FABP-5基因沉默对宫颈癌SiHa细胞生物学特性的影响
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摘要
背景:研究发现肿瘤相关基因以及肿瘤基因通路的改变在肿瘤的形成与发展中起重要作用。目的:探讨siRNA干扰技术沉默FABP-5基因表达对人宫颈癌SiHa细胞生物学特性的影响。方法:以人宫颈癌SiHa细胞为研究对象,根据FABP-5 mRNA编码序列设计并合成干扰si RNA序列,瞬时转染SiHa细胞。用RT-PCR和Western blot法分别从mRNA水平和蛋白水平检测FABP-5基因的表达;流式细胞技术检测各组细胞周期的变化;CCK-8法测定细胞体外增殖能力;细胞侵袭小室法检测各组细胞的体外侵袭力;TUNEL染色检测各组细胞的凋亡情况。结果与结论:与转染空病毒阴性对照组和未转染空白对照组比较,FABP-5 siRNA组FABP-5 mRNA和蛋白表达明显下降,细胞生长速度明显减慢,细胞周期阻滞在G_0/G_1期,S期细胞数减少,细胞侵袭力显著下降,细胞凋亡率明显升高。结果说明siRNA技术可以成功诱导FABP-5基因沉默,从而影响宫颈癌SiHa细胞的的生长、增殖和凋亡。
BACKGROUND: Cancer related genes and pathways play a critical role in formation and development of cancer. OBJECTIVE: To investigate the silencing epidermal fatty acid binding protein 5(FABP-5) by siRNA interference on biological characteristics of the cervical carcinoma cell line SiHa. METHODS: Design and synthesis of siRNA interference sequence used to transiently transfect SiHa cells was performed according to FABP-5 mRNA coding sequence. mRNA and protein expressions of FABP-5 were detected by RT-PCR and western blot assay, respectively. Meanwhile, cell cycle, proliferation, invasion and apoptosis were detected by flow cytometry, cell counting bit-8 assay, Boyden chamber assay, and TUNEL staining, respectively. RESULTS AND CONCLUSION: FABP-5 mRNA and protein expressions, cell growth speed, cell number at S phase(cell cycle was arrested at the G_0/G_1 phase) and cell invasion were significantly decreased, while the cell apoptosis was significantly increased in FABP-5 siRNA group compared with the negative control and blank control groups. Our findings indicate that the specific silencing FABP-5 gene expression by siRNA interference can inhibit SiHa cervical cell growth and proliferation, but accelerate cell apoptosis.
BACKGROUND: Cancer related genes and pathways play a critical role in formation and development of cancer. OBJECTIVE: To investigate the silencing epidermal fatty acid binding protein 5(FABP-5) by siRNA interference on biological characteristics of the cervical carcinoma cell line SiHa. METHODS: Design and synthesis of siRNA interference sequence used to transiently transfect SiHa cells was performed according to FABP-5 mRNA coding sequence. mRNA and protein expressions of FABP-5 were detected by RT-PCR and western blot assay, respectively. Meanwhile, cell cycle, proliferation, invasion and apoptosis were detected by flow cytometry, cell counting bit-8 assay, Boyden chamber assay, and TUNEL staining, respectively. RESULTS AND CONCLUSION: FABP-5 mRNA and protein expressions, cell growth speed, cell number at S phase(cell cycle was arrested at the G_0/G_1 phase) and cell invasion were significantly decreased, while the cell apoptosis was significantly increased in FABP-5 siRNA group compared with the negative control and blank control groups. Our findings indicate that the specific silencing FABP-5 gene expression by siRNA interference can inhibit SiHa cervical cell growth and proliferation, but accelerate cell apoptosis.
引文
[1]陆晓楣,李晶,刘畅浩,等.年轻宫颈癌患者的病理特点和预后分析[J]中国妇产科临床杂志,2011,12(1):10-13.
