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G蛋白偶联受体激酶3在口腔鳞状细胞癌细胞增殖、迁移和侵袭中的作用及其可能的机制

张晗 罗清琼 朱丽萍 陈福祥

张晗, 罗清琼, 朱丽萍, 陈福祥. G蛋白偶联受体激酶3在口腔鳞状细胞癌细胞增殖、迁移和侵袭中的作用及其可能的机制[J]. 中国肿瘤生物治疗杂志, 2021, 28(5): 435-442. doi: 10.3872/j.issn.1007-385x.2021.05.003
引用本文: 张晗, 罗清琼, 朱丽萍, 陈福祥. G蛋白偶联受体激酶3在口腔鳞状细胞癌细胞增殖、迁移和侵袭中的作用及其可能的机制[J]. 中国肿瘤生物治疗杂志, 2021, 28(5): 435-442. doi: 10.3872/j.issn.1007-385x.2021.05.003
ZHANG Han, LUO Qingqiong, ZHU Liping, CHEN Fuxiang. Role of G protein-coupled receptor kinase 3 in the proliferation, migration and invasion of oral squamous carcinoma cells and its possible mechanism[J]. Chin J Cancer Biother, 2021, 28(5): 435-442. doi: 10.3872/j.issn.1007-385x.2021.05.003
Citation: ZHANG Han, LUO Qingqiong, ZHU Liping, CHEN Fuxiang. Role of G protein-coupled receptor kinase 3 in the proliferation, migration and invasion of oral squamous carcinoma cells and its possible mechanism[J]. Chin J Cancer Biother, 2021, 28(5): 435-442. doi: 10.3872/j.issn.1007-385x.2021.05.003

G蛋白偶联受体激酶3在口腔鳞状细胞癌细胞增殖、迁移和侵袭中的作用及其可能的机制

doi: 10.3872/j.issn.1007-385x.2021.05.003
基金项目: 上海市“医苑新星”青年医学人才培养计划:青年医学人才类−临床检验项目资助(No. 沪卫人事[2020] 087号);上海交通大学医学院附属第九人民医院院级基金−基础研究助推计划资助(No. JYZZ040)
详细信息
    作者简介:

    张晗(1987–),女,硕士,主管技师,主要从事肿瘤免疫相关研究,E-mail:zhanghan4015@163.com

    通讯作者:

    陈福祥(CHEN Fuxiang,corresponding author),博士,主任技师,教授,博士生导师,主要从事肿瘤发生发展机制的研究,E-mail:chenfx@sjtu.edu.cn

  • 中图分类号: R739.85;R730.2

Role of G protein-coupled receptor kinase 3 in the proliferation, migration and invasion of oral squamous carcinoma cells and its possible mechanism

Funds: Project supported by the Young Medical Talents-Clinical Laboratory Projects of Shanghai “Rising Stars of Medical Talent” Youth Development Program (No. Shanghai Municipal Health Personnel [2020] 087), and the Basic Research Boost Program of the Fundamental Research Program Funding of Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. JYZZ040)
  • 摘要:   目的:  探讨沉默G蛋白偶联受体激酶3(G protein-coupled receptor kinase 3,GRK3)对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞增殖、迁移和侵袭的影响及其可能的机制。  方法:  利用Oncomine数据库分析GRK3在正常口腔组织及OSCC组织中的表达水平。用RNA干扰技术敲降GRK3在OSCC细胞WSU-HN6和CAL27中的表达,用qPCR法验证干扰效率后,采用CCK-8法和流式细胞术分别检测敲降GRK3对OSCC细胞增殖和凋亡的影响,Transwell小室法检测对OSCC细胞迁移、侵袭能力的影响,qPCR法检测对OSCC细胞周期、上皮间质转化(epithelial to mesenchymal transition,EMT)和基质金属蛋白酶(matrix metallopeptidase,MMP)相关分子mRNA水平表达的影响,WB法检测EMT及MMP相关分子的蛋白表达水平变化。  结果:  OSCC组织中GRK3的表达水平显著高于正常口腔组织(P<0.01)。转染si-GRK3后,OSCC细胞中GRK3 mRNA表达水平均下调70%以上。敲降GRK3可显著抑制OSCC细胞的增殖、迁移和侵袭能力(均P<0.01),对细胞凋亡无显著影响(P>0.05)。敲降GRK3表达后,OSCC细胞的G0/G1期比例显著增高(P<0.01),细胞周期蛋白D1(Cyclin D1)、Cyclin D3、细胞周期蛋白依赖性激酶2(cyclin-dependent kinases 2,CDK2)和CDK4基因的mRNA表达降低(均P<0.05);EMT相关分子波形蛋白(Vimentin)、Zeb1和Slug表达降低,E-钙黏蛋白(E-cadherin)表达升高(均P<0.05);MMP3和MMP9表达降低(均P<0.05),MMP2和MMP7表达无明显变化(均P>0.05)。  结论:  GRK3可通过调节细胞周期促进OSCC细胞的增殖能力,并通过调控EMT和MMP增强细胞的迁移和侵袭能力。
  • 图  1  GRK3在OSCC组织中的表达及敲降GRK3对OSCC细胞增殖的影响

