HSF2基因在肺癌中的表达意义

HSF2基因在肺癌中的表达意义
钟云华;范敏娟
【摘要】Objective In order to investigate the significance and expressioon of heat shock factor 2 (HSF2)gene in lung cancer. Methods We collected
50lung cancer tissue and adjacent cancer normal tissue
samples.Meanwhile,the expression of HSF2 was inves-tigated by Western blotting and RT -qPCR.ResultsWhen compared the lung cancer tissue and adjacent cancer normal tissue sam-ples,we found that 76% (38 of 50)of the HSF2 protein in cancer tissues were upregulated compared to the normal lung tissues (P <0.05).However,in normal tissue,the rare of mRNA down -regulation was 80%.In normal control,expression of HSF2 gene was lower than lung cancer tissue.In addition,there was no significant difference in age status,gender status,tumor size status,histologi-cal differentiation status and histologic type of lung cancer.Conclusion The result of HSF2 gene upregulation,which indicate HSF2 may participate information and development of lung cancer.%目的：检测肺癌组织中 HSF2基因的表达和意义。

方法：分别采用 Western blot 和 RT －qPCR 技术，检测50例肺癌组织及其对
应癌旁组织中 HSF2基因蛋白质和 mRNA 变化。

结果：50例肺癌患者组织及其
正常癌旁组织对比，38例的蛋白质水平明显升高（P ＜0.05），而正常肺组织中HSF2基因的 mRNA 缺失率达80％，正常组织中 HSF2基因表达明显低于肺癌组织（P ＜0.05）。

不同年龄、性别、肿瘤大小、分化程度、组织类型中 HSF2基因表达无显著差异。

结论：肺癌中 HSF2基因表达水平的升高，暗示 HSF2基因在
肺癌的发生发展中起一定作用。

【期刊名称】《中国民族民间医药》
【年(卷),期】2016(025)012
【总页数】3页(P177-178,180)
【关键词】HSF2;肺癌;蛋白质;mRNA
【作者】钟云华;范敏娟
【作者单位】云南省第一人民医院，昆明理工大学生命科学院，云南昆明650032;昆明医科大学第二附属医院干疗科，云南昆明 650101
【正文语种】中文
【中图分类】R734.2
医药论坛
热休克蛋白(heat shock protein，HSP)作为一种分子伴侣，主要功能在于稳定细胞结构，使细胞维持正常的生理功能。

真核生物中已发现的HSFs有四种亚型: HSF1、HSF2、HSF3和HSF4[1]，具有的共同结构为一个保守的DNA结合区域(DNA binding domain，DBD)和一个三聚区域(Trimerization domain，TD)，
以及存在于N-端和C-端的非保守的转录激活功能域及调控区域。

目前认为HSF2对热刺激耐受, 对生长、发育和分化的信号更敏感，可以调控胚胎发育[2]。

研究发现，在睾丸中，HSF2表达上升且在精子形成中具有重要的作用通过敲除HSF2的小鼠在神经系统发育和生殖上出现异常，暗示HSF2在发育中起重要作用[1; 3-11]。

国内亦有相关报道，HSF2 mRNA水平在生精细胞凋亡早期略有降低，而在凋亡
高峰期之后其表达急剧下降。

HSF2蛋白可能调控Hsp70基因的表达，而且可能
通过多种方式影响精子的形成并抑制生精细胞的凋亡。

目前HSF2的作用机制国内
外均无统一定论。

有研究认为HSP2作为细胞内的保护蛋白，对肺组织上皮细胞的修复具有重要的作用[12]。

因此本研究用Western blot和RT-qPCR技术检测肺癌组织和癌旁组织HSF2基因的蛋白质和mRNA的变化，分析HSF2表达水平和肺癌发生发展的关系。

1.1 一般资料选取2015年1月至2015年12月本院收治的肺癌患者50例(取距离肿块 5 cm 以上作为正常对照组织，50 例)，男30例，女20例；年龄36～70岁，平均年龄(56±0.4)岁；其中高分化23例，中分化15例，低分化12例；小细胞肺癌18例，非小细胞肺癌32例。

