YAP1在食管癌KYSE30和EC9706细胞侵袭和转移中的作用机制探讨
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摘要
目的探讨Yes相关蛋白(YAP)在食管癌KYSE30和EC9706细胞侵袭转移中的作用。方法将不做任何处理空白食管癌KYSE30和EC9706细胞分别作为A组和E组,将只添加Lipofectamine 2000转染试剂的食管癌KYSE30和EC9706细胞分别作为B组和F组,将转染无义干扰小RNA(siRNA)和YAP1 siRNA的食管癌KYSE30细胞和EC9706细胞分别作为C组、D组和G组、H组。逆转录聚合酶链反应(RT-PCR)和蛋白质印迹(Western blot)法检测基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)、血管内皮生长因子(VEGF)和YAP1 mRNA及其对应蛋白的相对表达量,CCK8法、Transwell小室和划痕实验检测各组细胞增殖、侵袭和迁移能力。结果 D组细胞中MMP2、VEGF、YAP1 m RNA和蛋白的相对表达量均低于A、B和C组细胞(P﹤0.05),H组细胞MMP2、VEGF、YAP1 mRNA和蛋白的相对表达量均低于E、F和G组细胞(P﹤0.05)。转染48、72 h后,D组细胞OD值低于A、B和C组(P﹤0.05),H组细胞OD值低于E、F和G组细胞(P﹤0.05)。D组穿膜细胞数少于A、B和C组细胞(P﹤0.05),划痕愈合距离小于A、B和C组细胞(P﹤0.05);H组穿膜细胞数少于E、F和G组细胞(P﹤0.05),划痕愈合距离小于E、F和G组细胞(P﹤0.05)。结论 YAP1可能通过抑制MMP2和VEGF的表达,抑制食管癌细胞的侵袭和转移,有望成为抑制食管癌恶性进程的有效靶点。
Objective To explore the role of Yes-associated protein 1(YAP1) in the invasion and metastasis of esophageal cell lines KYSE30 and EC9706. Method White esophageal cancer KYSE30 and EC9706 cells were used as group A and group E, respectively. Esophageal cancer KYSE30 and EC9706 cells supplemented with Lipofectamine 2000 transfection reagent were used as group B and F, respectively. Nonsense interfering small interfering RNA(siRNA) and YAP1 siRNA were transfected into esophageal cell lines KYSE30(as group C and D) and EC9706(as Group G and H), respectively. Reverse transcription polymerase chain reaction(RT-PCR) and Western blot method were employed to detect the mRNA and relative protein expression of matrix metalloproteinase 2(MMP2), MMP9, vascular endothelial growth factor(VEGF), and YAP1,besides, CCK8, Transwell chamber assay, and scratch wound healing assay were applied to detect the proliferation, invasion, and migration ability of KYSE30 and EC9706 cells. Result The relative expressions of MMP2 mRNA, VEGF mRNA, YAP1 mRNA, MMP2, VEGF and YAP1 proteins in group D were lower than those in group A, B and C(P<0.05). The relative expressions of MMP2 m RNA, VEGF mRNA, YAP1 mRNA, MMP2, VEGF and YAP1 proteins in groupH were lower than those in group E, F and G(P<0.05). After 48 and 72 h of transfection, the optical density(OD) in cells of group D were lower than those of group A, B and C(P<0.05), and the OD values of groupH were lower than those of group E, F and G(P<0.05). The counts of invading cells in group D was less than that in groups A, B and C(P<0.05), the healing distance of scratches was smaller than that of groups A, B and C(P<0.05). The number of perforated cells in groupH was lower than that of E, F and G(P<0.05), and the scratch wound healing assay of the cells in H was smaller than that of the E, F and G(P<0.05). Conclusion YAP1 may, through inhibiting the expression of MMP2 and VEGF, restrain the invasion and metastasis of esophageal cells, and thus may become an effective potential target for blocking the progression of malignant esophageal cancer.
Objective To explore the role of Yes-associated protein 1(YAP1) in the invasion and metastasis of esophageal cell lines KYSE30 and EC9706. Method White esophageal cancer KYSE30 and EC9706 cells were used as group A and group E, respectively. Esophageal cancer KYSE30 and EC9706 cells supplemented with Lipofectamine 2000 transfection reagent were used as group B and F, respectively. Nonsense interfering small interfering RNA(siRNA) and YAP1 siRNA were transfected into esophageal cell lines KYSE30(as group C and D) and EC9706(as Group G and H), respectively. Reverse transcription polymerase chain reaction(RT-PCR) and Western blot method were employed to detect the mRNA and relative protein expression of matrix metalloproteinase 2(MMP2), MMP9, vascular endothelial growth factor(VEGF), and YAP1,besides, CCK8, Transwell chamber assay, and scratch wound healing assay were applied to detect the proliferation, invasion, and migration ability of KYSE30 and EC9706 cells. Result The relative expressions of MMP2 mRNA, VEGF mRNA, YAP1 mRNA, MMP2, VEGF and YAP1 proteins in group D were lower than those in group A, B and C(P<0.05). The relative expressions of MMP2 m RNA, VEGF mRNA, YAP1 mRNA, MMP2, VEGF and YAP1 proteins in groupH were lower than those in group E, F and G(P<0.05). After 48 and 72 h of transfection, the optical density(OD) in cells of group D were lower than those of group A, B and C(P<0.05), and the OD values of groupH were lower than those of group E, F and G(P<0.05). The counts of invading cells in group D was less than that in groups A, B and C(P<0.05), the healing distance of scratches was smaller than that of groups A, B and C(P<0.05). The number of perforated cells in groupH was lower than that of E, F and G(P<0.05), and the scratch wound healing assay of the cells in H was smaller than that of the E, F and G(P<0.05). Conclusion YAP1 may, through inhibiting the expression of MMP2 and VEGF, restrain the invasion and metastasis of esophageal cells, and thus may become an effective potential target for blocking the progression of malignant esophageal cancer.
