APE1基因沉默对人肝癌MHCC97-H体内成瘤能力及AP-1信号通路的影响
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摘要
目的观察APE1基因表达沉默后对人高侵袭肝癌细胞株MHCC97-H在裸鼠体内成瘤能力的影响,及其在AP-1信号通路的作用,为进一步阐明肝细胞癌增殖及侵袭转移的分子机制提供实验依据。
方法培养人高侵袭肝癌细胞株MHCC97-H,使用慢病毒载体介导的siRNA技术,用构建的LV-APE1-shRNA(实验组)表达载体感染MHCC97-H细胞,并设LV-APE1-NC(空载体)组和control(空白对照)组,随后将3组细胞分别接种于裸鼠皮下,第21d处死裸鼠,剥瘤称重,计算抑瘤率,绘制肿瘤生长曲线。制作移植瘤组织HE切片,观察肿瘤细胞形态。采用RT-PCR及Western blot方法分别检测各组裸鼠移植瘤组织中APE1及其下游AP-1信号通路相关基因c-Fos、c-Jun、caspase-3和MMP-2的mRNA及蛋白表达水平。采用TUNEL法检测各组肝癌细胞的凋亡情况,计算凋亡指数。
结果
1.LV-APE1-shRNA组裸鼠移植瘤生长缓慢,肿瘤体积(0.467±0.052)cm3及质量(0.267±0.082)g均小于LV-APE1-NC组(1.700±0.063)cm3、(1.018±0.160)g和control组(1.780±0.095)cm3、(1.107±0.178)g,差异有统计学意义(P<0.01); LV-APE1-shRNA组肿瘤抑制率为73.772%。
2. HE染色镜下形态显示,LV-APE1-shRNA组肿瘤组织坏死少见,较多肿瘤细胞凋亡小体,病理性核分裂<5个/HPF。LV-APE1-NC组和control组肿瘤组织坏死明显,可见散在肿瘤细胞凋亡小体,病理性核分裂>5个/HPF。
3.RT-PCR结果显示,LV-APE1-shRNA组APE1、c-Fos、c-Jun、caspase-3及MMP-2的mRNA表达水平明显低于LV-APE1-NC组和control组,差异有统计学意义(P<0.01)。APE1基因沉默后可下调90.622%APE1、47.580%c-Fos、
47.402%c-Jun、65.355%caspase-3、64.369%MMP-2的mRNA表达,APE1与c-Fos、c-Jun、caspase-3、MMP-2 mRNA的表达呈正相关性。
4.Western blot结果显示,LV-APE1-shRNA组APE1、c-Fos、c-Jun、caspase-3及MMP-2的蛋白表达水平明显低于LV-APE1-NC组和control组,差异有统计学意义(P<0.01)。APE1基因沉默后可下调72.439%APE1、71.872%c-Fos、80.399%c-Jun、75.616%caspase-3、88.393%MMP-2的蛋白表达,APE1与c-Fos、c-Jun、caspase-3、MMP-2蛋白的表达呈正相关性。
5.TUNEL法凋亡检测实验发现,LV-APE1-shRNA组肿瘤组织凋亡细胞数明显高于LV-APE1-NC组和control组,差异有统计学意义(P<0.01)。LV-APE1-shRNA组肿瘤细胞凋亡指数为5.503%,LV-APE1-NC组为2.911%,control组为2.569%。
结论
1.成功构建人高侵袭肝癌细胞株MHCC97-H裸鼠移植瘤动物模型,慢病毒载体介导的APE1基因沉默可显著抑制MHCC97-H细胞株在裸鼠体内的生长。
2. RT-PCR及Western blot实验表明,APE1、c-Fos、c-Jun、caspase-3和MMP-2基因在移植瘤组织中的mRNA及蛋白表达明显受到抑制,APE1对c-Fos、c-Jun、caspase-3、MMP-2的表达具有正向调控作用。APE1可通过调控AP-1信号通路参与肝癌的增殖及侵袭转移。
3.肿瘤细胞形态学观察结果和TUNEL法凋亡检测实验分析显示,APE1基因沉默可抑制肝癌细胞增殖,促进肝癌细胞凋亡。
Objective to investigate the influences of APE1 gene silence on forming tumor in vitro and AP-1 signal transduction pathway of Human Hepatocellular Carcinoma Cell line MHCC97-H ,which can settle the foundation for the father clarify of the molecular mechanism on proliferation ,invasion and metastasis of human hepatocellular carcinoma(HCC).