所有患者术前均未经过任何治疗。

取材时将所取组织用预冷的 DEPC 处理后的双蒸水冲洗组织两遍后，置于液氮中冷冻保存备用。

1.2 HSF2基因蛋白质的检测将肺癌组织及正常癌旁组织加入组织裂解液后，于浆器冰上研磨及超声破碎，离心后收集上清，吸取上清用烤蓝法定量。

然后，以各泳道50μg总蛋白，用 12%SDS 不连续聚丙烯酰胺凝胶进行电泳分离，转膜，经封闭，标一抗和二抗，洗膜和显影后，即可进行结果分析。

1.3 HSF2基因mRNA的检测组织总RNA按照RNAprep Pure动物组织总RNA提取试剂盒DP431(TIANGEN，北京)说明书进行。

引物上海生物公司合成，序列为：
HSF2：扩增片段为139bp
Forward:5′- AAGTTCAGGCAGTGATGGCA -3′
Reverse: 5′- TGCACAGAACTAGTGAAAAGATCA -3′
GAPDH：扩增片段为185bp
Forward: 5′-GAAGGTCGGAGTCAACGGAT-3′
Reverse: 5′-GAGGGATCTCGCTCCTGGAAG-3′
1.4 统计学方法采用 SPSS 20.0 统计软件进行数据处理和分析，HSF2基因的
表达用χ2检验和方差分析，以P<0.05为差异有统计学意义。

2.1 Western blot检测HSF2蛋白的表达为了了解HSF2蛋白在肺癌组织及癌旁组织中表达情况，将肺癌组织及癌旁组织的蛋白提取后用western blot检测HSF2的表达。

结果显示，38例肺癌组织中HSF2蛋白呈现高表达，而在所有正常癌旁组织中呈现低表达(图1A)。

两组比较差异具有统计学意义
(χ2=61.29,P<0.05)。

2.2 RT-qPCR检测HSF2 mRNA 为观察HSF2基因在肺癌组织及正常癌旁组织的表达情况，采用RT-PCR技术检测HSF2基因mRNA的表达情况。

结果显示，在50例肺癌组织中，80%存在高表达；而正常癌旁组织中，低表达率达100%(图1B)。

两组对比，差异有统计学意义(χ2=66.67,P<0.05)。

2.3 HSF2基因表达与肺癌患者临床病理特征的关系结果显示，HSF2基因表达和性别、年龄、肿瘤大小、肺癌分化程度以及肺癌组织类型都没有关系，见表1。

研究表明，HSF2能调节HSP25、HSP40、HSP110尤其是HSP70、HSP90的转录表达[13]，且其所诱导HSP27、HSP90的表达在HSF2受RNA干扰后都下降[14]，提示HSF2参与了热休克反应的调控。

研究证实，G1期细胞，HSF2可结合热休克蛋白基因的启动子上，热休克蛋白的转录进而诱导下游基因的表达，有利于基因在G1期快速高效的表达[15]。

然而，HSF2并不是热休克反应独立的调控因子，只有与HSF1结合形成HSF1-HSF2复合物后才有激活调控转录的作用。

以上研究暗示，HSF2可能参与癌症的发生发展。

为研究HSF2基因表达情况在肺癌发生发展中作用，本研究采用western blot和RT-qPCR技术，检测肺癌组织及其对应正常癌旁组织中HSF2蛋白情况和mRNA 情况。

