The cataract-linked RNA-binding protein Celf1 post-transcriptionally controls the spatiotemporal expression of the key homeodomain transcription factors Pax6 and Prox1 in lens development.

The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells-directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3' UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.

Mir-206 partially rescues myogenesis deficiency by inhibiting CUGBP1 accumulation in the cell models of myotonic dystrophy.

Objectives: In this study, we aim to determine how CUG-expansion and the abundance of Celf1 regulates normal myocyte differentiation and reveal the role ofmiR-206 in myotonic dystrohy and explore a possible gene therapy vector. Methods: we set up CUG-expansion and Celf1 overexpression C2C12 cell models to imitate the myocyte differentiation defects of DM1, then transfected AdvmiR-206 into cell models, tested the level of myogenic markers MyoD, MyoG, Mef2c, Celf1 by RT-PCRand Western Blotting, detected myotube formation by myosin heavy chain immunostaining. Result: 3'-UTR CUG-expansion leads to myotube defects and impaired myoblasts differentiation. Overexpression of Celf1 inhibits myoblast differentiation and impairs differentiation. Knockdown of Celf1 partially rescues differentiation defects of myoblasts harboring CUG-expansion. miR-206 incompletely reverses myoblast differentiation inhibition induced by CUG-expansion and partially recuses myoblast differentiation defects induced by Celf1 overexpression. Conclusions: Ectopic miR-206 mimicking the endogenous temporal patterns specifically drives a myocyte program that boosts myoblast lineages, likely by promoting the expression of MyoD to rectify the myogenic deficiency by stimulating the accumulation of Celf1. Abbreviations: DMPK: (dystrophia myotonica protein kinase); 3'-UTR: (3'-untranslated region); MBNL1: (muscleblind-like [Drosophila]); DM1: (myotonic dystrophy type 1); GFP: (green fluorescent protein); RT-PCR: (quantitative reverse transcriptase-polymerase chain reaction); shRNA: (short hairpin RNA).

Inhibition of CUG-binding protein 1 and activation of caspases are critically involved in piperazine derivative BK10007S induced apoptosis in hepatocellular carcinoma cells.

Though piperazine derivative BK10007S was known to induce apoptosis in pancreatic cancer xenograft model as a T-type CaV3.1 a1G isoform calcium channel blocker, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the antitumor mechanism of BK10007S was elucidated in hepatocellular carcinoma cells (HCCs). Herein, BK10007S showed significant cytotoxicity by 3-[4,5-2-yl]-2,5-diphenyltetra-zolium bromide (MTT) assay and anti-proliferative effects by colony formation assay in HepG2 and SK-Hep1 cells. Also, apoptotic bodies and terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) positive cells were observed in BK10007S treated HepG2 and SK-Hep1 cells by 4',6-diamidino-2-phenylinodole (DAPI) staining and TUNEL assay, respectively. Consistently, BK10007S increased sub G1 population in HepG2 and SK-Hep1 cells by cell cycle analysis. Furthermore, Western blotting revealed that BK10007S activated the caspase cascades (caspase 8, 9 and 3), cleaved poly (ADP-ribose) polymerase (PARP), and downregulated the expression of cyclin D1, survivin and for CUG-binding protein 1 (CUGBP1 or CELF1) in HepG2 and SK-Hep1 cells. Conversely, overexpression of CUGBP1 reduced cleavages of PARP and caspase 3, cytotoxicity and subG1 population in BK10007S treated HepG2 cells. Overall, these findings provide scientific evidences that BK10007S induces apoptosis via inhibition of CUGBP1 and activation of caspases in hepatocellular carcinomas as a potent anticancer candidate.

Olive Oil-Supplemented Lipid Emulsion Induces CELF1 Expression and Promotes Apoptosis in Caco-2 Cells.

BACKGROUND AND AIMS: Parenterally-administered lipid emulsion (LE) is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1) has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro. METHODS: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting. RESULTS: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE)-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF) was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation. CONCLUSION: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1.