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[4]朱素文.子宫颈病变筛查的研究进展[J].医学理论与实践,2011,24(7):774-775.
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[7]Thumser AE,Moore JB,Plant NJ.Fatty acid binding proteins:tissue-specific functions in health and disease.Curr Opin Clin Nutr Metab Care.2014;17(2):124-129.
[8]王欢,刘富萍.VEGF-C在宫颈癌中的研究现状与展望[J].四川解剖学杂志,2011,19(3):30-33.
[9]曾广平.子宫颈恶性肿瘤介入治疗及护理进展[J].齐齐哈尔医学院学报,2011,32(9):1454-1456.
[10]Armstrong EH,Goswami D,Griffin PR,et al.Structural basis for ligand regulation of the fatty acid-binding protein 5,peroxisome proliferator-activated receptorβ/δ(FABP5-PPARβ/δ)signaling pathway.J Biol Chem.2014;289(21):14941-14954.
[11]王伟,殷红梅.宫颈癌组织中Fhit、survivin和Bcl-2的表达变化及意义[J].山东医药,2011,51(30):42-43.
[12]Fang LY,Wong TY,Chiang WF,et al.Fatty-acid-binding protein 5 promotes cell proliferation and invasion in oral squamous cell carcinoma.J Oral Pathol Med.2010;39(4):342-348.
[13]陈燕,林莺莺,陈岩松,等.原发性肝癌早期诊断中甲胎蛋白异质体检测的意义[J].福建医药杂志,2012,34(1):3-6.
[14]Gu TT,Liu SY,Zheng PS.Cytoplasmic NANOG-positive stromal cells promote human cervical cancer progression.Am J Pathol.2012;181(2):652-661.
[15]谢玲玲,卢淮武,钟沅月,等.分割剂量射线照射富集宫颈癌肿瘤干细胞的研究[J].中山大学学报:医学科学版,2012,33(3):330-335.
[16]Gu W,Yeo E,Mc Millan N,et al.Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability.Cancer Gene Ther.2011;18(12):897-905.
[17]李淑青,陈亚萍,杨恭,等.宫颈癌干细胞相关标记物的研究进展[J].现代妇产科进展,2014,23(11):931-936.
[18]肖松舒,蒋建发.宫颈癌干细胞的研究进展[J].现代妇产科进展,2013,22(9):756-758.
[19]Zhang SL,Wang YS,Zhou T,et al.Isolation and characterization of cancer stem cells from cervical cancer He La cells.Cytotechnology.2012;64(4):477-484.
[20]刘晨,安晓霞,张凡,等.肿瘤干细胞相关基因ABCB1、ABCG2表达与宫颈上皮恶变及发展关系的研究[J].现代妇产科进展,2012,21(4):294-297.
[21]李德冠,王小春,孟爱民,等.肿瘤干细胞靶向治疗[J].中国药理学通报,2009,25(6):701-703.
[22]张松灵,张立红,钟艳平,等.人宫颈癌组织中CK17和P63蛋白的表达及意义[J].吉林大学学报:医学版,2009,35(5):914-917.
[23]Choijamts B,Jimi S,Kondo T,et al.CD133+cancer stem cell-like cells derived from uterine carcinosarcoma(malignant mixed Müllerian tumor).Stem Cells.2011;29(10):1485-1495.
[24]Kato K,Takao T,Kuboyama A,et al.Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage Am J Pathol.2010;176(1):381-392.
[25]G?tte M,Greve B,Kelsch R,et al.The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/CIP1.Int J Cancer.2011;129(8):2042-2049.
[26]路春华,盛修贵.宫颈癌相关干细胞的研究进展[J].中华肿瘤防治杂志,2013,20(4):317-320.
[27]D’Andrea FP.Intrinsic radiation resistance of mesenchymal cancer stem cells and implications for treatment response in a murine sarcoma model.Dan Med J.2012;59(2):B4388.
