Glucocorticoid receptor regulates expression of microRNA-22 and downstream signaling pathway in apoptosis of pancreatic acinar cells.

AIM: To elucidate the underlying mechanism that microRNA-22 (miR-22) promotes the apoptosis of rat pancreatic acinar cells (AR42J) and the elements that regulate the expression of miR-22. METHODS: One hundred nanomoles per liter of caerulein (Cae) was administrated to induce the apoptosis of AR42J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis, chromatin immunoprecipitation (ChIP) and ChIP-qPCR assays. Then, a mimic of miR-22, Nr3c1 plasmid encoding the glucocorticoid receptor (GR), and si-Nr3c1 were used to transfect AR42J cells, respectively. The mRNA expression of miR-22, Nr3c1, and Erb-b2 receptor tyrosine kinase 3 (ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3k, PI3k-p85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3. RESULTS: After inducing apoptosis of AR42J cells in vitro, the expression of miR-22 was significantly increased by 2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at 3 h and 6 h in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group, accompanied with an obviously increased acinar cell apoptosis rate (32.53 ± 1.15 vs 18.07 ± 0.89, P = 0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and site-directed mutagenesis indicated that the binding site (GACAGCCATGTACA) of the GR, which is encoded by the Nr3c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42J cells. CONCLUSION: GR transcriptionally represses the expression of miR-22, which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.

Single-Cell Non-coding RNA in Embryonic Development.

Non-coding RNAs (ncRNAs) have significant regulatory functions on the regulation of gene expression of various life activities after transcription, even though they do not encode proteins. During the development of embryos, ncRNAs, such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs), circular RNAs (circRNAs), small nucleolar RNAs (snoRNAs), and Piwi-interacting RNAs (piRNAs), have been widely proven as key regulators. The emerging single-cell RNA sequencing technique is powerful for profiling "cell-to-cell" variability at the genomic level. It has been applied to detect the expression of ncRNAs during embryo development. In this chapter, we pay close attention to single-cell ncRNA expression and summarize their roles in embryo development.