Methods culture Human Hepatocellular Carcinoma Cell line MHCC97-H . there are three groups:transfected group LV-APE1-shRNA,empty vector transfected group LV-APE1-NC and non-transfected group (control group),then establish an xenograft model of the human hepatocellular carcinoma by respectively inoculated above-mentioned three groups cells . Take the intact subcutaneous tumor tissues and measure their volumes and weight.calulate tumor growth inhibition ratio. Make paraffin section by hematoxylin-eosin staining ,observe the morphology of tumors under light microscope. Then,RT-PCR assay and Western blot assay were performed to detect the mRNA and protein expression levels of APE1gene and genes from AP-1 signal transduction pathway ,which involve c-Fos,c-Jun,caspase-3and MMP-2 .To detection the apoptosis difference among the three groups by TUNEL method,and calculate the apoptosis index of Hepatocarcinoma.
Results
1.The transplantable tumors of LV-APE1-shRNA group grew slowly, the volume(0.467±0.052)cm3 and weight(0.267±0.082)g were lower cmpared with LV-APE1-NC group (1.700±0.063)cm3、(1.018±0.160)g and control group(1.780±0.095)cm3、(1.107±0.178)g (P<0.01). The tumor growth inhibitory rate was 73.772%.
2. necrosis was observed infrequently in LV-APE1-shRNA group by HE dye under light microscope,but observed frequently in LV-APE1-NC group and control group. Apoptosis small body was more compared with the other two groups. Pathologic fission phasefive per high power field in LV-APE1-NC group and control group.
3.It was shower that the mRNA expression levels of APE1, c-Fos,c-Jun,caspase-3and MMP-2 were lower cmpared with LV-APE1-NC group and control group (P<0.01). After APE1 gene silence , mRNA expression levels of APE1 decreased by 90.622% ,c-Fos decreased by47.580%,c-Jun decreased by 47.402%,caspase-3 decreased by 65.355%,MMP-2 decreased by 64.369%。And their expressions were positive correlation.
4.It was showed that the protein expression levels of APE1, c-Fos,c-Jun,caspase-3and MMP-2 were lower cmpared with LV-APE1-NC group and control group (P<0.01). After APE1 gene silence , protein expression levels of APE1 decreased by 72.439% ,c-Fos decreased by71.872%,c-Jun decreased by 80.399%,caspase-3 decreased by 75.616%,MMP-2 decreased by 88.393%。And their expressions were positive correlation.
5. It was found that apoptosis count of LV-APE1-shRNA group was higher compared with the other two groups ( P<0.01 ), the apoptosis index of LV-APE1-shRNA group was 5.503%,LV-APE1-NC group was 2.911% and control group was 2.569% by TUNEL method. Results
Conclusions
1.It was successful to establish an xenogrft model of the Human Hepatocellular Carcinoma Cell line MHCC97-H . APE1 gene silence of MHCC97-H by infected lentivirus vector can inhibit observably the growth of transplant tumor.
2.RT-PCR assay and Western blot assay showed that the mRNA and protein expression levels of APE1, c-Fos,c-Jun,caspase-3 and MMP-2 were inhibited observably,and APE1 can regulat expression of c-Fos,c-Jun,caspase-3 and MMP-2 positively. APE1 could have a hand in proliferation ,invasion and metastasis of human hepatocellular carcinoma by AP-1 signal transduction pathway.
