Huang Limin, Hu Chaoquan, Cao Hui, Wu Xiaoliang, Wang Rongpin, Lu He, Li Hong, Chen Hui
Department of Oncology, Guizhou Provincial People's Hospital, Guiyang, China.
Department of Surgery, Affiliated Hospital, GuiZhou Medical University, Guiyang, China.
Cell Physiol Biochem. 2018;47(2):747-758. doi: 10.1159/000490027. Epub 2018 May 22.
BACKGROUND/AIMS: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro.
We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c.
miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis.
Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.
背景/目的：胰腺癌（PC）是一种侵袭性恶性肿瘤，生存率低。尽管PC治疗取得了进展，但化疗耐药性的产生限制了治疗效果。在此，我们研究了微小RNA-29c（miR-29c）的作用以及自噬和凋亡在PC细胞体内外化疗耐药中的参与情况。
我们采用qRT-PCR、蛋白质免疫印迹法和免疫荧光法检测miR-29c、泛素特异性蛋白酶22（USP22）和自噬相关蛋白的表达水平。此外，我们使用MTT法检测细胞增殖，使用Transwell法测量迁移和侵袭能力。通过流式细胞术使用膜联蛋白V-异硫氰酸荧光素/碘化丙啶凋亡检测试剂盒测定凋亡情况。荧光素酶报告基因检测证实了USP22与miR-29c之间的关系。
PC细胞系PANC-1中miR-29c的过表达增强了吉西他滨降低细胞活力和诱导凋亡的作用，并抑制了自噬，蛋白质免疫印迹法、免疫荧光染色、集落形成试验和流式细胞术均显示了这一点。泛素特异性蛋白酶（USP）-22是一种已知可诱导自噬并促进PC细胞存活的去泛素化酶，被确定为miR-29c的直接靶点。USP22敲低实验表明，USP22通过促进自噬抑制吉西他滨诱导的凋亡，从而增加PC细胞的化疗耐药性。荧光素酶报告基因检测证实USP22是miR-29c的直接靶点。异种移植小鼠模型表明，miR-29c通过下调USP22增加PC在体内的化疗敏感性，导致自噬受到抑制并诱导凋亡。
综上所述，这些发现揭示了miR-29c通过调节USP22介导的自噬介导PC细胞化疗耐药的潜在机制，提示了PC的潜在靶点和治疗策略。