发现，与正常癌旁组织对比，HSF2蛋白在肺癌组织中高表达率为76%，mRNA为80%，差异具有统计学意义，表明HSF2参与肺癌的发生发展。

在肺癌患者的临床病理特征中，HSF2基因表达和性别、年龄、肿瘤大小、癌症分化程度
以及病理类型不存在关系。

综上所述，HSF2基因在肺癌中为高表达，而在正常旁组织中为低表达。

本研究结果表明，HSF2基因表达参与肺癌的发生发展，但和肺癌的临床病理特征，如性别、年龄、肿瘤大小、癌症分化程度以及病理类型不相关。

因本研究样本量少，以及HSF2在细胞中相关机制研究较少，仍需进一步的研究。

[1]Kallio M, Chang YH, Manuel M, Alastalo TP, Rallu M, Gitton Y, Pirkkala L, Loones MT, Paslaru L, Larney S, Hiard S, Morange M, Sistonen L, Mezger
V. Brain abnormalities, defective meiotic chromosome synapsis and female subfertility in HSF2 null mice. Embo Journal[J]. 2002, 21(11): 2591-2601. [2]Nakai A TM, Kawazoe Y, Inazawa J, Morimoto Ri, Nagata K. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol Cell Biol[J]. 1997, 17(1): 469-481.
[3]Akerfelt M, Henriksson E, Laiho A, Vihervaara A, Rautoma K, Kotaja N, Sistonen L. Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2. Proceedings of the National Academy of Sciences of the United States of America[J]. 2008, 105(32): 11224-11229. [4]Ostling P, Bjork JK, Roos-Mattjus P, Mezger V, Sistonen L. Heat shock factor 2 (HSF2) contributes to inducible expression of hsp genes through interplay with HSF1. Journal of Biological Chemistry[J]. 2007, 282(10):
7077-7086.
[5]Akerfelt M, Trouillet D, Mezger V, Sistonen L. Heat shock factors at a crossroad between stress and development. Stress Responses in Biology and Medicine[J]. 2007, 1113: 15-27.
[6]Chang YH, Ostling P, Akerfelt M, Trouillet D, Rallu M, Gitton Y, El Fatilmy R, Fardeau V, Le Crom S, Morange M, Sistonen L, Mezger V. Role of heat-shock factor 2 in cerebral cortex formation and as a regulator of p35 expression. Genes & Development[J]. 2006, 20(7): 836-847.
[7]Xing HY, Wilkerson DC, Mayhew CN, Lubert EJ, Skaggs HS, Goodson ML, Hong YL, Park-Sarge OK, Sarge KD. Mechanism of hsp70i gene bookmarking. Science[J]. 2005, 307(5708): 421-423.
[8]Birch-Machin I, Gao S, Huen D, Mcgirr R, White RaH, Russell S. Genomic analysis of heat-shock factor targets in Drosophila. Genome Biology[J]. 2005, 6(7).
[9]Horikoshi M, Yura T, Tsuchimoto S, Fukumori Y, Kanemori M. Conserved region 2.1 of Escherichia coli heat shock transcription factor sigma(32) is required for modulating both metabolic stability and transcriptional activity. Journal of Bacteriology[J]. 2004, 186(22): 7474-7480.[10]Wang G, Zhang J, Moskophidis D, Mivechi NF. Targeted disruption of the heat shock transcription factor (hsf)-2 gene results in increased embryonic lethality, neuronal defects, and reduced spermatogenesis. Genesis[J]. 2003, 36(1): 48-61.
[11]Castillo-Davis CI, Hartl DL. GeneMerge - post-genomic analysis, data mining, and hypothesis testing. Bioinformatics[J]. 2003, 19(7): 891-892. [12]Malago JJ, Koninkx JFJG, Van Dijk JE. The heat shock response and cytoprotection of the intestinal epithelium. Cell Stress & Chaperones[J]. 2002, 7(2): 191-199.[13]Alastalo T, Hellesuo M, Sandqvist A, Hietakangas V, Kallio M, Sistonen L. Formation of nuclear stress granules involves HSF2
and coincides with the nucleolar localization of Hsp70. Journal of cell science[J]. 2003, 116(Pt 17): 3557.
[14]Wilkerson D, Skaggs H, Sarge K. HSF2 binds to the Hsp90, Hsp27, and c-Fos promoters constitutively and modulates their expression. Cell Stress & Chaperones[J]. 2007, 12(3): 283.
[15]Nakai A, Ishikawa T. Cell cycle transition under stress conditions controlled by vertebrate heat shock factors. The EMBO Journal[J]. 2001, 20(11): 2885-2895.
基金项目：省应用基础研究(昆医联合专项)(2013FB197)；云南省教育厅重点项目(2014Z066)。

