人口腔鳞状细胞癌干细胞中microRNAs差异表达研究
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摘要
目的:探究微小RNA(miRNAs)在人口腔鳞状细胞癌(OSCC)干细胞中的表达及其意义。方法:以CD133为表面标志,应用免疫磁珠法分选TCA-8113细胞中的干细胞、流式细胞术检测,通过克隆形成实验和transwell侵袭实验检测干细胞特性。应用二代测序检测并筛选差异表达的miRNAs。对差异表达miRNAs运用生物信息学方法进行靶基因预测和功能分析。结果:筛选分析得到TCA-8113中的CD133+细胞,较CD133-TCA-8113细胞表达上调超过1.5倍的miRNA有2个;表达下调的有10个。miRanda和RNAhybrid靶基因预测共得到2 490个靶基因,GO注释显示他们主要涉及干细胞分化、血管生成、细胞增殖等功能。Pathway注释显示主要参与Wnt信号途径、癌症途径、FC受体信号途径等信号通路。结论:miRNAs在口腔鳞癌干细胞中存在特异性的表达谱,差异表达miRNAs调控多种具有不同细胞功能和参与不同信号通路的基因。
Objective: To identify microRNAs(miRNAs) differentially expressed in the stem cells of human oral squamous cell carcinoma(OSCC)TCA-8113 cells. Methods: CD133+TCA-8113 cells were isolated by immunomagnetic bead cell sorting from TCA-8113 cells, and further identified by flow cytometry. The characteristics of CD133+TCA-8113 cells were examined by colony formation assay, transwell migration assay. Differentialy expressed miRNAs were screened by the Next generation sequencing. Bioinformatics analyses were used to investigate the functions of aberrantly expressed miRNAs and their associated target miRNAs. Results: Compared with CD133-TCA-8113 cells, in CD133+TCA-8113 cells 2 miRNAs were upregulated by more than 1.5 fold, and 10 microRNAs were downregulated. 2 490 target genes were predicted by miRanda and RNAhybrid database, and these genes were mainly involved in the events, such as stem cell differentiation, blood vessel morphogenesis, cell proliferation, and in pathways such as Wnt signaling pathway and pathways in cell cycle. Conclusion: MiRNAs in OSCC stem cells exists specific expression profile. Their target genes are involved in a variety of biological events and in multiple signal pathways.
Objective: To identify microRNAs(miRNAs) differentially expressed in the stem cells of human oral squamous cell carcinoma(OSCC)TCA-8113 cells. Methods: CD133+TCA-8113 cells were isolated by immunomagnetic bead cell sorting from TCA-8113 cells, and further identified by flow cytometry. The characteristics of CD133+TCA-8113 cells were examined by colony formation assay, transwell migration assay. Differentialy expressed miRNAs were screened by the Next generation sequencing. Bioinformatics analyses were used to investigate the functions of aberrantly expressed miRNAs and their associated target miRNAs. Results: Compared with CD133-TCA-8113 cells, in CD133+TCA-8113 cells 2 miRNAs were upregulated by more than 1.5 fold, and 10 microRNAs were downregulated. 2 490 target genes were predicted by miRanda and RNAhybrid database, and these genes were mainly involved in the events, such as stem cell differentiation, blood vessel morphogenesis, cell proliferation, and in pathways such as Wnt signaling pathway and pathways in cell cycle. Conclusion: MiRNAs in OSCC stem cells exists specific expression profile. Their target genes are involved in a variety of biological events and in multiple signal pathways.
引文
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[2] Patel SS,Shah KA,Shah MJ,et al.Cancer stem cells and stemness markers in oral squamous cell carcinomas[J].Asian Pac J Cancer Prev,2014,15(20):8549-8556.
[3] Frank NY,Schatton T,Frank MH.The therapeutic promise of the cancer stem cell concept[J] J Clin Invest,2010,120(1):41-50.
[4] 林诗晗,胡雪刚,吕红兵.人牙髓干细胞与根尖牙乳头干细胞miRNAs表达谱的差异分析[J].实用口腔医学杂志,2018,34(2):239-243.
[5] Huang JT,Wang J,Srivastava V,et al.MicroRNA machinery genes as novel biomarkers for cancer[J].Front Oncol,2014,4:113.
[6] Ferlay J,Soerjomataram I,Dikshit R,et al.Cancer incidence and mortality worldwide:Sources,methods and major patterns in GLOBOCAN 2012[J].Int J Cancer,2015,136(5):E359-E386.
[7] Lippman SM,Hong WK.Molecular markers of the risk of oral cancer[J].N Engl J Med,2001,344(17):1323-1326.
[8] Fan X,Chen X,Deng W,et al.Up- regulated microRNA- 143 in cancer stem cells differentiation promotes prostate cancer cells metastasis by modulating FNDC3B expression[J].BMC Cancer,2013,13:61.
[9] Chikamatsu K,Ishii H,Takahashi G,et al.Resistance to apoptosis- inducing stimuli in CD44+ head and neck squamous cell carcinoma cells[J].Head Neck,2012,34(3):336-343.
[10] Bai X,Zhou Y,Chen P,et al.MicroRNA- 142- 5p induces cancer stem cell- like properties of cutaneous squamous cell carcinoma via inhibiting PTEN[J].J Cell Biochem,2018,119(2):2179-2188.
[11] El Helou R,Pinna G,Cabaud O,et al.miR- 600 acts as a bimodal switch that regulates breast cancer stem cell fate through WNT signaling[J].Cell Rep,2017,18(9):2256-2268.
[12] Zhou X,Yue Y,Wang R,et al.MicroRNA- 145 inhibits tumorigenesis and invasion of cervical cancer stem cells[J].Int J Oncol,2017,50(3):853-862.
[13] Liu C,Liu R,Zhang D,et al.MicroRNA- 141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro- metastasis genes[J].Nat Commun,2017,8:14270.
[14] Yu CC,Chen PN,Peng CY,et al.Suppression of miR- 204 enables oral squamous cell carcinomas to promote cancer stemness,EMT traits,and lymph node metastasis[J].Oncotarget,2016,7(15):20180-20192.
[15] Ghosh RD,Ghuwalewala S,Das P,et al.MicroRNA profiling of cisplatin- resistant oral squamous cell carcinoma cell lines enriched with cancer- stem- cell- like and epithelial- mesenchymal transition- type features[J].Sci Rep,2016,6:23932.
[16] Ries J,Baran C,Wehrhan F,et al.Prognostic significance of altered miRNA expression in whole blood of OSCC patients[J].Oncol Rep,2017,37(6):3467-3474.
[17] Pellatt DF,Stevens JR,Wolff RK,et al.Expression profiles of miRNA subsets distinguish human colorectal carcinoma and normal colonic mucosa[J].Clin Transl Gastroenterol,2016,7:e152.
[18] Fang F,Chang RM,Yu L,et al.MicroRNA- 188- 5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma[J].J Hepatol,2015,63(4):874-885.
[19] Zhang H,Qi S,Zhang T,et al.miR- 188- 5p inhibits tumour growth and metastasis in prostate cancer by repressing LAPTM4B expression[J].Oncotarget,2015,6(8):6092-6104.
[20] Huang W,Li J,Guo X,et al.miR- 663a inhibits hepatocellular carcinoma cell proliferation and invasion by targeting HMGA2[J].Biomed Pharmacother,2016,81:431-438.