引文
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[2]霍小东. ZEB2在食管癌侵袭转移中的作用及其机制研究[D].天津:天津医科大学, 2017.
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[8]卢洪飞,万福生. Hippo-YAP信号通路在结直肠癌发生发展中的作用[J].生命的化学, 2014, 34(4):506-511.
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[10] Lamar JM, Stern P, Liu H, et al. The Hippo pathway tar-get, YAP, promotes metastasis through its TEAD-interaction domain[J]. Proc Natl Acad Sci U S A, 2012, 109(37):E2441-E2450.
[11]贾灿灿,黄飞,王远志,等. LPS刺激对肝细胞YAP和pYAP表达的影响[J].热带医学杂志, 2016, 16(5):557-560.
[12]周西广. IKKε通过YAP1对胶质瘤细胞的细胞学行为影响的初步研究[D].天津:天津医科大学, 2016.
[13]魏晓龙. YAP1/KLF5转录激活Ascl2调控结肠癌CSCs自更新的分子机制[D].重庆:第三军医大学, 2017.
[14]张红珠,张翠萍,张琪,等. MST1与YAP1在食管疾病中的表达及意义[J].青岛大学医学院学报, 2014, 50(1):62-63; 67.
[15]宋杰.干扰YAP1对食管癌Eca-109细胞生物学功能的影响[D].重庆:重庆医科大学, 2015.
[16]郭业兵.α-SMA、MMP-2、b FGF在子宫颈微小浸润癌间质中的表达与意义[D].济南:山东大学, 2017.
[17]袁静萍,陈创,袁修学,等.乳腺癌中VEGF和MMP9的免疫组织化学表达及其与肿瘤转移复发的关系[J].中国组织化学与细胞化学杂志, 2016, 25(5):431-436.
[18]段泽星,谢立群. VEGF在肿瘤生长和血管生成中的作用[J].世界华人消化杂志, 2010, 18(27):2894-2900.
[2]霍小东. ZEB2在食管癌侵袭转移中的作用及其机制研究[D].天津:天津医科大学, 2017.
[3]宋娟,叶小群. Hippo-YAP信号通路为靶点的肿瘤治疗研究进展[J].中国肿瘤临床, 2015, 42(17):876-880.
[4]李道胜,班媛媛,侯刚. YAP通过Hippo信号通路在肿瘤发生发展中的作用[J/OL].中华临床医师杂志(电子版), 2016, 10(7):1016-1020[2017-12-01]. http://med.wanfangdata.com.cn/Paper/Detail/PeriodicalP aper_zhlcyszz201607023.
[5] Zygulska AL, Krzemieniecki K, Pierzchalski P. Hippo pathway-brief overview of its relevance in cancer[J]. J Physiol Pharmacol, 2017, 68(3):311-335.
[6]韩新影,孙杰,李艳艳,等. YAP、VGLL4蛋白在胃癌组织中的表达及对侵袭和转移的影响[J].现代肿瘤医学,2018, 26(2):237-240.
[7]张素青. FOXP1和YAP基因在肝细胞肝癌细胞增殖中的作用研究[D].苏州:苏州大学, 2015.
[8]卢洪飞,万福生. Hippo-YAP信号通路在结直肠癌发生发展中的作用[J].生命的化学, 2014, 34(4):506-511.
[9] Shi P, Feng J, Chen C. Hippo pathway in mammary gland development and breast cancer[J]. Acta Biochim Biophys Sin(Shanghai), 2015, 47(1):53-59.
[10] Lamar JM, Stern P, Liu H, et al. The Hippo pathway tar-get, YAP, promotes metastasis through its TEAD-interaction domain[J]. Proc Natl Acad Sci U S A, 2012, 109(37):E2441-E2450.
[11]贾灿灿,黄飞,王远志,等. LPS刺激对肝细胞YAP和pYAP表达的影响[J].热带医学杂志, 2016, 16(5):557-560.
[12]周西广. IKKε通过YAP1对胶质瘤细胞的细胞学行为影响的初步研究[D].天津:天津医科大学, 2016.
[13]魏晓龙. YAP1/KLF5转录激活Ascl2调控结肠癌CSCs自更新的分子机制[D].重庆:第三军医大学, 2017.
[14]张红珠,张翠萍,张琪,等. MST1与YAP1在食管疾病中的表达及意义[J].青岛大学医学院学报, 2014, 50(1):62-63; 67.
[15]宋杰.干扰YAP1对食管癌Eca-109细胞生物学功能的影响[D].重庆:重庆医科大学, 2015.
[16]郭业兵.α-SMA、MMP-2、b FGF在子宫颈微小浸润癌间质中的表达与意义[D].济南:山东大学, 2017.
[17]袁静萍,陈创,袁修学,等.乳腺癌中VEGF和MMP9的免疫组织化学表达及其与肿瘤转移复发的关系[J].中国组织化学与细胞化学杂志, 2016, 25(5):431-436.
[18]段泽星,谢立群. VEGF在肿瘤生长和血管生成中的作用[J].世界华人消化杂志, 2010, 18(27):2894-2900.