沉默重组蛋白2对NCI-H1688细胞的抑制作用及其Wnt/β连环蛋白信号通路机制

doi:10.13481/j.1671-587x.20200414

摘要/Abstract

摘要： 目的：探讨T细胞淋巴瘤常见重排蛋白2（Frat2）通过Wnt/β连环蛋白（β-catenin）信号通路对小细胞肺癌NCI-H1688细胞增殖的影响，阐明其可能机制。方法：选取生长状态良好的人小细胞肺癌NCI-H1688细胞和正常支气管上皮样细胞HBE细胞，采用Western blotting法和逆转录实时荧光定量-聚合酶链反应（Real-Time RT-qPCR）法分别检测NCI-H1688细胞和HBE细胞中Frat2蛋白及mRNA表达水平。NCI-H1688细胞随机分为空白对照组、阴性对照组、Frat2-siRNA组和Frat2-siRNA+XAV组。空白对照组细胞不转染，阴性对照组细胞转染阴性对照慢病毒，Frat2-siRNA组转染Frat2-siRNA慢病毒，Frat2-siRNA+XAV组转染Frat2-siRNA慢病毒并同时加入4 μmol·L^-1Wnt信号通路抑制剂XAV939。采用Western blotting法和Real-Time RT-qPCR法分别检测转染后各组细胞中Frat2蛋白和mRNA表达水平，采用MTT法检测各组细胞增殖活性，采用流式细胞术检测不同细胞周期细胞百分率，采用Western blotting法检测细胞中增殖细胞核抗原（PCNA）、c-myc、细胞周期蛋白D1（Cyclin D1）、β-catenin和磷酸化糖原合成酶激酶3（pGSK-3β）蛋白表达水平。结果：与HBE细胞比较，NCI-H1688细胞中Frat2蛋白和mRNA表达水平明显升高（P<0.01）。与空白对照组和阴性对照组比较，Frat2-siRNA组NCI-H1688细胞中Frat2蛋白和mRNA表达水平明显升高（P<0.05）；空白对照组与阴性对照组NCI-H1688细胞中Frat2蛋白和mRNA表达水平比较差异无统计学意义（P>0.05）。与空白对照组和阴性对照组比较，Frat2-siRNA组细胞增殖活性降低（P<0.05），G₀/G₁期细胞百分率升高（P<0.05），S期和G₂/M期细胞百分率降低（P<0.05），PCNA、c-myc、Cyclin D1、β-catenin和pGSK-3β蛋白表达水平降低（P<0.05）；与Frat2-siRNA组比较，Frat2-siRNA+XAV组细胞增殖活性降低（P<0.05），G₀/G₁期细胞百分率升高（P<0.05），S期和G₂/M期细胞百分率降低（P<0.05），PCNA、c-myc、Cyclin D1、β-catenin和pGSK-3β蛋白表达水平降低（P<0.05）；空白对照组与阴性对照组上述各指标比较差异无统计学意义（P>0.05）。结论：沉默Frat2可抑制小细胞肺癌细胞增殖，其机制可能与抑制Wnt/β-catenin信号通路有关。

