咪达唑仑调节YAP/TAZ信号通路对骨肉瘤细胞增殖凋亡和侵袭的影响OACSTPCD
Effects of Midazolam on Proliferation Apoptosis and Invasion of Osteosarcoma Cells by Regulating the YAP/TAZ Signaling Pathway
目的:探究咪达唑仑(MDZ)调节 Yes 相关蛋白(YAP)/PDZ 结合基序转录共激活因子(TAZ)信号通路对骨肉瘤(OS)细胞增殖、凋亡和侵袭的影响.方法:qRT-PCR、Western blot 检测人正常成骨细胞系、OS细胞系YAP、TAZ mRNA 和蛋白的表达,筛选最佳干预细胞系;以不同浓度的 MDZ(0、12.5、25、50、100、200μmoL/L)干预OS细胞,MTT 法检测细胞增殖活性,筛选最佳干预浓度;将 OS细胞随机分成control组、MDZ组、pcDNA3.1 组、pcDNA3.1-YAP/TAZ 组,qRT-PCR 法检测转染效率;EdU染色、流式细胞仪和Transwell小室分别检测细胞增殖、凋亡和侵袭;Western blot方法检测增殖细胞核抗原(PCNA)、Bcl-2 相关 X 蛋白(Bax)、N-钙黏蛋白(N-cadherin)、E-钙黏蛋白(E-cadherin)及YAP、TAZ蛋白表达;构建 OS 裸鼠模型,分为对照组和 MDZ 组,免疫组化法检测移植瘤组织 Ki-67、YAP、TAZ蛋白表达,TUNEL染色检测凋亡.结果:OS细胞系中YAP、TAZ mRNA 及蛋白表达水平显著升高(P<0.05);MDZ显著降低PCNA、N-cadherin、YAP、TAZ 表达,抑制 MG63、U20S 细胞增殖和侵袭,升高Bax、E-cadherin表达,促进细胞凋亡.过表达 YAP/TAZ 可逆转 MDZ 对 MG63、U20S 细胞增殖和侵袭的抑制作用,对MG63、U20S细胞凋亡的促进作用(P<0.05);体内实验表明,MDZ显著降低移植瘤质量、体积及 Ki-67、YAP、TAZ 表达水平,促进移植瘤细胞凋亡(P<0.05).结论:MDZ 可能通过抑制YAP/TAZ信号通路,抑制OS细胞增殖和侵袭,促进凋亡.
Objective:To investigate the effects of midazolam(MDZ)on the proliferation,apoptosis and invasion of osteosarcoma(OS)cells by regulating the Yes associated protein(YAP)/PDZ transcriptional activator of transcription(TAZ)signaling pathway.Methods:qRT-PCR and western blot were applied to de-tect the expression of YAP,TAZ mRNA,and proteins in normal osteoblast and OS cell lines,and to screen the optimal intervention cell line.OS cells were intervened with different concentrations of MDZ(0,12.5,25,50,100,200 μmoL/L),MTT method was applied to detect cell proliferation activity and screen the opti-mal intervention concentration;OS cells were randomly divided into control group,MDZ group,pcDNA3.1 group,and pcDNA3.1-YAP/TAZ group,and transfection efficiency was detected using qRT-PCR method;EdU staining,flow cytometry,and Transwell cells were applied to detect cell proliferation,apoptosis,and in-vasion,respectively;Western blot method was applied to detect the expression of proliferating cell nuclear an-tigen(PCNA),Bcl-2 associated X protein(Bax),N-cadherin,E-cadherin,and YAP,TAZ proteins;OS nude mouse model was constructed,and grouped into a control group and an MDZ group,immunohistochemi-cal methods were applied to detect the expression of Ki-67,YAP,and TAZ proteins in transplanted tumor tis-sue,and TUNEL staining was applied to detect apoptosis.Results:The expression levels of YAP and TAZ mRNA and protein in OS cell lines were obviously increased(P<0.05);MDZ obviously reduced the expres-sion of PCNA,N-cadherin,YAP,and TAZ,inhibitd the proliferation and invasion of MG63 and U20S cells,increased the expression of Bax and E-cadherin,and promoted cell apoptosis.Overexpression of YAP/TAZ was able to reverse the inhibitory effects of MDZ on the proliferation and invasion of MG63 and U20S cells,and promote the apoptosis of MG63 and U20S cells(P<0.05);in vivo experiments showed that MDZ obvi-ously reduced the mass and volume of transplanted tumors,and the expression levels of Ki-67,YAP,and TAZ,promoted apoptosis of transplanted tumor cells(P<0.05).Conclusion:MDZ may inhibit the YAP/TAZ signaling pathway,inhibit the proliferation and invasion of OS cells,and promote apoptosis.
奥婷婷;涂梦佳;张熙
湖北省十堰市人民医院麻醉科,湖北 十堰 442099
咪达唑仑YAP/TAZ骨肉瘤增殖凋亡侵袭
MidazolamYAP/TAZOsteosarcomaProliferationApoptosisInvasion
《河北医学》 2024 (001)
1-8 / 8
湖北省卫生健康委科研项目,(编号:WJ2023F090)
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