GSTπ基因在宫颈癌中生物学作用的研究
摘要
目的:研究外源性Gstπ基因的高表达对宫颈癌细胞生长、转移、浸润和辐射敏感性的影响,初步探讨Gstπ基因的生物学功能及作用机制。
方法:(1)根据GeneBank中GSTπ的基因序列,自行设计扩增Gstπ基因全长cDNA的引物,将人类全长GSTπcDNA定向地克隆到真核细胞表达载体pcDNA3中,并采用限制性内切酶酶切分析和DNA序列分析鉴定建立好的pcDNA3.0-GSTπ重组质粒;(2)采用阳离子脂质体介导的质粒转染法,完成pcDNA3/GSTπ载体和pcDNA3“空”载体(作为阴性对照)转染,在含有G418的筛选培养基中进一步筛选阳性克隆。采用RT-PCR和Western Blot检测克隆细胞中GSTπmRNA和蛋白表达水平;(3)采用细胞计数法观察GSTπ转染后,对体外培养的宫颈癌HeLa细胞生长和增殖的影响;(4)划痕实验和Boyden小室法用于观察GSTπ转染后宫颈癌细胞系HeLa转移及侵袭能力的改变;(5)细胞接受不同剂量的χ射线照射后,应用克隆形成实验检测GSTπ对HeLa细胞辐射敏感性的影响;(6)采用流式细胞术分析GSTπ对HeLa细胞周期进程的影响;Western Blot法检测GSTπ对调控细胞周期、DNA损伤修复相关基因或蛋白表达水平的影响。
结果:(1)经限制性内切酶酶切和DNA序列分析证实GSTπ真核表达载体构建成功;(2)经RT-PCR和Western Blot证实G418筛选得到的阳性克隆中GSTπ高表达,表明GSTπ高表达的细胞株筛选成功;(3)细胞计数结果表明GSTπ高表达可以明显抑制宫颈癌HeLa细胞的生长和增殖;(4)划痕实验和Boyden小室法结果显示高水平GSTπ明显降低宫颈癌HeLa细胞的转移和侵袭能力;(5)克隆形成实验的结果说明GSTπ高水平表达可降低宫颈癌HeLa细胞对χ射线的辐射敏感性;(6)流式细胞术分析结果表明GSTπ高表达可导致HeLa细胞在G0/G1期的阻滞。Western Blot法检测出GSTπ明显增加细胞周期G1期调控因子cyclin D1和细胞转移相关抑制蛋白PTEN的表达。
结论:本实验发现外源性Gstπ基因转染提高Gstπ表达水平可以抑制宫颈癌HeLa细胞的生长,其中的机制可能与其降低cyclin D1的蛋白表达,从而引发细胞周期阻滞有关。GSTπ高水平表达还可明显抑制HeLa细胞的转移和侵袭能力,这可能与其上调肿瘤转移抑制基因PTEN的蛋白表达水平有关。此外,GSTπ高表达使HeLa细胞的辐射敏感性降低。其机制可能是通过调控细胞周期调节蛋白cyclinB1的表达水平,诱导明显的G2期阻滞,以降低宫颈癌细胞的辐射敏感性。
这些实验结果表明GSTπ是影响宫颈癌细胞的生长,转移和侵袭以及放疗一重要的调节基因,为将来进一步深入研究GSTπ的生物学功能提供了必要的研究基础和实验依据。
Objective:To study the effects of overexpression of exogenous GSTπgene on cell growth、migration、invasion and radiosensitivity in human cervix cancer cell line HeLa,and to preliminarily investigate the underlying mechanisms.
Methods:(1) A full-length sequence of human Gstπgene was obtained by a polymerase chain reaction using the primers based on to the GSTπsequence in the GeneBank, and inserted into a recombinant eukaryotic expression plasmid by molecular cloning, and identified by restriction analysis and DNA sequencing. (2) The HeLa cells with a high expression of GSTπwere obtained by stable transfection of plasmids by using lipofectamine and G418 resistant screening. The mRNA and protein expression of GSTπwere detected by RT-PCR assay and Western Blot assay. (3) Cell counting was used to evaluate the effects of GSTπon HeLa cell growth. (4) In vitro scratch assay and Boyden chamber assay were used to identify the functions of GSTπin cell migration and invasion. (5) Colony formation assay was used to detect the influence of GSTπgene on HeLa cells’radiosensitivity ofχray. (6) Flow cytometry assay was performed to explore the impacts of GSTπon cell cycle prograssion. And the protein expression of cell cycle regulating factor was detected by Western Blot assay.
