P75NTR基因在舌鳞状细胞癌细胞凋亡中的作用及其机制

doi:10.3760/cma.j.issn.1673422X.2016.07.001

摘要/Abstract

摘要： 目的探求p75神经营养因子受体（P75NTR）基因在舌鳞状细胞癌细胞凋亡中的作用及其可能的分子作用机制。方法 p75NTR特异性小干扰RNA（siRNA）转染p75NTR阳性舌鳞状细胞癌细胞；P75NTR阳性舌鳞状细胞癌株Tca8113细胞分为空白对照组、阴性对照组、实验组776（转染siRNAP75NTR776）、实验组1234（转染siRNAP75NTR1234）。流式细胞仪双染色标记法检测转染率及细胞凋亡；荧光定量PCR检测P75NTR基因；Western blotting检测各组P75NTR蛋白的表达；四甲基偶氮唑蓝法检测siRNA对细胞增殖的影响；细胞免疫荧光标记法观察核转录因子κB（NFκB）在细胞质及细胞核中的分布变化。结果测得转染效率为30%；实验组776、实验组1234与阴性对照组的凋亡率分别为（20.35±0.18）%、（12.32±1.51）%、（2.63±0.10）%，前两者与阴性对照组相比，差异有统计学意义（t=177.20，P＜0.005；t=37.12，P＜0.005）。P75NTR 基因干扰成功，实验组776组的P75NTR蛋白抑制率达31%。P75NTRsiRNA干扰后实验组776 、实验组1234 与阴性对照组Tca8113细胞存活率分别为70.02%、78.01%和95.81%，前两者与阴性对照组相比，差异有统计学意义（χ2=235.3,P＜0.001；χ2=117.5, P＜0.005）。实验组内NFκB在细胞质内分布明显增多。结论 P75NTR能促进舌鳞状细胞癌细胞增殖或抑制其凋亡，其分子机制可能与阻碍NFκB核内转运密切相关。

关键词: 细胞凋亡, 神经生长因子, NF-&kappa, B

Abstract: Objective To investigate the molecular mechanism of P75NTR geneinduced apoptosis in tongue squamous cell carcinoma Tca8113 cell lineage. Methods P75NTR specific siRNA was transferred into P75NTR positive tongue squamous cell carcinoma Tca8113 cells. P75NTR positive Tca8113 cells were divided into 4 groups: blank group (without transfection), negative control group (transfected with negative control siRNA), experiment group776 (transfected with siRNAP75NTR776) and experiment group1234 (transfected with siRNAP75NTR1234). Transfection efficiency and cell apoptosis were detected by flow cytometry. The interference effect of P75NTR mRNA expression was detected by fluorescence quantitative PCR. 3(4,5dimethyl2thiazoly)2,5diphenyl2Htetrazolium bromide assay was applied in measuring cell proliferation. The protein changes of P75NTR were detected by Western blotting. The distributions of nuclear factor-κB（NF-κB） of cells were observed by cell immunofluorescence labeling method. Results The transfection efficiency was 30%. The apoptosis rate of experiment group776, experiment group1234 and negative control group was (20.35±0.18) %, (12.32±1.51)% and (2.63±0.10)% respectively. Compared with the negative control group, the differences of the former two group had statistical significance (t=177.20, P<0.005; t=37.12, P<0.005). The P75NTR gene interference was successful. The inhibition rate of P75NTR protein reached 31% in experiment group776. The cell viability of Tca8113 cells after P75NTRsiRNA interference was 70.02%, 78.01% and 95.81% in experiment group776, experiment group1234 and negative control group. And there were significant differences between experiment group776 and negative control group(χ2= 235.3, P＜0.010), and between experiment group1234 and negative control group (χ2= 117.5, P＜0.005). NF-κB distribution was increased in cell cytoplasm in the interference group than that in control group.Conclusion P75NTR may promote the proliferation or inhibit the apoptosis of tongue squamous cell carcinoma, and the molecular mechanism may be correlated with hindering the transportion of NFκB into cell nuclear.

Key words: Apoptosis, Nerver growth factor, NF-Kappa B

张兆弢, 张风河, 佟冬冬, 李青, 王进兵, 张新莲. P75NTR基因在舌鳞状细胞癌细胞凋亡中的作用及其机制[J]. 国际肿瘤学杂志, 2016, 43(7): 481-485.

