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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(P<0.0001);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。
Objective: To investigate the effect of metformin on the activity of tumor-associated fibroblasts (CAF) in ovarian cancer and the possible regulatory mechanisms. Methods: The mRNA expression levels of inflammatory factors (IL-6, OPN, IL-1b, COX-2, Cyr61) after metformin treatment in SKOV3 cell line and primary CAF were detected by RT-PCR. The expression of MRC5 in SKOV3-CM ) Cultured for 7 to 10 days to obtain activated MRC5 MRC5-CAF, primary CAF and MRC5-CAF were identified by immunofluorescence α-SMA expression; Western blot detection of the inflammatory cytokine protein levels. Correlation of inflammatory cytokines with CAF-related genes was verified in 557 cases of ovarian cancer in the TCGA database. Effect of Metformin on α-SMA and IL-6 Expression in Activated MRC5 Cells by Immunofluorescence. The effect of metformin or IL-6 on CAF-related genes was further verified in the MRC5-CAF cell line. Proliferation and Transwell experiments were performed to compare the effects of metformin or IL-6 on the proliferation and migration of SKOV3 induced by MRC5-CAF. Collagen retraction test compares the effect of metformin or IL-6 on ECM of contractile extracellular matrix of MRC5-CAF. Results: Metformin down-regulated CAF inflammatory cytokines, especially IL-6, and down-regulated the level of α-SMA in CAF. There were significant positive correlations between IL-6 mRna and CAF-related genes (FAP, PDGFRB, FN1, COLL6A6) in TCGA database (P <0.0001) SMA down-regulated. Metformin reversed the ability of IL-6 to promote tumor proliferation and metastasis and collagen contraction in CAF of ovarian cancer. CONCLUSION: IL-6 in ovarian cancer may be involved in the maintenance of interstitial CAF cell activity and interstitial properties. Metformin may inhibit the proliferation of ovarian cancer CAF cells by down-regulating the IL-6 / P-STAT3 pathway.