The role of miR-101 and miR-135a in reprogramming of somatic cells into induced pluripotent stem cells

Abstract

The groundbreaking use of transcription factors (Oct4, Sox2, Klf4, c-Myc) in reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) provides novel ways in regenerative medicine and disease modeling. The reprogramming process is a stepwise process involving global epigenetic remodeling. In recent years, small molecules like DNA methyltransferase inhibitor that alter the epigenetic status of cells were shown to enhance the reprogramming efficiency. It was postulated that chromatin modifying enzymes played an important role during the reprogramming process, and microRNAs (miRNAs) were the upstream regulators. The objectives of this study involve the identification of potential miRNAs regulating the expression of chromatin modifying enzymes and the study of their roles during reprogramming.

Primary mouse embryonic fibroblasts (1o MEFs) were used for the establishment of a reprogramming system, where the delivery of transcription factors Oct4, Sox2, klf4 and cMyc was mediated by lentivirus. Another established secondary MEFs (2o MEFs) reprogramming system was also included in the study. Mouse iPSCs (miPSCs) derived from both systems were shown to express pluripotent markers. In-silico analysis predicted a set of miRNAs (miR-101, miR-135a, miR-148a and miR-148b) commonly targeted the chromatin modifying enzymes in mouse genome. Among them, miR-101 and miR-135a overexpression were found to inhibit the reprogramming efficiency significantly in both 1o and 2o MEFs. Conversely, the inhibition of miR-135a but not miR-101 expression significantly enhanced the reprogramming efficiency in both systems.

In this study, it was postulated that miR-101 regulated enhancer of zeste homolog 2 (Ezh2) during reprogramming. Ezh2 was confirmed to be negatively regulated by miR-101 at protein level. The expression of Ezh2 was high in mouse embryonic stem cells (mESCs) but time dependently depressed during mESC differentiation, while its expression was increased during reprogramming of MEFs. Ezh2 expression was found to negatively correlate with miR-101 expression in these conditions. In addition, the knockdown of Ezh2 mimicked the inhibitory effect of miR-101 overexpression on reprogramming efficiency.

The inhibitory role of miR-135a on reprogramming was linked to its potential target, Sirtuin 1 (Sirt1). Sirt1 was negatively regulated by miR-135a. The expression of miR-135a was upregulated upon mESC differentiation and decreased during reprogramming. Together with the previous finding in this laboratory, miR-135a expression was negatively correlated with Sirt1. Furthermore, miR-135a inhibition increased the proliferation rate of MEFs. More importantly, miPSCs reprogrammed from miR-135a knockdown MEFs maintained the pluripotent state.

To further analyze the pluripotency of the miPSCs, the tetraploid complementation assay was established. Preliminary studies were performed to optimize the conditions for electrofusion. Although single electrofusion with a lower field strength (1000V/cm) resulted in lower fusion rate, the development of the mESC aggregated embryo was the best when compared to higher field strength and those with double electrofusion. Lastly, the mESCs aggregated into tetraploid embryo were mainly localize in the inner cell mass of the embryo.

In conclusion, negative correlations were found between miR-101/Ezh2, and miR-135a/Sirt1 during somatic cell reprogramming. The identification of small molecules in reprogramming helps to understand the molecular mechanisms of reprogramming.