SHCBP1高表达促进鼻咽癌细胞上皮间质转化过程的分析

High expression of SHCBP1 promotes epithelial-mesenchymal transition in nasopharyngeal carcinoma cells

摘要:

目的 探究SHC SH2结构域结合蛋白1(SHCBP1)在鼻咽癌组织及鼻咽癌细胞系中的表达情况，分析其对鼻咽癌细胞系的增殖、侵袭能力及上皮间质转化(EMT)过程的影响。方法 利用TCGA数据库分析SHCBP1在头颈鳞状细胞癌(HNSC)中的表达情况，分析SHCBP1表达程度与肿瘤免疫细胞浸润程度的关系。同时收集2019年9月—2021年9月于贵州医科大学附属医院及贵州医科大学附属肿瘤医院经病理确诊的31例鼻咽癌初诊患者临床组织样本，所有患者就诊前均未接受任何放化疗，并收集同期就诊的31例疑似鼻咽癌但病检示慢性鼻咽炎组织作为对照组。利用逆转录实时荧光定量PCR(RT-qPCR)检测临床样本及鼻咽癌细胞系5-8F中SHCBP1的表达情况。细胞实验以鼻咽癌细胞系5-8F、正常鼻咽部永生化上皮细胞系NP69作为研究对象。采用利用慢病毒载体shRNA-SHCBP1干扰技术沉默5-8F细胞中SHCBP1表达，并分为NC组(空载慢病毒LV-Zs-Green-PURO-NC感染5-8F)、SHCBP1-shRNA组(LV-SHCBP1-shRNA-Zs-Green-PURO沉默5-8F细胞SHCBP1表达)。CCK-8法、克隆形成实验检测SHCBP1对鼻咽癌细胞增殖的影响；划痕实验、Transwell迁移、侵袭实验检测SHCBP1对鼻咽癌细胞的转移、侵袭能力的影响。Western blot检测NC组、SHCBP1-shRNA组中E-cadherin、N-cadherin、基质金属蛋白9(MMP9)、Vimentin的表达情况。结果 在HNSC中SHCBP1高表达，表达量与Th2细胞浸润程度相关。与慢性鼻咽炎组织相比，鼻咽癌组织中SHCBP1明显高表达，差异具有统计学意义(P<0.05)。与NC组相比，SHCBP1-shRNA组鼻咽癌细胞增殖、迁移、侵袭能力均减弱，差异具有统计学意义(P<0.05)。SHCBP1-shRNA组E-cadherin表达较NC组升高，N-cadherin、MMP9、Vimentin表达较NC组降低，差异具有统计学意义(P<0.05)。结论 SHCBP1在鼻咽癌中高表达，并可促进鼻咽癌细胞增殖、侵袭、迁移及激活EMT过程。

关键词:鼻咽癌;转移;侵袭;上皮间质转化

Abstract:

Objective To investigate the expression of Shc SH2-domain binding protein 1 (SHCBP1) gene in nasopharyngeal carcinoma (NPC) tissues and NPC cell lines, and its effect on the proliferation, invasion ability and epithelial-mesenchymal transition (EMT) process of NPC cell lines.Methods The expression of SHCBP1 in head and neck squamous cell carcinoma (HNSC) was analyzed by TCGA database, and the relationship between the expression of SHCBP1 and the degree of tumor immune cell infiltration was analyzed. The clinical tissue samples of 31 patients with NPC diagnosed by pathology in the Affiliated Hospital of Guizhou Medical University and the Affiliated Cancer Hospital of Guizhou Medical University from Sept 2019 to Sept 2021 were collected. At the same time, 31 cases of chronic nasopharyngitis were selected as the control group. The expression of SHCBP1 in clinical samples and NPC cell line 5-8F was detected by reverse transcriptase real-time fluorescence quantitative PCR (RT-qPCR). In the cell experiment, NPC cell line 5-8F and normal nasopharyngeal immortalized epithelial cell line NP69 were used as the subjects. SHCBP1 expression in 5-8F cells was silenced by lentiviral vector shRNA-SHCBP1 interference technique and divided into normal control (NC) group (empty viral vector infected with 5-8F) and SHCBP1-shRNA group (silencing SHCBP1 expression in 5-8F cells). The effect of SHCBP1 on the proliferation of NPC cells was detected by CCK-8 assay and clone formation assay, and the effect of SHCBP1 on the metastasis and invasion of NPC cells was detected by scratch test, Transwell migration and invasion test. Western blot was used to detect the expression of E-cadherin, N-cadherin, MMP9 and Vimentin in the NC group and SHCBP1-shRNA group.Results SHCBP1 was highly expressed in HNSC, and the expression was related to the degree of Th2 cell infiltration. Compared with the chronic nasopharyngitis, the expression of SHCBP1 in NPC was significantly elevated (P<0.05). The proliferation, migration and invasion of NPC cells in the SHCBP1-shRNA group were significantly lower than those in the NC group (P<0.05). The expression of E-cadherin in SHCBP1-shRNA group was higher than that in the NC group, while the expressions of N-cadherin, MMP9 and Vimentin were lower than those in the NC group.Conclusion SHCBP1 is highly expressed in NPC and can promote the proliferation, invasion, migration and activation of EMT in NPC cells.

Key words:Nasopharyngeal carcinoma;Metastasis;Invasion;Epithelial-mesenchymal transition