[28]Shishido Y,Ueno S,Yamazaki R,et al.ABCG2 inhibitor YHO-13351 sensitizes cancer stem/initiating-like side population cells to irinotecan.Anticancer Res.2013;33(4):1379-1386.
[29]张松灵,于小伟,魏振佟,等.宫颈癌干细胞表面标记物的筛选及Bcrpl(+)表型He La细胞[J].中华妇产科杂志,2012,45(7):525-529.
[30]Huang LW,Pan HS,Lin YH,et al.P16 methylation is an early event in cervical carcinogenesis.Int J Gynecol Cancer.2011;21(3):452-456.
[31]陈平平,中贵隽.天然抗癌药物活性成分研究进展[J].吉林中医药,2005,25(3):97-100.
[32]宋菁华,王克芳,李斌,等.人宫颈癌侧群细胞的分选及其生物学特性的研究[J].首都医科大学学报,2012,33(1):5-10.
[33]项峰,任利萍,刘克勤,等.子宫颈癌肿瘤干细胞筛选及体外敏感药物试验的临床对比研究[J].河北医药,2014,36(2):285-287.
[34]Li Z,Min W,Huang C,et al.Proteomics-based approach identified differentially expressed proteins with potential roles in endometrial carcinoma.Int J Gynecol Cancer.2010;20(1):9-15.
[35]张庆,赵文超,韩娜.肿瘤干细胞在人宫颈癌细胞株Si Ha耐顺铂效应中的作用[J].中国妇幼保健,2015,30(12):1908-1911.
[36]张铭,张一鸣,左伟,等.干细胞标志物SALL4在宫颈癌中的表达研究[J].重庆医学,2014,43(3):285-287.
[37]陈建琼,蒋艳,毛熙光,等.宫颈癌侧群细胞与肿瘤干细胞的研究进展[J].西南军医,2013,15(3):284-288.
[38]Jemal A,Bray F,Center MM,et al.Global cancer statistics.CA Cancer J Clin.2011;61(2):69-90.
[39]雷张涛.宫颈癌肿瘤干细胞的研究进展[J].医学综述,2012,18(8):1156-1158.
[40]曾令成,叶飞,韩林,等.脂肪酸结合蛋白5和细胞维甲酸结合蛋白2在脑胶质瘤中的表达[J].肿瘤防治研究,2015,42(3):242-245.
[41]白刚,罗林,王行桥,等.CRABP-Ⅱ、FABP-5在颅咽管瘤中的表达[J].昆明医科大学学报,2013,34(6):75-78.
[42]刘子后,李培军,陈庆良,等.心肌型脂肪酸结合蛋白的研究进展及在心脏外科中的应用[J].天津医药,2010,38(7):637-640.
[43]凌侠,姜艳梅.心型脂肪酸结合蛋白在急性冠脉综合征早期诊断与危险分层中的应用探讨[J].国际检验医学杂志,2010,31(9):961.963.
[44]李华,吕青,薛晖,等.肝型脂肪酸结合蛋白和脂肪酸合成酶在乳腺浸润性导管癌的表达变化及其意义[J].细胞与分子免疫学杂志,2009,25(7):600-602.
[45]董立华,李华,王凡,等.肝型脂肪酸结合蛋白及血管内皮生长因子在原发性肝癌中的表达以及相互关系[J].南方医科大学学报,2007,27(3):318-321.
[46]董解菊,肖颖彬.脂肪酸结合蛋白测定及临床应用研究进展[J].国外医学:临床生物化学与检验学分册,2001,22(2):74-76.
[2]关冰,李道明.3种常用抗癌药物对宫颈癌Hela细胞和肿瘤干细胞抑制作用研究[J].河南医学研究,2013,22(5):641-645.
[3]李海侠.影响子宫颈癌根治术后并发症的因素分析[J].中国中医药咨讯,2011,13(3):353.
[4]朱素文.子宫颈病变筛查的研究进展[J].医学理论与实践,2011,24(7):774-775.
