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

Hedyotis diffusa Willd selectively promotes mesenchymal-epithelial transition of human renal cancer ACHN cells by inhibiting RAP1-JNK signaling pathway

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 andβ-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.