Wang Yuanchuan, Yin Xiaohong, Zhao Long, Li Shun, Duan Jie, Kuang Renzhao, Duan Junwei
Department of Neurosurgery, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P.R. China.
Exp Ther Med. 2017 Aug;14(2):1706-1714. doi: 10.3892/etm.2017.4681. Epub 2017 Jun 27.
The present study aimed to investigate the expression of miR-200b and protein kinase Cα (PKCα) in pituitary tumors and to determine whether miR-200b may inhibit proliferation and invasion of pituitary tumor cells. The regulation of PKCα expression was targeted in order to find novel targets for the treatment of pituitary tumors. In total, 53 pituitary tumor tissue samples were collected; these included 28 cases of invasive pituitary tumors and 25 cases of non-invasive tumors, in addition to 5 normal pituitaries. The expression level of miR-200b in the pituitary tumor tissue was detected by quantitative polymerase chain reaction (qPCR) and the expression of PKCα protein was detected by immunohistochemistry. A PKCα 3'untranslated region (UTR) luciferase vector was constructed and a dual luciferase reporter gene assay was employed in order to examine the effect of miR-200b on the PKCα 3'UTR luciferase activity. AtT-20 cells were transfected with miR-200b mimics, PKCα siRNA and miR-200b mimics + PKCα, and the changes in cellular proliferation, invasion and apoptosis were observed via MTT, Transwell assay and flow cytometric analysis. Furthermore, PKCα mRNA expression was determined by qPCR, and Western blotting was performed to detect the expression of PKCα protein. miR-200b revealed downregulation in invasive pituitary tumor tissue, and the expression level was significantly down-regulated compared with normal and non-invasive pituitary tumor tissue (P<0.01). In addition, the positive rate of PKCα protein expression in invasive pituitary tumor tissues was significantly higher than in normal and non-invasive tissues (P<0.01). PKCα protein levels are inversely correlated with miR-200b levels in invasive pituitary tumor tissues (r=-0.436, P=0.021). The dual luciferase reporter gene assay revealed that miR-200b could specifically bind to the 3'UTR of PKCα and significantly inhibit the luciferase activity by 39% (P<0.01). Upregulation of miR-200b or downregulation of PKCα could suppress cell proliferation and invasion, and increase apoptosis of AtT-20 cells. It was revealed that PKCα siRNA could suppress both proliferation and invasion of AtT-20 cells and partially simulate the function of miR-200b. Expression of PKCα mRNA and protein decreased significantly in AtT-20 cells overexpressing miR-200b. Additionally, miR-200b was significantly down-regulated in invasive pituitary tumor tissue and inversely correlated with PKCα protein levels. In conclusion, miR-200b inhibited proliferation and invasiveness and promoted the apoptosis of pituitary tumor cells by targeting PKCα. The observations of the present study indicate that miR-200b and PKCα may serve as promising therapeutic targets for invasive pituitary tumors.
本研究旨在探讨miR-200b和蛋白激酶Cα（PKCα）在垂体肿瘤中的表达情况，并确定miR-200b是否可抑制垂体肿瘤细胞的增殖和侵袭。通过靶向PKCα表达的调控来寻找治疗垂体肿瘤的新靶点。总共收集了53例垂体肿瘤组织样本，其中包括28例侵袭性垂体肿瘤和25例非侵袭性肿瘤，另外还有5例正常垂体组织。采用定量聚合酶链反应（qPCR）检测垂体肿瘤组织中miR-200b的表达水平，采用免疫组织化学检测PKCα蛋白的表达。构建PKCα 3'非翻译区（UTR）荧光素酶载体，并采用双荧光素酶报告基因检测法检测miR-200b对PKCα 3'UTR荧光素酶活性的影响。用miR-200b模拟物、PKCα siRNA和miR-200b模拟物+PKCα转染AtT-20细胞，通过MTT法、Transwell实验和流式细胞术分析观察细胞增殖、侵袭和凋亡的变化。此外，通过qPCR测定PKCα mRNA表达，并进行蛋白质印迹法检测PKCα蛋白的表达。miR-200b在侵袭性垂体肿瘤组织中呈下调表达，与正常和非侵袭性垂体肿瘤组织相比，其表达水平显著下调（P<0.01）。此外，侵袭性垂体肿瘤组织中PKCα蛋白表达的阳性率显著高于正常和非侵袭性组织（P<0.01）。在侵袭性垂体肿瘤组织中，PKCα蛋白水平与miR-200b水平呈负相关（r=-0.436，P=0.021）。双荧光素酶报告基因检测显示，miR-200b可特异性结合PKCα的3'UTR，并显著抑制荧光素酶活性达39%（P<0.01）。上调miR-200b或下调PKCα均可抑制AtT-20细胞的增殖和侵袭，并增加其凋亡。结果显示，PKCα siRNA可抑制AtT-20细胞的增殖和侵袭，并部分模拟miR-200b的功能。在过表达miR-200b的AtT-20细胞中，PKCα mRNA和蛋白的表达显著降低。此外，miR-200b在侵袭性垂体肿瘤组织中显著下调，并与PKCα蛋白水平呈负相关。总之，miR-200b通过靶向PKCα抑制垂体肿瘤细胞的增殖和侵袭，并促进其凋亡。本研究的观察结果表明，miR-200b和PKCα可能是侵袭性垂体肿瘤有前景的治疗靶点。
