RBBP6 maintains glioblastoma stem cells through CPSF3-dependent alternative polyadenylation.

Glioblastoma is one of the most lethal malignant cancers, displaying striking intratumor heterogeneity, with glioblastoma stem cells (GSCs) contributing to tumorigenesis and therapeutic resistance. Pharmacologic modulators of ubiquitin ligases and deubiquitinases are under development for cancer and other diseases. Here, we performed parallel in vitro and in vivo CRISPR/Cas9 knockout screens targeting human ubiquitin E3 ligases and deubiquitinases, revealing the E3 ligase RBBP6 as an essential factor for GSC maintenance. Targeting RBBP6 inhibited GSC proliferation and tumor initiation. Mechanistically, RBBP6 mediated K63-linked ubiquitination of Cleavage and Polyadenylation Specific Factor 3 (CPSF3), which stabilized CPSF3 to regulate alternative polyadenylation events. RBBP6 depletion induced shortening of the 3'UTRs of MYC competing-endogenous RNAs to release miR-590-3p from shortened UTRs, thereby decreasing MYC expression. Targeting CPSF3 with a small molecular inhibitor (JTE-607) reduces GSC viability and inhibits in vivo tumor growth. Collectively, RBBP6 maintains high MYC expression in GSCs through regulation of CPSF3-dependent alternative polyadenylation, providing a potential therapeutic paradigm for glioblastoma.

New chromones from the roots of Saposhnikovia divaricata (Turcz.) Schischk with anti-inflammatory activity.

Fifteen new chromones, sadivamones A-E (1-5), cimifugin monoacetate (6), sadivamones F-N (7-15), together with fifteen known chromones (16-30), were isolated from the ethyl acetate portions of 70% ethanol extract of Saposhnikovia divaricata (Turcz.) Schischk roots. The structures of the isolates were determined using 1D/2D NMR data and electron circular dichroism (ECD) calculations. Meanwhile, LPS induced RAW264.7 inflammatory cell model was used to determine the potential anti-inflammatory activity of all the isolated compounds in vitro. The results showed that compounds 2, 8, 12-13, 18, 20-22, 24, and 27 significantly inhibited the production of lipopolysaccharide (LPS)-induced NO in macrophages. To determine the signaling pathways involved in the suppression of NO production by compounds 8, 12 and 13, we investigated ERK and c-Jun N-terminal protein kinase (JNK) expression by western blot analysis. Further mechanistic studies demonstrated that compounds 12 and 13 inhibited the phosphorylation of ERK and the activation of ERK and JNK signaling in RAW264.7 cells via MAPK signaling pathways. Taken together, compounds 12 and 13 may be valuable candidates for the treatment of inflammatory diseases.

Effects of demethylase KDM4B on the biological characteristics and function of yak cumulus cells in vitro.

The present study aims to investigate the expression and function of lysine-specific demethylase 4B (KDM4B) in yak cumulus cells (CCs) in order to reveal the mechanisms by which KDM4B regulates biological characteristics and function of CCs. The cellular location of KDM4B and the methylation pattern of H3K9 were detected using immunofluorescence (IF) staining in CCs. The mRNA expression levels of apoptosis-related genes (BCL-2, HAX1 and BAX) and genes related to the estrogen pathway (ESR2, CYP17 and 3B-HSD) were estimated by qRT-PCR after knockdown of KDM4B expression by siRNA in yak CCs. Then, a proliferation assay, Annexin V-FITC staining, and ELISA were utilized to explore the effects of KDM4B silencing on CCs proliferation, apoptosis, and estrogen (E2) secretion, respectively. The results showed that KDM4B is located in the nuclei of yak CCs and is distributed in a dotted pattern. Knockdown KDM4B induced a decrease in cell proliferation, an increase in apoptotic rate and a reduction in the levels of E2 secretion of CCs. Additionally, the methylation patterns of H3K9me2 and H3K9me3 were significantly increased in CCs transfected with KDM4B siRNA-1 (P < 0.05). The mRNA expression level of apoptosis promoting BAX genes was significantly upregulated, but 3B-HSD, ESR2 and anti-apoptotic HAX1 genes were significantly downregulated in transfected CCs (P < 0.05). Furthermore, the rate of embryos developing from the 2-cell stage to blastocysts was lower in the siRNA-1 transfection group than that of the control group (28.6 ± 2.9% vs 40.4 ± 2.4%, P < 0.05). In conclusion, our study indicates that KDM4B regulates the biological characteristics and physiological function of yak CCs mainly through changing the methylation patterns of H3K9 and related gene expression levels.