目的 探讨miR-148a对大鼠急性胰腺炎细胞模型中细胞自噬的影响。方法 选取培养AR42J细胞,细胞分为4组,即正常对照组、模型组、miR-148a mimics组及miR-148a阴性对照组。利用Lipofectamine 2000转染miR-148a mimics及阴性对照miR-148a至AR42J细胞,继续培养48 h后,利用浓度为200 μmol的牛磺胆酸钠盐(TLCs)刺激以上两组及模型组AR42J细胞20 min,正常对照组不做处理,然后提取各组细胞蛋白及RNA。利用RT-qPCR检测各组细胞中miR-148a的表达;利用CCK8实验检测各组细胞的活性;利用ELISA法检测各组细胞培养液中炎性因子IL-6,IL-1β及TNF-α的含量;利用Western blot检测自噬相关的基因Beclin1、LC3Ⅰ、 LC3Ⅱ的表达。结果 RT-qPCR结果显示,与正常对照组相比较,模型组心肌细胞中miR-148a mRNA的表达降低,而miR-148a mimics组细胞中miR-148a mRNA的表达显著升高;CCK-8实验结果显示,转染miR-148a mimics至细胞后,可提高模型细胞的活性;ELISA实验结果显示,与模型组相比较,转染miR-148a mimics至细胞后,细胞培养液中炎性因子IL-6,IL-1β及TNF-α的含量显著降低;Western blot结果显示,与模型组相比较,转染miR-148a mimics至细胞后,可降低细胞中Beclin1的表达,降低LC3Ⅱ/LC3Ⅰ的比率。结论 利用miR-148a mimics提高TLCs刺激的细胞模型中的miR-148a表达后,细胞中Beclin1的表达降低,LC3Ⅱ/LC3Ⅰ的比率降低,抑制了细胞自噬,降低了炎性因子IL-6、IL-1β、TNF-α的释放,从而提高了细胞的活性,miR-148a可通过调节模型细胞的自噬而发挥细胞保护作用。

Objective To investigate the effect of miR-148a on autophagy in rat acute pancreatitis cell model. Methods AR42J cells were cultured and divided into 4 groups: normal control group, model group, miR-148a mimics group and miR-148a negative control group. miR-148a mimics and miR-148a negative control were transfected to AR42J cells with Lipofectamine 2 000, then cells were cultured for 48 h. The AR42J cells were stimulated with sodium taurocholate (TLCs) at a concentration of 200 μmol for 20 min, the normal control group was not treated, then the protein and RNA were extracted in each group. The expression of miR-148a was detected by RT-qPCR in each group. The activity of cells was detected by CCK8 assay in each group. The contents of IL-6, IL-1β and TNF-α in the cell culture medium were detected by ELISA. Western blot was used to detect the expression of autophagy related genes Beclin1, LC3Ⅰ and LC3Ⅱ. Results RT-qPCR results showed that the expression of miR-148a mRNA in model group was significantly lower than that in normal control group, while the expression of miR-148a mRNA in miR-148a mimics group was significantly higher than that in normal control group. The results of CCK-8 assay showed that miR-148a could significantly increase the activity of model cells stimulated by TLCs. The results of ELISA showed that the contents of IL-6, IL-1β and TNF-α in cell culture medium were significantly decreased after miR-148a mimics transfection, compared with the model group. Western blot showed that miR-148a mimics could significantly decrease the expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ, compared with the model group. Conclusion miR-148a mimics was used to enhance the expression of miR-148a in cells model stimulated by TLCs, the expression of Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ were decreased, and the autophagy was inhibited. The release of IL-6, IL-1β and TNF-α was decreased and the activity of cells was increased. miR-148a plays a cellular protective role by regulating autophagy in model cells.