作者简介：钟云华(1972-)，女，云南昆明人，硕士，副主任医师，研究方向：心肺系统疾病。

【中图分类号】R734.2
【文献标志码】 A
【文章编号】1007-8517(2016)12-0177-02
(收稿日期：2016.05.21)
Significance and Expression of HSF2 Gene in Lung Cancer
ZHONG yunhua1 FAN minjuan2*
1.First People’s Hospital of Yunnan Province; Faculty of Life Science and Technology, Kunming University of Science and Technology,Kunming 650032 China；
2.The Second Affiliated Hospital of Kunming Medical University, Kunming 650101,China
Abstract:Objective In order to investigate the significance and expressioon of heat shock factor 2 (HSF2) gene in lung cancer.Methods We collected
50lung cancer tissue and adjacent cancer normal tissue samples. Meanwhile, the expression of HSF2 was investigated by Western blotting and RT-qPCR.ResultsWhen compared the lung cancer tissue and adjacent cancer normal tissue samples, we found that 76% (38 of 50) of the HSF2 protein in cancer tissues were upregulated compared to the normal lung tissues (P<0.05). However, in normal tissue, the rare of mRNA down-regulation was 80%. In normal control, expression of HSF2 gene was lower than lung cancer tissue. In addition, there was no significant difference in age status, gender status, tumor size status, histological differentiation status and histologic type of lung cancer. Conclusion The result of HSF2 gene upregulation, which indicate HSF2 may participate information and development of lung cancer.
Key words:HSF2; Lung Cancer; Protein; mRNA
V. Brain abnormalities, defective meiotic chromosome synapsis and female subfertility in HSF2 null mice. Embo Journal[J]. 2002, 21(11): 2591-2601. [2]Nakai A TM, Kawazoe Y, Inazawa J, Morimoto Ri, Nagata K. HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. Mol Cell Biol[J]. 1997, 17(1): 469-481.
[3]Akerfelt M, Henriksson E, Laiho A, Vihervaara A, Rautoma K, Kotaja N, Sistonen L. Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2. Proceedings of the National Academy of Sciences of the United States of America[J]. 2008, 105(32): 11224-11229.
[4]Ostling P, Bjork JK, Roos-Mattjus P, Mezger V, Sistonen L. Heat shock
factor 2 (HSF2) contributes to inducible expression of hsp genes through interplay with HSF1. Journal of Biological Chemistry[J]. 2007, 282(10): 7077-7086.
[5]Akerfelt M, Trouillet D, Mezger V, Sistonen L. Heat shock factors at a crossroad between stress and development. Stress Responses in Biology and Medicine[J]. 2007, 1113: 15-27.
[6]Chang YH, Ostling P, Akerfelt M, Trouillet D, Rallu M, Gitton Y, El Fatilmy R, Fardeau V, Le Crom S, Morange M, Sistonen L, Mezger V. Role of heat-shock factor 2 in cerebral cortex formation and as a regulator of p35 expression. Genes & Development[J]. 2006, 20(7): 836-847.
[7]Xing HY, Wilkerson DC, Mayhew CN, Lubert EJ, Skaggs HS, Goodson ML, Hong YL, Park-Sarge OK, Sarge KD. Mechanism of hsp70i gene bookmarking. Science[J]. 2005, 307(5708): 421-423.
[8]Birch-Machin I, Gao S, Huen D, Mcgirr R, White RaH, Russell S. Genomic analysis of heat-shock factor targets in Drosophila. Genome Biology[J]. 2005, 6(7).
[9]Horikoshi M, Yura T, Tsuchimoto S, Fukumori Y, Kanemori M. Conserved region 2.1 of Escherichia coli heat shock transcription factor sigma(32) is required for modulating both metabolic stability and transcriptional activity. Journal of Bacteriology[J]. 2004, 186(22): 7474-7480.[10]Wang G, Zhang J, Moskophidis D, Mivechi NF. Targeted disruption of the heat shock transcription factor (hsf)-2 gene results in increased embryonic lethality, neuronal defects, and reduced spermatogenesis. Genesis[J]. 2003, 36(1): 48-61.
[11]Castillo-Davis CI, Hartl DL. GeneMerge - post-genomic analysis, data mining, and hypothesis testing. Bioinformatics[J]. 2003, 19(7): 891-892. [12]Malago JJ, Koninkx JFJG, Van Dijk JE. The heat shock response and cytoprotection of the intestinal epithelium. Cell Stress & Chaperones[J]. 2002, 7(2): 191-199.[13]Alastalo T, Hellesuo M, Sandqvist A, Hietakangas V, Kallio M, Sistonen L. Formation of nuclear stress granules involves HSF2 and coincides with the nucleolar localization of Hsp70. Journal of cell science[J]. 2003, 116(Pt 17): 3557.
[14]Wilkerson D, Skaggs H, Sarge K. HSF2 binds to the Hsp90, Hsp27, and c-Fos promoters constitutively and modulates their expression. Cell Stress & Chaperones[J]. 2007, 12(3): 283.
[15]Nakai A, Ishikawa T. Cell cycle transition under stress conditions controlled by vertebrate heat shock factors. The EMBO Journal[J]. 2001, 20(11): 2885-2895.。