关键词: 重组蛋白2, Wnt/β连环蛋白信号通路, 小细胞肺癌, 细胞增殖, 增殖细胞核抗原, 细胞周期蛋白D1, 糖原合成酶激酶3

Abstract: Objective: To investigate the effect of frequently rearranged in advanced T-cell lymphomas 2 (Frat2) on the proliferation of small cell lung cancer NCI-H1688 cells via Wnt/β-catenin signaling pathway,and to elucidate its possible mechanism. Methods: The human small cell lung cancer NCI-H1688 cells with good growth status and the normal bronchial epithelial-like cells HBE were selected;Western blotting method and Real-Time RT-qPCR were used to detect the expression levels of Frat2 mRNA and protein in the NCI-H1688 cells and the HBE cells.The NCI-H1688 cells were randomly divided into blank control group, negative control group,Frat2-siRNA group and Frat2-siRNA+XAV group.The cells in blank control group were not transfected.The cells in negative control group were transfected with negative control lentivirus,the cells in Frat2-siRNA group were transfected with Frat2-siRNA lentivirus and the cells in Frat2-siRNA+XAV group were transfected with the Frat2-siRNA lentivirus and treated with 4 μmol·L^-1of Wnt signaling pathway inhibitor XAV939.The expression levels of Frat2 protein and mRNA in the cells in various groups were determined by Western blotting method and Real-Time RT-qPCR method.The proliferation activities of the cells in various groups were determined by MTT method.The percentages of cells in different cell cycles were measured by flow cytometry.The expression levels of Frat2, proliferating cell nuclear antigen (PCNA), c-myc, cyclin D1, β-catenin and phosphorylated glycogen synthase kinase 3 (pGSK-3β) proteins in the cells were determined by Western blotting method. Results: Compared with the normal bronchial epithelial-like cells HBE, the levels of Frat2 protein and mRNA in the NCI-H1688 cells were elevated (P<0.01).Compared with blank control group and negative control group, the expression levels of protein and Frat2 mRNA in Frat2-siRNA group were increased (P<0.05).There were no significant difference in the expression levels of Frat2 protein and mRNA in the NCI-H1688 cells between blank control group and negative control group (P>0.05).Compared with blank control group and negative control group, the proliferation activity of the cells in Frat2-siRNA group was decreased (P<0.05), the percentage of cells in G₀/G₁ phase was increased (P<0.05),the percentages of cells in S phase and G₂/M phase were decreased (P<0.05), and the expression levels of PCNA, c-myc, Cyclin D1, β-catenin and pGSK-3β proteins were decreased (P<0.05).Compared with Frat2-siRNA group,the proliferation activity of cells in Frat2 mRNA+XAV group was decreased(P<0.05),the percentage of cells in G₀/G₁ phase was increased (P<0.05), the percentages of cells in S phase and G₂/M phase were decreased (P<0.05),and the expression levels of PCNA, c-myc, Cyclin D1, β-catenin and pGSK-3β proteins were decreased (P<0.05).There were no significant differences in the indexes mentioned above between blank control group and negative control group (P>0.05). Conclusion: Silencing Frat2 can inhibit the proliferation of small cell lung cancer cells, and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.

Key words: frequently rearranged in advanced T-cell lymphomas 2, Wnt/β-catenin signaling pathway, small cell lung cancer, proliferation, proliferating cell nuclear antigen, cyclin D1, glycogen synthase kinase 3

中图分类号:

R734.2

陈艳, 潘殿柱, 刘忠, 马艳梅, 张岚. 沉默重组蛋白2对NCI-H1688细胞的抑制作用及其Wnt/β连环蛋白信号通路机制[J]. 吉林大学学报(医学版), 2020, 46(04): 751-758.

CHEN Yan, PAN Dianzhu, LIU Zhong, MA Yanmei, ZHANG Lan. Inhibitory effect of sileneing Frat2 on NCI-H1688 cells and its Wnt/β catenin signaling pathway mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 751-758.

