Shen Li-Jun, Qi Cheng-Lin, Yang Rui, Huang Mao-Ling, Zou You, Jiang Yang, Sheng Jian-Fei, Kong Yong-Gang, Hua Qing-Quan, Chen Shi-Ming
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University 238 Jie-Fang Road, Wuhan 430060, Hubei, P. R. China.
Institute of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University 238 Jie-Fang Road, Wuhan 430060, Hubei, P. R. China.
Am J Transl Res. 2021 Jun 15;13(6):5985-6000. eCollection 2021.
In vitro cell experiments showed that knocking out the placenta-specific protein 8 (PLAC8) gene significantly increased the sensitivity of tumor cells to radiation. This study used two nude mouse models of nasopharyngeal carcinoma (NPC) to investigate the radio-sensitization and molecular mechanism of PLAC8 knockout . The expression of PLAC8 in 120 NPC tissues and 30 nasopharyngitis (NPG) tissues was detected by immunohistochemistry (IHC) to analyze the relationship between PLAC8 and neck lymph node metastasis and prognosis in NPC patients. The mRNA expression level of PLAC8 in several NPC cell lines was detected by semi-quantitative RT-PCR. The PLAC8 gene was knocked out in CNE-2 cells using CRISPR/Cas9. The effect of PLAC8 gene knockout on the radiotherapy sensitivity of NPC cells was analyzed by establishing model 1 and model 2 tumor-bearing nude mouse models with two different irradiation methods. The expression of γH2AX, Bax, Bcl-2, Caspase-3 and cleaved Caspase-3 was detected by immunofluorescence (IF), IHC and western blot analysis. PLAC8 expression was significantly increased in NPC tissue samples and NPC cell lines compared with NPG tissue samples and normal cell lines (P<0.01). PLAC8 upregulation was associated with lymph node metastasis and a poor prognosis in patients with NPC (P<0.01). Both animal models showed that radiotherapy after PLAC8 knockout significantly slowed tumor growth and reduced tumor volume, with tumor inhibition rates of 100% and 66.04%, respectively. In model 2, PLAC8 knockout with radiotherapy increased the expressions of γH2AX, Bax, Caspase-3 and cleaved Caspase-3 but decreased the expression of Bcl-2 (P<0.01). In model 1, there was no tumor formation at the site where the cancer cells were injected. The expression levels of γH2AX, Bax, Caspase-3 and cleaved Caspase-3 in skin tissues taken at the injection site were lower than those in NPC tissues treated with radiotherapy, while the expression level of Bcl-2 was higher (P<0.01). PLAC8 expression is closely related to neck metastasis and the prognosis of NPC. PLAC8 gene knockout significantly increases the radio-sensitivity of NPC cells by promoting apoptosis, which is an effective strategy for the radiotherapy sensitization of NPC.
体外细胞实验表明，敲除胎盘特异性蛋白8（PLAC8）基因可显著提高肿瘤细胞对辐射的敏感性。本研究采用两种鼻咽癌（NPC）裸鼠模型，探讨PLAC8基因敲除的放射增敏作用及分子机制。通过免疫组织化学（IHC）检测120例NPC组织和30例鼻咽炎（NPG）组织中PLAC8的表达，分析PLAC8与NPC患者颈部淋巴结转移及预后的关系。采用半定量逆转录-聚合酶链反应（RT-PCR）检测几种NPC细胞系中PLAC8的mRNA表达水平。利用CRISPR/Cas9技术在CNE-2细胞中敲除PLAC8基因。通过建立两种不同照射方法的模型1和模型b荷瘤裸鼠模型，分析PLAC8基因敲除对NPC细胞放疗敏感性的影响。通过免疫荧光（IF）、IHC和蛋白质免疫印迹分析检测γH2AX、Bax、Bcl-2、Caspase-3和裂解型Caspase-3的表达。与NPG组织样本和正常细胞系相比，NPC组织样本和NPC细胞系中PLAC8表达显著增加（P<0.01）。PLAC8上调与NPC患者的淋巴结转移和不良预后相关（P<0.01）。两种动物模型均显示，PLAC8基因敲除后放疗显著减缓肿瘤生长并减小肿瘤体积，肿瘤抑制率分别为100%和66.04%。在模型2中，PLAC8基因敲除联合放疗可增加γH2AX、Bax、Caspase-3和裂解型Caspase-3的表达，但降低Bcl-2的表达（P<0.01）。在模型1中，注射癌细胞的部位未形成肿瘤。注射部位皮肤组织中γH2AX、Bax、Caspase-3和裂解型Caspase-3的表达水平低于接受放疗的NPC组织，而Bcl-2的表达水平较高（P<0.01）。PLAC8表达与NPC颈部转移及预后密切相关。PLAC基因敲除通过促进细胞凋亡显著提高NPC细胞的放射敏感性，是NPC放疗增敏的有效策略。
