中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (2): 135-141.doi: 10.35541/cjd.20210416

• 论著 • 上一篇    下一篇

表皮生长因子受体shRNA联合西罗莫司对Colo-16细胞体外增殖、凋亡的研究

王慧1    刘董2    田芳3    魏志平4   

  1. 1青岛市城阳区人民医院皮肤科，青岛  266109；2青岛市城阳区人民医院药剂科，青岛  266109；3青岛市城阳区人民医院门诊部，青岛  266109；4徐州医科大学附属医院皮肤科，徐州  221002
  • 收稿日期:2021-05-28 修回日期:2021-12-08 发布日期:2022-01-27
  • 通讯作者: 魏志平 E-mail:xzwzp1961@aliyun.com

In vitro effect of a short hairpin RNA targeting epidermal growth factor receptor combined with sirolimus on proliferation and apoptosis of the human cutaneous squamous cell carcinoma cell line Colo-16

Wang Hui1, Liu Dong2, Tian Fang3, Wei Zhiping4   

  1. 1Department of Dermatology, Qingdao Chengyang People′s Hospital, Qingdao 266109, Shandong, China; 2Department of Pharmacy, Qingdao Chengyang People′s Hospital, Qingdao 266109, Shandong, China; 3Outpatient Department, Qingdao Chengyang People′s Hospital, Qingdao 266109, Shandong, China; 4Department of Dermatology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou 221002, Jiangsu, China
  • Received:2021-05-28 Revised:2021-12-08 Published:2022-01-27
  • Contact: Wei Zhiping E-mail:xzwzp1961@aliyun.com

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摘要： 【摘要】 目的 探讨表皮生长因子受体（EGFR）shRNA联合西罗莫司对人皮肤鳞状细胞癌Colo-16细胞增殖、凋亡的影响及机制。方法 Colo-16细胞分为5组：正常细胞组（常规培养 + 磷酸盐缓冲液）、阴性对照组（转染shRNA-NC质粒 + 磷酸盐缓冲液）、西罗莫司组（常规培养 + 西罗莫司）、EGFR shRNA组（转染EGFR shRNA质粒）、联合组（转染EGFR shRNA质粒＋西罗莫司）。采用MTT法检测各组24 ~ 96 h各时间点细胞增殖活性，流式细胞仪分析处理48 h后各组细胞凋亡情况。RT-PCR检测Bcl-2、Bax mRNA的表达，Western印迹法检测凋亡相关蛋白cleaved caspase3、cleaved caspase9、Bcl-2、Bax、细胞增殖相关蛋白p-mTOR、p-AKT、p-P70S6K及细胞周期蛋白D1（cyclin D1）的表达。多组间比较采用单因素方差分析，组间两两多重比较采用SNK-q检验。结果 MTT检测显示，24 ~ 96 h西罗莫司组、EGFR shRNA组及联合组Colo-16细胞增殖活性均显著低于正常细胞组（均P < 0.05），且联合组细胞增殖活性显著低于西罗莫司组和EGFR shRNA组（均P < 0.001），正常细胞组与阴性对照组各时间点细胞增殖活性差异均无统计学意义（均P > 0.05）。流式细胞实验结果显示，西罗莫司组、EGFR shRNA组和联合组细胞凋亡率分别为9.52% ± 0.25%、12.65% ± 0.23%、19.81% ± 0.31%，均显著高于正常细胞组（3.33% ± 0.18%，q值分别为60.07、78.08、122.81，均P < 0.001）和阴性对照组（3.42% ± 0.19%，q值分别为59.90、77.91、122.64，均P < 0.001），且联合组凋亡率最高。RT-PCR和Western印迹法显示，与正常细胞组相比，西罗莫司组、EGFR shRNA组和联合组Bcl-2 mRNA及cyclin D1、p-AKT、p-mTOR、p-P70S6K、Bcl-2蛋白表达均显著降低（均P < 0.05），Bax mRNA及cleaved caspase3、cleaved caspase9、Bax蛋白表达均显著增加（均P < 0.05），其中联合组Bcl-2 mRNA及cyclin D1、p-AKT、p-mTOR、p-P70S6K、Bcl-2蛋白表达显著低于西罗莫司组及EGFR shRNA组（均P < 0.05），而Bax mRNA及cleaved caspase3、cleaved caspase9、Bax蛋白表达均显著高于西罗莫司组及EGFR shRNA组（均P < 0.01）。结论 EGFR shRNA联合西罗莫司在抑制Colo-16细胞增殖、促进其凋亡方面有协同增效的作用，其机制可能与抑制PI3K/AKT/mTOR通路有关。

