胰腺腺泡细胞凋亡相关微小RNA-22和微小RNA-135a调控机制研究

Acute pancreatitis is a common clinical condition with a watchable morbidity and mortality. There were two patterns of pancreatic acinar cell death: apoptosis and necrosis in acute pancreatitis. The apoptosis has protecting effect in acute pancreatitis, and the mechanism of phenomena is unclear. MicroRNAs are short non-coding RNAs and play important regulating genes expression roles in multiply processes including development, cell proliferation, cell differentiation, apoptosis, and metabolism. The finding of apoptosis associated miRNA (AAmiRNA) might be a new breakthrough for the treatment programs. The results of pilot studies indicated that miR-22 and miR-135a were identified as apoptosis associated miRNA. We also demonstrated that miR-22 and miR-135a might promote the apoptosis of pancreatic acinar cell by repressing their target genes ERBB3 and Ptk2 in vitro. But the up-stream and down-stream of the signal pathway of miR-22 and miR-135a are still unknown. In this project, We would find out the regulation mechanism that promote the apoptosis of pancreatic acinar cell of miR-22 and miR-135a in vivo and in vitro, which could provide a new therapeutic targets for the prevention and treatment of acute pancreatitis.

急性胰腺炎(AP)是严重危害人类健康的疾病，在AP过程中腺泡细胞凋亡具有保护作用，保护机制尚未明确。微小RNA（microRNA）作为基因转录后表达调控因子，寻找凋亡相关miRNA（AAmiRNA）可能是研究AP过程中凋亡保护机制及治疗的新突破口。我们前期研究鉴定了凋亡相关 miR-22和miR-135a，鉴定靶基因分别为ERBB3 和Ptk2，miR-22和miR-135a可能通过调控靶基因诱导AP腺泡细胞凋亡，进一步研究应用染色质免疫共沉淀（ChIP）、凝胶迁移实验(EMSA)、腺相关病毒等技术研究miR-22和miR-135a 调控网络中上游转录因子、下游信号通路基因的表达，以及体内实验中诱导胰腺腺泡细胞的凋亡发挥保护机制。本项目首次从miRNA水平研究AP发病中腺泡细胞AAmiRNA，阐明miR-22和miR-135a对AP腺泡细胞凋亡的调控机制，为AP的治疗开辟了新的方向.

我们前期的研究表明miRNA-22（miR-22）通过靶向调控ErbB3基因促进胰腺腺泡细胞凋亡，其上下游调控机制尚未完全阐明。本研究进一步证实miR-22通过靶向ErbB3进一步调控下游PI3K/Akt信号通路促进AR42J细胞凋亡。我们使用在线工具预测了miR-22的潜在转录因子和结合位点，并通过荧光素酶定量实验进行验证，证实Nr3c1基因编码的糖皮质激素受体（GR）可能与位点（GACAGCCATGTACA）结合从而调节miR-22启动子活性，并通过ChIP和ChIP-qPCR结果进一步验证。Nr3c1（GR）通过结合miR-22启动子转录起始位点转录抑制miR-22的表达。总之，本研究的结果表明，下调Nr3c1（GR）的表达可以上调miR-22的表达。miR-22的上调通过靶向ErbB3并进一步抑制PI3K/Akt信号通路促进Cae诱导的AR42J细胞凋亡。