摘要
AIM To elucidate the underlying mechanism that microRNA-22(miR-22) promotes the apoptosis of rat pancreatic acinar cells(AR42 J) 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 AR42 J 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 ChIPqP CR assays. Then, a mimic of miR-22, Nr3 c1 plasmid encoding the glucocorticoid receptor(GR), and siNr3 c1 were used to transfect AR42 J cells, respectively.The mRNA expression of miR-22, Nr3 c1, and Erb-b2 receptor tyrosine kinase 3(ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42 J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3 k, PI3 kp85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3.RESULTS After inducing apoptosis of AR42 J cells in vitro, the expression of miR-22 was significantly increased by2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at3 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 PI3 k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and sitedirected mutagenesis indicated that the binding site(GACAGCCATGTACA) of the GR, whi
AIM To elucidate the underlying mechanism that microRNA-22(miR-22) promotes the apoptosis of rat pancreatic acinar cells(AR42 J) 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 AR42 J 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 ChIPqP CR assays. Then, a mimic of miR-22, Nr3 c1 plasmid encoding the glucocorticoid receptor(GR), and siNr3 c1 were used to transfect AR42 J cells, respectively.The mRNA expression of miR-22, Nr3 c1, and Erb-b2 receptor tyrosine kinase 3(ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42 J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3 k, PI3 kp85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3.RESULTS After inducing apoptosis of AR42 J cells in vitro, the expression of miR-22 was significantly increased by2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at3 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 PI3 k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and sitedirected mutagenesis indicated that the binding site(GACAGCCATGTACA) of the GR, whi
基金
National Natural Science Foundation of China,No.31671440
