miR-509-3p对头颈鳞状细胞癌细胞CAL27增殖的影响
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摘要
目的:探讨miR-509-3p对头颈鳞状细胞癌(HNSCC)细胞CAL27增殖的调控作用。方法:实时定量RT-PCR检测HNSCC系HN4、HN6、HN13、CAL27和正常口腔上皮细胞系HOK中miR-509-3p的表达差异。应用miR-509-3p mimic和mimic NC转染CAL27细胞,实时定量RT-PCR检测miR-509-3p表达水平;使用CCK8、克隆形成和Edu等实验技术验证miR-509-3p表达水平变化对CAL27细胞增殖能力的影响;Western blot法检测miR-509-3p表达水平变化对EGFR/PI3K/AKT/MTOR信号通路中各蛋白水平的影响。结果:与HOK细胞株相比,miR-509-3p在HNSCC系内表达量明显下调;miR-509-3p mimic显著提高CAL27中miR-509-3p的表达;过表达miR-509-3p能明显抑制CAL27细胞的增殖能力;Western blot结果显示,转染miR-509-3p mimic的CAL27中,表皮生长因子受体(EGFR)、磷酸化表皮生长因子受体(p-EGFR)、磷酸肌醇3激酶(PI3K)、磷酸化丝氨酸/苏氨酸蛋白激酶(p-AKT)、磷酸化的哺乳动物雷帕霉素靶蛋白(p-MTOR)的表达水平均明显降低。结论:miR-509-3p可能通过调控EGFR/PI3K/AKT/MTOR信号通路影响HNSCC细胞CAL27的增殖能力。
Objective: To investigate the regulation of miR-509-3 p on the proliferation of CAL27. Methods: Real-time quantitative RT-PCR was used to detected the expression of miR-509-3 p in head and neck squamous cell carcinoma cell lines HN4,HN6,HN13,CAL27 and normal oral epithelial cell line HOK. The CAL27 cells were transfected with miR-509-3 p mimic and mimic NC,the expression of miR-509-3 p was detected by real-time PCR. Cell proliferation experiments including CCK8,colony formation and Edu analysis have been used to verified the effect of miR-509-3 p on proliferation. Results: The expression of miR-509-3 p was significantly down-regulated in head and neck squamous cell carcinoma cell lines compared with HOK cells; miR-509-3 p mimic can significantly increase the expression of miR-509-3 p in CAL27. CCK8,colony formation and Edu analysis have shown that overexpression of miR-509-3 p can significantly inhibit the proliferation of CAL27 cells. Western blot results showed that the expression levels of EGFR,p-EGFR,PI3 K,pAKT and p-MTOR were significantly decreased in cells transfected with miR-509-3 p mimic. Conclusions: MiR-509-3 p can affect the proliferation of CAL27 by regulating the EGFR/PI3 K/AKT/MTOR signal pathway.
Objective: To investigate the regulation of miR-509-3 p on the proliferation of CAL27. Methods: Real-time quantitative RT-PCR was used to detected the expression of miR-509-3 p in head and neck squamous cell carcinoma cell lines HN4,HN6,HN13,CAL27 and normal oral epithelial cell line HOK. The CAL27 cells were transfected with miR-509-3 p mimic and mimic NC,the expression of miR-509-3 p was detected by real-time PCR. Cell proliferation experiments including CCK8,colony formation and Edu analysis have been used to verified the effect of miR-509-3 p on proliferation. Results: The expression of miR-509-3 p was significantly down-regulated in head and neck squamous cell carcinoma cell lines compared with HOK cells; miR-509-3 p mimic can significantly increase the expression of miR-509-3 p in CAL27. CCK8,colony formation and Edu analysis have shown that overexpression of miR-509-3 p can significantly inhibit the proliferation of CAL27 cells. Western blot results showed that the expression levels of EGFR,p-EGFR,PI3 K,pAKT and p-MTOR were significantly decreased in cells transfected with miR-509-3 p mimic. Conclusions: MiR-509-3 p can affect the proliferation of CAL27 by regulating the EGFR/PI3 K/AKT/MTOR signal pathway.
引文
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[2] Jou A,Hess J. Epidemiology and Molecular Biology of Head and Neck Cancer[J/OL]. Oncol Res Treat,2017,40(6):328-332[2018-05-15]. http://dx.doi.org/10.1159/000477127.
[3] Poenitzsch Strong AM,Berry SM,Beebe DJ,et al. mi FAST:A novel and rapid microRNA target capture method[J/OL]. Mol Carcinog,2018,57(4):559-566[2018-05-15]. http://dx. doi.org/10.1002/mc.22780.
[4] Jafri MA,Al-Qahtani MH,Shay JW. Role of miRNAs in human cancer metastasis:Implications for therapeutic intervention[J/OL]. Semin Cancer Biol,2017,44:117-131[2018-05-15].http://dx.doi.org/10.1016/j.semcancer.2017.02.004.
[5]杜莉,曹伟靖,田莹,等. miR-140-5p对口腔鳞状细胞癌细胞增殖及凋亡的影响[J].口腔生物医学,2017,8(4):186-190.
