Down-regulation of long non-coding RNA SNHG14 protects against acute lung injury induced by lipopolysaccharide through microRNA-34c-3p-dependent inhibition of WISP1.

BACKGROUND: Accumulating evidence has shown the important roles of long non-coding RNAs (lncRNAs) in acute lung injury (ALI). This study aimed to investigate the potential role of lncRNA small nucleolar RNA host gene 14 (SNHG14) in lipopolysaccharides (LPS)-induced ALI. METHODS: Expression of SNHG14, microRNA-34c-3p (miR-34c-3p) and Wnt1 inducible signaling pathway protein 1 (WISP1) in LPS-exposed mouse alveolar macrophages (MH-S) and lung tissues from mice with LPS-induced ALI was determined by reverse transcription quantitative polymerase chain reaction. The interactions among SNHG14, miR-34c-3p and WISP1 were analyzed by dual-luciferase reporter and RIP assays. Using gain-of-function or loss-of-function approaches, the contents of proinflammatory proteins were determined and MH-S cell viability was assessed to evaluate the in vitro functions of SNHG14, miR-34c-3p and WISP1, and wet/dry weight ratio and proinflammatory proteins in lung tissues were determined to assess their in vivo effects. RESULTS: SNHG14 and WISP1 expression was increased, while miR-34c-3p was decreased in ALI models. SNHG14 bound to miR-34c-3p, resulting in impaired miR-34c-3p-dependent down-regulation of WISP1. Both SNHG14 silencing and miR-34c-3p over-expression reduced the levels of proinflammatory proteins IL-18, IL-1ß, TNF-α and IL-6 and inhibited MH-S cell viability. SNHG14 silencing or miR-34c-3p over-expression decreased the wet/dry weight ratio in lung tissues from ALI mice. The reductions induced by SNHG14 silencing or miR-34c-3p over-expression were rescued by WISP1 over-expression. CONCLUSION: This study demonstrated that lncRNA SNHG14 silencing alleviated inflammation in LPS-induced ALI through miR-34c-3p-mediated inhibition of WISP1. Our findings suggest that lncRNA SNHG14 may serve as a therapeutic target for ALI.

Global assessment of its network dynamics reveals that the kinase Plk1 inhibits the phosphatase PP6 to promote Aurora A activity.

Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. The subcellular localization and substrate interactions of Plk1 are tightly controlled and require its binding to phosphorylated residues. To identify phosphorylation-dependent interactions within the Plk1 network in human mitotic cells, we performed quantitative proteomics on HeLa cells cultured with kinase inhibitors or expressing a Plk1 mutant that was deficient in phosphorylation-dependent substrate binding. We found that many interactions were abolished upon kinase inhibition; however, a subset was protected from phosphatase opposition or was unopposed, resulting in persistent interaction of the substrate with Plk1. This subset includes phosphoprotein phosphatase 6 (PP6), whose activity toward Aurora kinase A (Aurora A) was inhibited by Plk1. Our data suggest that this Plk1-PP6 interaction generates a feedback loop that coordinates and reinforces the activities of Plk1 and Aurora A during mitotic entry and is terminated by the degradation of Plk1 during mitotic exit. Thus, we have identified a mechanism for the previously puzzling observation of the Plk1-dependent regulation of Aurora A.

Assessment of Bromodomain Target Engagement by a Series of BI2536 Analogues with Miniaturized BET-BRET.

Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.

Thoughts on the current assessment of Polo-like kinase inhibitor drug discovery.

The Polo-like kinase 1 (Plk1) plays a key role in regulating a broad spectrum of critical cell cycle events. Plk1 is a marker of cellular proliferation and has prognostic potential in different types of human tumors. In a series of preclinical studies, Plk1 has been validated as a cancer target. This prompted many pharmaceutical companies to develop small-molecule inhibitors targeting the classical ATP-binding site of Plk1 for anticancer drug development. Recently, FDA has granted a Breakthrough Therapy designation to the Plk inhibitor BI 6727 (volasertib), which provided a survival benefit for patients suffering from acute myeloid leukemia. Remarkably, a new generation of Plk1 inhibitors that target the second druggable domain of Plk1, the Polo-box domain, is currently being tested preclinically. Since various ATP-competitive compounds of Plk1 inhibit also the activities of Plk2 and Plk3, which act as tumor suppressors, the roles of closely related Plk-family members in cancer cells need to be considered carefully. In this article, the authors highlight recent insights into the biology of Plks in cancer cells and discuss the progress in the development of small-molecule Plk1 inhibitors. The authors believe that the greatest therapeutic benefit might come through leukemic cells that are in direct contact with the inhibitor in the blood stream. The identification of biomarkers and studies that document Plk activities in treated patients would also be beneficial to better understand the role of Plk inhibition in tumor development and anticancer therapy.