血管抑制素-2通过调控上皮细胞间质化促进宫颈癌细胞的增殖和转移

Vasohibin-2 promotes proliferation and metastasis of cervical cancer cells by regulating epithelial-mesenchymal transition

目的探讨血管抑制素-2(VASH2)能否促进宫颈癌细胞在体外的增殖和转移及其可能的分子机制。方法在外源性过表达和干扰内源性flotillin-1表达的宫颈癌细胞中,通过公共数据库结合RNA-seq挖掘分析差异表达基因;在宫颈正常上皮细胞(HcerEpic细胞)和宫颈癌细胞(HeLa、C-33A、Ca ski、SiHa和MS751)以及不同淋巴结转移状态的新鲜宫颈癌组织中检测VASH2的表达;运用慢病毒稳定表达载体构建外源性过表达VASH2和干扰内源性VASH2表达的宫颈癌细胞及对照细胞,并检测其增殖、迁移、侵袭和淋巴管形成的情况;在上述细胞模型中检测上皮细胞间质化(EMT)过程关键分子及TGF-β的表达水平。结果RNA-seq发现在外源性过表达flotillin-1的宫颈癌细胞中VASH2的表达上调(P<0.05),而在抑制内源性flotillin-1表达的细胞中VASH2表达下调(P<0.05),并在公共数据库中证实VASH2在有淋巴结转移的宫颈癌组织中相对于无淋巴结转移的癌组织呈高表达(P<0.01)。相对于HcerEpic细胞和淋巴结未转移的宫颈癌组织,VASH2在Ca Ski、SiHa、MS751细胞和有淋巴结转移的宫颈癌组织中表达上调(P<0.05);相对于对照细胞,外源性过表达VASH2的宫颈癌细胞增殖、迁移、侵袭和淋巴管形成能力增强,而抑制内源性VASH2表达的宫颈癌细胞上述能力则受到抑制(P<0.05);相对于对照细胞,外源性过表达VASH2的宫颈癌细胞中EMT过程的关键分子E-cadherin下调,N-cadherin、Vimentin和VEGF-C上调,而抑制内源性VASH2表达的宫颈癌细胞中E-cadherin上调,N-cadherin、Vimentin和VEGF-C下调(P<0.05);外源性过表达VASH2的宫颈癌细胞中TGF-β的mRNA表达水平上调,而抑制内源性VASH2表达的宫颈癌细胞中TGF-β的mRNA表达水平下调(P<0.001)。结论flotillin-1可能通过下游靶基因VASH2参与TGF-β信号通路诱导EMT过程在体外促进宫颈癌细胞的增殖、迁移、侵袭和淋巴管形成。

Objective To explore the role of vasohibin-2(VASH2)in regulation of proliferation and metastasis of cervical cancer cells.Methods We analyzed the differentially expressed genes between cervical cancer cells with flotillin-1 overexpression and knockdown by RNA-seq combined with analysis of public databases.The expression levels of VASH2 were examined in normal cervical epithelial cells(HcerEpic),cervical cancer cell lines(HeLa,C-33A,Ca ski,SiHa and MS751)and fresh cervical cancer tissues with different lymph node metastasis status.We further tested the effects of lentivirus-mediated overexpression and interference of VASH2 on proliferation,migration,invasion and lymphatic vessel formation of the cervical cancer cells and detected the expression levels of key epithelial-mesenchymal transition(EMT)markers and TGF-βmRNA.Results RNA-seq and analysis of public databases showed that VASH2 expression was significantly upregulated in cervical cancer cells exogenously overexpressing flotillin-1(P<0.05)and downregulated in cells with flotillin-1 knockdown(P<0.05),and was significantly higher in cervical cancer tissues with lymph node metastasis than in those without lymph node metastasis(P<0.01).In cervical cancer cell lines Ca Ski,SiHa,and MS751 and cervical cancer tissue specimens with lymph node metastasis,VASH2 expression was also significantly upregulated as compared with HcerEpic cells and cervical cancer tissues without lymph node metastasis(P<0.05).Exogenous overexpression of VASH2 significantly promoted proliferation,migration,invasion and lymphatic vessel formation of cervical cancer cells,whereas these abilities were significantly inhibited in cells with VASH2 knockdown(P<0.05).The cervical cancer cells overexpressing VASH2 showed significant down-regulation of e-cadherin and up-regulation of N-cadherin,Vimentin and VEGF-C,while the reverse changes were detected in cells with VASH2 knockdown(P<0.05).TGF-βmRNA expression was significantly up-regulated in cervical cancer cells overexpressing VASH2 and down-regulated in cells with VASH2 knockdown(P<0.001).Conclusion Flotillin-1 may participate in TGF-βsignaling pathway mediated EMT through its down-stream target gene VASH2 to promote the proliferation,migration,invasion and lymphatic vessel formation of cervical cancer cells in vitro.