生物钟基因与MEN2型甲状腺髓样癌相关性分析

Correlation analysis of clock genes and MEN2 medullary thyroid carcinoma

目的探讨生物钟基因CLOCK、BMAL1与多发性内分泌腺瘤病2型(multiple endocrine neoplasia 2,MEN2型)甲状腺髓样癌(medullary thyroid carcinoma,MTC)的相关性。方法回顾性分析2013年1月至2021年9月在青岛大学附属烟台毓璜顶医院病理确诊为MEN2型MTC的病例13例(男性8例,女性5例,年龄15~69岁)及同期随机选取的非MEN2型MTC病例13例(男性7例,女性6例,年龄33~74岁)。分析比较2组患者的临床指标,并利用免疫组织化学、实时荧光定量PCR(quantitative real time PCR,qPCR)等技术检测CLOCK、BMAL1在MEN2型MTC组织与癌旁组织、非MEN2型MTC组织间的表达差异;分析淋巴结转移数量与CLOCK、BMAL1表达相关性;蛋白互作(protein-protein interaction,PPI)网络分析结合qPCR和相关性分析挖掘MEN2常见突变基因RET表达与生物钟基因间的表达调控关系;利用地塞米松节律同步化后脂多糖细胞刺激试验验证MEN2细胞节律紊乱特点。采用独立样本t检验对数据进行分析。结果本研究MEN2型MTC病例呈现典型RET基因突变;临床相关指标分析发现MEN2型MTC血清降钙素水平、肿瘤直径及转移淋巴结数量,均较非MEN2型MTC升高,差异均有统计学意义(t值分别为2.76、2.53、2.26,P值均<0.05)。免疫组织化学结果显示,生物钟基因CLOCK、BMAL1在MEN2型MTC癌组织中高表达,而在癌旁正常甲状腺滤泡内阴性表达,且表达水平高于非MEN2型MTC;qPCR结果显示,在MEN2型MTC癌组织中CLOCK基因表达量高于非MEN2型MTC以及癌旁正常组织的表达量,差异均有统计学意义(t值分别为2.68和2.86,P值均<0.05);BMAL1基因表达量高于非MEN2型MTC以及癌旁正常组织的表达量,差异均有统计学意义(t值分别为2.21和2.35,P值均<0.05)。相关性分析发现MEN2型MTC患者CLOCK、BMAL1基因表达水平与淋巴结转移数量呈正相关(r=0.65,P<0.001;r=0.52,P=0.005)。PPI网络分析发现RET和CLOCK间存在调控关系,依据qPCR结果做进一步的相关性分析,提示CLOCK基因的表达和RET基因的异常表达呈正相关(r=0.96,P<0.001)。地塞米松节律同步化后再用脂多糖刺激离体培养的细胞发现同步化12 h时生物钟基因CLOCK、BMAL1在MEN2型MTC细胞中的表达(0.47±0.22、2.60±1.48)显著低于癌旁组织细胞中的表达(1.70±1.62、8.23±2.52),差异有统计学意义(t=5.04,P=0.007;t=3.34,P=0.029)。结论生物钟基因CLOCK、BMAL1与MEN2型MTC的发生发展存在相关性,可能是MEN2型MTC新治疗策略开发的潜在靶点。

Objective To investigate the correlation between CLOCK and BMAL1 genes and MEN2 medullary thyroid carcinoma(MTC).Methods Thirteen cases with MEN2 MTC and thirteen cases with non-MEN2 MTC were selected who were treated in the Yantai Yuhuangding Hospital between January 2013 and September 2021.Clinical indicators such as blood calcitonin level,tumor diameter and metastatic lymph node of patients were collected.The expression differences of CLOCK and BMAL1 between MEN2 MTC and para-carcinoma tissue as well as between MEN2 MTC and non-MEN2 MTC were detected by immunohistochemistry and qPCR.The correlation between lymph node metastasis and CLOCK or BMAL1 expression was analyzed.Protein-protein interaction(PPI)network analysis combined with qPCR and correlation analysis was used to explore the expression regulation relationship between RET and circadian clock genes.The rhythm disorder of MEN2 cells was verified by lipopolysaccharide cell stimulation experiment after dexamethasone rhythm synchronization.Results MEN2 MTC exhibited typical RET gene mutation.The mean blood calcitonin level,the tumor diameter and the number of metastatic lymph nodes of patients with MEN2 MTC were higher than those of patients with non-MEN2 MTC(t value was 2.76,2.53,2.26,all P<0.05).Immunohistochemical results showed that the expression levels of CLOCK and BMAL1 in MEN2 MTC were higher than those in non-MEN2 MTC,while negatively expressed in para-cancerous thyroid follicle.qPCR displayed that the expression of CLOCK gene in cancer tissues was higher than that in non-MEN2 MTC and para-cancerous tissues(t value was 2.68 and 2.86,all P<0.05);the expression of BMAL1 gene in MEN2 MTC was higher than that in non-MEN2 MTC and para-cancerous tissues(t value was 2.21 and 2.35,all P<0.05).Correlation analysis showed that the expression levels of CLOCK and BMAL1 genes were positively correlated with the number of lymph node metastases in patients with MEN2 MTC(r=0.65,P<0.001;r=0.52,P=0.005).PPI network analysis indicated that the expression of CLOCK gene was positively correlated with the abnormal expression of RET gene(r=0.96,P<0.001).With lipopolysaccharide to stimulate cultured cells in vitro after dexamethasone rhythm synchronization,the expressions of CLOCK and BMAL1 in MEN2 MTC cells(0.47±0.22 and 2.60±1.48)at 12 hours of synchronization were significantly lower than those in para-cancerous tissues(1.70±1.62 and 8.23±2.52),the difference was statistically significant(t=5.04,P=0.007;t=3.34,P=0.029).Conclusion CLOCK and BMAL1 are correlated with the occurrence and development of MEN2 MTC,and may be potential targets for the development of new therapeutic strategies for MEN2 MTC.