Results:(1) The recombinant eukaryotic expression plasmid of GSTπgene (pcDNA3/GSTπ) was successfully constructed. (2) A HeLa cell line stably expressing high level GSTπwas obtained,as demonstrated by Western blot and RT-PCR assay. (3) A significant inhibition of cell growth was observed in GSTπoverexpressing HeLa cells compared to untransfected HeLa parent cells and HeLa/Neo cells transfected with control“empty”pcDNA3 vector. (4) Increased expression of GSTπsignificantly inhibited the migration and invasion of HeLa cells. (5) HeLa/GSTπcells were more resistant to radiotherapy compared to HeLa/Neo cells; (6) Analyzed by flow cytometry assay, GSTπdelayed cell cycle progression by arresting cells at G0/G1 phase. And the expression level of cyclin D1 and PTEN was increased in HeLa/GSTπcells compared to HeLa/Neo cells as determined by Western blot assay.
Conclusion: These results demonstrated that the increased expression of Gstπgene inhibited HeLa cell growth through decreasing the protein level of G0/G1 phase controller-cyclin D1. GSTπsuppressed migration and invasion of HeLa cells, and the mechanisms may be related to its ability to increase the expression of metastasis suppressor PTEN. GSTπalso reduced cell sensitivity to radiotherapy, and the mechanisms may be related to its ability to increase the expression of G2/M phase controller-cyclin B1. These findings will provide a basis and evidence for further investigation of biological activities of GSTπin cervical cancer.
方法:(1)根据GeneBank中GSTπ的基因序列,自行设计扩增Gstπ基因全长cDNA的引物,将人类全长GSTπcDNA定向地克隆到真核细胞表达载体pcDNA3中,并采用限制性内切酶酶切分析和DNA序列分析鉴定建立好的pcDNA3.0-GSTπ重组质粒;(2)采用阳离子脂质体介导的质粒转染法,完成pcDNA3/GSTπ载体和pcDNA3“空”载体(作为阴性对照)转染,在含有G418的筛选培养基中进一步筛选阳性克隆。采用RT-PCR和Western Blot检测克隆细胞中GSTπmRNA和蛋白表达水平;(3)采用细胞计数法观察GSTπ转染后,对体外培养的宫颈癌HeLa细胞生长和增殖的影响;(4)划痕实验和Boyden小室法用于观察GSTπ转染后宫颈癌细胞系HeLa转移及侵袭能力的改变;(5)细胞接受不同剂量的χ射线照射后,应用克隆形成实验检测GSTπ对HeLa细胞辐射敏感性的影响;(6)采用流式细胞术分析GSTπ对HeLa细胞周期进程的影响;Western Blot法检测GSTπ对调控细胞周期、DNA损伤修复相关基因或蛋白表达水平的影响。
结果:(1)经限制性内切酶酶切和DNA序列分析证实GSTπ真核表达载体构建成功;(2)经RT-PCR和Western Blot证实G418筛选得到的阳性克隆中GSTπ高表达,表明GSTπ高表达的细胞株筛选成功;(3)细胞计数结果表明GSTπ高表达可以明显抑制宫颈癌HeLa细胞的生长和增殖;(4)划痕实验和Boyden小室法结果显示高水平GSTπ明显降低宫颈癌HeLa细胞的转移和侵袭能力;(5)克隆形成实验的结果说明GSTπ高水平表达可降低宫颈癌HeLa细胞对χ射线的辐射敏感性;(6)流式细胞术分析结果表明GSTπ高表达可导致HeLa细胞在G0/G1期的阻滞。Western Blot法检测出GSTπ明显增加细胞周期G1期调控因子cyclin D1和细胞转移相关抑制蛋白PTEN的表达。
结论:本实验发现外源性Gstπ基因转染提高Gstπ表达水平可以抑制宫颈癌HeLa细胞的生长,其中的机制可能与其降低cyclin D1的蛋白表达,从而引发细胞周期阻滞有关。GSTπ高水平表达还可明显抑制HeLa细胞的转移和侵袭能力,这可能与其上调肿瘤转移抑制基因PTEN的蛋白表达水平有关。此外,GSTπ高表达使HeLa细胞的辐射敏感性降低。其机制可能是通过调控细胞周期调节蛋白cyclinB1的表达水平,诱导明显的G2期阻滞,以降低宫颈癌细胞的辐射敏感性。
这些实验结果表明GSTπ是影响宫颈癌细胞的生长,转移和侵袭以及放疗一重要的调节基因,为将来进一步深入研究GSTπ的生物学功能提供了必要的研究基础和实验依据。
Objective:To study the effects of overexpression of exogenous GSTπgene on cell growth、migration、invasion and radiosensitivity in human cervix cancer cell line HeLa,and to preliminarily investigate the underlying mechanisms.
