FOXP3过表达对HPV感染阳性宫颈癌细胞增殖和凋亡的影响
|
摘要
目的:探讨叉状头/翅膀状螺旋转录因子(Foxp3)过表达对人乳头状瘤病毒(HPV)感染阳性宫颈癌细胞增殖、凋亡及细胞免疫的影响。方法:利用脂质体介导转染方法,建立Foxp3过表达HPV感染阳性SiHa细胞株,作为Foxp3过表达阳性细胞,同时利用pcDNA3.1空载体转染SiHa细胞株,作为本研究阴性对照,同时设置空白对照组,采用实时荧光定量聚合酶链式反应(qRT-PCR)方法及Western blot法检测三组Foxp3mRNA及蛋白表达水平,采用酶联免疫吸附试验(ELISA)检测SiHa细胞中白细胞介素8(IL-8)、白细胞介素10(IL-10)水平,采用四甲基偶氮唑蓝(MTT)实验检测SiH a细胞增殖能力,采用利用流式细胞仪(FCM)检测SiHa细胞周期及凋亡率情况。结果:过表达组Foxp3 mRNA及蛋白表达水平明显高于阴性对照组及空白对照组,差异有统计学意义(P<0.05),阴性对照组及空白对照组Foxp3 mRNA及蛋白表达水平比较,差异无统计学意义(P>0.05);与阴性对照组及空白对照组比较,各时间点过表达组IL-8水平明显降低,36 h、48 h IL-10水平明显升高;MTT结果显示,Foxp3过表达可明显促进SiHa细胞增殖,FCM检测显示,Foxp3过表达可明显抑制SiHa细胞凋亡,抑制G_0/G_1期阻滞。结论:Foxp3过表达可促进HPV感染阳性宫颈癌细胞增殖,抑制细胞凋亡。
Objective:To explore the influence of forkhead/winged helix transcription factor(FOXP3) overexpression on cell proliferation and apoptosis of human papilloma virus( HPV) infected positive cervical cancer cells. Methods:Foxp3 overexpression of HPV infected positive Si Ha cell lines were developed by using liposome mediated transfection method,as Foxp3 overexpression positive cell,and transfected Si Ha cell lines by using pcDNA3. 1 empty carrier were as the negative control group in the study,and meanwhile,the blank control group were set. Foxp3 mRNA and protein expression levels were detected in the three groups by using real-time fluorescence quantitative polymerase chain reaction( qRT-PCR) and Western blot method. Interleukin 8( IL-8) and interleukin 10( IL-10) levels in Si Ha cells were tested by using Enzyme linked immunosorbent assay( ELISA) method. Si Ha cell proliferation ability were tested by using methylthiazolyldiphenyl-tetrazolium bromide( MTT) experiment,and Si Ha cell cycle and apoptosis rate were detected by using flow cytometry( FCM). Results:The expression levels of Foxp3,mRNA and protein in the overexpression group were significantly higher than those in the negative control group and the blank control group,with statistically significant differences( P < 0. 05). There were no significant differences in the expression levels between the negative control group and the blank control group( P > 0. 05). Compared with those in the negative control group and the blank control group,IL-8 level decreased significantly in different time points,and IL-10 level increased significantly at 36 h and 48 h in the overexpression group. MTT result showed that Foxp3 overexpression could significantly promote the proliferation of Si Ha cells. FCM showed that Foxp3 overexpression could significantly inhibit the apoptosis of Si Ha cells,and inhibit G_0/G_1 blocking. Conclusion:FOXP3 overexpression can promote the proliferation of HPV infected positive cervical cancer cells,and inhibit the apoptosis.