Zhang-Zhao-Tao, ZHANG Feng-He, TONG Dong-Dong, LI Qing, WANG Jin-Bing, ZHANG Xin-Lian. Molecular mechanism of P75NTR geneinduced apoptosis in tongue squamous cell carcinoma cell[J]. Journal of International Oncology, 2016, 43(7): 481-485.

参考文献

1］ Johnston AL, Lun X, Rahn JJ, et al. The p75 neurotrophin receptor is a central regulator of glioma invasion［J］. PLoS Biol, 2007, 5(8): e212. DOI: 10.1371/journal.pbio.0050212. ［2］吴修胤, 佟冬冬, 张风河. P75神经营养因子受体在口腔鳞状细胞癌中的表达及意义［J］. 上海口腔医学, 2011, 20(4): 405408. ［3］佟冬冬, 张风河, 姚瑶, 等. p75神经营养蛋白受体阳性舌鳞状细胞癌细胞的生物学特性研究［J］. 华西口腔医学杂志, 2014, 32(1): 1822. ［4］ Boskovic Z, Alfonsi F, Rumballe BA, et al. The role of p75NTR in cholinergic basal forebrain structure and function［J］. J Neurosci, 2014, 34(39): 1303313038. DOI: 10.1523/JNEUROSCI.236414.2014. ［5］ Beutel G, Meyer J, Ma L, et al. Expression of the p75 neurotrophin receptor in acute leukaemia［J］. Br J Haematol, 2005, 131(1): 6770. DOI: 10.1111/j.13652141.2005.05717.x. ［6］ Micera A, Puxeddu I, Balzamino BO, et al. Chronic nerve growth factor exposure increases apoptosis in a model of in vitro induced conjunctival myofibroblasts［J］. PLoS One, 2012, 7(10): e47316. DOI: 10.1371/journal.pone.0047316. ［7］ Tomellini E, Lagadec C, Polakowska R, et al. Role of p75 neurotrophin receptor in stem cell biology: more than just a marker［J］. Cell Mol Life Sci, 2014, 71(13): 24672481. DOI: 10.1007/s0001801415649. ［8］ Dai JW, Yuan H, Shen SY, et al. p75 neurotrophin receptor positive dental pulp stem cells: new hope for patients with neurodegenerative disease and neural injury［J］. Shanghai Kou Qiang Yi Xue, 2013, 22(4): 469472. ［9］ Okumura T, Tsunoda S, Mori Y, et al. The biological role of the lowaffinity p75 neurotrophin receptor in esophageal squamous cell carcinoma［J］. Clin Cancer Res, 2006, 12(17): 50965103. DOI: 10.1158/10780432.CCR052852. ［10］ Foehr ED, Lin X, O′mahony A, et al. NFkappa B signaling promotes both cell survival and neurite process formation in nerve growth factorstimulated PC12 cells［J］. J Neurosci, 2000, 20(20): 75567563. ［11］ Fritz MD, Mirnics ZK, Nylander KD, et al. p75NTR enhances PC12 cell tumor growth by a nonreceptor mechanism involving downregulation of cyclin D2［J］. Exp Cell Res, 2006, 312(17): 32873297. DOI: 10.1016/j.yexcr.2006.06.029. ［12］ Matsuno T, Kariya R, Yano S, et al. Diethyldithiocarbamate induces apoptosis in HHV8infected primary effusion lymphoma cells via inhibition of the NFκB pathway［J］. Int J Oncol, 2012, 40(4): 10711078. DOI: 10.3892/ijo.2011.1313. ［13］ Tanaka K, Babic I, Nathanson D, et al. Oncogenic EGFR signaling activates an mTORC2NFκB pathway that promotes chemotherapy resistance［J］. Cancer Discov, 2011, 1(6): 524538. DOI: 10.1158/21598290.CD110124. ［14］ Shen RR, Zhou AY, Kim E, et al. TRAF2 is an NFκBactivating oncogene in epithelial cancers［J］. Oncogene, 2015, 34(2): 209216. DOI: 10.1038/onc.2013.543. ［15］ Madge LA, May MJ. The NFκB paradox: RelB induces and inhibits gene expression［J］. Cell Cycle, 2011, 10(1): 67.

