Wnt/β-catenin信号通路在卵巢癌干细胞增殖、迁移和侵袭中的作用
|
摘要
背景:在肿瘤细胞中,β-catenin降解障碍导致胞浆内游离的β-catenin聚集并与TCF/LEF结合进入细胞核内,启动下游基因转录,诱发Wnt/β-catenin信号通路异常改变。因此,研究β-catenin与卵巢癌干细胞的关系,可为特异性抑制卵巢癌干细胞提供新的思路。目的:观察β-catenin基因沉默后卵巢癌干细胞增殖、迁移与侵袭能力的变化。方法:采用免疫磁珠法从人卵巢癌细胞A2780中分离CD133阳性卵巢癌干细胞。选取70%-80%融合的CD133阳性细胞,分别转染慢病毒p LKO.1-sh RNA-β-catenin(实验组)与p LKO.1-sh RNA-ctrl(对照组)质粒,转染后72 h,采用Western blot检测两组细胞β-catenin、上皮表型分子E-cadherin和间质表型分子Vimentin的表达,MTT法检测两组细胞增殖活性,Transwell小室实验检测两组细胞迁移与侵袭能力。结果与结论:(1)转染后72 h,实验组细胞β-catenin蛋白明显低于对照组(P<0.05);(2)实验组培养2,3 d的细胞增殖能力低于对照组(P<0.05);(3)转染后72 h,实验组细胞迁移能力、侵袭能力明显低于对照组(P<0.05);(4)转染后72 h,实验组上皮表型分子E-cadherin蛋白表达高于对照组(P<0.05),间质表型分子Vimentin蛋白表达低于对照组(P<0.05);(5)结果表明,Wnt/β-catenin信号通路参与了卵巢癌干细胞的迁移与侵袭,沉默β-catenin基因表达可抑制卵巢癌干细胞的增殖、迁移及侵袭能力。
BACKGROUND: Inability of degradation makes free cytosolic β-catenin in tumor cells aggregate and bind to TCF/LEF into the nuclei, which initiates transcription of downstream genes, and eventually induces abnormal changes in Wnt/β-catenin signaling pathways. Therefore, investigations on the relationship between β-catenin and ovarian cancer stem cells can provide a new insight into the specific inhibition of ovarian cancer stem cells.OBJECTIVE: To observe the proliferation, migration and invasion abilities of ovarian cancer stem cells after β-catenin gene silencing. METHODS: CD133 positive tumor stem cells were isolated from human ovarian cancer cell line A2780 by immunomagnetic beads. CD133 positive cells with 70%-80% confluence were selected and transfected with p LKO.1-sh RNA-β-catenin(experimental group) and p LKO.1-sh RNA-ctrln(control group), respectively. The expression of β-catenin, E-cadherin and Vimentin were detected by western blot at 72 hours after transfection. Cell proliferation was detected by MTT, and cell migration and invasion ability was detected by Transwell chamber assay. RESULTS AND CONCLUSION:(1) The expression of β-catenin protein in the experimental group was significantly lower than that in the control group at 72 hours after transfection(P < 0.05).(2) With the extension of culture time, the proliferation ability of the two groups increased gradually, but the cell proliferation ability of the experimental group was lower than that of the control group at 2 and 3 days after transfection(P < 0.05).(3) After 72 hours of transfection, the cell migration and invasion abilities of the experimental group were significantly lower than those of the control group(P < 0.05).(4) At 72 hours after transfection, the expression of E-cadherin protein in the experimental group was significantly higher than that of the control group(P < 0.05), and the expression of Vimentin protein was lower than that of the control group(P < 0.05). To conclude, the Wnt/β-catenin signaling pathway is involved in the migration and invasion of ovarian cancer stem cells. β-catenin silencing can inhibit the proliferation, migration and invasion of ovarian cancer stem cells.