3.Tumor morphology observation and apoptosis detection assay by TUNEL method showed that APE1 gene silence can suppress proliferation and promote apoptosis of hepatocarcinoma. Conclusions
方法培养人高侵袭肝癌细胞株MHCC97-H,使用慢病毒载体介导的siRNA技术,用构建的LV-APE1-shRNA(实验组)表达载体感染MHCC97-H细胞,并设LV-APE1-NC(空载体)组和control(空白对照)组,随后将3组细胞分别接种于裸鼠皮下,第21d处死裸鼠,剥瘤称重,计算抑瘤率,绘制肿瘤生长曲线。制作移植瘤组织HE切片,观察肿瘤细胞形态。采用RT-PCR及Western blot方法分别检测各组裸鼠移植瘤组织中APE1及其下游AP-1信号通路相关基因c-Fos、c-Jun、caspase-3和MMP-2的mRNA及蛋白表达水平。采用TUNEL法检测各组肝癌细胞的凋亡情况,计算凋亡指数。
结果
1.LV-APE1-shRNA组裸鼠移植瘤生长缓慢,肿瘤体积(0.467±0.052)cm3及质量(0.267±0.082)g均小于LV-APE1-NC组(1.700±0.063)cm3、(1.018±0.160)g和control组(1.780±0.095)cm3、(1.107±0.178)g,差异有统计学意义(P<0.01); LV-APE1-shRNA组肿瘤抑制率为73.772%。
2. HE染色镜下形态显示,LV-APE1-shRNA组肿瘤组织坏死少见,较多肿瘤细胞凋亡小体,病理性核分裂<5个/HPF。LV-APE1-NC组和control组肿瘤组织坏死明显,可见散在肿瘤细胞凋亡小体,病理性核分裂>5个/HPF。
3.RT-PCR结果显示,LV-APE1-shRNA组APE1、c-Fos、c-Jun、caspase-3及MMP-2的mRNA表达水平明显低于LV-APE1-NC组和control组,差异有统计学意义(P<0.01)。APE1基因沉默后可下调90.622%APE1、47.580%c-Fos、
47.402%c-Jun、65.355%caspase-3、64.369%MMP-2的mRNA表达,APE1与c-Fos、c-Jun、caspase-3、MMP-2 mRNA的表达呈正相关性。
4.Western blot结果显示,LV-APE1-shRNA组APE1、c-Fos、c-Jun、caspase-3及MMP-2的蛋白表达水平明显低于LV-APE1-NC组和control组,差异有统计学意义(P<0.01)。APE1基因沉默后可下调72.439%APE1、71.872%c-Fos、80.399%c-Jun、75.616%caspase-3、88.393%MMP-2的蛋白表达,APE1与c-Fos、c-Jun、caspase-3、MMP-2蛋白的表达呈正相关性。
5.TUNEL法凋亡检测实验发现,LV-APE1-shRNA组肿瘤组织凋亡细胞数明显高于LV-APE1-NC组和control组,差异有统计学意义(P<0.01)。LV-APE1-shRNA组肿瘤细胞凋亡指数为5.503%,LV-APE1-NC组为2.911%,control组为2.569%。
结论
1.成功构建人高侵袭肝癌细胞株MHCC97-H裸鼠移植瘤动物模型,慢病毒载体介导的APE1基因沉默可显著抑制MHCC97-H细胞株在裸鼠体内的生长。
2. RT-PCR及Western blot实验表明,APE1、c-Fos、c-Jun、caspase-3和MMP-2基因在移植瘤组织中的mRNA及蛋白表达明显受到抑制,APE1对c-Fos、c-Jun、caspase-3、MMP-2的表达具有正向调控作用。APE1可通过调控AP-1信号通路参与肝癌的增殖及侵袭转移。
3.肿瘤细胞形态学观察结果和TUNEL法凋亡检测实验分析显示,APE1基因沉默可抑制肝癌细胞增殖,促进肝癌细胞凋亡。
Objective to investigate the influences of APE1 gene silence on forming tumor in vitro and AP-1 signal transduction pathway of Human Hepatocellular Carcinoma Cell line MHCC97-H ,which can settle the foundation for the father clarify of the molecular mechanism on proliferation ,invasion and metastasis of human hepatocellular carcinoma(HCC).
Methods culture Human Hepatocellular Carcinoma Cell line MHCC97-H . there are three groups:transfected group LV-APE1-shRNA,empty vector transfected group LV-APE1-NC and non-transfected group (control group),then establish an xenograft model of the human hepatocellular carcinoma by respectively inoculated above-mentioned three groups cells . Take the intact subcutaneous tumor tissues and measure their volumes and weight.calulate tumor growth inhibition ratio. Make paraffin section by hematoxylin-eosin staining ,observe the morphology of tumors under light microscope. Then,RT-PCR assay and Western blot assay were performed to detect the mRNA and protein expression levels of APE1gene and genes from AP-1 signal transduction pathway ,which involve c-Fos,c-Jun,caspase-3and MMP-2 .To detection the apoptosis difference among the three groups by TUNEL method,and calculate the apoptosis index of Hepatocarcinoma.
Results
1.The transplantable tumors of LV-APE1-shRNA group grew slowly, the volume(0.467±0.052)cm3 and weight(0.267±0.082)g were lower cmpared with LV-APE1-NC group (1.700±0.063)cm3、(1.018±0.160)g and control group(1.780±0.095)cm3、(1.107±0.178)g (P<0.01). The tumor growth inhibitory rate was 73.772%.
