乳腺癌转移的实验研究γ-干扰素联合顺铂通过调控xCT抑制三阴性乳腺癌转移的实验研究

Experimental Study on IFN-γCombined with Cisplatin Inhibiting Triple-Negative Breast Cancer Metastasis by Regulating xCT

[目的]探讨γ-干扰素(interferon-gamma,IFN-γ)联合顺铂(cisplatin,DDP)抑制三阴性乳腺癌转移的机制。[方法]采用BALB/c小鼠构建三阴性乳腺癌模型,观察IFN-γ联合DDP的治疗效果。用流式细胞术检测细胞凋亡水平;CCK-8实验检测细胞增殖;实时荧光定量逆转录PCR(qRT-PCR)检测相关基因的表达;UALCAN和Kaplan-Meier Plotter数据平台分析目的基因表达与乳腺癌预后的相关性;免疫组化检测肿瘤组织中目的蛋白的表达。[结果]各治疗组小鼠原位肿瘤体积均显著性缩小(P<0.05),而IFN-γ与DDP联合治疗组小鼠肺转移灶数目显著减少(P<0.01)。IFN-γ联合DDP促进小鼠三阴性乳腺癌细胞4T1凋亡(P<0.001),并显著性抑制肿瘤细胞增殖(P<0.001)。成纤维细胞L929上清能促进4T1细胞增殖(P<0.01),且该作用被IFN-γ抑制(P<0.01)。谷胱甘肽促进4T1细胞增殖(P<0.001),并且消除了IFN-γ对L929上清促肿瘤增殖的抑制作用(P<0.01)。qRT-PCR结果显示,L929中胱氨酸/谷氨酸反转运体x(xCT)mRNA表达水平显著性下调(P<0.05)。数据库分析结果显示,乳腺癌患者的xCT表达水平明显高于正常乳腺组织(P<0.001),xCT高表达与较差的无远处转移生存期存在显著性相关(P<0.01)。免疫组化结果表明,发生肺转移的乳腺癌患者肿瘤组织中xCT表达水平显著性高于未发生肺转移者(P<0.001)。[结论]IFN-γ联合DDP可能通过抑制L929 xCT的表达减少谷胱甘肽分泌,从而抑制三阴性乳腺癌转移。研究有望为三阴性乳腺癌转移的临床治疗提供新思路。

[Objective]To explore the mechanism of interferon-gamma(IFN-γ)and cisplatin(DDP)on inhibiting triple-negative breast cancer metastasis.[Methods]BALB/c mouse was used to construct triple-negative breast cancer model to evaluate the combined effect of IFN-γand DDP.Cell apoptosis level was measured by flow cytometry and proliferation ability was measured by CCK-8 assay.Reverse transcriptial quantitative real-time PCR(qRT-PCR)was used to detect expression levels of relative genes.UALCAN and Kaplan-Meier plotter data platforms were used to analyze the correlation between prognosis and expression level of target gene.Immunohistochemistry staining was performed to detect protein expression level in clinical tumor samples.[Results]Tumor volume in situ greatly decreased in all groups(P<0.05).Combined therapy of IFN-γand DDP significantly inhibited lung metastasis(P<0.01).IFN-γcombined with DDP promoted apoptosis of triple-negative breast cancer cell 4T1(P<0.001),and greatly restrained cell proliferation(P<0.001).Fibroblast L929 supernatant actived 4T1 cell proliferation(P<0.01),which was obviously suppressed by IFN-γ(P<0.01).Glutathione notably increased 4T1 proliferation ability(P<0.001)and relieved the inhibition of IFN-γon L929 supernatant’s promoting 4T1 proliferation(P<0.01).qRT-PCR results revealed that mRNA expression level of cystine-glutamate trunsporter(xCT)in L929 incredibly declined(P<0.05).Database analysis results showed that xCT expression in cancer tissue was apparently higher than that in normal breast tissue(P<0.001).High level of xCT expression had incredible relevance with poor distant metastasis-free survival in patients(P<0.01).Results from immunohistochemistry demonstrated that xCT expression level in breast cancer tissues was greatly higher in patients with lung metastasis than that without lung metastasis(P<0.001).[Conclusion]Combined therapy of IFN-γand DDP decreases glutathione secretion by inhibiting xCT expression in L929,thereby suppressing triple-negative breast cancer metastasis.Our study is supposed to provide a new perspective for clinical treatment of triple-negative breast cancer metastasis.