OPEN Experimental & Molecular Medicine (2018) 50, e447; doi:10.1038/emm.2017.278 Official journal of the Korean Society for Biochemistry and Molecular Biology www.nature.com/emm ORIGINAL ARTICLE The long non-coding RNA uc.4 influences cell differentiation through the TGF-beta signaling pathway Zijie Cheng1,5, Qijun Zhang2,5, Anwen Yin1,5, Mengwen Feng1, Hua Li1, Hailang Liu3, Yun Li4 and Lingmei Qian1 In a previous study, we screened thousands of long non-coding RNAs (lncRNAs) to assess their potential relationship with congenital heart disease (CHD). In this study, uc.4 attracted our attention because of its high level of evolutionary conservation and its antisense orientation to the CASZ1 gene, which is vital for heart development. We explored the function of uc.4 in cells and in zebrafish, and describe a potential mechanism of action. P19 cells were used to investigate the function of uc.4. We studied the effect of uc.4 overexpression on heart development in zebrafish. The overexpression of uc.4 influenced cell differentiation by inhibiting the TGF-beta signaling pathway and suppressed heart development in zebrafish, resulting in cardiac malformation. Taken together, our findings show that uc.4 is involved in heart development, thus providing a potential therapeutic target for CHD. Experimental & Molecular Medicine (2018) 50, e447; doi:10.1038/emm.2017.278; published online 16 February 2018 INTRODUCTION Heart development involves formation of the early primitive Congenital heart disease (CHD) is the most common birth heart tube, cardiomyocyte differentiation and heart morpho- defect, with an estimated incidence of 2–3%.1,2 CHD can lead genesis. The role and mechanism of lncRNA in cardiac to both fetal growth retardation as well as spontaneous development have been primarily described in three reports on abortion and stillbirth. CHD can seriously affect the quality Braveheart,13 and Fendrr,14 which are involved in mouse of life of children and puts heavy economic and social burdens cardiac development, and Terminator,15 a vertebrate lncRNA on the families and society.3 Despite the remarkable progress in that is important for cardiovascular development. There is no scientific research and clinical treatment, the mechanism of doubt that exploring the role and mechanism of lncRNA in 16 CHD remains poorly understood. cardiac development will deepen our understanding of CHD. In recent years, new evidence has shown that ~ 95% of However, the relationship between UCEs and CHD remains human genome transcripts are non-coding RNAs.4 Long non- unknown. coding RNAs (lncRNAs) are a subtype of non-coding RNAs In our previous study, we screened thousands of lncRNAs to investigate their relationship with CHD.17 Among those (ncRNAs) with transcripts longer than 200 nucleotides. lncRNAs, uc.4 attracted our attention due to its high level of LncRNAs are involved in several biological and pathological conservation and its antisense orientation to the CASZ1 gene, processes, including vascularization,5 osteoarthritis,6 diabetes 7 which is essential for cardiac morphogenesis and development. and tumorigenesis.8 Ultraconserved elements are specific In the present study, we performed bioinformatics analyses and lncRNAs that are completely conserved among the human, 9 assessed the effect of uc.4 on the differentiation of P19 cells as rat and mouse genomes. Recent studies have delineated the well as the potential relationship between uc.4 and CASZ1. We essential role of ultraconserved elements (UCEs) that regulate also assessed the effects of uc.4 on heart development in 10 11,12 adipogenesis and tumor formation. All of these lines of zebrafish. We showed that uc.4 exerts its activity through the evidence emphasizes the relevance of UCEs in disease etiology. TGF-beta signaling pathway. These data suggested that uc.4 1Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China; 2Department of Cardiology, YinZhou Hospital Affiliated to Medical School of Ningbo University, Ningbo, China; 3Huai An First People’s Hospital, HuaiAn, China and 4Department of Pharmacy, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, China 5These authors contributed equally to this work. Correspondence: Dr Y Li, Department of Pharmacy, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing 210029, China. E-mail: [email protected] or Professor L Qian, Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. E-mail: [email protected] Received 3 May 2017; revised 26 August 2017; accepted 31 August 2017 uc.4 influences cell differentiation through TGF-beta signaling pathway ZChenget al 2 may play an important role in heart development and provide a acquisition (10 000 events collected per sample) was