2. necrosis was observed infrequently in LV-APE1-shRNA group by HE dye under light microscope,but observed frequently in LV-APE1-NC group and control group. Apoptosis small body was more compared with the other two groups. Pathologic fission phase
3.It was shower that the mRNA expression levels of APE1, c-Fos,c-Jun,caspase-3and MMP-2 were lower cmpared with LV-APE1-NC group and control group (P<0.01). After APE1 gene silence , mRNA expression levels of APE1 decreased by 90.622% ,c-Fos decreased by47.580%,c-Jun decreased by 47.402%,caspase-3 decreased by 65.355%,MMP-2 decreased by 64.369%。And their expressions were positive correlation.
4.It was showed that the protein expression levels of APE1, c-Fos,c-Jun,caspase-3and MMP-2 were lower cmpared with LV-APE1-NC group and control group (P<0.01). After APE1 gene silence , protein expression levels of APE1 decreased by 72.439% ,c-Fos decreased by71.872%,c-Jun decreased by 80.399%,caspase-3 decreased by 75.616%,MMP-2 decreased by 88.393%。And their expressions were positive correlation.
5. It was found that apoptosis count of LV-APE1-shRNA group was higher compared with the other two groups ( P<0.01 ), the apoptosis index of LV-APE1-shRNA group was 5.503%,LV-APE1-NC group was 2.911% and control group was 2.569% by TUNEL method. Results
Conclusions
1.It was successful to establish an xenogrft model of the Human Hepatocellular Carcinoma Cell line MHCC97-H . APE1 gene silence of MHCC97-H by infected lentivirus vector can inhibit observably the growth of transplant tumor.
2.RT-PCR assay and Western blot assay showed that the mRNA and protein expression levels of APE1, c-Fos,c-Jun,caspase-3 and MMP-2 were inhibited observably,and APE1 can regulat expression of c-Fos,c-Jun,caspase-3 and MMP-2 positively. APE1 could have a hand in proliferation ,invasion and metastasis of human hepatocellular carcinoma by AP-1 signal transduction pathway.
3.Tumor morphology observation and apoptosis detection assay by TUNEL method showed that APE1 gene silence can suppress proliferation and promote apoptosis of hepatocarcinoma. Conclusions
引文
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[1] Giorgio M,Trinei M, Migliaccio E, Pelicci PG. Hydrogen peroxide:A metabolic by-product or a common mediator of ageing signals? [J]. Nat Rev Mol Cell Biol ,2007,8(9):722-728.
[2] Akiyama K,Seki S , Oshida T ,et al. Structure , promoter analysis and chromosomal assignment of the human APEX gene [J]. BiochimBiophys Act ,1994, 1219(1):15.
[3] Puglisi F,Barbone F,Tell G,et al. Prognostic role of Ape / Ref-1 subcellar expression in stage I-III breast carcinomas [J]. Oncol Rep , 2002,9 (1):11.
[4] Gorman MA ,Morera S ,Tothwell DG. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extrahelical deoxyribose at DNA a basic sites1 [J]. EMBO J ,1997,16(21):6548.
[5] Xanthoudakis S,Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity [J]. EMBO J ,1992,11(2):653-665.
[6] Kuninger DT,Izumi T,Papaconstantinou J,et al. Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter [J]. Nucleic Acids Res ,2002,30(3):823-829.
[7] Chung U,Igarashi T, Nishishita T,et al. The interaction between Ku antigen and REF1 protein mediates negative gene regulation by extracellular calcium [J]. J Biol Chem,1996,271(15):8593-8598.
[8] YamamoriT,DericcoJ,NaqviA,et al. SIRT1 deacetylates APE1 and regulates cellular base excision repair [J]. Nucleic Acids Res ,2010 ,38(3):832-845.
[9] Fung H,Demple B. A vital role for APE1/Ref1 protein in repairing spontaneous DNA damage in human cells [J]. Mol Cell , 2005,17(3):463-470.
[10] He T, Weintraub NL, Goswami PC, et al. Redox factor-1 contributes to the regulation of progression from G0/G1 to S by PDGF in vascular smooth muscle cells [J]. Am J Physiol Heart Circ Physiol ,2003,285(2):H804-812.
