六味消癥方通过调节TRIM65表达抑制肝癌细胞侵袭和迁移机制研究
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摘要
目的:研究六味消癥方含药血清对肝癌细胞侵袭和迁移能力的影响及其作用机制。方法:采用Transwell侵袭实验和划痕迁移实验,考察不同剂量六味消癥方和TRIM65表达对肝癌细胞侵袭和迁移能力的影响;采用RT-PCR实验和Western blot实验,考察不同剂量六味消癥方对肝癌细胞中TRIM65表达的影响,以及六味消癥方和TRIM65表达对Wnt/β-catenin信号通路中关键蛋白表达的影响。结果:六味消癥方含药血清显著抑制肝癌细胞通过基质胶的数量(P<0.01)、伤痕愈合率(P<0.01)、TRIM65的表达(P<0.01),以及β-catenin、c-myc和MMP9的表达(P<0.01)。TRIM65基因干扰加强了含药血清对肝癌细胞侵袭迁移能力的抑制,TRIM65过表达则减弱了含药血清对肝癌细胞侵袭迁移能力的抑制,同时减弱了含药血清对β-catenin、c-myc和MMP9的表达的抑制。结论:六味消癥方含药血清能够抑制肝癌细胞的侵袭和迁移能力,其作用机制可能是通过调控TRIM65的表达水平,进而抑制Wnt/β-catenin信号通路异常激活。
Objective:To study the influence and mechanism of Liuwei Xiaozheng(LWXZ)Decoction on invasion and migration of hepatic carcinoma cells. Methods:The effects of different doses of LWXZ Decoction and expression levels of TRIM65 on invasion and migration of hepatic carcinoma cells were explored by transwell assay and wound healing assay. It was also explored by RT-PCR and Western blot including the influences of different doses of LWXZ Decoction on expression level of TRIM65,and the effects of LWXZ Decoction and TRIM65 on expression levels of key proteins in Wnt/β-catenin signaling pathway. Results:LWXZ Decoction significantly inhibited invaded cell number(P<0.01),wound closure(P<0.01),expressions of TRIM65(P<0.01),β-catenin,c-myc and MMP9(P<0.01). TRIM65 siRNA significantly strengthened the effect of LWXZ Decoction,while TRIM65 overexpression weakened the effect.Conclusion:LWXZ Decoction could inhibit the invasion and migration of hepatic carcinoma cells,and the mechanisms might be involved in regulating expression of TRIM65 and inhibiting abnormal activation of Wnt/β-catenin signaling pathway.
Objective:To study the influence and mechanism of Liuwei Xiaozheng(LWXZ)Decoction on invasion and migration of hepatic carcinoma cells. Methods:The effects of different doses of LWXZ Decoction and expression levels of TRIM65 on invasion and migration of hepatic carcinoma cells were explored by transwell assay and wound healing assay. It was also explored by RT-PCR and Western blot including the influences of different doses of LWXZ Decoction on expression level of TRIM65,and the effects of LWXZ Decoction and TRIM65 on expression levels of key proteins in Wnt/β-catenin signaling pathway. Results:LWXZ Decoction significantly inhibited invaded cell number(P<0.01),wound closure(P<0.01),expressions of TRIM65(P<0.01),β-catenin,c-myc and MMP9(P<0.01). TRIM65 siRNA significantly strengthened the effect of LWXZ Decoction,while TRIM65 overexpression weakened the effect.Conclusion:LWXZ Decoction could inhibit the invasion and migration of hepatic carcinoma cells,and the mechanisms might be involved in regulating expression of TRIM65 and inhibiting abnormal activation of Wnt/β-catenin signaling pathway.
引文
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[15]王新颖,王菁,龚伟,等.β-catenin、磷酸化GSK-3β、c-myc与大肠侧向发育型肿瘤关系的研究[J].广东医学,2010,31(15):1973-1975.
[16]苏娟,张庆瑜,康春生,等.miR-200a通过β-catenin/TCF-4信号通路抑制胃癌细胞系生长侵袭能力的体外研究[J].中国肿瘤临床,2012,39(2):65-69.
[17]陈茹,王海燕,苏莹,等.白藜芦醇通过β-catenin/TCF-4信号通路对胃癌细胞增殖活性剂侵袭迁移能力的影响[J].肿瘤药学,2018,8(1):20-25.