[5]Thun MJ,De Lancey JO,Center MM,et al.The global burden of cancer:priorities for prevention.Carcinogenesis.2010;31(1):100-110.
[6]吴宁,朱博慧,薛英姿,等.干细胞生长因子反义寡核苷酸对肝癌细胞中原癌基因蛋白质c-kit及碱性成纤维细胞生长因子表达的影响[J].中华临床医师杂志:电子版,2011,5(12):3449-3454.
[7]Thumser AE,Moore JB,Plant NJ.Fatty acid binding proteins:tissue-specific functions in health and disease.Curr Opin Clin Nutr Metab Care.2014;17(2):124-129.
[8]王欢,刘富萍.VEGF-C在宫颈癌中的研究现状与展望[J].四川解剖学杂志,2011,19(3):30-33.
[9]曾广平.子宫颈恶性肿瘤介入治疗及护理进展[J].齐齐哈尔医学院学报,2011,32(9):1454-1456.
[10]Armstrong EH,Goswami D,Griffin PR,et al.Structural basis for ligand regulation of the fatty acid-binding protein 5,peroxisome proliferator-activated receptorβ/δ(FABP5-PPARβ/δ)signaling pathway.J Biol Chem.2014;289(21):14941-14954.
[11]王伟,殷红梅.宫颈癌组织中Fhit、survivin和Bcl-2的表达变化及意义[J].山东医药,2011,51(30):42-43.
[12]Fang LY,Wong TY,Chiang WF,et al.Fatty-acid-binding protein 5 promotes cell proliferation and invasion in oral squamous cell carcinoma.J Oral Pathol Med.2010;39(4):342-348.
[13]陈燕,林莺莺,陈岩松,等.原发性肝癌早期诊断中甲胎蛋白异质体检测的意义[J].福建医药杂志,2012,34(1):3-6.
[14]Gu TT,Liu SY,Zheng PS.Cytoplasmic NANOG-positive stromal cells promote human cervical cancer progression.Am J Pathol.2012;181(2):652-661.
[15]谢玲玲,卢淮武,钟沅月,等.分割剂量射线照射富集宫颈癌肿瘤干细胞的研究[J].中山大学学报:医学科学版,2012,33(3):330-335.
[16]Gu W,Yeo E,Mc Millan N,et al.Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability.Cancer Gene Ther.2011;18(12):897-905.
[17]李淑青,陈亚萍,杨恭,等.宫颈癌干细胞相关标记物的研究进展[J].现代妇产科进展,2014,23(11):931-936.
[18]肖松舒,蒋建发.宫颈癌干细胞的研究进展[J].现代妇产科进展,2013,22(9):756-758.
[19]Zhang SL,Wang YS,Zhou T,et al.Isolation and characterization of cancer stem cells from cervical cancer He La cells.Cytotechnology.2012;64(4):477-484.
[20]刘晨,安晓霞,张凡,等.肿瘤干细胞相关基因ABCB1、ABCG2表达与宫颈上皮恶变及发展关系的研究[J].现代妇产科进展,2012,21(4):294-297.
[21]李德冠,王小春,孟爱民,等.肿瘤干细胞靶向治疗[J].中国药理学通报,2009,25(6):701-703.
[22]张松灵,张立红,钟艳平,等.人宫颈癌组织中CK17和P63蛋白的表达及意义[J].吉林大学学报:医学版,2009,35(5):914-917.
[23]Choijamts B,Jimi S,Kondo T,et al.CD133+cancer stem cell-like cells derived from uterine carcinosarcoma(malignant mixed Müllerian tumor).Stem Cells.2011;29(10):1485-1495.
[24]Kato K,Takao T,Kuboyama A,et al.Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage Am J Pathol.2010;176(1):381-392.
[25]G?tte M,Greve B,Kelsch R,et al.The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/CIP1.Int J Cancer.2011;129(8):2042-2049.
