miR-19a调控血小板反应蛋白在结肠癌淋巴转移中的机制
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摘要
目的探讨miR-19a调控血小板反应蛋白(THBS1)在结肠癌淋巴转移中的机制。方法采用实时定量聚合酶链反应(qRT-PCR)的方法检测不同结肠癌细胞株(LOVO、HT116、SW480、HT29)中的miR-19a的表达水平;蛋白免疫印迹法(Western blot)/免疫共沉淀(CO-IP)技术检测LOVO与HT29细胞中THBS1及血管内皮生长因子C/D(VEGF-C/D)蛋白结合及表达情况;Western blot法检测转染后细胞中THBS1、VEGF-C/D的表达情况;细胞划痕法检测转染后细胞的迁移能力。取shRNA、shRNA-NC转染后的LOVO细胞以及正常LOVO细胞分别接种于裸鼠皮下,建立裸鼠移植瘤模型观察成瘤情况,免疫组化法检测肿瘤组织中CD34的表达,Western blot法检测瘤体中THBS1、VEGF-C、VEGF-D蛋白的表达。结果细胞株LOVO和HT29的miR-19a的表达量分别为最高和最低;THBS1蛋白在HT29细胞中高表达,VEGF-C/D蛋白在LOVO细胞中高表达;LOVO和HT29细胞中THBS1和VEGF-C免疫共沉淀反应为阳性;转染miR-19a后,LOVO细胞的迁移能力显著下降,而HT29细胞迁移能力有显著上升。注射了miR-NA抑制物组的裸鼠瘤块明显变小;瘤块组织中THBS1的蛋白表达显著上升,VEGF-C的蛋白表达显著下降,CD34的阳性表达降低。结论 miR-19a能够通过下调THBS1促进VEGF-C的表达,从而促进结肠癌淋巴结转移。
Objective To investigate the role of miR-19 ain the regulation of thrombospondin(THBS1)in lymphatic metastasis of colon cancer.Methods qRT-PCR was used to detect the expression level of miR-19 ain different colon cancer cell lines(LOVO,HT116,SW480,HT29)and Western blotting/co-immunoprecipitation(CO-IP)to detect the binding and expression of THBS1 and vascular endothelial growth factor C/D(VEGF-C/D)protein in LOVO and HT29 cells.Western blotting was employed to detect the expression of THBS1 and VEGF-C/D in the transfected cells.The cell scratching method was used to detect the migratory ability of the cells after transfection.The nude mouse xenograft model was established by subcutaneously inoculating normal LOVO cells or LOVO cells transfected with shRNA or shRNA-NC.The expression of CD34 in tumor tissues was detected by immunohistochemistry and the expression of THBS1,VEGF-C and VEGF-D proteins by Western blotting.Results The expression of miR-19 awas the highest in LOVO cells and the lowest in the HT29 cells.The THBS1 protein was highly expressed in HT29 cells,and the VEGF-C/D protein highly expressed in LOVO cells.The co-immunoprecipitation reaction of THBS1 and VEGF-C was positive in LOVO and HT29 cells.After transfection of miR-19 a,the migratory ability of LOVO cells was significantly decreased,while the migratory ability of HT29 cells was significantly increased.The tumor of nude mice injected with miRNA inhibitor was significantly smaller,the protein expression of THBS1 in tumor tissues was significantly increased,and the expression levels of VEGF-C and CD34 were significantly decreased.Conclusion miR-19 acan promote the lymph node metastasis of colon cancer by upregulating the expression of VEGF-C and downregulating THBS1.
Objective To investigate the role of miR-19 ain the regulation of thrombospondin(THBS1)in lymphatic metastasis of colon cancer.Methods qRT-PCR was used to detect the expression level of miR-19 ain different colon cancer cell lines(LOVO,HT116,SW480,HT29)and Western blotting/co-immunoprecipitation(CO-IP)to detect the binding and expression of THBS1 and vascular endothelial growth factor C/D(VEGF-C/D)protein in LOVO and HT29 cells.Western blotting was employed to detect the expression of THBS1 and VEGF-C/D in the transfected cells.The cell scratching method was used to detect the migratory ability of the cells after transfection.The nude mouse xenograft model was established by subcutaneously inoculating normal LOVO cells or LOVO cells transfected with shRNA or shRNA-NC.The expression of CD34 in tumor tissues was detected by immunohistochemistry and the expression of THBS1,VEGF-C and VEGF-D proteins by Western blotting.Results The expression of miR-19 awas the highest in LOVO cells and the lowest in the HT29 cells.The THBS1 protein was highly expressed in HT29 cells,and the VEGF-C/D protein highly expressed in LOVO cells.The co-immunoprecipitation reaction of THBS1 and VEGF-C was positive in LOVO and HT29 cells.After transfection of miR-19 a,the migratory ability of LOVO cells was significantly decreased,while the migratory ability of HT29 cells was significantly increased.The tumor of nude mice injected with miRNA inhibitor was significantly smaller,the protein expression of THBS1 in tumor tissues was significantly increased,and the expression levels of VEGF-C and CD34 were significantly decreased.Conclusion miR-19 acan promote the lymph node metastasis of colon cancer by upregulating the expression of VEGF-C and downregulating THBS1.
