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邓心怡, 高子煦, 王璐, 等. 环指蛋白157通过下调小凹蛋白-1表达参与ERK通路激活促进黑素瘤细胞增殖[J]. 中国临床医学, 2023, 30(2): 257-264. DOI: 10.12025/j.issn.1008-6358.2023.20230530
引用本文: 邓心怡, 高子煦, 王璐, 等. 环指蛋白157通过下调小凹蛋白-1表达参与ERK通路激活促进黑素瘤细胞增殖[J]. 中国临床医学, 2023, 30(2): 257-264. DOI: 10.12025/j.issn.1008-6358.2023.20230530
DENG Xin-yi, GAO Zi-xu, WANG Lu, et al. RNF157 participates in the ERK pathway activation to promote melanoma cells proliferation by down-regulating the expression of caveolin-1[J]. Chinese Journal of Clinical Medicine, 2023, 30(2): 257-264. DOI: 10.12025/j.issn.1008-6358.2023.20230530
Citation: DENG Xin-yi, GAO Zi-xu, WANG Lu, et al. RNF157 participates in the ERK pathway activation to promote melanoma cells proliferation by down-regulating the expression of caveolin-1[J]. Chinese Journal of Clinical Medicine, 2023, 30(2): 257-264. DOI: 10.12025/j.issn.1008-6358.2023.20230530

环指蛋白157通过下调小凹蛋白-1表达参与ERK通路激活促进黑素瘤细胞增殖

RNF157 participates in the ERK pathway activation to promote melanoma cells proliferation by down-regulating the expression of caveolin-1

  • 摘要:
    目的 探讨环指蛋白157(RING finger protein 157, RNF157)在黑素瘤细胞增殖中的作用及相关机制。
    方法 采用慢病毒转染技术构建RNF157过表达黑素瘤细胞株,小干扰RNA(siRNA)技术构建敲减RNF157的黑素瘤细胞株,采用实时荧光定量聚合酶链反应(qRT-PCR)和Western印迹实验验证转染效果。采用MTT实验、克隆形成实验检测RNF157过表达黑素瘤细胞和敲减RNF157黑素瘤细胞的细胞增殖能力。采用裸鼠皮下成瘤实验及免疫组化染色检测RNF157过表达黑素瘤细胞体外增殖能力。采用免疫共沉淀(Co-IP)结合质谱分析、蛋白质组学和泛素化组学,探究RNF157在ERK通路中的潜在作用机制。
    结果 免疫组化染色结果显示,RNF157在黑素瘤组织中的表达高于瘤旁组织(P<0.01)。qRT-PCR和Western印迹实验结果显示,过表达组的RNF157表达水平显著高于对照组(P<0.000 1);si3转染效率最高,si3组的RNF157表达水平显著低于对照组(P<0.01)。MTT实验、克隆形成实验结果显示,RNF157过表达可促进黑素瘤细胞增殖,敲减RNF157抑制黑素瘤细胞增殖。进一步检测发现,RNF157过表达可以激活黑素瘤细胞的ERK通路。Co-IP结合质谱分析、蛋白质组学和泛素化组学结果显示,RNF157可结合小凹蛋白-1(caveolin-1,CAV1)并下调其表达水平,从而调控ERK通路。
    结论 RNF157可促进黑素瘤细胞的体内外增殖;在黑素瘤细胞中,RNF157可能通过结合并下调CAV1表达水平,参与细胞内ERK通路激活。

     

    Abstract:
    Objective To explore the role and mechanism of RING finger protein 157 (RNF157) in melanoma cells proliferation.
    Methods The RNF157 overexpression melanoma cell line was constructed by lentivirus transfection technique, and the RNF157 knockout melanoma cell line was constructed by small interfering RNA (siRNA). The transfection effect was verified by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and Western blotting. MTT assay and clone formation assay were used to analyze the cell proliferation ability of RNF157 overexpression melanoma cells and knockdown RNF157 melanoma cells. The proliferation ability of RNF157 overexpressing melanoma cells in vitro was detected by subcutaneous tumorigenesis test and immunohistochemical staining in nude mice. The potential mechanism of RNF157 in ERK pathway was explored by co-immunoprecipitation (Co-IP) combined with mass spectrometry, proteomics and ubiquitinomics.
    Results Immunohistochemical staining showed that the expression of RNF157 in melanoma tissue was higher than that in adjacent normal tissue (P < 0.01). The results of qRT-PCR and Western blotting showed that the expression level of RNF157 in overexpression group was significantly higher than that in control group (P < 0.000 1), the transfection efficiency of si3 was the highest, and the expression level of RNF157 in si3 group was significantly lower than that in control group (P < 0.01). The results of MTT assay and colony formation assay showed that overexpression of RNF157 could promote the proliferation of melanoma cells, and knockdown RNF157 could inhibit the proliferation of melanoma cells. The results of Western blotting showed that overexpression of RNF157 could activate ERK pathway in melanoma cells. Co-IP combined with mass spectrometry, proteomics and ubiquitinomics analysis showed that RNF157 could bind to caveolin-1 (CAV1) and down-regulate its expression level, thus regulating the ERK pathway.
    Conclusions RNF157 can promote the proliferation of melanoma cells in vitro and in vivo. In melanoma cells, RNF157 participates in the activation of ERK pathway by binding and down-regulating the expression of CAV1.

     

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