Distinct and Additive Effects of Long Noncoding RNA-expression and Retinoic Acid-treatment During Neuronal Differentiation of Human Neuroblastoma Cells.

Retinoic Acid (RA) induces differentiation and regulates gene expression through three subtypes (α, β, γ) of the nuclear retinoid receptor heterodimer (RAR/RXR), which also function as transcription factors. Earlier, we reported the long intergenic noncoding RNAs (LINC-RBE and LINC-RSAS) induced by All-Trans Retinoic Acid (ATRA) in cultured primary hippocampal neurons from adult rat brain at transcriptional and post-transcriptional levels, respectively. In this study, we report that ~ 25% of the human neuroblastoma (SH-SY5Y) cells were differentiated by 1 µM ATRA-treatment within 72 h showing extension of neurites from spindle-shaped cells demonstrating neuronal differentiation. Expression of RARβ and interferon regulatory factor-1 (IRF-1) mRNAs was significantly upregulated up to 16xfold at 10 h and 2.6xfold at 8 h by 1 µM ATRA-treatment, respectively. This indicated activation of the RA-signaling pathway in these cells. With a transfection efficiency of ~ 40%, overexpression of LINC-RBE and LINC-RSAS caused ~ 34% and ~ 33% inhibition of cell proliferation, respectively, with an increase in cell death and ~ 10% reduction in number of cells in G1-phase of cell cycle. ATRA-treatment alone caused ~ 40% inhibition of cell proliferation, and induced ~ 66% of cells to G1-phase arrest. Combined effect of LINC-RSAS + ATRA further enhanced inhibition of cell proliferation by additional ~ 32%, whereas LINC-RBE, ATRA and LINC-RBE + ATRA showed similar effects indicating distinct effects and mechanisms of their actions. Moreover, overexpression of these lncRNAs led to fourfold increase in genomicDNA breakage/damage in these cells. Thus it showed an unique relationship between lncRNA and RA during neuronal differentiation, most likely involving regulation of gene expression.