BRCC3/NLRP3促进子宫内膜异位症炎癌转化中的机制

The mechanism of BRCC3/NLRP3 in promoting the transformation of endometriosis to endometriosis-associated ovarian carcinoma

目的:探讨NOD样受体蛋白3(NLRP3)炎症小体的活化在子宫内膜异位症(EMT)进展为EMT相关性卵巢癌(EAOC)过程中的作用及其机制。方法:选取2018年4月至2019年6月上海市长宁区幼保健院收治的EAOC、EMT、正常子宫内膜(CON组)组织标本各15例及患者的临床资料,利用免疫组织化学染色法、WB法检测EAOC、EMT和CON组织中NLRP3、caspase-1和IL-1β及含BRCA1/BRCA2的复杂亚基3(BRCC3)的表达水平。构建过表达BRCC3质粒和si-NLRP3质粒并转染EMT细胞CRL-7566,通过WB法检测转染后细胞中BRCC3蛋白的表达水平,利用MTT法、流式细胞术及Transwell实验分别检测转染后细胞增殖、凋亡、迁移与侵袭能力的变化。对过表达BRCC3组细胞进行干扰NLRP3实验,通过WB法检测干扰后BRCC3和NLRP3蛋白的表达水平,检测干扰后细胞增殖、凋亡、迁移与侵袭能力的变化。结果:EAOC和EMT组织中NLRP3、caspase-1、IL-1β和BRCC3的表达水平较CON组均呈明显升高(均P<0.01),且EAOC组织中NLRP3与BRCC3的表达呈正相关(r=0.65,P<0.01)。在CRL-7566细胞中过表达BRCC3显著促进细胞的增殖、迁移和侵袭并抑制细胞凋亡(均P<0.01),敲减NLRP3则抑制CRL-7566细胞的上述表型(均P<0.01),过表达BRCC3增强NLRP3的表达水平(P<0.01),而干扰BRCC3则抑制NLRP3表达(P<0.01);干扰NLRP3可以部分逆转BRCC3对细胞凋亡的抑制作用(P<0.01)、对细胞迁移(P<0.05)和侵袭(P<0.01)的促进作用。结论:EAOC和EMT组织中NLRP3和BRCC3均呈高表达,过表达BRCC3可促进CRL-7566细胞的增殖、迁移和侵袭并抑制细胞凋亡,与EMT向EAOC转化有关,BRCC3/NLRP3是潜在的EAOC炎癌转化预测标志物及治疗靶点。

Objective:To investigate the role of NOD-like receptor protein 3(NLRP3)inflammatory vesicle activation in the progression of endometriosis(EMT)to endometriosis-associated ovarian cancer(EAOC)and the mechanism.Methods:EOAC tissues(n=15),EMT tissues(n=15),and normal endometrium tissues(CON,n=15)resected from patients during April 2018 and June 2019,as well as the clinical data of patients,were collected from the Shanghai Changning Maternity and Infant Health Hospital.Expression of NLRP3,BRCA1/BRCA2 containing complex subunit 3(BRCC3),caspase-1 and IL-1βin the tissues of each group was detected by immunohistochemistry and WB methods.BRCC3 overexpression plasmids and si-BRCC3 plasmids were constructed and transfected into EMT CRL-7566 cells.The expression level of BRCC3 protein in the transfected cells was detected by WB method.The changes in cell proliferation,apoptosis,migration and invasion of transfected cells were detected by MTT method,flow cytometry and Transwell test,respectively.The BRCC3-overexpressing cells were further interfered with NLRP3.The expression levels of BRCC3 and NLRP3proteins after the interference were detected by WB method,and the changes in cell proliferation,apoptosis,migration and invasion after interference were also detected.Results:The expression levels of NLRP3,caspase-1,IL-1βand BRCC3 in EAOC and EMT tissues were higher than those in CON group(all P<0.01),and there was a positive correlation between the expression of NLRP3 and BRCC3 in EAOC tissues(r=0.65,P<0.01).Overexpression of BRCC3 significantly promoted cell proliferation,migration and invasion and inhibited cell apoptosis in CRL-7566 cells(all P<0.01);While knockdown of BRCC3 had the opposite effects(all P<0.01).Overexpression of BRCC3 increased the expression level of NLRP3(P<0.01),while interference with BRCC3 inhibited the expression of NLRP3(P<0.01).Interference with NLRP3 partially attenuated the inhibition on cell apoptosis(P<0.01)and promotion on cell migration(P<0.05)and invasion(P<0.01)brought by BRCC3 overexpression.Conclusion:Both NLRP3 and BRCC3 are highly expressed in EAOC and EMT tissues.Over-expression of BRCC3 can promote the proliferation,migration and invasion but inhibit apoptosis of CRL-7566 cells.BRCC3/NLRP3 is a potential predictive marker and treatment target for the transformation of EMT to EAOC.