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[12] Chattopadhyay R, Wiederhold L, Szczesny B, et al. Identification and characterization of mitochondrial abasic(AP)-endonuclease in mammalian cells [J]. Nucleic Acids Res ,2006,34(7):2067-2076.
[13] Fung H, Bennett RA, Demple B. Key role of a downstream specificity protein 1 site in cell cycleregulated transcription of the AP endonuclease gene APE1/APEX in NIH3T3 cells [J]. J Biol Chem , 2001,276(45):42011-42017.
[14] Tell G, Crivellato E, Pines A,et al. Mitochondrial localization of APE/Ref-1 in thyroid cells [J]. Mutat Res ,2001,485(2):143-152.
[15] Duguid JR,Eble JN,Wilson TM,et al. Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme [J]. Cancer Res ,1995,55(24):6097-6102.
[16] Zou GM,Luo MH,Reed A,et a1.APEl regulates hematopoietic differentiation of embryonic stem cells through its redox functional domain[J].Blood , 2007,109(5):1917.
[17] Tell G, Damante G, Caldwell D,et al.The intracellular localization of APE1/Ref-1: More than a passive phenomenon? [J]. Antioxid Redox Signal ,2005,7(3-4):367-384.
[18] Di Maso V, Avellini C, CrocèLS, et al. Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance [J]. Mol Med , 2007,13(1-2):89-96.
[19] Davydov V, Hansen LA, Shackelford DA. Is DNA repair compromised in Alzheimer’s disease? [J].Neurobiol Aging 2003,24(7):953-968.
[20] Moradpour D, Blum HE. Pathogenesis of hepatocellular carcinoma [J]. Eur J Gastroenterol Hepatol , 2005,17(5):477-483
[21] Gu D , Wang M , Zhang Z ,et al. The DNA repair gene APE1 T1349G polymorphism and cancer risk: a meta-analysis of 27 case-control studies [J]. Mutagenesis , 2009,24(6):507-512.
[22] Moore DH,Michael H,Tritt R,et a1.Alterations in the expression of the DNA repair/redox enzyme APE/ ref-1 in epithelial ovarian cancers[J].Clin Cancer Res,2000,6(2):602-609.
[23]张颖,王东,辛晓燕,王建,向德兵,杨宇馨APE1在卵巢上皮性癌中的表达及其临床意义[J]第四军医大学学报2008,29 (19):1803-1806.
[24] Kelley MR, Cheng L, Foster R,et al. Elevated and altered expression of the multifunctional DNA base excision repair and redox enzyme Ape1/ref-1 in prostate cancer [J]. Clin Cancer Res ,2001,7(4):824-830.
[25]郑智华,黄爱民,刘景丰,等. APEl在肝细胞癌组织芯片的表达及临床病理意义[J].福建医科大学学报, 2008,42(2):100-103
[26]张云嵩,范士志,王东,等. APE1在非小细胞肺癌中的表达特点及其与预后的关系[J].第三军医大学报,2007,29(9):776-778.
[27] Lu J, Zhang S, Chen D, et al. Functional characterization of a promoter polymorphism in APE1/Ref-1 that contributes to reduced lung cancer susceptibility [J]. FASEB J ,2009,23(10):3459-3469.
[28] TellG,Pellizzari L, Pucillo C, et al .TSH controlsRef-1 nuclear translocation in thyroid cells [J]. J Mol Endocrinol, 2000,24(3):389-390.
[29] Russo D,Arturi F,Bulotta S,et al. ApeI/Ref- I expression and cellular localization in human thyroid carcinoma cell lines [ J]. J Endocrinol Invest,2001,24(3):10-12.
[30]谢家印,仲召阳,李增鹏,等. APE1在鼻腔NK /T细胞淋巴瘤中的表达及其意义[ J].解放军医学杂志,2005, 30(12 ):1105-1107.
[31]王东,仲召阳,李增鹏,等.双功能基因APE1敲低后人体骨肉瘤细胞基因表达谱变化的研究[ J].第三军医大学报,2007,29(1)1-4.
[32] Helleday T, Petermann E, Lundin C,et al. DNA repair pathways as targets for cancer therapy [J]. Nat Rev Cancer ,2008,8(3):193-204.
[33] Madhusudan S, Middleton MR. The emerging role of DNA repair proteins aspredictive, prognostic and therapeutic targets in cancer [J]. Cancer Treat Rev , 2005,31(8):603-617.
[34] Bryant HE , Schultz N , Thomas HD , et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase [J]. Nature ,2005,434(7035):913-917.