白花蛇舌草通过抑制RAP1-JNK信号通路选择性促进人肾癌ACHN细胞间质-上皮转化
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摘要
目的 :研究白花蛇舌草(Hedyotis di usa Willd,HDW)对人肾癌细胞增殖、细胞周期、凋亡、迁移和侵袭的影响,并探讨其可能的作用机制。方法 :人肾细胞腺癌ACHN和人肾近曲小管HK-2细胞经HDW处理24 h后,采用CCK-8法检测HDW对ACHN和HK-2细胞增殖的影响,FCM法检测HDW对细胞周期和凋亡的影响。光学显微镜下及罗丹明染色法观察HDW处理对ACHN和HK-2细胞形态的影响;采用免疫荧光染色法和蛋白质印迹法检测间质-上皮转化(mesenchymal-epithelial transition,MET)相关蛋白表达的变化。划痕愈合实验和Transwell小室法检测ACHN和HK-2细胞的迁移和侵袭能力,RNA测序法检测HDW对ACHN细胞转录组的影响;实时荧光定量PCR法检测RAP1信号通路相关基因RAP1GAP、RASGRP2、RAPGEF3、MAGI1及GNAI1 mRNA的表达水平;蛋白质印迹法检测丝裂原活化蛋白激酶(mitogen-activate protein kinase,MAPK)通路相关蛋白c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、磷酸化JNK(phospho-JNK,p-JNK)、蛋白激酶B(protein kinase B,PKB,又称Akt)、磷酸化Akt(phospho-Akt,p-Akt)、细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)和磷酸化ERK(phospho-ERK,p-ERK)表达水平的改变。结果:HDW可选择性抑制ACHN细胞增殖(P <0.01),将细胞周期阻滞于S期(P <0.01),诱导细胞凋亡(P <0.01)。HDW处理后ACHN细胞的形态发生了明显变化,HDW可促进ACHN细胞中E-钙黏蛋白1(E-cadherin 1)和β-连环蛋白(β-catenin)的表达,并抑制波形蛋白(Vimentin)和Snail1的表达(P值均<0.01)。HDW能显著抑制细胞的迁移和侵袭能力(P值均<0.01),且ACHN和HK-2细胞之间没有明显差异。RNA测序结果分析显示,HDW可影响细胞周期相关基因以及控制细胞生长的转录因子E2F和Myc靶基因的表达,并激活p53信号通路;HDW可显著下调ACHN细胞中RAP1GAP、RASGRP2、RAPGEF3、MAGI1及GNAI1 mRNA(P值均<0.000 1)和p-JNK蛋白的表达水平。结论 :HDW可能通过抑制RAP1-JNK信号通路来选择性抑制肾癌细胞ACHN的增殖,促进ACHN细胞MET、周期阻滞和凋亡。
Objective: To investigate the effects of Hedyotis diffusa Willd(HDW) on the proliferation, cell cycle, apoptosis, migration and invasion of human renal carcinoma cells, and to explore the possible mechanisms.Methods: After human renal adenocarcinoma ACHN cells and human renal proximal tubule HK-2 cells were treated with HDW for 24 h, the cell proliferation was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by FCM assay. The phenotypes of ACHN and HK-2 cells treated with HDW were observed by light microscopy and rhodamine staining. The expressions of mesenchymal-epithelial transition(MET)-related proteins were detected by immunofluorescence staining and Western blotting, respectively. The effects of HDW on the migration and invasion of ACHN and HK-2 cells were detected by wound-healing and Transwell chamber assay. RNAsequencing was used to detect the effect of HDW on the transcriptome in ACHN cells. The expression levels of RAP1 pathway-related genes [RAP1 GTPase activating protein(RAP1 GAP), RAS guanyl releasing protein 2(RASGRP2), RAP guanine nucleotide exchange factor 3(RAPGEF3), membrane-associated guanylate kinase, WW and PDZ domain containing 1(MAGI1) and G protein subunit alpha i1(GNAI1)] were detected by real-time fluorescent quantitative PCR. The expression levels of mitogen-activate protein kinase(MAPK) pathway-related proteins [c-Jun N-terminal kinase(JNK), phospho-JNK(p-JNK), protein kinase B(PKB, Akt), phospho-Akt(p-Akt), extracellular signalregulated kinase(ERK) and phospho-ERK(p-ERK)] were detected by Western blotting. Results: HDW selectively inhibited the proliferation of ACHN cells(P < 0.01), blocked cell cycle at S-phase(P < 0.01), and induced apoptosis(P < 0.01). After treatment with HDW, the morphology of ACHN cells significantly changed. HDW promoted the expressions of MET-related proteins(E-cadherin 1 and β-catenin) in ACHN cells, and inhibited the expressions of epithelial-mesenchymal transition(EMT)-related proteins(Vimentin and Snail1)(all P < 0.01). HDW inhibited the migration and invasion of ACHN and HK-2 cells(all P < 0.01), and there was no significant difference between ACHN and HK-2 cells. HDW affected the expressions of cell cycle-related genes and transcription factor E2 F and Myc target genes, and activated the p53 signaling pathway. HDW significantly downregulated the expression levels of RAP1 GAP, RASGRP2, RAPGEF3, MAGI1 and GNAI1(all P < 0.000 1) and the phosphorylation level of JNK in ACHN cells.Conclusion: HDW may selectively inhibit cell proliferation, and promote MET, cell cycle arrest and apoptosis of ACHN cells by inhibiting RAP1-JNK signaling pathway.
