三阴性乳腺癌IL-17基因Real-time RT-qPCR检测法的建立与应用
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摘要
目的:建立Real-time RT-qPCR(实时荧光定量RT-PCR)检测IL-17的方法,应用该方法检测三阴性乳腺癌(three negative breast cancer,TNBC)组织IL-17 mRNA的水平。方法:以TNBC组织为实验组,纤维腺瘤旁正常乳腺组织为对照组,并经HE染色病理分析确诊,Trizol法提取总RNA并反转录为c DNA。选β-actin作为内参,建立SYBR GreenⅠReal-time RT-qPCR检测法。利用该方法检测两组IL-17和β-actin的初始模板量,IL-17/β-actin计算IL-17 mRNA的相对表达量。结果:IL-17扩增效率为98.6%,相关系数0.997,溶解曲线为特异单峰,变异系数小于2.0%,IL-17 mRNA在TNBC组[(0.64±0.12)×10~(-2)]的相对表达高于正常对照组[(0.43±0.07)×10~(-2)],差异有统计学意义(P=0.025)。结论:成功建立了人源IL-17的Real-time RT-qPCR检测方法,TNBC组IL-17的高表达提示其可能与TNBC有一定关系,为研究TNBC的发病机制奠定了理论基础。
Objective: To establish a Real-time quantitative RT-qPCR detection method of IL-17 and detect the IL-17 mRNA level of human three negative breast cancer( TNBC). Methods: TNBC was the experimental group while the fibroadenoma adjacent normal breast was control group. Both groups were stained by HE in order to confirme. Total RNA was extracted by Trizol and reverse transcribed into c DNA. β-actin was chose as an internal control. Establish SYBR Green I Real-time RT qPCR method and detect the initial template amount of two groups of IL-17. The relative expression of mRNA IL-17 was calculated by IL-17/β-actin. Results: IL-17 amplification efficiency was 98. 6%,correlation coefficient was 0. 997. Melting curve was specific unimodal. Coefficient of variation was less than 2. 0%. IL-17 mRNA expressed in each group as follow: TNBC group [( 0. 64 ± 0. 12) × 10~(-2)] was higher than normal control group [( 0. 43 ± 0. 07) × 10~(-2)]. The difference was statistically significant( P = 0. 025).Conclusion: The RT-qPCR Real-time detection method was successfully established. The high expression of IL-17 in the TNBC group suggested that it may be related to the TNBC,which laid a theoretical foundation for the study of the pathogenesis of TNBC.
Objective: To establish a Real-time quantitative RT-qPCR detection method of IL-17 and detect the IL-17 mRNA level of human three negative breast cancer( TNBC). Methods: TNBC was the experimental group while the fibroadenoma adjacent normal breast was control group. Both groups were stained by HE in order to confirme. Total RNA was extracted by Trizol and reverse transcribed into c DNA. β-actin was chose as an internal control. Establish SYBR Green I Real-time RT qPCR method and detect the initial template amount of two groups of IL-17. The relative expression of mRNA IL-17 was calculated by IL-17/β-actin. Results: IL-17 amplification efficiency was 98. 6%,correlation coefficient was 0. 997. Melting curve was specific unimodal. Coefficient of variation was less than 2. 0%. IL-17 mRNA expressed in each group as follow: TNBC group [( 0. 64 ± 0. 12) × 10~(-2)] was higher than normal control group [( 0. 43 ± 0. 07) × 10~(-2)]. The difference was statistically significant( P = 0. 025).Conclusion: The RT-qPCR Real-time detection method was successfully established. The high expression of IL-17 in the TNBC group suggested that it may be related to the TNBC,which laid a theoretical foundation for the study of the pathogenesis of TNBC.
引文
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[2]Zhao Yan,Sun Yali,Nie Xin.The characteristics of gene,pathology and imaging manifestations in triple-negative breast cancer[J].Modern Oncology,2016,24(16):2656-2659.[赵妍,孙雅丽,聂鑫.三阴性乳腺癌的基因、蛋白表达特点及病理、影像学表现[J].现代肿瘤医学,2016,24(16):2656-2659.]
