二甲双胍通过下调IL-6抑制卵巢癌肿瘤相关成纤维细胞活性的研究

目的:探讨二甲双胍对卵巢癌肿瘤相关成纤维细胞(CAF)活性的影响,以及可能的调控机制。方法:RT-PCR法检测二甲双胍作用于SKOV3细胞系及原代CAF后炎症因子(IL-6、OPN、IL-1b、COX-2、Cyr61)mRNA水平变化;将MRC5于SKOV3-CM(condition medium)培养7~10天得到活化的MRC5即MRC5-CAF,原代CAF及MRC5-CAF经免疫荧光鉴定α-SMA表达;Western blot检测上述炎症因子蛋白水平。在TCGA数据库557例卵巢癌中验证炎症因子与CAF属性相关基因的相关性。免疫荧光验证活化的MRC5中二甲双胍对α-SMA及IL-6表达的影响。在MRC5-CAF细胞系中进一步验证了二甲双胍或IL-6对CAF属性相关基因的影响。增殖实验和Transwell实验比较二甲双胍或IL-6对MRC5-CAF促进SKOV3增殖及迁移能力的影响。胶原回缩试验比较二甲双胍或IL-6对MRC5-CAF收缩细胞外基质ECM的影响。结果:二甲双胍下调CAF中炎症因子尤其是IL-6,同时下调CAF中α-SMA水平。TCGA数据库中IL-6 mRna与CAF属性相关基因(FAP、PDGFRB、FN1、COLL6A6)呈显著正相关(P0.0001);二甲双胍、IL-6共刺激组较IL-6组,STAT3通路和α-SMA下调;二甲双胍能逆转IL-6对卵巢癌CAF促肿瘤增殖转移及收缩胶原的能力。结论:卵巢癌中IL-6可能参与维持了间质CAF细胞活性和间质属性,二甲双胍可能通过下调CAF中IL-6/P-STAT3通路从而抑制卵巢癌CAF对肿瘤细胞的支持作用。

Metformin suppresses the activity of ovarian cancer associated fibroblast through the ;downregulation of IL-6

Objective:To explore the influence of metformin on ovarian cancer associat-ed fibroblast ( CAF ) and the involved mechanism. Methods:RT-PCR was done to detect the effect of metformin on mRNA levels of inflammatory cytokines ( IL-6, OPN, IL-1b, COX-2, Cyr61) in SKOV3 and primary CAFs. Activated MRC5 was gotten from that cultured with SK-OV3-CM for 7 to 10 days and was named MRC5-CAF. MRC5-CAF and primary CAFs were i-dentified by immunofluorescence ofα-SMA. Then the effect of metformin on the aforementioned cytokines was detected in MRC5-CAF using immunoblotting. Online TCGA data base ( http://www. cbioportal. org ) was employed to analyze the mRNA correlation between IL-6 and the CAFs characteristic moleculars among 557 ovarian cancers. Immunofluorescence was adopted to further verify the impact of metformin on IL-6 andα-SMA expressions of activated MRC5 . More-over,we performed the same treatment in MRC5-CAF to validate the influence of metformin or IL-6 on CAF characteristic genes. In vitro proliferation assay and transwell were employed to ex-amine the effects of metformin or IL-6 on CAFs' support for cancer cells' proliferaton and inva-sion. Collagen contraction experiment was done to check the alteration of CAFs in contraction of the ECM substrate under the influence of metformin or IL-6 . Result:Metformin was noted to re-duce the inflammatory cytokines expression,especially IL-6 and it could attenuate the character-istic a-SMA level in CAFs. The mRNA levels of IL-6 and CAF associated genes ( FAP, PDG-FRB,FN1,COLL6A6)were dramatically positively-correlated in TCGA datasets(P<0. 0001). Metformin and IL-6 co-stimulation regulated the activation of STAT3 signaling by IL-6 and its subsequent effect on CAF properties comparing to IL-6 group. It was confirmed that metformin could impair the capacities of CAF in promoting the proliferation,invasion and collagen contrac-tion induced by IL-6 . Conclusions:IL-6 is positively correlated with CAFs charactersitc genes and probably sustain the CAFs property in ovarian cancer. Metformin can probably downregulate IL-6 expression and the subsequent STAT3 activation mediated maintainence of CAFs property, thus diminishing the ability of CAFs in supporting of ovarian cancer cells' proliferation and in-vasion.