摘要
目的探讨生物钟基因Period2在鼻咽癌中下调ERK/MAPK磷酸化水平的分子机制。方法用慢病毒对细胞进行感染,构建细胞系。采用Label-free蛋白质组学检测方法对PER2蛋白表达进行验证。采用不同浓度的ERK通路激活剂Ceramide C6干预各组细胞,MTT检测各组细胞的增殖能力,筛选最佳药物浓度和作用时间。采用ERK通路激活剂Ceramide C6干预细胞,正常对照组采用等剂量药物溶剂进行处理,分为6组。Western blot检测各组细胞PER2、ERK、p38MAPK、p-ERK、p-p38MAPK蛋白的表达。流式细胞术检测细胞周期,Transwell实验检测各组细胞侵袭能力,进一步明确PER2调控ERK、MAPK磷酸化的机制。用免疫组化检测PER2、p-ERK在人体鼻咽癌样本中的表达,并分析PER2、p-ERK与鼻咽癌临床特点的相关性。结果蛋白质组学检测结果示PER2过表达组的PER2蛋白表达明显高于阴性病毒对照组和空白对照组(P<0.05)。Top 20差异蛋白显示MAPK3在PER2过表达组和对照组差异有统计学意义(P<0.05)。MTT实验结果:不同药物浓度处理后,根据每组细胞抑制率,得出最佳的药物作用浓度10μmol/L,最佳处理时间24 h。WB结果显示PER2过表达下调p-ERK、p-p38MAPK蛋白表达,ERK通路激活剂Ceramide C6干预后,在PER2-OE组中ERK、p38MAPK蛋白水平无明显变化,但p-ERK、p-p38MAPK蛋白表达水平上调,干预前后差异有统计学意义(P<0.01)。细胞周期实验结果显示:ERK通路激活剂Ceramide C6干预各组细胞后,使细胞在G1期的比例明显增加,而在G2期数量减少。Transwell实验结果显示PER2过表达抑制细胞侵袭能力,此种现象不能被磷酸激酶逆转。在鼻咽癌组织、鼻咽部黏膜中PER2蛋白、p-ERK蛋白表达阳性率差异有统计学意义(P<0.05)。PER2蛋白表达与肿瘤的T分期相关(P<0.05)。在鼻咽癌组织中PER2蛋白与p-ERK蛋白表达相关(P<0.05)。结论PER2过表达下调ERK/MAPK磷酸化水平,可能不是直接作用于ERK和p38MAPK的�
Objective To investigate the molecular mechanism by which the circadian clock gene Period2(PER2)downregulates extracellular signal-regulated kinase/mitogen-activated protein kinase(ERK/MAPK)phosphorylation levels in nasopharyngeal carcinoma(NPC).Methods Cells were infected with lentivirus to construct cell lines.The expression of PER2 protein was validated using label-free proteomic analysis.Different concentrations of the ERK pathway activator Ceramide C6 were used to intervene with the cells,and the optimal drug concentration and treatment time were determined based on cell proliferation assessed by MTT assay.Western blotting was performed to detect the expression of PER2,ERK,p38MAPK,p-ERK,and p-p38MAPK proteins in cells after intervention with Ceramide C6,with the normal control group treated with an equal amount of drug solvent.Flow cytometry was used to analyze the cell cycle,and Transwell assay was conducted to evaluate cell invasion ability,further elucidating the mechanism by which PER2 regulates ERK and MAPK phosphorylation.Immunohistochemistry was employed to detect the expression of PER2 and p-ERK proteins in human NPC samples,and the correlation between PER2,p-ERK expression,and clinical characteristics of NPC was analyzed.Results Proteomic analysis showed that the expression of PER2 protein was significantly higher in the PER2 overexpression(OE)group compared to the negative virus control group and blank control group(P<0.05).The top 20 differentially expressed proteins revealed that MAPK3 exhibited significant differences between the PER2 OE group and control group(P<0.05).MTT assay results indicated that the optimal drug concentration was 10μM,with the optimal treatment time being 24 hours.Western blotting results showed that PER2 overexpression downregulated the expression of p-ERK and p-p38MAPK proteins,and intervention with Ceramide C6 led to upregulation of p-ERK and p-p38MAPK protein expression levels in the PER2-OE group,with significant differences before and after intervention(P<0.01).Cell
作者
张志娟
马政
康晶
杨敬
徐倩茹
牛欣冉
罗小丫
王婧媛
李海亮
侯丽
ZHANG Zhi-juan;MA Zheng;KANG Jing;YANG Jing;XU Qian-ru;NIU Xin-ran;LUO Xiao-ya;WANG Jing-yuan;LI Hai-liang;HOU Li(Department of Otolaryngology,General Hospital,Ningxia Medical University,Yinchuan 750004,Ningxia,China)
出处
《广东医学》
CAS
2024年第10期1255-1265,共11页
Guangdong Medical Journal
基金
宁夏自然科学基金项目(2019AAC03210,2019AAC03215)
宁夏医科大学校级课题(XM20211041)
宁夏重点研发项目(2022BEG03105)。