引文
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[21]周晓黎,舒磊,廖艳,等.PI3K/AKT通路在低氧环境下对结肠癌细胞HIF-1α及糖酵解的作用[J].华中科技大学学报:医学版,2018,47(2):203-206.
[22]杨庚,李昱,贺亚峰.miRNA-19a对结肠癌细胞增殖活性的影响[J].中国老年学杂志,2018,38(23):5804-5807.
[2]李道娟,李倩,贺宇彤.结直肠癌流行病学趋势[J].肿瘤防治研究,2015,42(3):305-310.
[3]凌旭坤,陈创奇.淋巴结转移评估与结直肠癌预后关系的研究进展[J].消化肿瘤杂志(电子版),2016,8(2):116-120.
[4]Yang J,Hao Y,Xi J J.Therapeutic application of MicroRNAs against human cancers[J].J Lab Autom,2013,18(1):30-33.
[5]Tutar Y.miRNA and cancer;computational and experimental approaches[J].Curr Pharm Biotechnol,2014,15(5):429.
[6]Liu Y,Liu R,Yang F,et al.miR-19apromotes colorectal cancer proliferation and migration by targeting TIA1[J].Mol Cancer,2017,16(1):53.
[7]Liu Z,Morgan S,Ren J,et al.Thrombospondin-1(TSP1)contributes to the development of vascular inflammation by regulating monocytic cell motility in mouse models of abdominal aortic aneurysm[J].Circ Res,2015,117(2):129-141.
[8]刘涛.miR-19a下调THBS1促进结直肠癌转移的作用及机制研究[D]:重庆:第三军医大学,2016.
[9]郑树,张苏展,黄彦钦.结直肠癌研究30年回顾和现状[J].实用肿瘤杂志,2016,31(1):2-5.
[10]Arnold M,Sierra M S,Laversanne M,et al.Global patterns and trends in colorectal cancer incidence and mortality[J].Gut,2017,66(4):683-691.
[11]张林西,郭颖,白美玲.新生血管、淋巴管在结直肠癌进展中的作用[J].中国老年学杂志,2016,36(5):1116-1118.
[12]Naxerova K,Reiter J G.Origins of lymphatic and distant metastases in human colorectal cancer[J].Science,2017,357(6346):55-60.
[13]Jin M,Frankel W L.Lymph node metastasis in colorectal cancer[J].Surg Oncol Clin N Am,2018,27(2):401-412.
[14]王桂梅,王彩霞,聂美楠,等.RegⅣ、VEGF-C在结肠癌中的表达及意义[J].黑龙江医药科学,2016,39(4):91-92,94.
[15]Szajewski M,Kruszewski W J,Lakomy J,et al.VEGF-C expression is not a prognostic factor in locally advanced colon adenocarcinoma[J].Contemporary Oncology(Poznan,Poland),2015,19(6):446-450.
[16]Su F,Li X,You K,et al.Expression of VEGF-D,SMAD4,and SMAD7and their relationship with lymphangiogenesis and prognosis in colon cancer[J].J Gastrointest Surg,2016,20(12):2074-2082.
[17]Li X,Li X,Lv X,et al.Smad4inhibits VEGF-A and VEGF-Cexpressions via enhancing Smad3 phosphorylation in colon cancer[J].Anat Rec(Hoboken),2017,300(9):1560-1569.
[18]Maness L,Goktepe I,Chen H,et al.Impact of phytolacca americana extracts on gene expression of colon cancer cells[J].Phytother Res,2014,28(2):219-223.
[19]Soto-Pantoja D R,Sipes J M,Martin-Manso G,et al.Dietary fat overcomes the protective activity of thrombospondin-1signaling in the Apc(Min/+)model of colon cancer[J].Oncogenesis,2016,5(5):e230.
[20]Wang T,Xu X,Xu Q,et al.miR-19apromotes colitis-associated colorectal cancer by regulating tumor necrosis factor alphainduced protein 3-NF-kappaB feedback loops[J].Oncogene,2017,36(23):3240-3251.
[21]周晓黎,舒磊,廖艳,等.PI3K/AKT通路在低氧环境下对结肠癌细胞HIF-1α及糖酵解的作用[J].华中科技大学学报:医学版,2018,47(2):203-206.
[22]杨庚,李昱,贺亚峰.miRNA-19a对结肠癌细胞增殖活性的影响[J].中国老年学杂志,2018,38(23):5804-5807.