YAP-TEAD转录活性对Hippo通路突变的恶性胸膜间皮瘤细胞增殖的影响

Effect of YAP-TEAD transcriptional activity on proliferation of Hippo mutant malignant pleural mesothelioma cell lines

目的研究YAP(Yes-associated protein,YAP)、TEAD(TEA domain transcription factor,TEAD)在恶性胸膜间皮瘤(malignant pleural mesothelioma,MPM)组织中的表达水平,以及YAP-TEAD转录活性对Hippo通路突变的MPM细胞增殖的影响。方法利用高通量基因表达数据库(Gene Expression Omnibus,GEO)通过生物信息学方法分析YAP及TEAD蛋白家族成员在MPM及正常胸膜组织中mRNA差异表达。免疫组化法检测36例MPM及15例正常胸膜组织中YAP、TEAD4、CTGF及Ki-67的蛋白表达水平。选取3株Hippo通路突变的MPM细胞株(H2373、MESO25及H2052)及2株Hippo通路野生型细胞株(MCF10A及293T)。通过慢病毒分别感染上述细胞株构建dnTEAD4过表达稳转细胞系及其对照EV细胞系,双荧光素酶报告基因实验检测YAP-TEAD转录活性的变化,实时荧光定量PCR检测下游靶分子CTGF及CYR61 mRNA表达水平的差异,平板克隆形成实验通过比较两组细胞克隆形成的大小、数目及克隆形成指数检验过表达dnTEAD4对MPM细胞增殖的影响。结果生信分析(GSE2549)显示,恶性胸膜间皮瘤组织中YAP及TEAD4 mRNA表达水平高于正常组织(P<0.01)。免疫组化结果显示,恶性胸膜间皮瘤组织中YAP、TEAD4及CTGF的蛋白表达水平高于正常胸膜组织(P<0.05),YAP蛋白表达阳性的恶性胸膜间皮瘤组织较YAP蛋白表达阴性的恶性胸膜间皮瘤组织具有更高的Ki-67指数(P<0.001)。相比EV组,dnTEAD4过表达组YAP-TEAD转录活性降低(P<0.01),下游靶分子CTGF及CYR61的mRNA表达水平降低(P<0.05)。同时,在Hippo通路突变的MPM细胞株中,相比EV组,dnTEAD4过表达组细胞克隆大小减小、克隆数目减少且克隆形成指数减小(P<0.001)。在Hippo通路野生型细胞株中,dnTEAD4过表达组细胞的克隆大小、克隆数目及克隆形成指数较EV组无明显变化。结论YAP及TEAD4在恶性胸膜间皮瘤组织中呈高表达。YAP-TEAD转录活性对Hippo通路突变的恶性胸膜间皮瘤细胞增殖至关重要,Hippo通路突变的恶性胸膜间皮瘤细胞的增殖依赖高水平的YAP-TEAD转录活性。

Objective To investigate the expression status of YAP and TEAD in malignant pleural mesothelioma(MPM)and the effect of YAP-TEAD transcriptional activity on Hippo mutant MPM proliferation.Methods GEO dataset was used to analyze the differential expression of YAP and TEAD family members between MPM tumor and normal samples.The expression levels of YAP,TEAD4,CTGF and Ki-67 in 36 MPM tissues and 15 normal hyperplasia pleural tissues were tested by immunohistochemistry.Three Hippo mutant MPM cell lines(H2373,MESO25 and H2052)and two Hippo wild-type cell lines(MCF10A and 293T)were infected with lentivirus stably expressing dominant negative TEAD4(dnTEAD4)and empty vector,respectively.Dual-luciferase assays were used to detect the effect of dnTEAD4 on YAP-TEAD transcriptional activity.The effects of dnTEAD4 on YAP-TEAD targeted genes CTGF and CYR61 mRNA expression levels were tested through quantitative PCR.The colony sizes,the numbers and the colony formation rates of dnTEAD overexpression cell lines and empty vector cell lines were detected by colony formation assays to evaluate the effect of dnTEAD4 on the proliferation of MPM cell lines.Results Bioinformatic results showed the mRNA expression levels of YAP and TEAD4 in MPM tissues were higher than those in normal pleural tissues(P<0.01).Immunohistochemistry results showed that the protein levels of YAP,TEAD4 and CTGF in MPM tissues were higher than those in normal pleural tissues(P<0.05).Ki-67 level was significantly higher in YAP positive MPM tissues than that in YAP negative MPM tissues(P<0.001).YAP-TEAD transcriptional activity and mRNA expression levels of targeted genes CTGF and CYR61 were significantly lower in dnTEAD overexpression cell lines than those in empty vector(EV)cell lines(P<0.01 or P<0.05).And in Hippo mutant MPM cell lines,the colonies of dnTEAD4 overexpression cell lines were smaller and fewer than those of EV cell lines,and the colony formation rate was lower(P<0.001).In Hippo wild-type cell liens,there was no significant change in colony sizes,numbers and colony formation rates between dnTEAD overexpression cell lines and EV cell lines.Conclusion The expression levels of YAP and TEAD are higher in MPM tissues.YAP-TEAD transcriptional activity is critically important for the proliferation of Hippo mutant MPM cell lines.The proliferation of MPM cell lines is dependent on high level of YAP-TEAD transcriptional activity.