时钟基因CLOCK在鼻咽癌中的表达及其临床意义

The expression of clock gene CLOCK and its clinical significance in nasopharyngeal carcinoma

目的探讨CLOCK mRNA及蛋白表达水平与鼻咽癌患者临床特征的关系。方法收集2018—2019年贵州医科大学附属肿瘤医院33例鼻咽癌患者的冰冻组织标本,收集2019年贵州医科大学附属医院17例鼻咽部慢性炎症组织标本,收集2008—2014年贵州医科大学附属肿瘤医院鼻咽癌组织蜡块68例及鼻咽慢性炎症组织蜡块37例。采用实时荧光定量PCR方法和Western blot法检测CLOCK mRNA和蛋白的表达情况。培养鼻咽癌细胞CNE1、CNE2、5-8F与正常鼻咽上皮细胞NP69,采用实时荧光定量PCR法检测各细胞中CLOCK mRNA在ZT2、ZT6、ZT10、ZT14、ZT18、ZT22时间点的表达水平,采用余弦法拟合CLOCK mRNA在鼻咽癌中表达的节律性。采用免疫组化法检测68例鼻咽癌和37例鼻咽慢性炎症组织蜡块中CLOCK蛋白的表达。生存分析采用Kaplan-Meier法和Log rank检验,影响因素分析采用Cox回归模型。结果CNE1、CNE2和5-8F细胞中CLOCK mRNA表达水平(分别为0.63±0.07、0.91±0.02和0.33±0.04)均低于NP69细胞(1.00±0.00,均P<0.05)。CNE1、CNE2和5-8F细胞中CLOCK蛋白表达水平(分别为0.79±0.06、0.57±0.05和0.74±0.10)均低于NP69细胞(1.00±0.00,均P<0.05)。鼻咽癌细胞CEN1、CNE2、5-8F和正常鼻咽上皮细胞NP69中CLOCK mRNA在不同时间点表达不同,存在时相性波动。CLOCK mRNA在CNE1、CNE2、5-8F、NP69细胞中的波动周期分别为16、14、22和24 h,其波峰和波谷时间分别为ZT10:40和ZT18:40、ZT10和ZT3、ZT14:30和ZT3:30、ZT12:39和ZT0:39。鼻咽癌组织中CLOCK mRNA和蛋白表达水平(分别为0.37±0.20和0.20±0.26)均低于鼻咽慢性炎症组织(分别为1.00±0.00和0.51±0.41,均P<0.05)。CLOCK蛋白高表达组(CLOCK蛋白表达水平≥0.178)患者1、3、5年生存率分别为96.2%、92.1%和80.1%,高于低表达组(CLOCK蛋白表达水平<0.178,分别为92.9%、78.6%和57.1%,P=0.009)。CLOCK蛋白高表达组患者1、3、5年无进展生存率分别为96.2%、87.8%和87.7%,高于低表达组(分别为92.7%、82.2%和70.8%,P=0.105)。CLOCK蛋白高表达组患者1、3、5年无复发生存率(分别为100.0%、95.7%和95.7%)与低表达组(分别为100.0%、96.9%和90.0%)比较,差异无统计学意义(P=0.514)。CLOCK蛋白高表达组1、3、5年无远转转移生存率(分别为96.2%、92.0%和92.0%)与低表达组(分别为92.7%、82.2%和79.3%)比较,差异无统计学意义(P=0.136)。CLOCK蛋白表达和T分期为总生存的独立影响因素(均P<0.05)。结论鼻咽癌细胞及鼻咽癌组织中的CLCOK基因均为低表达,时钟基因CLOCK在鼻咽癌细胞与正常鼻咽上皮细胞中均呈节律性表达;相比于正常鼻咽上皮细胞,鼻咽癌细胞的波动周期均缩短,CLOCK蛋白高表达组的总生存情况优于低表达患者,CLOCK蛋白的表达是影响总生存的独立影响因素,CLOCK基因在鼻咽癌中可能是一个潜在的抑癌基因。