Objective:To explore the influence of forkhead/winged helix transcription factor(FOXP3) overexpression on cell proliferation and apoptosis of human papilloma virus( HPV) infected positive cervical cancer cells. Methods:Foxp3 overexpression of HPV infected positive Si Ha cell lines were developed by using liposome mediated transfection method,as Foxp3 overexpression positive cell,and transfected Si Ha cell lines by using pcDNA3. 1 empty carrier were as the negative control group in the study,and meanwhile,the blank control group were set. Foxp3 mRNA and protein expression levels were detected in the three groups by using real-time fluorescence quantitative polymerase chain reaction( qRT-PCR) and Western blot method. Interleukin 8( IL-8) and interleukin 10( IL-10) levels in Si Ha cells were tested by using Enzyme linked immunosorbent assay( ELISA) method. Si Ha cell proliferation ability were tested by using methylthiazolyldiphenyl-tetrazolium bromide( MTT) experiment,and Si Ha cell cycle and apoptosis rate were detected by using flow cytometry( FCM). Results:The expression levels of Foxp3,mRNA and protein in the overexpression group were significantly higher than those in the negative control group and the blank control group,with statistically significant differences( P < 0. 05). There were no significant differences in the expression levels between the negative control group and the blank control group( P > 0. 05). Compared with those in the negative control group and the blank control group,IL-8 level decreased significantly in different time points,and IL-10 level increased significantly at 36 h and 48 h in the overexpression group. MTT result showed that Foxp3 overexpression could significantly promote the proliferation of Si Ha cells. FCM showed that Foxp3 overexpression could significantly inhibit the apoptosis of Si Ha cells,and inhibit G_0/G_1 blocking. Conclusion:FOXP3 overexpression can promote the proliferation of HPV infected positive cervical cancer cells,and inhibit the apoptosis.
引文
[1]Zhou Hui,Lu Huaiwu,Peng Yongpai,et al.The explain of《NCCN clinical practice guidelines in cervical cancer 2015》[J].Chinese Journal of Practical Gynecology and Obstetrics,2015,3(31):185-191.[周晖,卢淮武,彭永排,等.《2015年NCCN宫颈癌临床实践指南》解读[J].中国实用妇科与产科杂志,2015,3(31):185-191.]
[2]Liu Huiqiang.Analysis of the epidemiological characteristics and risk factors of cervical cancer in China[J].Maternal and Child Health Care of China,2016,31(6):1258-1260.[刘慧强.我国宫颈癌流行病学特征和高危因素分析[J].中国妇幼保健,2016,31(6):1258-1260.]
[3]Nehar Belaid D,Courau T,Dérian N,et al.Regulatory T cells orchestrate similar immune evasion of fetuses and tumors in mice[J].Journal of Lmmunology,2016,196(2):678-680.
[4]Yao Xiuhua,Wang Zhonghai.The relationship between regulatory T cells,FOXP3 and tumors[J].Chinese Journal of Reproductive Health,2017,28(3):314-317.[姚秀华,王中海.调节性T细胞、叉状头螺旋转录因子与肿瘤的关系[J].中国生育健康杂志,2017,28(3):314-317.]
[5]Ferlay J,Soerjomataram I,Dikshit R,et al.Cancer incidence and mortality worldwide:Sources,methods and major patterns in GLOBOCAN 2012[J].International Journal of Cancer,2015,136(5):E359-E386.
[6]Jing Jiayu,Mou Jingyi,Wang Yiying,et al.Screening methods for cervical cancer and precancerous lesions[J].Journal of Chinese Practical Diagnosis and Therapy,2017,31(2):203-205.[井佳雨,牟婧祎,王轶英,等.宫颈癌及癌前病变筛查方法研究进展[J].中华实用诊断与治疗杂志,2017,31(2):203-205.]
[7]Zou Zhengyun,Liu Baorui.Immune escape and immunotherapy strategies of malignant tumor[J].International Journal of Immunology,2015,38(3):280-283.[邹征云,刘宝瑞.恶性肿瘤免疫逃逸与免疫治疗策略[J].国际免疫学杂志,2015,38(3):280-283.]
[8]Chen Xiang.Hot points and development trend of research on the immune escape and immune therapy of hepatocellular carcinoma[J].Chinese Journal of Cancer Biotherapy,2017,24(1):73-82.[陈祥.肝细胞癌的免疫逃逸和免疫治疗研究热点与发展趋势[J].中国肿瘤生物治疗杂志,2017,24(1):73-82.]
[9]Neharbelaid D,Courau T,Dérian N,et al.Regulatory T cells orchestrate similar immune evasion of fetuses and tumors in mice[J].Journal of Immunology,2016,196(2):678-680.
[10]Garib FY,Rizopulu AP.T-regulatory cells as part of strategy of immune evasion by pathogens[J].Biochemistry,2015,80(8):957-971.
[11]Song Yan,Zhu Xike.Current progress of the mechanism of tumor immune escape[J].Modern Oncology,2011,19(6):1230-1234.[宋彦,朱喜科.肿瘤免疫逃逸机制的研究新进展[J].现代肿瘤医学,2011,19(6):1230-1234.]