参考文献

[1] 刘景瑜,曹欣,郭庚,等.FRAT1生物学特性的研究进展[J].国际遗传学杂志,2015,38(1):35-40,46.
[2] ZHANG B, LI N, ZHANG H.Knockdown of homeobox B5(HOXB5) inhibits cell proliferation, migration, and invasion in non-small cell lung cancer cells through inactivation of the Wnt/β-catenin pathway[J].Oncol Res,2018,26(1):37-44.
[3] 张勇, 曹红一, 王恩华. Frat1与TCF4在非小细胞肺癌(NSCLC)中表达的相关性及意义[J]. 现代肿瘤医学, 2012, 20(6):1175-1178.
[4] PAN F, SHEN F Z, YANG L J, et al. Inhibitory effects of XAV939 on the proliferation of small-cell lung cancer H446 cells and Wnt/β-catenin signaling pathway in vitro[J]. Oncol Lett, 2018, 16(2):1953-1958.
[5] 李高兵,蔡东平,毛张凡.川芎嗪通过调控Wnt信号通路抑制肺癌细胞增殖、侵袭和迁移的机制研究[J].现代肿瘤医学,2019,27(12):2035-2040.
[6] DIMAKAKOS E, LIVANIOS K, GKIOZOS I,et al.New data for venous thromboembolism in patients with small cell lung cancer:A review[J].Phlebology, 2018,33(8):517-522.
[7] 吕婷婷,吕欣,郭庚.FRAT1与肿瘤的相关性研究进展[J].医学综述,2014,20(20):3691-3693.
[8] WERNER T V, HART M, NICKELS R, et al. MiR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro and is directly targeting SMAD4, FRAT1 and BCL2[J]. Aging (Albany NY), 2017, 9(3):932-954.
[9] ZHU K X, GUO J Q, WANG H J, et al. FRAT1 expression regulates proliferation in colon cancer cells[J]. Oncol Lett, 2016, 12(6):4761-4766.
[10] YU Q G, SHANG L, YU H B, et al. Silencing of FRAT1 by siRNA inhibits the proliferation of SGC7901 human gastric adenocarcinoma cells[J]. Biomed Rep, 2016, 4(2):223-226.
[11] FAN W H, DU F J, LIU X J, et al. Knockdown of FRAT1 inhibits hypoxia-induced epithelial-to-mesenchymal transition via suppression of the Wnt/β-catenin pathway in hepatocellular carcinoma cells[J]. Oncol Rep, 2016, 36(5):2999-3004.
[12] 王苏雅, 于庆功, 谷伟, 等. 沉默FRAT1基因对结肠癌HT-29细胞增殖与凋亡的影响及其机制研究[J]. 中国实验诊断学, 2014, 18(2):195-199.
[13] ZHANG W, XIONG H, ZOU Y M,et al.Frequently rearranged in advanced T cell lymphomas-1 demonstrates oncogenic properties in prostate cancer[J].Mol Med Rep,2016,14(4):3551-3558.
[14] STOOTHOFF W H, CHO J H, MCDONALD R P,et al.FRAT-2 preferentially increases glycogen synthase kinase 3 beta-mediated phosphorylation of primed sites, which results in enhanced tau phosphorylation[J].J Biol Chem,2005,280(1):270-276.
[15] 卢迎宏, 王丹, 井海云. 苦参碱对oxLDL诱导的血管平滑肌细胞炎症反应及增殖凋亡的影响及分子机制研究[J]. 中国免疫学杂志, 2018, 34(4):537-543.
[16] JIA W, WU W, YANG D,et al.Histone demethylase JMJD3 regulates fibroblast-like synoviocyte-mediated proliferation and joint destruction in rheumatoid arthritis[J].FASEB J,2018,32(7):4031-4042.
[17] WANG Y, CONG W, WU G,et al.MiR-376a suppresses the proliferation and invasion of non-small-cell lung cancer by targeting c-Myc[J].Cell Biol Int, 2018, 42(1):25-33.
[18] ZOU J, LI X L, SHI Z M,et al.Effects of C-myc gene silencing on interleukin-1β-induced rat chondrocyte cell proliferation, apoptosis and cytokine expression[J].J Bone Miner Metab,2018,36(3):286-296.
[19] WU J H, ZHANG Y H, SUN L, et al. Cyclin D1 expression by histiocytes may mimic cyclin D1-positive proliferation centres of chronic lymphocytic leukaemia/small lymphocytic lymphoma[J]. Pathol Res Pract, 2018, 214(1):72-75.
[20] CHEN J, LI X, CHENG Q,et al.Effects of cyclin D1 gene silencing on cellproliferation, cell cycle, and apoptosis of hepatocellular carcinoma cells[J].J Cell Biochem,2018,119(2):2368-2380.
[21] WANG B, SUN L W, LI J D, et al. MiR-577 suppresses cell proliferation and epithelial-mesenchymal transition by regulating the WNT2B mediated Wnt/β-catenin pathway in non-small cell lung cancer[J]. Mol Med Rep, 2018, 18(3):2753-2761.
[22] WANG D P, GU L L, XUE Q, et al. CtBP2 promotes proliferation and reduces drug sensitivity in non-small cell lung cancer via the Wnt/β-catenin pathway[J]. Neoplasma, 2018, 65(6):888-897.
[23] CHEN Z Y, DU Y, WANG L,et al.iR-543 promotes cell proliferation and metastasis of renal cell carcinoma by targeting Dickkopf 1 through the Wnt/β-catenin signaling pathway[J].J Cancer,2018,9(20):3660-3668.
[24] GUO W X, SHEN F Z, XIAO W J, et al. Wnt inhibitor XAV939 suppresses the viability of small cell lung cancer NCI-H446 cells and induces apoptosis[J]. Oncol Lett, 2017, 14(6):6585-6591.