关键词: 癌, 鳞状细胞, 受体, 表皮生长因子, RNA干扰, 西罗莫司, 细胞增殖, 细胞凋亡

Abstract: 【Abstract】 Objective To investigate the effect of a short hairpin RNA （shRNA） targeting epidermal growth factor receptor （EGFR） combined with sirolimus on proliferation and apoptosis of the human cutaneous squamous cell carcinoma cell line Colo-16, and to explore underlying mechanisms. Methods Cultured Colo-16 cells were divided into 5 groups: normal cell group receiving conventional culture and treatment with phosphate-buffered saline （PBS）, negative control group transfected with a shRNA-NC-expressing plasmid and treated with PBS, sirolimus group receiving conventional culture and sirolimus treatment, EGFR shRNA group transfected with an EGFR shRNA-expressing plasmid and treated with PBS, and combined group transfected with an EGFR shRNA-expressing plasmid and treated with sirolimus. Methyl thiazol tetrazolium （MTT） assay was performed to evaluate cellular proliferative activity in the above groups from 24 to 96 hours, and flow cytometry to detect cell apoptosis after 48-hour treatment. Semiquantitative RT-PCR was conducted to determine the mRNA expression of Bcl-2 and Bax, and Western blot analysis to determine the expression of apoptosis-related proteins cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, cell proliferation-related proteins phosphorylated mammalian target of rapamycin （p-mTOR）, phosphorylated protein kinase B （p-AKT）, phosphorylated 70-kDa ribosomal protein S6 kinase （p-P70S6k）, and cyclin D1. Comparisons among groups were carried out by using one-way analysis of variance, and multiple comparisons between 2 groups by using Student-Newman-Keuls q test. Results MTT assay showed that the proliferative activity of Colo-16 cells was significantly lower in the sirolimus group, EGFR shRNA group and combined group during 24 - 96 hours than in the normal cell group （all P < 0.05）, and higher in the combined group than in the sirolimus group and EGFR shRNA group at 24 - 96 hours （all P < 0.001）, and there was no significant difference in the cellular proliferative activity at any time points between the normal cell group and negative control group （all P > 0.05）. Flow cytometry showed that the apoptosis rate was significantly higher in the sirolimus group, EGFR shRNA group and combined group （9.52% ± 0.25%, 12.65% ± 0.23%, 19.81% ± 0.31%, respectively） than in the normal cell group （3.33% ± 0.18%, q = 60.07, 78.08, 122.81, respectively, all P < 0.001） and negative control group （3.42% ± 0.19%, q = 59.90, 77.91, 122.64, respectively, all P < 0.001）, and was highest in the combined group. As RT-PCR and Western blot analysis revealed, the sirolimus group, EGFR shRNA group and combined group showed significantly decreased mRNA expression of Bcl-2 and protein expression of cyclin D1, p-AKT, p-mTOR, p-P70S6K and Bcl-2, but significantly increased mRNA expression of Bax and protein expression of cleaved caspase-3, cleaved caspase-9 and Bax compared with the normal cell group（all P < 0.05）. Compared with the sirolimus group and EGFR shRNA group, the combined group showed significantly decreased mRNA expression of Bcl-2 and protein expression of cyclin D1, p-AKT, p-mTOR, p-P70S6K and Bcl-2 （all P < 0.05）, but significantly increased mRNA expression of Bax and protein expression of cleaved caspase-3, cleaved caspase-9 and Bax （all P < 0.01）. Conclusion EGFR shRNA and sirolimus exerted a synergistic effect in inhibiting the proliferation and promoting the apoptosis of Colo-16 cells, which may be related to the inhibition of the phosphoinositide 3-kinase （PI3K）/AKT/mTOR pathway.

Key words: Carcinoma, squamous cell, Receptor, epidermal growth factor, RNA interference, Sirolimus, Cell proliferation, Apoptosis

中图分类号: 

  • R739.5