Objective To explore the relationship between expression levels of CLOCK mRNA and protein and the clinical characteristics of patients with nasopharyngeal carcinoma.Methods The frozen tissue specimens from 33 patients with nasopharyngeal carcinoma in the Affiliated Tumor Hospital of Guizhou Medical University from 2018 to 2019 were collected.Seventeen cases of tissue specimens from patients with nasopharyngeal chronic inflammation in the Affiliated Hospital of Guizhou Medical University in 2019 were collected.From 2008 to 2014,68 cases of formalin-fixed paraffin-embedding(FFPE)nasopharyngeal carcinoma tissue and 37 cases of FFPE nasopharyngeal chronic inflammation tissue were collected from the Affiliated Tumor Hospital of Guizhou Medical University.Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and western blot(WB)were used to detect the mRNA and protein expression levels of CLOCK.The nasopharyngeal carcinoma cells including CNE1,CNE2,5-8F and the normal nasopharyngeal epithelial cell NP69 were cultured.qRT-PCR was used to detect the expression level of CLOCK mRNA in each cell line at the time points of ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22.The cosine method was used to fit the rhythm of CLOCK gene in nasopharyngeal carcinoma.The protein expression of CLOCK protein was detected by using immunohistochemical method in 68 cases of nasopharyngeal carcinoma and 37 cases of nasopharyngeal chronic inflammation tissue.Survival was analyzed by Kaplan-Meier method and Log rank test,and the influencing factors was analyzed by Cox regression model.Results The expression levels of CLOCK mRNA in CNE1,CNE2 and 5-8F cells(0.63±0.07,0.91±0.02 and 0.33±0.04,respectively)were lower than that in NP69 cell(1.00±0.00,P<0.05).The expression levels of CLOCK protein in CNE1,CNE2 and 5-8F cells(0.79±0.06,0.57±0.05 and 0.74±0.10,respectively)were lower than that of NP69 cells(1.00±0.00,P<0.05).The expressions of CLOCK mRNA in nasopharyngeal carcinoma cells including CEN1,CNE2,5-8F and normal nasopharyngeal epithelial cell NP69 were different at different time points,with temporal fluctuations.The fluctuation periods of CLOCK mRNA in CNE1,CNE2,5-8F,and NP69 cells were 16,14,22 and 24 hours,respectively.The peak and trough times were ZT10:40 and ZT18:40,ZT10 and ZT3,ZT14:30 and ZT3:30,ZT12:39 and ZT0:39,respectively.CLOCK mRNA and protein expression levels in nasopharyngeal carcinoma tissues(0.37±0.20 and 0.20±0.26,respectively)were lower than those in nasopharyngeal chronic inflammation tissues(1.00±0.00 and 0.51±0.41,respectively,P<0.05).The 1,3,and 5-year survival rates of patients in the CLOCK protein high expression group(CLOCK protein expression level≥0.178)were 96.2%,92.1%,and 80.1%,respectively,which were higher than those in the low expression group(CLOCK protein expression level<0.178,92.9%,78.6%and 57.1%,respectively,P=0.009).The 1,3,and 5-year progression-free survival(PFS)rates of patients in the CLOCK protein high expression group were 96.2%,87.8%,and 87.7%,respectively,which were higher than those in the low expression group(92.7%,82.2%,and 70.8%,respectively,P=0.105).Compared with the low-expression group(100.0%,96.9%,and 90.0%,respectively),the 1,3,and 5-year recurrence-free survival rates of patients in the CLOCK protein high expression group(100.0%,95.7%,and 95.7%,respectively)were not statistically significant(P=0.514).Compared with the low-expression group(92.7%,82.2%,and 79.3%),the 1,3,and 5-year survival rates without metastasis in the CLOCK protein high expression group(96.2%,92.0%,and 92.0%,respectively)were not statistically significant(P=0.136).CLOCK protein expression and T stage were independent prognostic factors of overall survival(P<0.05).Conclusions The expression of CLCOK is downregulated in the nasopharyngeal carcinoma cell and nasopharyngeal carcinoma tissues.Clock gene CLOCK is rhythmically expressed in the nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells.Compared with normal nasopharyngeal epithelial cells,the fluctuation period of CLOCK in nasopharyngeal carcinoma cells is shortened.The overall survival of patients in the CLOCK protein high expression group is better than that of low expression group.The expression of CLOCK protein is an independent influencing factor for overall survival.CLOCK gene may be a potential tumor suppressor gene in the nasopharyngeal carcinoma.