目的 探讨环指蛋白RNF181与Hippo/YAP对脑胶质瘤SHG44细胞恶性程度的影响及其可能机制。方法 选取人脑胶质瘤细胞系SHG44细胞体外培养,腺病毒表达载体转染过表达RNF181,对比分析RNF181 过表达对肿瘤细胞生长增殖能力、细胞干性和体外侵袭及转移能力;通过RT-PCR和Western blot实验检测RNF181及Hippo/YAP的表达,明确RNF181与Hippo/YAP的关系;最后通过免疫荧光共定位分析RNF181和YAP在SHG44细胞表达情况。结果 转染后SHG44中RNF181 mRNA及蛋白相对表达量1.95±0.06、0.39±0.06,明显高于对照组1.05±0.03、0.23±0.06和Negative组1.03±0.04、0.24±0.07;YAP mRNA及蛋白相对表达量2.63±0.12、0.71±0.06,与对照组1.02±0.04、0.29±0.05和Negative组1.03±0.05、0.31±0.07比较,表达水平明显升高,差异有统计学意义(P<0.05)。MTT、免疫荧光和Transwell 实验证实, RNF181过表达组细胞数、细胞增值率及SHG-44穿膜细胞数明显高于对照组和Negative组,差异均有统计学意义(P<0.05)。免疫荧光共定位检测表明RNF181和YAP在SHG44细胞核内共表达。结论 RNF181过表达增加胶质瘤细胞的增殖和侵袭能力,可能是通过Hippo/YAP信号途径实现的。
Abstract
Objective To explore the effect of overexpression of RNF181 on the malignancy of human glioma SHG44 cells and the possible mechanism. Methods Human glioma cell line SHG44 was cultured in vitro. The adenovirus expression vector was transfected to overexpress RNF181. The effects of overexpression of RNF181 on the growth and proliferation of tumor cells, cell stemness, invasion and metastasis in vitro were compared. The expressions of RNF181 and hippo / Yap were detected by Western blot, and the relationship between RNF181 and hippo / Yap was clarified. Finally, the expressions of RNF181 and Yap in SHG44 cells were analyzed by immunofluorescence co-localization. Results After transfection, the relative expression levels of RNF181 mRNA and protein in SHG44 were [(1.95±0.06) and (0.39±0.06)], which were much higher than those of the control group [(1.05±0.03) and (0.23±0.06) ] and the negative group[ (1.03±0.04) and (0.24±0.07)], and the difference was statistically significant (P<0.05). The relative expression levels of YAP mRNA and protein were (1.02±0.04), and (0.29±0.05) in the control group, compared with[(1.03±0.05) and (0.31±0.07)] in the negative group, and [(2.63±0.12) and (0.71±0.06)]in the overexpression group, so the difference was statistically significant (P<0.05). MTT, immunofluorescence and Transwell assay confirmed that the proliferation rate of the RNF181 overexpression group was significantly higher than that of the control group and the negative group. The cell number of the RNF181 overexpression group was significantly larger than that of control group and negative group, so was the number of SHG-44 transmembrane cells. Immunofluorescence co-localization showed that RNF181 and YAP were co-expressed in the nucleus of SHG44. Conclusions Overexpression of RNF181 may contribute to the proliferation and invasion of glioma cells along the Hippo/Yap signaling pathway.
关键词
神经胶质瘤细胞 /
环指蛋白181 /
Hippo/YAP /
恶性度
Key words
glioma cells /
RING finger protein 181 /
Hippo/Yap /
malignancy
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Bush N A, Chang S M, Berger M S. Current and future strategies for treatment of glioma[J]. Neurosurg Rev, 2017,40(1):1-14.
[2] 武卫周, 钟士江, 王 莉. 脑胶质瘤移植模型的研究进展[J].武警医学,2019,30(3):253-256.
[3] Ouyang T, Meng W, Li M, et al. Recent advances of the hippo/YAP signaling pathway in brain development and glioma[J]. Cell Mol Neurobiol, 2020,40(4):495-510.
[4] Wang L, Zhang Z, Yu X, et al. Unbalanced YAP-SOX9 circuit drives stemness and malignant progression in esophageal squamous cell carcinoma[J]. Oncogene,2019,38(12):2042-2055.
[5] Zhou R, Ding Y, Xue M, et al. RNF181 modulates hippo signaling and triple negative breast cancer progression[J]. Cancer Cell Int,2020,20:291.
[6] Pedersen S M, Chan W, Jattani R P, et al. Negative regulation of CARD11 signaling and lymphoma cell survival by the E3 ubiquitin ligase RNF181[J]. Mol Cell Biol, 2015,36(5):794-808.
[7] Knudsen K M, Moen G, Fluge O, et al. Pseudoprogression in high-grade glioma[J]. Acta Neurol Scand Suppl, 2013(196):31-37.
[8] Wang Y, Pan P, Wang Z, et al. β-catenin-mediated YAP signaling promotes human glioma growth[J]. J Exp Clin Cancer Res, 2017,36(1):136.
[9] Ouyang T, Meng W, Li M, et al. Recent advances of the hippo/YAP signaling pathway in brain development and glioma[J]. Cell Mol Neurobiol,2020,40(4):495-510.
[10] Kim M H, Kim J. Role of YAP/TAZ transcriptional regulators in resistance to anti-cancer therapies[J]. Cell Mol Life Sci,2017,74(8):1457-1474.
[11] Thompson B J.YAP/TAZ:drivers of tumor growth,metastasis,and resistance to therapy[J].Bioessays,2020,42(5):e1900162.
[12] Nguyen C D, Yi C. YAP/TAZ signaling and resistance to cancer therapy[J]. Trends Cancer, 2019,5(5):283-296.
[13] Yang Y K, Yang C, Chan W, et al. Molecular determinants of scaffold-induced linear ubiquitinylation of B cell lymphoma/leukemia 10 (Bcl10) during T cell receptor and oncogenic caspase recruitment domain-containing protein 11 (CARD11) signaling[J]. J Biol Chem,2016,291(50):25921-25936.
[14] Gupta A, Jain R, Wahi D, et al. Abrogation of auroraA-TPX2 by novel natural inhibitors: molecular dynamics-based mechanistic analysis[J]. J Recept Signal Transduct Res, 2015,35(6):626-33.
基金
1.福建自然科学基金(2020J011273);2.河北省重点研发计划项目(19272404D);3.天津市科技计划项目(16ZXHLSY00120)
PDF(1034 KB)
