Fem-1同系物C通过调节ELAVL1/OPA1轴介导的线粒体融合促进乳腺癌进展的机制研究

Mechanism study of FEM1C promoting breast cancer progression via the ELAVL1/OPA1 axis-mediated mitochondrial fusion

    摘要
  • 摘要:
    目的 探讨Fem-1同系物C（Fem-1 homolog C，FEM1C）对乳腺癌的影响及其潜在调节机制。
    方法 通过qPCR检测乳腺癌组织和细胞系中FEM1C的表达水平。利用在线数据库预测FEM1C与ELAVL1蛋白的结合，并通过CoIP分析进行验证；利用starBase数据库预测ELAVL1与OPA1 mRNA的结合，并通过RIP分析进行验证。采用FEM1C的shRNA（sh-FEM1C）和过表达载体（FEM1C）、ELAVL1的过表达载体（ELAVL1）、OPA1的过表达载体（OPA1）转染乳腺癌细胞MDA-MB-231，或用100 μM的DRP1抑制剂Mdivi-1或OPA1抑制剂MYLS22处理MDA-MB-231细胞。用sh-FEM1C慢病毒载体注射裸鼠构建异种移植瘤模型，监测肿瘤生长。
    结果 乳腺癌组织中FEM1C表达显著上调（P<0.01）。沉默FEM1C抑制MDA-MB-231细胞增殖，诱导细胞凋亡，促进自噬蛋白LC3Ⅱ/Ⅰ表达，抑制p62表达，上调线粒体PINK1蛋白水平，促进线粒体裂变蛋白DRP1和MIEF2表达，抑制融合蛋白OPA1和MFN1表达（P<0.01）。Mdivi-1处理抑制DRP1表达（P<0.01），对细胞活力无影响（P>0.05）；MYLS22处理抑制OPA1表达，抵消FEM1C过表达对MDA-MB-231细胞的影响（P<0.01）。机制研究显示FEM1C与ELAVL1蛋白结合，并促进其表达（P<0.01）；ELAVL1蛋白通过与OPA1 mRNA结合稳定其mRNA，上调OPA1蛋白水平（P<0.01）。过表达OPA1逆转了FEM1C沉默对乳腺癌细胞的影响（P<0.01）。体内结果显示，敲低FEM1C抑制体内肿瘤生长（P<0.01）。
    结论 FEM1C通过促进ELAVL1蛋白表达介导OPA1 mRNA稳定性，从而促进线粒体融合，抑制自噬，促进乳腺癌进展。

     

    Abstract:
    Objective To investigate the role of Fem-1 homolog C (FEM1C) in breast cancer progression and elucidate its underlying regulatory mechanism.
    Methods  The expression of FEM1C in breast cancer tissues and cells were detected with qPCR. The binding of FEM1C to ELAVL1 protein was predicted with an online database and validated by CoIP analysis; and the binding of ELAVL1 protein to OPA1 mRNA was predicted by using the starBase database and validated by RIP analysis. Next, breast cancer cell MDA-MB-231 was transfected with FEM1C shRNA (sh-FEM1C) or overexpression vector (FEM1C) or/and ELAVL1 overexpression vector (ELAVL1) or/and OPA1 overexpression vector (OPA1), or treated with 100 μM Mdivi-1, an DRP1 inhibitor, or MYLS22, an OPA1 inhibitor. Finally, nude mice were injected with sh-FEM1C lentiviral vectors to construct xenograft tumor models, and tumor growth was monitored.
    Results  The expression of FEM1C was upregulated in breast cancer tissues (P<0.01). Silencing FEM1C inhibited the proliferation, induced apoptosis, promoted the expression of autophagy protein LC3 Ⅱ/Ⅰ, inhibited p62 protein expression, upregulated the protein level of PINK1 in mitochondrial, promoted the expression of mitochondrial fission proteins DRP1 and MIEF2, and inhibited the expression of fusion proteins OPA1 and MFN1 in MDA-MB-231 cells (P<0.01). Mdivi-1 treatment inhibited DRP1 expression (P<0.01), but had no effect on cell viability (P>0.05); MYLS22 treatment inhibited OPA1 expression and counteracted the effect of FEM1C overexpression on MDA-MB-231 cells (P<0.01). Mechanistic studies revealed that FEM1C binds to ELAVL1 protein and promotes its expression (P<0.01); ELAVL1 protein stabilizes OPA1 mRNA by binding to it and upregulates OPA1 protein levels (P<0.01). Overexpression of OPA1 reversed the effect of FEM1C silencing on MDA-MB-231 cells (P<0.01). In vivo results showed that knockdown of FEM1C inhibited tumor growth in vivo (P<0.01).
    Conclusions  FEM1C promotes the stability of OPA1 mRNA by upregulation of ELAVL1 protein to promote mitochondrial fusion and inhibit autophagy, thereby promoting breast cancer progression.

     

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