[6] Nohata N,Hanazawa T,Kinoshita T,et al. Tumour-suppressive microRNA-874 contributes to cell proliferation through targeting of histone deacetylase 1 in head and neck squamous cell carcinoma[J/OL]. Br J Cancer,2013,108(8):1648-1658[2018-05-15].http://dx.doi.org/10.1038/bjc.2013.122.
[7] Kinoshita T,Nohata N,Hanazawa T,et al. Tumour-suppressivemicroRNA-29s inhibit cancer cell migration and invasion by targeting laminin-integrin signalling in head and neck squamous cell carcinoma[J/OL]. Br J Cancer,2013,109(10):2636-2645[2018-05-15]. http://dx.doi.org/10.1038/bjc.2013.607.
[8] Su Z,Chen D,Zhang E,et al. MicroRNA-509-3p inhibits cancer cell proliferation and migration by targeting the mitogen-activated protein kinase kinase kinase 8 oncogene in renal cell carcinoma[J/OL]. Mol Med Rep,2015,12(1):1535-1543[2018-05-15]. http://dx.doi.org/10.3892/mmr.2015.3498.
[9] Du P,Luan X,Liao Y,et al. MicroRNA-509-3p inhibits cell proliferation and invasion via downregulation of X-linked inhibitor of apoptosis in glioma[J/OL]. Oncol Lett,2018,15(1):1307-1312[2018-05-15]. http://dx.doi.org/10.3892/ol.2017.7390.
[10] Sun J,Li J,Zhang W,et al. MicroRNA-509-3p Inhibits Cancer Cell Proliferation and Migration via Upregulation of XIAP in Gastric Cancer Cells[J/OL]. Oncol Res,2017,25(3):455-461[2018-05-15]. http://dx.doi.org/10.3727/096504016X14747283032017.
[11] Amato F,Tomaiuolo R,Nici F,et al. Exploitation of a very small peptide nucleic acid as a new inhibitor of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression[J/OL].Biomed Res Int,2014,2014:610718[2018-05-15]. http://dx.doi.org/10.1155/2014/610718.
[12] Pandey R,Yang Y,Jackson L,et al. MicroRNAs regulating meis1expression and inducing cardiomyocyte proliferation[J/OL]. Cardiovasc Regen Med,2016,3:e1468[2018-05-15]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245919/.
[13] Zhai Q,Zhou L,Zhao C,et al.Identification of miR-508-3p and miR-509-3p that are associated with cell invasion and migration and involved in the apoptosis of renal cell carcinoma[J/OL]. Biochem Biophys Res Commun,2012,419(4):621-626[2018-05-15]. http://dx.doi.org/10.1016/j.bbrc.2012.02.060.
[14] Sun J,Li J,Zhang W,et al. MicroRNA-509-3p inhibits cancer cell proliferation and migration via upregulation of XIAP in gastric cancer cells[J/OL]. Oncol Res,2017,25(3):455-461[2018-05-15]. http://dx.doi.org/10.3727/096504016X14747283032017.
[15] Du P,Luan X,Liao Y,et al. MicroRNA-509-3p inhibits cell proliferation and invasion via downregulation of X-linked inhibitor of apoptosis in glioma[J/OL]. Oncol Lett,2018,15(1):1307-1312[2018-05-15]. http://dx.doi.org/10.3892/ol.2017.7390.
[16] Chen W,Du J,Li X,et al. miR-509-3p promotes cisplatin-induced apoptosis in ovarian cancer cells through the regulation of anti-apoptotic genes[J/OL]. Pharmacogenomics,2017,18(18):1671-1682[2018-05-15]. http://dx. doi. org/10.2217/pgs-2017-0115.
[17] Citri A,Yarden Y. EGF-ERBB signalling:towards the systems level[J/OL]. Nat Rev Mol Cell Biol,2006,7(7):505-516[2018-05-15]. http://dx.doi.org/10.1038/nrm1962.
[18] Zheng Y,Wang Z,Ding X,et al. Combined Erlotinib and PF-03084014 treatment contributes to synthetic lethality in head and neck squamous cell carcinoma[J/OL]. Cell Prolif,2017,51(3):e12424[2018-05-15]. http://dx.doi.org/10.1111/cpr.12424.
[19] Thelemann A,Petti F,Griffin G,et al. Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells[J/OL]. Mol Cell Proteomics,2005,4(4):356-376[2018-05-15]. http://dx. doi. org/10.1074/mcp. M400118-MCP200.
[20] Freudlsperger C,Burnett JR,Friedman JA,et al. EGFR-PI3KAKT-mTOR signaling in head and neck squamous cell carcinomas:attractive targets for molecular-oriented therapy[J/OL]. Expert Opin Ther Targets,2011,15(1):63-74[2018-05-15]. http://dx.doi.org/10.1517/14728222.2011.541440.
[21] Clark C,Shah S,Herman-Ferdinandez L,et al. Teasing out the best molecular marker in the AKT/m TOR pathway in head and neck squamous cell cancer patients[J/OL]. Laryngoscope,2010,120(6):1159-1165[2018-05-15]. http://dx. doi. org/10. 1002/lary.20917.