[18]宋厚盼,李如意,魏艳霞,等.四君子汤含药血清上调c-myc表达促进小肠上皮细胞迁移和增殖[J].中国实验方剂学杂志,2016,22(22):154-159.
[19]Li W,Ling W,Teng X,et al.Effect of advanced glycation end products,extracellular matrix metalloproteinase inducer and matrix metalloproteinases on type-Ⅰcollagen metabolism[J].Biomedicine Reports,2016,4(6):691-693.
[2]Chen JG,Egner PA,Ng D,et al.Reduced aflatoxin exposure presages decline in liver cancer mortality in an endemic region of China[J].Cancer Prevention Research,2013,6(10):1038-1045.
[3]Wang FS,Fan JG,Zhang Z,et al.The global burden of liver disease:the major impact of China[J].Hepatology,2014,60(6):2099-2108.
[4]石文静,谭佳妮,沈卫星,等.消癌解毒方含药血清对人结肠癌细胞增殖及糖酵解过程的影响[J].中国实验方剂学杂志,2017,23(20):120-125.
[5]陈磊峰,刘天德,杜晓红,等.原发性肝癌细胞中Rock2调控MMP2对其侵袭迁移的作用[J].肿瘤防治研究,2014,41(1):35-39.
[6]Reymond A,Meroni G,Fantozzi A,et al.The tripartite motif family identifies cell compartments[J].Embo J,2001,20(9):2140-2151.
[7]Li S,Wang L,Fu B,et al.Trim65:A cofactor for regulation of the microRNA pathway[J].RNA Biol,2014,11(9):1113-1121.
[8]Li S,Wang L,Fu B,et al.TRIM65 regulates microRNA activity by ubiquitination of TNRC6[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(19):6970-6975.
[9]Li Y,Ma CY,Zhou T,et al.TRIM65 negatively regulates p53through ubiquitination[J].Biochemical and Biophysical Research Communications,2016,473(1):278-282.
[10]Wang XL,Shi WP,Shi HC,et al.Knockdown of TRIM65inhibits lung cancer cell proliferation migration and invasion:Atherapeutic target in human lung cancer[J].Oncotarget,2016,7(49):81527-81540.
[11]Zhao L,Zhang P,Su XJ,et al.The ubiquitin ligase TRIM65inhibits ovarian cancer progression by targeting vimentin[J].Journal of Cellular Physiology,2018,233(3):2420-2425.
[12]汪彩霞,陈说,赵杨.XAV939对卵巢癌细胞侵袭迁移的影响及Wnt/β-catenin信号的抑制作用[J].武警医学,2016,27(2):170-174.
[13]Mariann B.β-catenin:A pivot between cell adhesion and wnt signaling[J].Current Biology,2004,15(2):2061-2069.
[14]许光伟,曹旭晨.β-catenin下调对乳腺癌MDA-MB-231细胞上皮间质转化及侵袭迁移能力的影响[J].天津医药,2015,43(9):981-984.
[15]王新颖,王菁,龚伟,等.β-catenin、磷酸化GSK-3β、c-myc与大肠侧向发育型肿瘤关系的研究[J].广东医学,2010,31(15):1973-1975.
[16]苏娟,张庆瑜,康春生,等.miR-200a通过β-catenin/TCF-4信号通路抑制胃癌细胞系生长侵袭能力的体外研究[J].中国肿瘤临床,2012,39(2):65-69.
[17]陈茹,王海燕,苏莹,等.白藜芦醇通过β-catenin/TCF-4信号通路对胃癌细胞增殖活性剂侵袭迁移能力的影响[J].肿瘤药学,2018,8(1):20-25.
[18]宋厚盼,李如意,魏艳霞,等.四君子汤含药血清上调c-myc表达促进小肠上皮细胞迁移和增殖[J].中国实验方剂学杂志,2016,22(22):154-159.
[19]Li W,Ling W,Teng X,et al.Effect of advanced glycation end products,extracellular matrix metalloproteinase inducer and matrix metalloproteinases on type-Ⅰcollagen metabolism[J].Biomedicine Reports,2016,4(6):691-693.