BACKGROUND: Inability of degradation makes free cytosolic β-catenin in tumor cells aggregate and bind to TCF/LEF into the nuclei, which initiates transcription of downstream genes, and eventually induces abnormal changes in Wnt/β-catenin signaling pathways. Therefore, investigations on the relationship between β-catenin and ovarian cancer stem cells can provide a new insight into the specific inhibition of ovarian cancer stem cells.OBJECTIVE: To observe the proliferation, migration and invasion abilities of ovarian cancer stem cells after β-catenin gene silencing. METHODS: CD133 positive tumor stem cells were isolated from human ovarian cancer cell line A2780 by immunomagnetic beads. CD133 positive cells with 70%-80% confluence were selected and transfected with p LKO.1-sh RNA-β-catenin(experimental group) and p LKO.1-sh RNA-ctrln(control group), respectively. The expression of β-catenin, E-cadherin and Vimentin were detected by western blot at 72 hours after transfection. Cell proliferation was detected by MTT, and cell migration and invasion ability was detected by Transwell chamber assay. RESULTS AND CONCLUSION:(1) The expression of β-catenin protein in the experimental group was significantly lower than that in the control group at 72 hours after transfection(P < 0.05).(2) With the extension of culture time, the proliferation ability of the two groups increased gradually, but the cell proliferation ability of the experimental group was lower than that of the control group at 2 and 3 days after transfection(P < 0.05).(3) After 72 hours of transfection, the cell migration and invasion abilities of the experimental group were significantly lower than those of the control group(P < 0.05).(4) At 72 hours after transfection, the expression of E-cadherin protein in the experimental group was significantly higher than that of the control group(P < 0.05), and the expression of Vimentin protein was lower than that of the control group(P < 0.05). To conclude, the Wnt/β-catenin signaling pathway is involved in the migration and invasion of ovarian cancer stem cells. β-catenin silencing can inhibit the proliferation, migration and invasion of ovarian cancer stem cells.
引文
[1]陈万青,郑荣寿,曾红梅,等.2011年中国恶性肿瘤发病和死亡分析[J].中国肿瘤,2015,24(1):1-10.
[2]施月琴.2007-2015年南通市三区育龄妇女恶性肿瘤死因分析[J].海南医学,2017,28(4):669-671.
[3]Van Hoesen K,Meynier S,Ribaux P,et al.Circulating GRP78antibodies from ovarian cancer patients:a promising tool for cancer cell targeting drug delivery system.Oncotarget.2017;8(63):107176-107187.
[4]Chien J,Poole EM.Ovarian Cancer Prevention,Screening,and Early Detection:Report From the 11th Biennial Ovarian Cancer Research Symposium.Int J Gynecol Cancer.2017;27(9S Suppl 5):S20-S22.
[5]Chien J,Mehta G.Meeting Report From the 2016 11th Biennial Ovarian Cancer Research Symposium:Mechanisms of Initiation and Progression of Ovarian Cancers.Int J Gynecol Cancer.2017;27(9S Suppl 5):S10-S13.
[6]曹颖,傅士龙.宫颈癌与肿瘤干细胞的研究进展[J].江苏医药,2017,43(5):356-359.
[7]宋东颖,王毅,孙岚,等.肿瘤干细胞理论及肿瘤干细胞分离和鉴定研究进展[J].中国药理学与毒理学杂志,2012,26(5):674-679.
[8]Jiang J,Ye F,Yang X,et al.Peri-tumor associated fibroblasts promote intrahepatic metastasis of hepatocellular carcinoma by recruiting cancer stem cells.Cancer Lett.2017;404:19-28.
[9]Wu L,Han L,Zhou C,et al.TGF-β1-induced CK17 enhances cancer stem cell-like properties rather than EMT in promoting cervical cancer metastasis via the ERK1/2-MZF1 signaling pathway.FEBS J.2017;284(18):3000-3017.
[10]郑帅,王晓东,崔岱,等.甲状腺未分化癌耐药和转移中肿瘤干细胞和上皮细胞间质的转化作用[J].南京医科大学学报:自然科学版,2014,34(12):1627-1631,1637.
[11]Whiteside TL.Exosome and mesenchymal stem cell cross-talk in the tumor microenvironment.Semin Immunol.2018;35:69-79.
[12]Zanjani LS,Madjd Z,Abolhasani M,et al.Expression of CD105 cancer stem cell marker in three subtypes of renal cell carcinoma.Cancer Biomark.2017 Dec 20.doi:10.3233/CBM-170755.[Epub ahead of print]
[13]杨森,赖钊,王学文,等.手术切除联合肝动脉化疗栓塞对术后血清肿瘤标志物含量及肿瘤复发过程中干细胞特性的影响[J].海南医学院学报,2017,23(9):1244-1247.
[14]刘蕾,郭宏强,杨树军.肿瘤干细胞信号通路[J].医药论坛杂志,2014,35(5):176.