Methods:(1) A full-length sequence of human Gstπgene was obtained by a polymerase chain reaction using the primers based on to the GSTπsequence in the GeneBank, and inserted into a recombinant eukaryotic expression plasmid by molecular cloning, and identified by restriction analysis and DNA sequencing. (2) The HeLa cells with a high expression of GSTπwere obtained by stable transfection of plasmids by using lipofectamine and G418 resistant screening. The mRNA and protein expression of GSTπwere detected by RT-PCR assay and Western Blot assay. (3) Cell counting was used to evaluate the effects of GSTπon HeLa cell growth. (4) In vitro scratch assay and Boyden chamber assay were used to identify the functions of GSTπin cell migration and invasion. (5) Colony formation assay was used to detect the influence of GSTπgene on HeLa cells’radiosensitivity ofχray. (6) Flow cytometry assay was performed to explore the impacts of GSTπon cell cycle prograssion. And the protein expression of cell cycle regulating factor was detected by Western Blot assay.
Results:(1) The recombinant eukaryotic expression plasmid of GSTπgene (pcDNA3/GSTπ) was successfully constructed. (2) A HeLa cell line stably expressing high level GSTπwas obtained,as demonstrated by Western blot and RT-PCR assay. (3) A significant inhibition of cell growth was observed in GSTπoverexpressing HeLa cells compared to untransfected HeLa parent cells and HeLa/Neo cells transfected with control“empty”pcDNA3 vector. (4) Increased expression of GSTπsignificantly inhibited the migration and invasion of HeLa cells. (5) HeLa/GSTπcells were more resistant to radiotherapy compared to HeLa/Neo cells; (6) Analyzed by flow cytometry assay, GSTπdelayed cell cycle progression by arresting cells at G0/G1 phase. And the expression level of cyclin D1 and PTEN was increased in HeLa/GSTπcells compared to HeLa/Neo cells as determined by Western blot assay.
Conclusion: These results demonstrated that the increased expression of Gstπgene inhibited HeLa cell growth through decreasing the protein level of G0/G1 phase controller-cyclin D1. GSTπsuppressed migration and invasion of HeLa cells, and the mechanisms may be related to its ability to increase the expression of metastasis suppressor PTEN. GSTπalso reduced cell sensitivity to radiotherapy, and the mechanisms may be related to its ability to increase the expression of G2/M phase controller-cyclin B1. These findings will provide a basis and evidence for further investigation of biological activities of GSTπin cervical cancer.
引文
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[5].朱键,谈莹,何兰,司马健,殷志敏.谷肤甘肤S-转移酶Pi在MAPK途径中的调节作用[R].细胞生物学杂志,2004; 26: 253-255.
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[13]Goto S, Hara Y, et al. Doxorubicin-induced DNA intercalation and scavenging by nuclear glutathione Stransferaseπ. FASEBJ, 2001, 15:2702-2714.
[14]Wang T L,Arifoglu P, et al. Glutathione S-transferase P1-1(GSTPI1-1) inhibits c-Jun N-terminal kinase 1 (JNK1) signaling through interaction with the C terminus. J Biol Chem, 2001, 276(24): 20999-21003.
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[16]殷志敏,刘爱华,姜勇.谷胱甘肽S-转移酶π通过抑制ASK1-MKK7-JNK通道保护血清撤离诱导的293细胞死亡.生物化学与生物物理学报2001, 33(2): 185-190.
[17]Dorion S, Lambert H. Activation of p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase mu from ASK1. J Biol Chem, 2002, 277(34): 30792-30797.
[18]Yin Z M, Ivanov V N, et al. Glutathione S-transferase P elicits protection against H2O2 induced cell death via coordinated regulation of stress kinases. Cancer Res, 2000, 60: 4053-4057.
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