稳定干扰癌相关成纤维细胞中YAP1表达可抑制乳腺癌MDAMB-231细胞迁移和侵袭

YAP1 knockdown in cancer-associated fibroblasts inhibits migration and invasion of breast cancer MDA-MB-231 cells

目的 :探讨稳定干扰癌相关成纤维细胞(cancer-associatedbroblast,CAF)中Yes相关蛋白1(Yes-associated protein 1,YAP1)表达对乳腺癌MDAMB-231细胞迁移和侵袭的影响。方法 :用生物信息学方法分析乳腺原代CAF与对应正常成纤维细胞(normal broblast,NF)的m RNA芯片数据中YAP1的表达,并采用实时荧光定量PCR和蛋白质印迹法检测乳腺原代及永生化CAF与NF细胞中YAP1m RNA和蛋白的表达水平,以验证芯片结果的真实性和准确性。用NF与CAF条件培养液分别培养乳腺癌MDA-MB-231细胞,然后采用Transwell小室法比较NF与CAF对乳腺癌细胞迁移和侵袭的影响。构建靶向YAP1基因的sh RNA重组质粒并转染入CAF,采用实时荧光定量PCR和蛋白质印迹法检测CAF细胞中YAP1 m RNA和蛋白的表达水平,然后采用划痕愈合实验和Transwell小室法分别检测干扰YAP1表达后的CAF条件培养液对乳腺癌MDA-MB-231细胞迁移和侵袭能力的影响。用Hippo通路抑制剂XMU-MP-1处理CAF,采用蛋白质印迹法检测CAF细胞中YAP1总蛋白和磷酸化蛋白水平,Transwell小室法检测XMU-MP-1处理后的CAF条件培养液对MDA-MB-231细胞侵袭能力的影响。采用实时荧光定量PCR法检测干扰YAP1表达后的CAF细胞中YAP1下游与迁移和侵袭密切相关的转化因子β1(transforminggrowthfactor-beta1,TGF-β1)和白细胞介素6(interleukin 6,IL6)的表达,并采用CollagenⅠ胶原凝胶收缩实验检测干扰YAP1表达对细胞外基质重塑的影响。结果 :m RNA芯片检测发现,与正常对照NF相比,乳腺原代CAF中YAP1高表达(P <0.05)。实时荧光定量PCR和蛋白质印迹法检测证实原代及永生化CAF中YAP1 m RNA(P <0.05)和蛋白(P <0.01)水平均明显高于NF。与NF相比较,CAF条件培养液可明显促进乳腺癌MDA-MB-231细胞侵袭(P <0.01)。成功构建YAP1sh RNA稳定转染的CAF细胞株,其中YAP1 m RNA和蛋白表达水平均明显下调(P值均<0.01)。与未干扰YAP1表达的CAF对照组相比,经稳定干扰YAP1的CAF条件培养液处理后,乳腺癌MDA-MB-231细胞的迁移(P <0.05)和侵袭(P <0.01)能力均明显减弱。用Hippo通路抑制剂XMU-MP-1处理后,CAF细胞中YAP1磷酸化蛋白水平明显降低(P <0.01),经该CAF条件培养液处理后MDA-MB-231细胞的侵袭能力明显增强(P <0.05)。干扰YAP1表达后的CAF细胞中TGF-β1和IL6m RNA表达水平均明显下调(P值均<0.05),且细胞外基质的胶原凝胶收缩能力明显减弱(P <0.05)。结论 :稳定干扰CAF细胞中YAP1表达可能通过下调细胞因子TGF-β1和IL6的表达,以及重塑细胞外基质,抑制乳腺癌MDA-MB-231细胞的迁移和侵袭。

Objective: To investigate the effects of Yes-associated protein 1(YAP 1) gene knockdown in cancer-associated fibroblast(CAF) on the migration and invasion of breast cancer MDAMB-231 cells.Methods: The expression of YAP1 in breast primary CAF and paired normal fibroblast(NF) in m RNA microarray was analyzed by bioinformatics methods. To verify the authenticity and accuracy of the microarray results, the expressions of YAP1 m RNA and protein in primary and immortalized NF and CAF were detected by real-time fluorescent quantitative PCR and Western blotting. A co-cultured system including breast cancer MDA-MB-231 cells and the conditioned medium of NF or CAF was used, and the effect of fibroblasts on the invasion ability of breast cancer cells was detected by Transwell chamber assay. CAF cells were transfected with the plasmids carrying the specific sh RNA targeting YAP1 gene, then the expressions of YAP1 m RNA and protein in CAF were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The effects of YAP 1 gene interference in CAF on the migration and invasion of MDA-MB-231 cells cultured with CAF conditioned medium were detected by scratch wound healing and Transwell chamber assay, respectively. CAF cells were treated with Hippo pathway inhibitor XMU-MP-1, then the expressions of total YAP1 protein and phosphorylated YAP1 protein in CAF were detected by Western blotting, as well as the invasion ability of MDA-MB-231 cells co-cultured with the CAF conditioned medium was measured by Transwell chamber assay. The expressions of transforming growth factor-beta 1(TGF-β 1) and interleukin 6(IL 6), two target genes of YAP1 and the cytokines associated with cell migration and invasion, in CAF with YAP 1 gene interference were detected by real-time fluorescent quantitative PCR. The effect of YAP1-knocked CAF on the extracellular matrix remodeling was tested by Collagen gel contraction assay.Results: Compared with primary NF, the expression of YAP 1 gene was significantly upregulated in primary CAF of breast cancer according m RNA microarray(P < 0.05). The higher expressions of YAP1 m RNA and protein in primary and immortalized CAF were confirmed by real-time fluorescent quantitative PCR(P < 0.05) and Western blotting(P < 0.01). The invasion ability of breast cancer MDA-MB-231 cells was significantly promoted in the coculture system with the conditioned medium of CAF as compared with NF(P < 0.01). The CAF cell line transfected with YAP1 sh RNA was successfully constructed, and the expressions of YAP1 m RNA and protein were successfully stably knocked down in CAF(both P < 0.01).Compared with the YAP1-unknocked CAF control group, the migration(P < 0.05) and invasion(P < 0.01) abilities of breast cancer MDA-MB-231 cells were significantly weakened after culture with the conditioned medium of YAP1-knocked CAF. The phosphorylation level of YAP1 protein was decreased in CAF treated with Hippo pathway inhibitor XMU-MP-1(P < 0.01), while the invasion ability of MDA-MB-231 cells was significantly enhanced after coculture with XMU-MP-1-treated CAF(P < 0.05). The expression levels of TGF-β1 and IL6 m RNAs were down-regulated in YAP1-knocked CAF(both P < 0.05), and the collagen gel shrinking ability of extracellular matrix was significantly decreased in YAP1-knocked CAF group(P < 0.05).Conclusion: The stable knockdown of YAP1 expression in CAF can inhibit the migration and invasion of breast cancer MDA-MB-231 cells through down-regulating the expressions of TGF-β1 and IL6 and remodeling the extracellular matrix.