MicroRNA-9对口腔鳞状细胞癌细胞增殖和凋亡的影响

MicroRNA-9 Inhibits cell proliferation and promotes cell apoptosis by targeting HMGA2 in oral squamous cell carcinoma

目的检测miR-9在口腔鳞状细胞癌(OSCC)组织和细胞中的表达水平,探讨miR-9对OSCC细胞增殖和凋亡的影响,并探讨其可能机制。方法收集42例OSCC患者肿瘤及癌旁组织标本。将SCC-9细胞分为3组:miR-9 mimics组,转染对照组(miR-NC)和未转染组(空白组)。通过实时荧光定量PCR(qRT-PCR)检测组织样本和细胞中miR-9的表达水平,体外实验通过转染miR-9 mimics至SCC-9细胞中,平板克隆形成试验和流式细胞术检测细胞增殖凋亡情况。Pearson相关分析分析miR-9与人高迁移率族AT Hook蛋白2(HMGA2)的相关性;荧光素酶试验,qRT-PCR和蛋白质印迹技术(Western blot)验证HMGA2是miR-9的靶基因。结果与癌旁组织和正常口腔角化细胞相比,miR-9在OSCC组织和细胞中的表达下调(P<0.05);体外实验发现,与空白组和miR-NC组相比,miR-9mimics组的细胞增殖受到抑制,而凋亡比率增加(P<0.05)。机制研究结果表明,OSCC组织中HMGA2 mRNA和蛋白的表达均增加(P<0.05),并与miR-9成显著负相关(r=-0.7701,P<0.001);荧光素酶结果显示,miR-9能直接与HMGA2-3'-UTR靶向结合;成功转染miR-9 mimics后能降低SCC-9细胞中HMGA2 mRNA和蛋白的表达(P<0.05)。结论在OSCC中,miR-9表达下调;miR-9能够降低HMGA2表达,抑制OSCC细胞增殖,促进其凋亡。

Objective To determine the expression signature of miR-9 in OSCC tissues and cell lines and to investigate the effects of miR-9 on cell proliferation and apoptosis and uncover its underlying mechanism. Methods Quantitative real-time PCR was performed to obtain miR-9 expression in 42 paired OSCC tissue specimens and three cell lines; After transfected with miR-9 mimics In vitro,SCC-9 cells were harvested for colony formation assay and flow cytometry. Pearson correlation analysis was employed to analyze the correlation between miR-9 and High mobility group AT-hook 2（ HMGA2） mRNA. Luciferase assay,as well as western blotting and qRT-PCR were performed to determine the target genes of miR-9 in OSCC. Results Compared with adjacent non-tumor tissues and normal oral keratinocytes,miR-9 expression was significantly lower in OSCC tissues and cell lines,respectively（ P〈0. 05）. In vitro experiment,the proliferative ability of SCC-9 cells was inhibited,whereas the cell apoptotic rates were increased after miR-9 overexpression（ P〈0. 05）. Meanwhile,HMGA2 mRNA and protein levels were significantly increased in OSCC tissues compared with the control groups（ P〈0. 05）. Pearson analysis showed a negative correlation between miR-9 and HMGA2 mRNA（ r =-0. 7701,P〈0. 01）. In addition,the Luciferase activity of the HMGA2-3＇-UTR plasmid was significantly suppressed by miR-9 binding（ P〈0. 05）. Furthermore,the expressions of HMGA2 were significantly decreased in the miR-9 mimics group both at mRNA and protein levels,compared with the control group（ P〈0. 05）. Conclusion The expression level of miR-9 is decreased in OSCC tissue and cells; miR-9 inhibits cell proliferation and promotes cell apoptosis,via targeting HMGA2 in OSCC.