[12]Ma Ningye,Zhang Shulan.Research progress of correlation between forkhead transcription 3 and malignant gynecology tumor[J].J Int Obstet Gynecol,2013,40(3):255-257.[马宁耶,张淑兰.叉头状/翼状螺旋转录因子3与妇科恶性肿瘤相关性研究进展[J].国际妇产科学杂志,2013,40(3):255-257.]
[13]O'Callaghan DS,Rexhepaj E,Gately K,et al.Tumour islet foxp3+T-cell infiltration predicts poor outcome in nonsmall cell lung cancer[J].European Respiratory Journal,2015,46(6):1762.
[14]Zhang Ke,Liu Yingying,Jin Chunhui,et al.The effects of CEACAM6 and FOXP3 on tumor infiltrating lymphocytes and prognosis in patients with pancreatic cancer[J].Chin J Cell Mol Immunol,2015,31(8):1103-1107.[张科,刘莹莹,金春晖,等.CEACAM6和FOXP3对胰腺癌肿瘤浸润淋巴细胞及预后的影响[J].细胞与分子免疫学杂志,2015,31(8):1103-1107.]
[15]Fan Qingbo,Liu Hongshan,Qin Bingyu,et al.Expression changes of regulatory T cells and forkhead/winged helix transcription factor P3 levels in patients with gastric cancer[J].Chin J Exp Surg,2015,32(8):1976-1978.[樊清波,刘红山,秦秉玉,等.调节性T细胞和叉状头/翅膀状螺旋转录因子在胃癌患者中的表达变化[J].中华实验外科杂志,2015,32(8):1976-1978.]
[16]Han Liping,Liu Liya,Sun Xiaohui,et al.Expression and correlation analysis of FOXP3,PD-1 and PD-L1 in the cervical cancer[J].Journal of Zhengzhou University(Medical Sciences),2017,52(1):83-88.[韩丽萍,刘丽雅,孙晓慧,等.宫颈癌组织中FOXP3、PD-1及PD-L1蛋白的表达[J].郑州大学学报(医学版),2017,52(1):83-88.]
[17]Huh WK,Ault KA,Chelmow D,et al.Use of primary high-risk human papillomavirus testing for cervical cancer screening:Interim clinical guidance[J].Gynecologic Oncology,2015,136(2):178-182.
[18]Xu F,Cao M,Shi Q,et al.Integration of the full-length HPV16genome in cervical cancer and Caski and Siha cell lines and the possible ways of HPV integration[J].Virus Genes,2015,50(2):210-220.
[19]Floudas A,Amu S,Fallon PG.New Insights into IL-10 dependent and IL-10 independent mechanisms of regulatory B cell immune suppression[J].Journal of Clinical Immunology,2016,36(1):25-33.
[20]Hou Zhenjiang,Mou Zhaoxin.Research progress in interleukin-10 and its receptor in autoimmune thyroid diseases[J].Medical Recapitulate,2016,22(24):4823-4827.[侯振江,牟兆新.白细胞介素10及其受体在自身免疫性甲状腺疾病中的研究进展[J].医学综述,2016,22(24):4823-4827.]
[2]Liu Huiqiang.Analysis of the epidemiological characteristics and risk factors of cervical cancer in China[J].Maternal and Child Health Care of China,2016,31(6):1258-1260.[刘慧强.我国宫颈癌流行病学特征和高危因素分析[J].中国妇幼保健,2016,31(6):1258-1260.]
[3]Nehar Belaid D,Courau T,Dérian N,et al.Regulatory T cells orchestrate similar immune evasion of fetuses and tumors in mice[J].Journal of Lmmunology,2016,196(2):678-680.
[4]Yao Xiuhua,Wang Zhonghai.The relationship between regulatory T cells,FOXP3 and tumors[J].Chinese Journal of Reproductive Health,2017,28(3):314-317.[姚秀华,王中海.调节性T细胞、叉状头螺旋转录因子与肿瘤的关系[J].中国生育健康杂志,2017,28(3):314-317.]
[5]Ferlay J,Soerjomataram I,Dikshit R,et al.Cancer incidence and mortality worldwide:Sources,methods and major patterns in GLOBOCAN 2012[J].International Journal of Cancer,2015,136(5):E359-E386.
[6]Jing Jiayu,Mou Jingyi,Wang Yiying,et al.Screening methods for cervical cancer and precancerous lesions[J].Journal of Chinese Practical Diagnosis and Therapy,2017,31(2):203-205.[井佳雨,牟婧祎,王轶英,等.宫颈癌及癌前病变筛查方法研究进展[J].中华实用诊断与治疗杂志,2017,31(2):203-205.]