performed possible mechanism of CHD. following the manufacturer’s instructions. MATERIALS AND METHODS Gene analysis and western blotting Cell culture and differentiation Total RNA was extracted from either cultured cells or frozen tissues fi Murine embryonal carcinoma cells (P19) were obtained from Amer- using TRIzol reagent (Thermo Fisher Scienti c). The concentration of ican Type Culture Collection (Manassas, VA, USA) and cultured in RNA was determined by measuring the absorbance ratio of 260- Alpha Modification of Eagle’s medium (Gibco, Grand Island, NY, /280 nm using a NanoDrop ND-1000 spectrophotometer (Thermo fi USA) containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin Fisher Scienti c), and the integrity of the RNA was checked using gel − 1 electrophoresis. The reverse transcription of RNA was performed and 100 μgml streptomycin in 5% CO2 at 37 °C. Following 1% DMSO induction, the cells were aggregated at days 3–4. After 7 days of using a PrimeScript RT reagent Kit with gDNA eraser (RR047A; aggregation in bacteriological dishes, the cell clusters were transferred Takara, Tokyo, Japan), and cDNA was analyzed by qRT-PCR using to six-well culture plates in Alpha Modification of Eagle’smedium SYBR Premix Ex Taq (RR420A; Takara). The data were normalized to − ΔΔCT containing 10% fetal bovine serum. The medium was replaced every the levels of GAPDH and further analyzed using the 2 method. 2 days. The cells were harvested on differentiation days 0, 4, 8 and 10. The sequences for all of the primers used for qPCR are listed in We observed and photographed the morphological changes in the P19 Supplementary Table 1. For the western blot analysis, protein was cells using an inverted microscope (Nikon, Tokyo, Japan). solubilized from cell lysates at four different time points using RIPA buffer and quantified using a BCA Protein Assay Kit (23229; Thermo fi μ Rapid amplification of cDNA ends Fisher Scienti c). A total of 40 g protein was separated on a 10% gel using SDS-PAGE and transferred to nitrocellulose membranes (Milli- 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed on pore, Billerica, MA, USA). Immunopositive bands were detected using RNA isolated from mouse heart using a SMARTer RACE 5′/3′Kit a FluorChem M System (ProteinSimple, San Jose, CA, USA). (Clontech, Mountain View, CA, USA), following the manufacturer’s Antibodies targeting cardiac troponin T (cTnT, ab92546), myocyte instructions. The RACE PCR products were separated on a 1% agarose enhancer factor 2C (MEF2C, ab197070), GATA-binding protein 4 gel and cloned into the pUC57 vector. The transcription start and end (GATA4, ab84593), phosphorylation of mothers against decapenta- sites of uc.4 were mapped by sequencing. plegic homolog 2 (smad2-P, ab188334), phosphorylation of mothers against decapentaplegic homolog 3 (smad3-P, ab52903), total mothers Nuclear and chromatin RNA fraction against decapentaplegic homolog 2 (smad2-T, ab33875) and total Nuclear and cytoplasmic fractions of P19 cells were partitioned using a mothers against decapentaplegic homolog 3 (smad3-T, ab40854) were fi 7 PARIS Kit (Thermo Fisher Scienti c, Carlsbad, CA, USA). Overall, 10 purchased from Abcam, Shanghai, China. cultured cells were collected, placed on ice and resuspended with μ 500 l of ice-cold cell fractionation buffer. The cells were then gently Zebrafish maintenance resuspended by vortexing or pipetting, and were incubated on ice for The Tübingen zebrafish strain used in this study was obtained from another 10 min. The samples were centrifuged at 500 g for 5 min, and the Model Animal Research Center of Nanjing University (Nanjing, the cytoplasmic fraction was then carefully aspirated away from the Jiangsu, China). Zebrafish were raised at 28 °C in a rotating system nuclear pellet fraction. Washing the pellet twice in ice-cold cell that continuously percolates, UV treats and aerates the circulating fractionation buffer prevented contamination of the nuclear fraction water. The embryos were acquired from the natural spawning of wild- with the cytoplasmic fraction. type (WT) adults and were raised at 28 ± 1 °C. Morphological features were used to determine the embryonic developmental stage.18 Lentivirus production and establishment of a stable cell line Embryos older than 24 h post fertilization (hpf) were incubated in The uc.4 lentiviral overexpression vector was constructed by Gene- 0.003% phenylthiourea to inhibit pigment formation. Pharma (Shanghai, China). Lentiviruses were amplified in 293T cells and concentrated using polyethylene glycol (System Biosciences, LLC, Expression vectors and microinjection Palo Alto, CA, USA). For the overexpression experiments, the The uc.4 overexpression