Xu Yan, Yang Junwen, Li Fujun, Lian Guanghui, Ouyang Miao
Health Management Center.
Gastroenterology Department, Xiangya Hospital, Central South University, Changsha, People's Republic of China.
Anticancer Drugs. 2018 Aug;29(7):652-659. doi: 10.1097/CAD.0000000000000636.
Ulcerative colitis (UC), with high morbidity has become one of the fastest-growing severe illnesses in the world. Although MiR-29a is highly expressed in the tissues of UC patients, the mechanism of miR-29a involved in the specific pathogenesis of UC is not known. In this study, a GFP-light chain 3 (LC3) immunofluorescence assay was used to observe the formation of the autophagic spot; qRT-PCR and western blotting analyses were carried out to detect the expression of autophagy-related proteins, including BECN1, Autophagy-related gene (ATG)5, ATG16L, and transcription factor EB. The dual-fluorescence reporter assay was used to analyze the direct effect of miR-29a on ATG9A; experimental dextran sulfate sodium-induced colitis in mice was used to establish the UC model. Our studies showed that the overexpression of miR-29a not only suppressed the production of GFP-LC3 autophagy spots but also inhibited the level of LC3II/LC3I and upregulated the expression of P62 in HT29 and HCT116 cells. Moreover, the results showed that miR-29a directly targeted the 3'UTR region of ATG9A mRNA to suppress the activation of HT29 and HCT116 cells' autophagy. Also, overexpression of ATG9A rescued rapamycin-induced autophagy that was inhibited by overexpression of miR-29a. In addition, miR-29a also affected the expression of autophagy-related proteins (BECN1, ATG5, ATG16L1, and transcription factor EB). Notably, miR-29a was upregulated, whereas ATG9A was downregulated in the experimental dextran sulfate sodium-induced colitis in mice. In effect, this study showed that miR-29a inhibits rapamycin-induced intestinal epithelial cells' autophagy partly by decreasing ATG9A in UC. These findings may provide new insights that may help control the inflammation in UC.
溃疡性结肠炎（UC）发病率高，已成为全球增长最快的严重疾病之一。尽管miR - 29a在UC患者组织中高表达，但其参与UC具体发病机制尚不清楚。本研究采用绿色荧光蛋白轻链3（LC3）免疫荧光检测法观察自噬斑点的形成；进行qRT - PCR和蛋白质印迹分析以检测自噬相关蛋白的表达，包括BECN1、自噬相关基因（ATG）5、ATG16L和转录因子EB。采用双荧光素酶报告基因检测法分析miR - 29a对ATG9A的直接作用；利用实验性葡聚糖硫酸钠诱导的小鼠结肠炎建立UC模型。我们的研究表明，miR - 29a的过表达不仅抑制了GFP - LC3自噬斑点的产生，还抑制了HT29和HCT116细胞中LC3II/LC3I的水平，并上调了P62的表达。此外，结果表明miR - 29a直接靶向ATG9A mRNA的3'UTR区域以抑制HT29和HCT116细胞自噬的激活。而且，ATG9A的过表达挽救了被miR - 29a过表达抑制的雷帕霉素诱导的自噬。此外，miR - 29a还影响自噬相关蛋白（BECN1、ATG5、ATG16L1和转录因子EB）的表达。值得注意的是，在实验性葡聚糖硫酸钠诱导的小鼠结肠炎中，miR - 29a上调，而ATG9A下调。实际上，本研究表明miR - 29a在UC中部分通过降低ATG9A来抑制雷帕霉素诱导肠上皮细胞的自噬。这些发现可能为控制UC中的炎症提供新的见解。