Objective: To investigate the effects of Hedyotis diffusa Willd(HDW) on the proliferation, cell cycle, apoptosis, migration and invasion of human renal carcinoma cells, and to explore the possible mechanisms.Methods: After human renal adenocarcinoma ACHN cells and human renal proximal tubule HK-2 cells were treated with HDW for 24 h, the cell proliferation was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by FCM assay. The phenotypes of ACHN and HK-2 cells treated with HDW were observed by light microscopy and rhodamine staining. The expressions of mesenchymal-epithelial transition(MET)-related proteins were detected by immunofluorescence staining and Western blotting, respectively. The effects of HDW on the migration and invasion of ACHN and HK-2 cells were detected by wound-healing and Transwell chamber assay. RNAsequencing was used to detect the effect of HDW on the transcriptome in ACHN cells. The expression levels of RAP1 pathway-related genes [RAP1 GTPase activating protein(RAP1 GAP), RAS guanyl releasing protein 2(RASGRP2), RAP guanine nucleotide exchange factor 3(RAPGEF3), membrane-associated guanylate kinase, WW and PDZ domain containing 1(MAGI1) and G protein subunit alpha i1(GNAI1)] were detected by real-time fluorescent quantitative PCR. The expression levels of mitogen-activate protein kinase(MAPK) pathway-related proteins [c-Jun N-terminal kinase(JNK), phospho-JNK(p-JNK), protein kinase B(PKB, Akt), phospho-Akt(p-Akt), extracellular signalregulated kinase(ERK) and phospho-ERK(p-ERK)] were detected by Western blotting. Results: HDW selectively inhibited the proliferation of ACHN cells(P < 0.01), blocked cell cycle at S-phase(P < 0.01), and induced apoptosis(P < 0.01). After treatment with HDW, the morphology of ACHN cells significantly changed. HDW promoted the expressions of MET-related proteins(E-cadherin 1 and β-catenin) in ACHN cells, and inhibited the expressions of epithelial-mesenchymal transition(EMT)-related proteins(Vimentin and Snail1)(all P < 0.01). HDW inhibited the migration and invasion of ACHN and HK-2 cells(all P < 0.01), and there was no significant difference between ACHN and HK-2 cells. HDW affected the expressions of cell cycle-related genes and transcription factor E2 F and Myc target genes, and activated the p53 signaling pathway. HDW significantly downregulated the expression levels of RAP1 GAP, RASGRP2, RAPGEF3, MAGI1 and GNAI1(all P < 0.000 1) and the phosphorylation level of JNK in ACHN cells.Conclusion: HDW may selectively inhibit cell proliferation, and promote MET, cell cycle arrest and apoptosis of ACHN cells by inhibiting RAP1-JNK signaling pathway.
引文
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[26]刘曙正,郭兰伟,曹小琴,等.中国2014年肾癌发病与死亡分析[J].中国流行病学杂志,2018,39(10):1346-1350
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[28]张林超,赵俊峰,孙继建,等.白花蛇舌草乙醇提取物对人肾癌细胞786-0体外增殖及小鼠肾癌细胞786-0移植瘤生长的影响[J].山东医药,2017,57(23):20-23.
[29]周进,赵朋.白花蛇舌草对Renca肾癌细胞模型小鼠凋亡相关蛋白Fas、caspase3及caspase7表达影响[J].中国生化药物杂志,2016,36(12):37-40,45.
[30]刘岩,张春阳,张大田,等.白花蛇舌草提取物诱导人肾癌GRC-1细胞凋亡及抑制血管生成的实验研究[J].山东医药,2011,51(8):97-98.
[2]THIERY JP,ACLOQUE H,HUANG RY,et al.Epithelial-mesenchymal transitions in development and disease[J].Cell,2009,139(5):871-890.
[3]MARCUCCI F,BELLONE M,CASERTACA,et al.Pushing tumor cells towards a malignant phenotype:stimuli from the microenvironment,intercellular communications and alternative roads[J].Int J Cancer,2014,135(6):1265-1276.
[4]SAHLGREN C,GUSTAFSSON MV,JIN S,et al.Notch signaling mediates hypoxiainduced tumor cell migration and invasion[J].Proc Natl Acad Sci U S A,2008,105(17):6392-6397.
[5]MARTíNEZ-ZAGUILáN R,SEFTOR EA,SEFTOR RE,et al.Acidic pH enhances the invasive behavior of human melanoma cells[J].Clin Exp Metastasis,1996,14(2):176-186.
