microRNA-181通过靶向PTEN对胰腺泡细胞自噬及凋亡的影响及作用机制研究

microRNA-181 Works on Pancreatic Acinar Cell Autophagy and Apoptosis via Target PTEN

目的探究对胰腺泡细胞自噬及凋亡的影响及作用机制。方法用100μmol/L的雨蛙素(cerulein)处理大鼠胰腺泡细胞AR42J, miR-181a mimic及miRNA mock分别转染细胞后,RT-PCR检测miR-181a、PTEN表达水平。生物信息预测两者关系并用荧光素酶报告实验验证。细胞分为Control、cerulein、miR-181a mimic、pc-PTEN 及 miR-181a mimic + pcDNA PTEN (pc-PTEN)组,miR-181a mimic 及 pc-PTEN 转染细胞后,CCK8检测细胞活性,Hoechst染色检测细胞凋亡,Western印迹检测PTEN, PCNA、Bcl-2, Bax、cleaved Caspase-9, LC3、Beclin1、P62、PI3K、p-PI3K、Akt、p-Akt, mTOR 和 p-mTOR 的表达,免疫荧光检测LC3表达。雷帕霉素与miR-181a mimic共处理细胞后,蛋白印迹检测LC3B、Beclin1及p62表达水平,CCK8检测细胞活性,Hoechst染色检测细胞凋亡。结果雨蛙素可下调miR-181a及上调PTEN表达。miR-181a mimic可显著降低PTEN wt的荧光素酶活性。miR-181a mimic + pc-PTEN能部分逆转pc-PTEN对PTEN和cleaved Caspase-9表达、凋亡细胞百分比、Bax/Bcl-2比值、PCNA表达、细胞存活率、LC3 Ⅱ/LC3 Ⅰ比值、Beclin1表达、LC3荧光值及p62表达的调节作用;同时,miR-181a mimic + pc-PTEN还能逆转pc-PTEN对PI3K/AKT/mTOR信号通路激活的抑制作用。最后,Rapamycin + miR-181a mimic能逆转miR-181a mimic对LC3B、Beclin1和细胞凋亡的抑制作用,及对p62和细胞存活率的促进作用。结论 miR-181a可通过靶向下调PTEN抑制胰腺泡细胞自噬及凋亡的发生。

Objective To investigate the mechanism of MicroRNA-181 a ( miR-181a) working on pancreatic acinar cell autophagy and apoptosis. Methods Rat pancreatic acinar cell were first treated with 100 pmol/L cerulein. After transfected with miR-181a mimic and miRNA mock spectively , RT-PCR was performed to evaluate the expression level of miR-181a and PTEN. The targeting relationship between them was bioinformatically predicted and confirmed by luciferase reporter assay. Cells were divided into control, cerulean, miR-181a mimic, pc-PTEN and miR-181 a mimic + pcDNA PTEN ( pc-PTEN ) groups. After cerulean-treated cells were transfected with miR-181 a and pc-PTEN, CCK8 was used to evaluate the cell viability;Hoechst was employed to evaluate cell apoptosis;Western blotting was performed to detect the expression of PTEN, PCNA, Bcl-2, Bax,cleaved Caspase-9, LC3, Beclinl, P62, PI3K, pPI3K, Akt, p-Akt, mTOR, pmTOR. Immunofluorescence test was done to determine the expression of LC3. After treatment with Rapamycin and miR-181a mimic, Western blotting was performed to measure the expression levels of LC3B, Beclinl and p62. CCK8 was utilized to detect the cell viability and Hoechst was used to evaluate cell apoptosis. Results Cerulein could down-regulate miR-181 a and upregulate PTEN. miR-181a mimic could decrease the luciferase activity of PTEN wt. MiR-181a mimic 4- pc-PTEN could partially reverse the effects of pc-PTEN on the expression of PTEN and cleaved Caspase-9 , apoptot ic rate of cells, Bax/Bel-2 ratio, PCNA, cell survival rate, LC3 II/LC3 I ratio, Beclinl expression , LC3 fluorescence value and p62 expression. At the same time, MiR-181a mimic + pc-PTEN could reverse the inhibitory effect of pc-PTEN on the activation of PI3 K/AKT/mTOR signaling pathway. Lastly, Rapamycin + microRNA-181 a mimic could reverse the inhibitory effect of microRNA- 181a mimic on the expression of LC3B, Beclinl and apoptosis rate, and could promote the expression of p62 and enhance cell survival. Conclusion miR-181a could inhibit pancreatic acinar cell autophagy and apoptosis via target PTEN.