[15]李月雅,李凯.Wnt/β-Catenin信号通路对肿瘤干细胞作用的研究进展[J].山东医药,2016,56(9):98-100.
[16]Wilms C,Kroeger CM,Hainzl AV,et al.MYSM1/2A-DUB is an epigenetic regulator in human melanoma and contributes to tumor cell growth.Oncotarget.2017;8(40):67287-67299.
[17]Li B,Yu F,Wu F,et al.EZH2 Impairs Human Dental Pulp Cell Mineralization via the Wnt/β-Catenin Pathway.J Dent Res.2017 Dec 1 doi:10.1177/0022034517746987.[Epub ahead of print]
[18]许峰,程巧莲,张忆雯,等.Let-7通过抑制Wnt信号通路激活而促进食管癌干细胞不对称分裂的研究[J].临床医学研究与实践,2017,2(16):8-12.
[19]马勇,王礼宁,郭杨,等.姜黄素通过调节Wnt/β-catenin信号通路促进软骨细胞增殖的研究[J].广州中医药大学学报,2017,34(1):90-95.
[20]王海全.转化生长因子β1通过Wnt/β-catenin信号通路诱导人肝癌细胞SMMC-7721上皮间质转化的实验研究[D].济南:山东大学,2016.
[21]熊姗姗.Wnt信号通路在维持鼻咽癌SP细胞“干”性的机制研究[D].广州:南方医科大学,2016.
[22]姜睿.Wnt/B-catenin信号通路与鼻咽癌肿瘤干细胞发生的关系[D].广州:南方医科大学,2011.
[23]贾冬丽,李红雨,秦巧红卵巢上皮性癌组织中SFRP5基因启动子区甲基化与β-catenin蛋白表达检测[J].郑州大学学报:医学版,2013,48(3):355-358.
[24]卢淮武,王东雁,刘昀昀,等.半乳糖凝集素-3与β-Catenin在上皮性卵巢癌中的表达和临床指标及预后关系[J].中山大学学报:医学科学版,2015,36(6):883-888.
[25]林盛,龙海霞,向橦,等.人卵巢癌组织肿瘤干细胞的分离和鉴定[J].中华肿瘤杂志,2011,33(12):896-899.
[26]宋东颖,王毅,孙岚,等.肿瘤干细胞理论及肿瘤干细胞分离和鉴定研究进展[J].中国药理学与毒理学杂志,2012,26(5):674-679.
[27]宋长山,谭家驹.干细胞与肿瘤干细胞[J].新乡医学院学报,2006,23(4):417-420.
[28]Zhong W,Chen S,Qin Y,et al.Doxycycline inhibits breast cancer EMT and metastasis through PAR-1/NF-κB/mi R-17/E-cadherin pathway.Oncotarget.2017;8(62):104855-104866.
[29]Zuo LL,Zhang J,Liu LZ,et al.Cadherin 6 is activated by Epstein-Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma.Oncogenesis.2017;6(12):402.
[2]施月琴.2007-2015年南通市三区育龄妇女恶性肿瘤死因分析[J].海南医学,2017,28(4):669-671.
[3]Van Hoesen K,Meynier S,Ribaux P,et al.Circulating GRP78antibodies from ovarian cancer patients:a promising tool for cancer cell targeting drug delivery system.Oncotarget.2017;8(63):107176-107187.
[4]Chien J,Poole EM.Ovarian Cancer Prevention,Screening,and Early Detection:Report From the 11th Biennial Ovarian Cancer Research Symposium.Int J Gynecol Cancer.2017;27(9S Suppl 5):S20-S22.
[5]Chien J,Mehta G.Meeting Report From the 2016 11th Biennial Ovarian Cancer Research Symposium:Mechanisms of Initiation and Progression of Ovarian Cancers.Int J Gynecol Cancer.2017;27(9S Suppl 5):S10-S13.
[6]曹颖,傅士龙.宫颈癌与肿瘤干细胞的研究进展[J].江苏医药,2017,43(5):356-359.
[7]宋东颖,王毅,孙岚,等.肿瘤干细胞理论及肿瘤干细胞分离和鉴定研究进展[J].中国药理学与毒理学杂志,2012,26(5):674-679.
[8]Jiang J,Ye F,Yang X,et al.Peri-tumor associated fibroblasts promote intrahepatic metastasis of hepatocellular carcinoma by recruiting cancer stem cells.Cancer Lett.2017;404:19-28.