    Figure  1.  The expression level of GRK3 in OSCC tissues and the effect of GRK3 silence on the proliferation of OSCC cells

    **P<0.01 vs si-NC group A: The mRNA level of GRK3 in normal oral tissues and OSCC tissues; B: Interference efficiency of GRK3 mRNA expression in OSCC cells was determined using qPCR; C and D: CCK-8 assay was performed to detect the proliferation of OSCC cells

    图  2  敲降GRK3对OSCC细胞迁移和侵袭能力的影响

    Figure  2.  The effects of migration knocking down GRK3 on the and invasion abilities of OSCC cells

    **P<0.01 vs si-NC group A: Migration ability of OSCC cells (×100); B: Invasion ability of OSCC cells (×100)

    图  3  敲降GRK3对OSCC细胞凋亡和细胞周期的影响

    Figure  3.  The effects of knocking down GRK3 on the apoptosis and cell cycle of OSCC cells

    *P<0.05, **P<0.01 vs si-NC group A: The apoptosis rate of WSU-HN6 cells; B: The percentage of WSU-HN6 cells at different phases of the cell cycle; C: The mRNA levels of molecules associated with cell cycle in WSU-HN6 cells

    图  4  敲降GRK3对OSCC细胞EMT及MMP相关分子表达的影响

    Figure  4.  The effects of knocking down GRK3 on the expression levels of molecules associated with EMT and MMPs in OSCC cells

    *P<0.05, **P<0.01 vs si-NC group A: The mRNA levels of EMT-associated molecules in WSU-HN6 cells; B: The mRNA levels of MMPs in WSU-HN6 cells; C: The protein levels of molecules associated with EMT and MMPs in WSU-HN6 cells

    表  1  引物序列

    Table  1.   Primer sequences

    GeneForward primer (5’-3’)Reverse primer (5’-3’)
    Cyclin D1 TCTACACCGACAACTCCATCCG TCTGGCATTTTGGAGAGGAAGTG
    Cyclin D3 AGATCAAGCCGCACATGCGGAA ACGCAAGACAGGTAGCGATCCA
    CDK2 ATGGATGCCTCTGCTCTCACTG CCCGATGAGAATGGCAGAAAGC
    CDK4 CCATCAGCACAGTTCGTGAGGT TCAGTTCGGGATGTGGCACAGA
    Vimentin AGGCAAAGCAGGAGTCCACTGA ATCTGGCGTTCCAGGGACTCAT
    Zeb1 GGCATACACCTACTCAACTACGG TGGGCGGTGTAGAATCAGAGTC
    E-Cadherin GCCTCCTGAAAAGAGAGTGGAAG TGGCAGTGTCTCTCCAAATCCG
    Slug ATCTGCGGCAAGGCGTTTTCCA GAGCCCTCAGATTTGACCTGTC
    MMP2 AGCGAGTGGATGCCGCCTTTAA CATTCCAGGCATCTGCGATGAG
    MMP3 CACTCACAGACCTGACTCGGTT AAGCAGGATCACAGTTGGCTGG
    MMP7 TCGGAGGAGATGCTCACTTCGA GGATCAGAGGAATGTCCCATACC
    MMP9 GCCACTACTGTGCCTTTGAGTC GGATCAGAGGAATGTCCCATACC
    GAPDH GTCTCCTCTGACTTCAACAGCG ACCACCCTGTTGCTGTAGCCAA
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  • 收稿日期:  2020-12-01
  • 修回日期:  2021-03-16
  • 刊出日期:  2021-05-01

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