Establishment and characterization of two human miR-34a knockout cell lines, HeLa and HEK293T, applying the CRISPR/Cas9 system.

Micro-RNAs (miRNAs) are small non-coding and regulatory RNAs of about 21 nucleotides that act as regulators of gene expression; generally, they have an inhibitory effect by either blocking protein translation or promoting mRNA degradation. These small RNAs have an important role both in physiological events like cell proliferation, differentiation, and development as well as in pathological conditions such as cancer and neurodegenerative diseases. One of the most studied miRNAs in cancer is the miR-34a that inhibits the expression of different pro-tumorigenic genes. In this study, we characterized HeLa and HEK293T miR-34a Knock-Out (KO) cell clones that were produced by using the CRISPR/Cas9 technology, in terms of proliferation rate, expression of already known and predicted targets, through quantitative PCR (qPCR), and Western blot analyzes. The results of the analysis of cell lines silenced with miR-34a indicate that: a) miR-34a is not indispensable for the control of cell proliferation; b) miR-34a may be involved in different signaling pathways and act differently according to cell type, suggesting a tissue-specific role of miR-34a; c) ONECUT2, a transcription factor that stimulates the expression of genes involved in the differentiation of melanocytes and hepatocytes, could be a novel target of miR-34a. Future functional studies on the pathways regulated by this transcription factor in miR-34a KO clones will help to clarify the role of miR-34a in cell differentiation and transformation.