[26]路春华,盛修贵.宫颈癌相关干细胞的研究进展[J].中华肿瘤防治杂志,2013,20(4):317-320.
[27]D’Andrea FP.Intrinsic radiation resistance of mesenchymal cancer stem cells and implications for treatment response in a murine sarcoma model.Dan Med J.2012;59(2):B4388.
[28]Shishido Y,Ueno S,Yamazaki R,et al.ABCG2 inhibitor YHO-13351 sensitizes cancer stem/initiating-like side population cells to irinotecan.Anticancer Res.2013;33(4):1379-1386.
[29]张松灵,于小伟,魏振佟,等.宫颈癌干细胞表面标记物的筛选及Bcrpl(+)表型He La细胞[J].中华妇产科杂志,2012,45(7):525-529.
[30]Huang LW,Pan HS,Lin YH,et al.P16 methylation is an early event in cervical carcinogenesis.Int J Gynecol Cancer.2011;21(3):452-456.
[31]陈平平,中贵隽.天然抗癌药物活性成分研究进展[J].吉林中医药,2005,25(3):97-100.
[32]宋菁华,王克芳,李斌,等.人宫颈癌侧群细胞的分选及其生物学特性的研究[J].首都医科大学学报,2012,33(1):5-10.
[33]项峰,任利萍,刘克勤,等.子宫颈癌肿瘤干细胞筛选及体外敏感药物试验的临床对比研究[J].河北医药,2014,36(2):285-287.
[34]Li Z,Min W,Huang C,et al.Proteomics-based approach identified differentially expressed proteins with potential roles in endometrial carcinoma.Int J Gynecol Cancer.2010;20(1):9-15.
[35]张庆,赵文超,韩娜.肿瘤干细胞在人宫颈癌细胞株Si Ha耐顺铂效应中的作用[J].中国妇幼保健,2015,30(12):1908-1911.
[36]张铭,张一鸣,左伟,等.干细胞标志物SALL4在宫颈癌中的表达研究[J].重庆医学,2014,43(3):285-287.
[37]陈建琼,蒋艳,毛熙光,等.宫颈癌侧群细胞与肿瘤干细胞的研究进展[J].西南军医,2013,15(3):284-288.
[38]Jemal A,Bray F,Center MM,et al.Global cancer statistics.CA Cancer J Clin.2011;61(2):69-90.
[39]雷张涛.宫颈癌肿瘤干细胞的研究进展[J].医学综述,2012,18(8):1156-1158.
[40]曾令成,叶飞,韩林,等.脂肪酸结合蛋白5和细胞维甲酸结合蛋白2在脑胶质瘤中的表达[J].肿瘤防治研究,2015,42(3):242-245.
[41]白刚,罗林,王行桥,等.CRABP-Ⅱ、FABP-5在颅咽管瘤中的表达[J].昆明医科大学学报,2013,34(6):75-78.
[42]刘子后,李培军,陈庆良,等.心肌型脂肪酸结合蛋白的研究进展及在心脏外科中的应用[J].天津医药,2010,38(7):637-640.
[43]凌侠,姜艳梅.心型脂肪酸结合蛋白在急性冠脉综合征早期诊断与危险分层中的应用探讨[J].国际检验医学杂志,2010,31(9):961.963.
[44]李华,吕青,薛晖,等.肝型脂肪酸结合蛋白和脂肪酸合成酶在乳腺浸润性导管癌的表达变化及其意义[J].细胞与分子免疫学杂志,2009,25(7):600-602.
[45]董立华,李华,王凡,等.肝型脂肪酸结合蛋白及血管内皮生长因子在原发性肝癌中的表达以及相互关系[J].南方医科大学学报,2007,27(3):318-321.
[46]董解菊,肖颖彬.脂肪酸结合蛋白测定及临床应用研究进展[J].国外医学:临床生物化学与检验学分册,2001,22(2):74-76.
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