[1]	刘娜, 寇介丽, 杨枫, 刘桃桃, 李丹萍, 韩君蕊, 杨立洲. 血清miR-106b-5p、miR-760联合低剂量螺旋CT诊断早期肺癌的临床价值[J]. 国际肿瘤学杂志, 2024, 51(6): 321-325.
[2]	袁健, 黄燕华. Hp-IgG抗体联合血清DKK1、sB7-H3对早期胃癌的诊断价值[J]. 国际肿瘤学杂志, 2024, 51(6): 338-343.
[3]	张宁宁, 杨哲, 檀丽梅, 李振宁, 王迪, 魏永志. 宫颈细胞DNA倍体分析联合B7-H4和PKCδ对宫颈癌的诊断价值[J]. 国际肿瘤学杂志, 2024, 51(5): 286-291.
[4]	任露, 谢晓丽, 张坤, 王丽娟. 双氢青蒿素联合卡非佐米对多发性骨髓瘤细胞活性、增殖、凋亡的影响及机制研究[J]. 国际肿瘤学杂志, 2024, 51(3): 129-136.
[5]	李丹, 李睿尧, 李膺函, 于秀艳, 吴雪峰. 血清miR-19b、miR-744-5p水平在非小细胞肺癌诊断中的临床价值[J]. 国际肿瘤学杂志, 2024, 51(2): 83-88.
[6]	张科平, 赵永生, 杨娟, 付茂勇. 绿原酸通过抑制PI3K-Akt信号通路诱导肺癌A549细胞线粒体功能障碍[J]. 国际肿瘤学杂志, 2024, 51(1): 21-28.
[7]	向玉玲, 谭佳杰, 熊远果, 赵丽蓉, 黎晨, 张洪. 脱水淫羊藿素对肝癌细胞增殖、迁移和凋亡的影响[J]. 国际肿瘤学杂志, 2023, 50(9): 513-519.
[8]	冯诚天, 黄芙蓉, 曹世玉, 王健宇, 南丁阿比雅思, 姜永冬, 朱娟英. HER2阳性乳腺癌患者HER2表达水平与影像学特征的关系[J]. 国际肿瘤学杂志, 2023, 50(9): 527-531.
[9]	叶彤彤, 吴泽宇, 席文一, 王芝微, 江晓春, 赵晨辉. ABRACL在恶性肿瘤发生发展中的作用[J]. 国际肿瘤学杂志, 2023, 50(9): 544-547.
[10]	冯东旭, 吴炜, 高平发, 王军, 施丽娟, 陈大伟, 李文兵, 张美峰. miR-451通过调控Rho/ROCK1信号通路对乳腺癌细胞糖酵解及凋亡的影响[J]. 国际肿瘤学杂志, 2023, 50(8): 449-456.
[11]	刘德宝, 孙子雯, 鲁守堂, 徐海东. ASB6在结直肠癌组织中的表达及临床意义[J]. 国际肿瘤学杂志, 2023, 50(8): 470-474.
[12]	吴佳丽, 张佳慧, 张萍, 肖昕悦, 李睿, 张红宇. Bcl-2 BH4选择性抑制剂BDA-366抑制NK/T细胞淋巴瘤细胞的机制研究[J]. 国际肿瘤学杂志, 2023, 50(7): 413-418.
[13]	杨娅, 王慧礼, 刘艳, 郭金凤, 王春霞, 吕敏, 山长平. GCSH基因在胃癌SNU-1细胞增殖和凋亡中的作用研究[J]. 国际肿瘤学杂志, 2023, 50(5): 257-262.
[14]	朱军, 黄美金, 李媛, 刘泽刚, 荀欣, 陈宏. HER2低表达乳腺癌的靶向治疗研究进展[J]. 国际肿瘤学杂志, 2023, 50(4): 236-240.
[15]	刘玉杰, 赵志强, 王子琤. 早期结直肠癌患者外周血单个核细胞中TOP2A、ERBB2的水平及其诊断价值[J]. 国际肿瘤学杂志, 2023, 50(12): 717-722.