[9]Wu L,Han L,Zhou C,et al.TGF-β1-induced CK17 enhances cancer stem cell-like properties rather than EMT in promoting cervical cancer metastasis via the ERK1/2-MZF1 signaling pathway.FEBS J.2017;284(18):3000-3017.
[10]郑帅,王晓东,崔岱,等.甲状腺未分化癌耐药和转移中肿瘤干细胞和上皮细胞间质的转化作用[J].南京医科大学学报:自然科学版,2014,34(12):1627-1631,1637.
[11]Whiteside TL.Exosome and mesenchymal stem cell cross-talk in the tumor microenvironment.Semin Immunol.2018;35:69-79.
[12]Zanjani LS,Madjd Z,Abolhasani M,et al.Expression of CD105 cancer stem cell marker in three subtypes of renal cell carcinoma.Cancer Biomark.2017 Dec 20.doi:10.3233/CBM-170755.[Epub ahead of print]
[13]杨森,赖钊,王学文,等.手术切除联合肝动脉化疗栓塞对术后血清肿瘤标志物含量及肿瘤复发过程中干细胞特性的影响[J].海南医学院学报,2017,23(9):1244-1247.
[14]刘蕾,郭宏强,杨树军.肿瘤干细胞信号通路[J].医药论坛杂志,2014,35(5):176.
[15]李月雅,李凯.Wnt/β-Catenin信号通路对肿瘤干细胞作用的研究进展[J].山东医药,2016,56(9):98-100.
[16]Wilms C,Kroeger CM,Hainzl AV,et al.MYSM1/2A-DUB is an epigenetic regulator in human melanoma and contributes to tumor cell growth.Oncotarget.2017;8(40):67287-67299.
[17]Li B,Yu F,Wu F,et al.EZH2 Impairs Human Dental Pulp Cell Mineralization via the Wnt/β-Catenin Pathway.J Dent Res.2017 Dec 1 doi:10.1177/0022034517746987.[Epub ahead of print]
[18]许峰,程巧莲,张忆雯,等.Let-7通过抑制Wnt信号通路激活而促进食管癌干细胞不对称分裂的研究[J].临床医学研究与实践,2017,2(16):8-12.
[19]马勇,王礼宁,郭杨,等.姜黄素通过调节Wnt/β-catenin信号通路促进软骨细胞增殖的研究[J].广州中医药大学学报,2017,34(1):90-95.
[20]王海全.转化生长因子β1通过Wnt/β-catenin信号通路诱导人肝癌细胞SMMC-7721上皮间质转化的实验研究[D].济南:山东大学,2016.
[21]熊姗姗.Wnt信号通路在维持鼻咽癌SP细胞“干”性的机制研究[D].广州:南方医科大学,2016.
[22]姜睿.Wnt/B-catenin信号通路与鼻咽癌肿瘤干细胞发生的关系[D].广州:南方医科大学,2011.
[23]贾冬丽,李红雨,秦巧红卵巢上皮性癌组织中SFRP5基因启动子区甲基化与β-catenin蛋白表达检测[J].郑州大学学报:医学版,2013,48(3):355-358.
[24]卢淮武,王东雁,刘昀昀,等.半乳糖凝集素-3与β-Catenin在上皮性卵巢癌中的表达和临床指标及预后关系[J].中山大学学报:医学科学版,2015,36(6):883-888.
[25]林盛,龙海霞,向橦,等.人卵巢癌组织肿瘤干细胞的分离和鉴定[J].中华肿瘤杂志,2011,33(12):896-899.
[26]宋东颖,王毅,孙岚,等.肿瘤干细胞理论及肿瘤干细胞分离和鉴定研究进展[J].中国药理学与毒理学杂志,2012,26(5):674-679.
[27]宋长山,谭家驹.干细胞与肿瘤干细胞[J].新乡医学院学报,2006,23(4):417-420.
[28]Zhong W,Chen S,Qin Y,et al.Doxycycline inhibits breast cancer EMT and metastasis through PAR-1/NF-κB/mi R-17/E-cadherin pathway.Oncotarget.2017;8(62):104855-104866.
[29]Zuo LL,Zhang J,Liu LZ,et al.Cadherin 6 is activated by Epstein-Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma.Oncogenesis.2017;6(12):402.