MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

miR-133b acts proapoptotically on HeLa cells stimulated with death receptor (DR) ligands.

<p>Cells were transfected with miR-133b alone or together with a control antimiR (ctrl αmiR) or a specific miR-133b inhibitor (αmiR-133b). After 48 h, cells were either left untreated (Unstim) or stimulated for 4 h with 20 ng/ml tumor necrosis factor-alpha (TNFα), 100 ng/ml of a cross-linking activating antiFas antibody (αFas/CD95) or 20 ng/ml recombinant human TRAIL (rhTRAIL). (A) Treated cells were harvested, stained and scanned by flow cytometry for the presence of cleaved active caspase 8 (upper graph) and 3 (lower graph). 7-Amino-actinomycin D (7-AAD) served for exclusion of cells with compromised membrane integrity from the caspase activation quantification assay. Cells transfected with ctrl miR alone were used as reference. (B) Western blot analysis of poly [ADP ribose] polymerase (PARP-1) in transfected, unstimulated cells (upper panel) and TNFα-, αFas/CD95- or rhTRAIL-treated cells (lower panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. Graphs are representative of at least three independent experiments. Asterisk represents p<0.01. Errors bars indicate standard deviation.</p

Samples from human prostate cancer patients have decreased expression of miR-133b.

<p>Scatter dot-plot of miR-133b expression. RT-qPCR was used to determine miR-133b expression in 69 primary prostate cancer tissues and corresponding normal adjacent tissue. Lines represent mean expression values. MiR-133b was normalized to miR-130b, since it shows a very stable expression pattern both in tumors and normal tissue.</p

Transfection of HeLa cells with miR-133b results in enhanced caspase-dependent loss of membrane integrity after DR ligation.

<p>Cells were transfected with miR-133b alone or together with ctrl miR. After 48 h, cells were left untreated (Unstim) or stimulated for 4 h with either 20 ng/ml TNFα, 100 ng/ml αFas/CD95 or 20 ng/ml rhTRAIL. In order to assess caspase dependency, 0.01% DMSO (vehicle) (left histogram column) or Z-VAD-FMK (50 µM) (right histogram column) were added to the cells. Following stimulation, both adherent and suspension cells were collected, stained with propidium iodide (PI) and analyzed by flow cytometry. Samples were compared to equally treated ctrl miR-transfected cells (black histogram). Results are representative of at least three independent experiments.</p

Genes downregulated after transfection of HeLa cells with miR-133b and TNFα stimulation and predicted to be potential miR targets.¹

1<p>According to the online database miRecords and predicted at least by three different algorithms.</p

miR-133b induces increased apoptosis in DR receptor stimulated PC3 cells.

<p>Cells were transfected with miR-133b (red) or a control miR (ctrl miR, black), as reference. After 48 h, cells were either left untreated (Unstim) or stimulated for 4 h with 20 ng/ml tumor necrosis factor-alpha (TNFα), 100 ng/ml of a cross-linking activating antiFas antibody (αFas/CD95) or 20 ng/ml recombinant human TRAIL (rhTRAIL). Treated cells were harvested, stained and scanned by flow cytometry for the presence of cleaved active caspase 8 (left) and caspase 3 (right). 7-Amino-actinomycin D (7-AAD) was used for exclusion of cells with compromised membrane integrity from the caspase activation quantification assay.</p