[7]Zou Zhengyun,Liu Baorui.Immune escape and immunotherapy strategies of malignant tumor[J].International Journal of Immunology,2015,38(3):280-283.[邹征云,刘宝瑞.恶性肿瘤免疫逃逸与免疫治疗策略[J].国际免疫学杂志,2015,38(3):280-283.]
[8]Chen Xiang.Hot points and development trend of research on the immune escape and immune therapy of hepatocellular carcinoma[J].Chinese Journal of Cancer Biotherapy,2017,24(1):73-82.[陈祥.肝细胞癌的免疫逃逸和免疫治疗研究热点与发展趋势[J].中国肿瘤生物治疗杂志,2017,24(1):73-82.]
[9]Neharbelaid D,Courau T,Dérian N,et al.Regulatory T cells orchestrate similar immune evasion of fetuses and tumors in mice[J].Journal of Immunology,2016,196(2):678-680.
[10]Garib FY,Rizopulu AP.T-regulatory cells as part of strategy of immune evasion by pathogens[J].Biochemistry,2015,80(8):957-971.
[11]Song Yan,Zhu Xike.Current progress of the mechanism of tumor immune escape[J].Modern Oncology,2011,19(6):1230-1234.[宋彦,朱喜科.肿瘤免疫逃逸机制的研究新进展[J].现代肿瘤医学,2011,19(6):1230-1234.]
[12]Ma Ningye,Zhang Shulan.Research progress of correlation between forkhead transcription 3 and malignant gynecology tumor[J].J Int Obstet Gynecol,2013,40(3):255-257.[马宁耶,张淑兰.叉头状/翼状螺旋转录因子3与妇科恶性肿瘤相关性研究进展[J].国际妇产科学杂志,2013,40(3):255-257.]
[13]O'Callaghan DS,Rexhepaj E,Gately K,et al.Tumour islet foxp3+T-cell infiltration predicts poor outcome in nonsmall cell lung cancer[J].European Respiratory Journal,2015,46(6):1762.
[14]Zhang Ke,Liu Yingying,Jin Chunhui,et al.The effects of CEACAM6 and FOXP3 on tumor infiltrating lymphocytes and prognosis in patients with pancreatic cancer[J].Chin J Cell Mol Immunol,2015,31(8):1103-1107.[张科,刘莹莹,金春晖,等.CEACAM6和FOXP3对胰腺癌肿瘤浸润淋巴细胞及预后的影响[J].细胞与分子免疫学杂志,2015,31(8):1103-1107.]
[15]Fan Qingbo,Liu Hongshan,Qin Bingyu,et al.Expression changes of regulatory T cells and forkhead/winged helix transcription factor P3 levels in patients with gastric cancer[J].Chin J Exp Surg,2015,32(8):1976-1978.[樊清波,刘红山,秦秉玉,等.调节性T细胞和叉状头/翅膀状螺旋转录因子在胃癌患者中的表达变化[J].中华实验外科杂志,2015,32(8):1976-1978.]
[16]Han Liping,Liu Liya,Sun Xiaohui,et al.Expression and correlation analysis of FOXP3,PD-1 and PD-L1 in the cervical cancer[J].Journal of Zhengzhou University(Medical Sciences),2017,52(1):83-88.[韩丽萍,刘丽雅,孙晓慧,等.宫颈癌组织中FOXP3、PD-1及PD-L1蛋白的表达[J].郑州大学学报(医学版),2017,52(1):83-88.]
[17]Huh WK,Ault KA,Chelmow D,et al.Use of primary high-risk human papillomavirus testing for cervical cancer screening:Interim clinical guidance[J].Gynecologic Oncology,2015,136(2):178-182.
[18]Xu F,Cao M,Shi Q,et al.Integration of the full-length HPV16genome in cervical cancer and Caski and Siha cell lines and the possible ways of HPV integration[J].Virus Genes,2015,50(2):210-220.
[19]Floudas A,Amu S,Fallon PG.New Insights into IL-10 dependent and IL-10 independent mechanisms of regulatory B cell immune suppression[J].Journal of Clinical Immunology,2016,36(1):25-33.
[20]Hou Zhenjiang,Mou Zhaoxin.Research progress in interleukin-10 and its receptor in autoimmune thyroid diseases[J].Medical Recapitulate,2016,22(24):4823-4827.[侯振江,牟兆新.白细胞介素10及其受体在自身免疫性甲状腺疾病中的研究进展[J].医学综述,2016,22(24):4823-4827.]