA addition, while quenching fluorescence phenomenon appeared for EB-DNA system when [Mn(Im)(6)](2+) was added. There were a couple quasi-reversible redox peaks of [Mn(Im)(6)](2+) from the cyclic voltammogram on the glassy carbon electrode. The peak current of [Mn(Im)(6)](2+) decreased with positive shift of the formal potential in the presence of DNA compared with that in the absence of DNA. All the experimental results indicate that [Mn(Im)(6)](2+) can bind to DNA mainly by intercalative binding mode. The binding ratio of the DNA-[Mn(Im)(6)](2+) association complex is calculated to be 1:1 and the binding constant is 4.44x10(3) M(-1). By using [Mn(Im)(6)](teph).4H(2)O as the electrochemical hybridization indicator, the DNA electrochemical sensor was prepared by covalent interaction and the selectivity of ssDNA modified electrode were described. The results demonstrate the use of electrochemical DNA biosensor in the determination of complementary ssDNA.     

Strategy for allosteric analysis based on protein-patterned stationary phase in microfluidic chip

An effective method is presented for the on-chip analysis of chiral interactions with a successful depression of nonspecific adsorption. The alumina gel-derived protein network on poly(methyl methacrylate) (PMMA) microchannel was explored to form a protein-stationary phase and then used to carry out electrophoresis for fast enantioseparation coupled with electrochemical detection. On the basis of the chemical modification of a synthesized copolymer containing silane-functionalized scaffold, alumina sol-gel could react readily with the silane groups and form steady microstructure on the chip surface achieving the encapsulation of functional biomolecules. Compared with the native PMMA microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and EOF of alumina-gel-derived PMMA substrate were 22 degrees and 4.3 x 10(-4) cm(2) V(-1) s(-1), compared to those of 73 degrees and 1.9 x 10(-4) cm(2) V(-1) s(-1) from the untreated one, respectively. Bovine serum albumin, acting as a target protein, could be stably and homogeneously immobilized in the modified PMMA microchannel to fabricate a protein-stationary phase. Under a mild condition, D- and L-tryptophan were efficiently separated with a resolution of 1.57. The as-prepared microchip can perform chiral separations within short time, indicating that the general protocol has the potential to provide a platform for high throughput screening of enantiomer candidates such as those biochemical drugs with protein targets and the research of receptor interactions.     

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Circulating miR‐145 is associated with plasma high‐sensitivity C‐reactive protein in acute ischemic stroke patients

Stroke is a major cerebrovascular disease threatening human health and life with high morbidity, disability and mortality. We aimed to find effective biomarkers for the early diagnosis on stroke. Nine previously reported stroke‐associated miRNAs (miR‐21, miR‐23a, miR‐29b, miR‐124, miR‐145, miR‐210, miR‐221, miR‐223 and miR‐483‐5p) were measured by quantitative real time‐PCR, and plasma high‐sensitivity C‐reactive protein (hs‐CRP) and serum interleukin 6 (IL‐6), the pro‐inflammation markers in brain injury, were examined by enzyme‐linked immunosorbent assay in 146 acute ischemic stroke patients and 96 healthy blood donors. We found that serum miR‐145 was significantly increased within 24 h after stroke onset and serum miR‐23a and miR‐221 were decreased in patients. Moreover, serum miR‐145 was strong positively correlated with plasma hs‐CRP and moderate positively correlated with serum IL‐6. Meanwhile, serum miR‐23a and miR‐221 were moderate negatively correlated with plasma hs‐CRP but not serum IL‐6. Importantly, the combination of hs‐CRP and serum miR‐145 gained a better sensitivity/spectivity for prediction of acute ischemia stroke (area under receiver operating characteristic curve from 0.794 to 0.896). Conclusively, our preliminary findings indicate that serum miR‐145 upregulated in acute ischemic stroke might be a new biomarker for acute ischemia stroke evaluation. Copyright © 2015 John Wiley & Sons, Ltd.     

Net sodium fluxes change significantly at anatomically distinct root zones of rice (Oryza sativa L.) seedlings

Casparian bands of endodermis and exodermis play crucial roles in blocking apoplastic movement of ions and water into the stele of roots through the cortex. These apoplastic barriers differ considerably in structure and function along the developing root. The present study assessed net Na⁺ fluxes in anatomically distinct root zones of rice seedlings and analyzed parts of individual roots showing different Na⁺ uptake. The results indicated that anatomically distinct root zones contributed differently to the overall uptake of Na⁺. The average Na⁺ uptake in root zones in which Casparian bands of the endo- and exo-dermis were interrupted by initiating lateral root primordia (root zone III) was significantly greater than that at the root apex, where Casparian bands were not yet formed (root zone I), or in the region where endo- and exo-dermis with Casparian bands were well developed (root zone II). The measurement of net Na⁺ fluxes using a non-invasive scanning ion-selective electrode technique (SIET) demonstrated that net Na⁺ flux varied significantly in different positions along developing rice roots, and a net Na⁺ influx was obvious at the base of young lateral root primordia. Since sodium fluxes changed significantly along developing roots of rice seedlings, we suggest that the significantly distinct net Na⁺ flux profile may be attributed to different apoplastic permeability due to lateral root primordia development for non-selective apoplastic bypass of ions along the apoplast.     

Small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-beta-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 microM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.