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Physiological and pathological cardiovascular processes are tightly regulated by several cellular mechanisms. Non-coding RNAs, including long non-coding RNAs (lncRNAs), represent one important class of molecules involved in regulatory processes within the cell. The lncRNA non-coding repressor of NFAT(NRON) was described as a repressor of the nuclear factor of activated T cells (NFAT) in different in vitrostudies. Although the calcineurin/NFAT-signaling pathway is one of the most important pathways in pathological cardiac hypertrophy, a potential regulation of hypertrophy by NRON in vivohas remained unclear. Applying subcellular fractionation and RNA fluorescence in situhybridization (RNA-FISH), we found that, unlike what is known from T cells, in cardiomyocytes, NRONpredominantly localizes to the nucleus. Hypertrophic stimulation in neonatal mouse cardiomyocytes led to a downregulation of NRON, while NRONoverexpression led to an increase in expression of hypertrophic markers. To functionally investigate NRON in vivo, we used a mouse model of transverse aortic constriction (TAC)-induced hypertrophy and performed NRONgain- and loss-of-function experiments. Cardiomyocyte-specific NRONoverexpression in vivoexacerbated TAC-induced hypertrophy, whereas cardiomyocyte-specific NRONdeletion attenuated cardiac hypertrophy in mice. Heart weight, cardiomyocyte cell size, hypertrophic marker gene expression, and left ventricular mass showed a NRON-dependent regulation upon TAC-induced hypertrophy. In line with this, transcriptome profiling revealed an enrichment of anti-hypertrophic signaling pathways upon NRON-knockout during TAC-induced hypertrophy. This set of data refutes the hypothesized anti-hypertrophic role of NRONderived from in vitrostudies in non-cardiac cells and suggests a novel regulatory function of NRONin the heart in vivo.

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.