二甲双胍通过AMPK/Smad3信号通路抑制卵巢癌腹腔间皮细胞的间皮-间质转化

Metformin suppresses the mesothelial-mesenchymal transition of ovarian cancer peritoneal mesothelial cells through AMPK/Smad3 signaling pathway

目的:探讨二甲双胍对人腹腔间皮细胞(human peritoneal mesothelial cells,HPMC)间皮-间质转化的影响及其可能机制,以及进一步对卵巢癌细胞系SKOV3系膜清除能力的影响。方法:搜集2018年02月至2021年02月襄阳市第一人民医院妇产科正常网膜组织和高级别浆液性卵巢癌网膜转移灶标本进行免疫组织化学检测Calretinin表达情况;分离、培养原代人腹腔间皮细胞并进行Calretinin流式鉴定;Western blot和Transwell实验检测二甲双胍或肿瘤上清处理正常患者来源HPMC后其间皮-间质转化相关蛋白(E-cadherin、Vimentin、α-SMA)及迁移能力改变情况;Western blot检测二甲双胍、Smad3抑制剂及AMPK干扰处理前后正常来源原代人腹腔间皮细胞中MMP2、E-cadherin、Vimentin、p-AMPK和p-Smad3的表达情况;SKOV3球体的系膜清除实验验证二甲双胍预处理人腹膜间皮细胞系HMrSV5对SKOV3系膜清除能力的影响。结果:正常网膜中Calretinin表达于腹腔侧表面的腹腔内皮细胞,卵巢癌网膜转移灶表面Calretinin表达缺失,肿瘤间质中高表达Calretinin;分离培养的肿瘤来源原代人腹腔间皮细胞流式检测表现为CD326^(-)CD45^(-)CD31^(-)Calretinin^(+);二甲双胍能够抑制肿瘤上清诱导的正常来源原代HPMC的间皮-间质转化及迁移;二甲双胍能够抑制肿瘤上清诱导的正常来源原代HPMC中Smad3/MMP2通路活化和E-cadherin表达,并依赖于AMPK信号活化;二甲双胍预处理HMrSV5通过AMPK信号的介导抑制了HMrSV5的间皮-间质转化进而间接抑制了SKOV3的系膜清除能力。结论:卵巢癌转移灶中的间质成分部分来源于腹腔间皮细胞,二甲双胍通过AMPK调控的Smad3信号通路抑制其间皮-间质转化,进而抑制卵巢癌肿瘤细胞的系膜清除。

Objective:To investigate the effect of metformin on the mesothelial-mesenchymal transition(MMT)of human peritoneal mesothelial cells(HPMC)and its possible mechanism,as well as the effect of metformin on mesangial clearance of ovarian cancer cell line SKOV3.Methods:Specimens of normal omentum and high-grade serous carcinoma(HGSC)omentum metastases were collected from the department of obstetrics and gynecology of Xiangyang No.1 People's Hospital from February 2018 to February 2021 for immunohistochemical(IHC)analysis of Calretinin expression.Primary HPMC was isolated,cocultured and identified via flow cytometry for further experiments.Western blot and Transwell assays were adopted to detect the influence of metformin on the MMT associated proteins(E-cadherin,Vimentin,α-SMA)expression and migration of HPMC from normal patients treated with metformin or tumor supernatant.Western blot assay was employed to detect the expression of MMP2,E-cadherin,Vimentin,p-AMPK and p-Smad3 in primary HPMC from normal origin before and after treatment with metformin,Smad3 inhibitors and AMPK interference.The mesangial clearance assay of SKOV3 sphere was conducted to confirm the effect of metformin pretreatment of HPMC cell line HMrSV5 on the mesangial clearance ability of SKOV3 cells.Results:Calretinin was mainly expressed in peritoneal mesothelial cell monolayer of normal omentum,which was missing on HGSC omentum metastases surface,but was highly expressed in the tumor stroma.Flow cytometry showed a specific phenotype(CD326^(-)CD45^(-)CD31^(-)Calretinin^(+))in primary tumor-derived HPMC.Metformin could inhibit the tumor supernatant-induced MMT and corresponding migration ability of normal HPMC.Metformin could suppress the tumor supernatant-induced Smad3/MMP2 signaling activation and E-cadherin expression of normal HPMC in an AMPK dependent manner.Metformin pretreatment of HMrSV5 inhibited the MMT of HMrSV5 through AMPK signal mediation,thereby indirectly inhibited the mesangial clearance of SKOV3 cells.Conclusion:Mesenchymal components in ovarian cancer metastasis are partially derived from peritoneal mesenchymal cells.Metformin inhibits the MMT of HPMC through Smad3 signaling pathway regulated by AMPK,thus inhibiting the mesangial clearance of ovarian cancer cells.