Identification of the hsa-miR-216/217 cluster in human embryonic stem cells neuronal differentiation

RODRÍGUEZ VARELA, MARÍA SOLEDAD; MUCCI, SOFÍA; ISAJA, LUCIANA; FERRIOL LAFFOUILLERE, SOFÍA LUJÁN; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; ROMORINI, LEONARDO

Buenos Aires

Congreso; Meeting of Biosciences Societies 2021, SAIC.SAI.AAFE.NANOMED.AR.; 2021

SAIC, SAI, AAFE, NANOMED.AR

Human embryonic stem cells (hESCs) are self-renewing cells that have the potential to differentiate into specialized cells. MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in many key processes, such as cell differentiation. Large fraction of them are key regulators throughout neural development. Due to this, we aimed to identify and characterize miRNAs involved in hESCs neuronal differentiation. To this end, we derived and characterized neural stem cells (NSCs) from H9 hESCs and differentiated these cells into neurons (NEU). We next performed a high throughput small RNA sequencing of these cell populations followed by bioinformat- ics analysis. First, we select miRNAs with no less than 10 reads and visualized them as a heat map to represent the miRNA expression profile. Then, we carried out a differential expression analysis (FDR 0.5) and found 728, 270 and 798 differentially expressed miRNAs between hESCs-NSCs; NSCs-NEU and hESCs-NEU, respectively. RNA-seq results were validated analyzing, by RT-qPCR with specific stem loop primers, expression levels of known miRNAs involved in neuronal differentiation (hsa-miR-9-3p/9-5p/124-3p/125a-5p/125b- 5p and 128-3p). From the list of differentially expressed miRNAs it caught our attention the hsa-miR-216a,b/217 cluster, which was only expressed in NSC and NEU. Moreover, this expression pat- tern was also validated by RT-qPCR. Furthermore, expression of this cluster was reported in published RNA-Seq bioinformatic data- sets of other neural cell types but not along mesoderm and endo- derm differentiations or in other species or types of pluripotent stem cells datasets. Besides, there is no bibliographic data of this family associated to neural differentiation or regulation of stemness. In a future, we aim to knock out the whole cluster using the CRISPR/ Cas9 technology as a loss-of-function strategy. This knowledge will enable us to characterize the function and targets of this cluster in a neurogenic context.