[3]Cui Wenfeng,Zhang Xuebin.Expression and significance of IL-17in breast cancer[J].Chin J Curr Adv Gen Surg,2015,7(7):575-577.[崔文峰,张学斌.IL-17在乳腺癌组织中的表达及其意义[J].中国现代普通外科进展,2015,7(7):575-577.]
[4]Welte T,Zhang XH.Interleukin-17 could promote breast cancer progression at several stages of the disease[J].Mediators Inflamm,2015,2015(1):1-6.
[5]Chen Guanghui,Chen Jian'an,Chen Hui,et al.Expression and significance of IL-17 in breast cancer[J].International J Laboratory Medicine,2014,35(20):2732-2733.[陈光辉,陈建安,陈慧,等.IL-17在乳腺癌中的表达及意义[J].国际检验医学杂志,2014,35(20):2732-2733.]
[6]Linghu Ruixia,Si Wen,Li Ying,et al.Epidemiological and clinicopathological characteristics of patients with breast cancer:A retrospective analysis of 3684 cases[J].Acad J Chin PLA Med Sch Oct,2015,36(10):1017-1021.[令狐锐霞,司文,李莹,等.3684例乳腺癌流行病学及临床病理学分析[J].解放军医学院学报,2015,36(10):1017-1021.]
[7]Zheng Ying,Wu Chunxiao,Zhang Minlu.The epidemic and characteristics of female breast cancer in China[J].China Oncology,2013,23(8):561-569.[郑莹,吴春晓,张敏璐.乳腺癌在中国的流行状况及疾病特征[J].中国癌症杂志,2013,23(8):561-569.]
[8]Lehmann BD,Pietenpol JA.Identification and use of biomarkers in treatment strategies for triple-negative breast cancer subtypes[J].J Pathol,2014,232(2):142-150.
[9]Tajadini M,Panjehpour M,Javanmard SH.Comparison of SYBR Green and Taq Man methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes[J].Adv Biomed Res,2014,3:85.
[10]Zhou Jieying,Lin Yingbiao,Cao Youde,et al.Establishment and application of Real-time quantitative PCR for human Metapneumovirus detection[J].Chinese J Experimental and Clinical Virology,2016,30(2):216-219.[周杰英,林应标,曹友德,等.偏肺病毒逆转录实时荧光定量PCR的建立及应用[J].中华实验和临床病毒学杂志,2016,30(2):216-219.]
[11]Wu Jiang,Li Tao,Zhao Jinming,et al.Establishment of real-time quantitative RT-PCR assays for detecting PD-L1 of liver transplantation in rat[J].China J Modern Medicine,2012,22(2):1-5.[巫姜,李涛,赵晋明,等.PD-L1基因实时荧光定量RTPCR法的建立及在大鼠肝移植中的应用[J].中国现代医学杂志,2012,22(2):1-5.]
[12]M Lucrecia Alvarez,Stefania Cotta Doné.SYBRGreen and TaqManQuantitative PCR Arrays:Expression profile of genes relevant to a pathway or a disease state[J].Methods Mol Biol,2014,1182:321-359.
[13]Sun Changrui,Deng Jun,Feng Lin,et al.Comparison of three different molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer[J].Chin J Lab Med,2015,38(10):666-671.[孙昌瑞,邓君,冯林,等.乳腺癌患者外周血循环肿瘤细胞分子学检测方法的对比研究[J].中华检验医学杂志,2015,38(10):666-671.]
[14]Qian Mingming,Xu Haiyan,Zong Qing,et al.Establishment of conventional RT-PCR and real time RT-PCR to detect norovirus and its application[J].Chinese J Zoonoses,2015,31(7):602-611.[钱明明,许海燕,宗睛,等.常规RT-PCR与荧光定量RT-PCR检测GI、GⅡ型诺如病毒方法的建立及其应用[J].中国人兽共患病学报,2015,31(7):602-611.]