[6]GJOREVSKI N,BOGHAERT E,NELSON CM.Regulation of epithelial-mesenchymal transition by transmission of mechanical stress through epithelial tissues[J].Cancer Microenviron,2012,5(1):29-38.
[7]GOETZ JG,MINGUET S,NAVARRO-LERIDA I,et al.Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis[J].Cell,2011,146(1):148-163.
[8]LIU R,WANG X,CHEN GY,et al.The prognostic role of a gene signature from tumorigenic breast-cancer cells[J].NEngl J Med,2007,356(3):217-226.
[9]POLYAK K,WEINBERG RA.Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits[J].Nat Rev Cancer,2009,9(4):265-273.
[10]LAMOUILLE S,XU J,DERYNCK R.Molecular mechanisms of epithelialmesenchymal transition[J].Nat Rev Mol Cell Biol,2014,15(3):178-196.
[11]杨丽娜,樊怡,马健飞.miR-29对腹膜间皮细胞EMT的作用[J].中国血液净化,2018,17(10):694-698.
[12]王锐,刘亚东,于时良,等.MG132对草酸钙肾结石模型TGF-β1及EMT的影响[J].现代泌尿外科杂志,2018,23(11):866-869.
[13]朱晨笛,郭慕真,蔡倩,等.MAPK信号通路在百草枯诱导上皮-间充质改变中的作用[J].中华劳动卫生职业病杂志,2018,36(8):561-567.
[14]MARCUCCI F,STASSI G,DE MARIA R.Epithelial-mesenchymal transition:a new target in anticancer drug discovery[J].Nat Rev Drug Discov,2016,15(5):311-325.
[15]钟立敏,周宏霞,刘丹.白花蛇舌草的药理和临床应用进展[J].中医药信息,2001,18(4):14-15.
[16]CHEN R,HE J,TONG X,et al.The hedyotis diffusa willd.(Rubiaceae):A review on phytochemistry,pharmacology,quality control and pharmacokinetics[J].Molecules,2016,21(6):E710.
[17]ZHANG Y,XIE RF,XIAO QG,et al.Hedyotis diffusa Willd extract inhibits the growth of human glioblastoma cells by inducing mitochondrial apoptosis via AKT/ERK pathways[J].J Ethnopharmacol,2014,158(Pt A):404-411.
[18]CAI Q,LIN J,WEI L,et al.Hedyotis diffusa Willd inhibits colorectal cancer growth in vivo via inhibition of STAT3signaling pathway[J].Int J Mol Sci,2012,13(5):6117-6128.
[19]LIN J,WEI L,SHEN A,et al.Hedyotis diffusa Willd extract suppresses Sonic hedgehog signaling leading to the inhibition of colorectal cancer angiogenesis[J].Int J Oncol,2013,42(2):651-656.
[20]LIU X,WU J,ZHANG D,et al.Network pharmacology-based approach to investigate the mechanisms of Hedyotis diffusa Willd.in the treatment of gastric cancer[J].Evid Based Complement Alternat Med,2018,2018:7802639.
[21]DAVE N,GUAITA-ESTERUELAS S,GUTARRA S,et al.Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition[J].J Biol Chem,2011,286(14):12024-12032.
[22]BARRALLO-GIMENO A,NIETO MA.The Snail genes as inducers of cell movement and survival:implications in development and cancer[J].Development,2005,132(14):3151-3161.
[23]DENT P.Crosstalk between ERK,AKT,and cell survival[J].Cancer Biol Ther,2014,15(3):245-246.
[24]LEMOS FO,EHRLICH BE.Polycystin and calcium signaling in cell death and survival[J].Cell Calcium,2018,69:37-45.
[25]JA?KIEWICZ A,PAJ?K B,ORZECHOWSKIA.The many faces of rap1 GTPase[J].Int J Mol Sci,2018,19(10).pii:E2848.
[26]刘曙正,郭兰伟,曹小琴,等.中国2014年肾癌发病与死亡分析[J].中国流行病学杂志,2018,39(10):1346-1350
[27]NICEE C,FABRI L,HAMMACHER A,et al.The purification of a Rap1 GTPase-activating protein from bovine brain cytosol[J].J Biol Chem,1992,267(3):1546-1553.
[28]张林超,赵俊峰,孙继建,等.白花蛇舌草乙醇提取物对人肾癌细胞786-0体外增殖及小鼠肾癌细胞786-0移植瘤生长的影响[J].山东医药,2017,57(23):20-23.
[29]周进,赵朋.白花蛇舌草对Renca肾癌细胞模型小鼠凋亡相关蛋白Fas、caspase3及caspase7表达影响[J].中国生化药物杂志,2016,36(12):37-40,45.
[30]刘岩,张春阳,张大田,等.白花蛇舌草提取物诱导人肾癌GRC-1细胞凋亡及抑制血管生成的实验研究[J].山东医药,2011,51(8):97-98.