环孢素A调控自噬诱导肿瘤相关巨噬细胞极化干预子宫内膜癌细胞的恶性生物学行为

Cyclosporin A regulates autophagy and induces tumor-associated macrophage polarization to interfere with the malignant biological behavior of endometrial cancer cells

目的:探究环孢素A(CsA)对肿瘤相关巨噬细胞极化的调控作用及其与子宫内膜癌(EC)细胞恶性生物学行为之间的联系,并初步探讨相关机制。方法:采用佛波酯(PMA)和白细胞介素-4(IL-4)诱导THP-1细胞向具有肿瘤相关巨噬细胞极化分型特点的M2样巨噬细胞分化,分别以50、100、150、200 ng/mL CsA处理诱导后的细胞24 h,CCK-8法检测细胞活性,流式细胞术检测CD86和CD206表达,实时荧光定量聚合酶链式反应(qRT-PCR)检测诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、重组人精氨酸酶1(Arg-1)的mRNA相对表达量。将人子宫内膜癌Ishikawa细胞随机分为4组并进行相应处理:对照组,Ishikawa细胞加入细胞培养液培养24 h;CsA组,200 ng/mL CsA干预Ishikawa细胞24 h;肿瘤相关巨噬细胞(TAM)组,肿瘤相关巨噬细胞培养液上清干预Ishikawa细胞24 h;CsA+TAM组,经200 ng/mL CsA处理肿瘤相关巨噬细胞的培养液上清干预Ishikawa细胞24 h。处理结束后,CCK-8法检测细胞活性,Transwell小室实验检测细胞迁移与侵袭,Annexin V-FITC/PI双染法检测细胞凋亡,Western Blotting检测自噬相关蛋白表达,免疫荧光染色检测自噬标记物LC3表达,透射电子显微镜观察自噬体形成。结果:经过不同浓度CsA处理肿瘤相关巨噬细胞后,细胞存活率未发生变化(P>0.05),而细胞中CD86表达增加、CD206表达下降,iNOS、TNF-α的mRNA相对表达量升高,TGF-β、Arg-1的mRNA相对表达量降低(P<0.01);与对照组比较,CsA组Ishikawa细胞存活率下降,迁移数目与侵袭数目减少,细胞凋亡率增加,Beclin-1蛋白表达下降、p62蛋白表达增加,LC3-Ⅱ/LC3-Ⅰ比值下调,LC3荧光表达减弱,细胞内未见自噬体,而TAM组细胞迁移数目与侵袭数目增加,细胞凋亡率减少,Beclin-1蛋白表达增加、p62蛋白表达下降,LC3-Ⅱ/LC3-Ⅰ比值上调,LC3荧光表达增强,细胞内自噬体数目较多(P<0.01);与TAM组比较,CsA+TAM组细胞存活率下降,迁移数目与侵袭数目减少,细胞凋亡率增加,Beclin-1蛋白表达下降、p62蛋白表达增加,LC3-Ⅱ/LC3-Ⅰ比值下调,同时,LC3荧光表达减弱,细胞内自噬体减少(P<0.01)。结论:CsA通过调控肿瘤相关巨噬细胞的极化,抑制子宫内膜癌细胞增殖、迁移与侵袭能力,其机制可能与细胞自噬水平相关。

Objective:To explore the regulation of Cyclosporin A(CsA)on tumor-associated macrophage polarization and its relationship with the malignant biological behavior of endometrial carcinoma(EC)cells.And preliminary discussion of related mechanisms.Methods:PMA and IL-4 were used to induce THP-1 cells to differentiate into M2-like macrophages with the characteristics of tumor-associated macrophage polarization typing,the induced cells were treated with 50,100,150,200 ng/mL CsA for 24 h,CCK-8 method to detect cell viability,flow cytometry to detect the expression of CD86 and CD206,real-time fluorescent quantitative PCR technology detects the relative expression mRNA of iNOS,TNF-α,TGF-βand Arg-1.Human endometrial cancer Ishikawa cells were randomly divided into 4 groups and processed accordingly:In the control group,Ishikawa cells were added to the cell culture medium and cultured for 24 h.In the CsA group,200 ng/mL CsA interfered with Ishikawa cells for 24 h.TAM group,tumor-associated macrophage culture supernatant intervention Ishikawa cells for 24 h.CsA+TAM group,200 ng/mL CsA treatment tumor-associated macrophage culture supernatant intervention Ishikawa cells 24 h,after the treatment,CCK-8 method was used to detect cell viability,Transwell chamber experiment detects cell migration and invasion,Annexin V-FITC/PI double staining method to detect cell apoptosis,Western Blotting detection of autophagy-related protein expression,immunofluorescence staining to detect the expression of autophagy marker LC3,transmission electron microscope observation of autophagosome formation.Results:After treating tumor-associated macrophages with different concentrations of CsA,the cell survival rate did not change(P>0.05),while the expression of CD86 increased and the expression of CD206 decreased,the relative expression mRNA of iNOS and TNF-αincreased,and the relative expression mRNA of TGF-βand Arg-1 decreased(P<0.01).Compared with the control group,the survival rate of Ishikawa cells in the CsA group decreased,the number of migration and invasion decreased,and the apoptosis rate increased,Beclin-1 protein expression decreased,p62 protein expression increased,the ratio of LC3-Ⅱ/LC3-Ⅰdecreased,the fluorescence expression of LC3 decreased,and no autophagosomes were seen in the cells.While the number of migration and invasion of the TAM group increased,and the apoptosis rate decreased,Beclin-1 protein expression increased,p62 protein expression decreased,LC3-Ⅱ/LC3-Ⅰratio increased,LC3 fluorescence expression increased,and the number of autophagosomes in the cell increased(P<0.01).Compared with the TAM group,the cell survival rate of the CsA+TAM group decreased,the number of migration and invasion decreased,and the rate of cell apoptosis increased,Beclin-1 protein expression decreased,p62 protein expression increased,the ratio of LC3-Ⅱ/LC3-Ⅰdecreased,at the same time,the fluorescence expression of LC3 decreased,and intracellular autophagosomes decreased(P<0.01).Conclusion:CsA can inhibit the proliferation,migration and invasion of endometrial cancer cells by regulating the polarization of tumor-associated macrophages.The mechanism may be related to the level of autophagy.