miR-98-5p对STAT3诱导的人舌鳞癌细胞SCC-25凋亡、增殖和成瘤性的影响

Effects of miR-98-5p on the apoptosis,proliferation and tumorigenesis of SCC-25 human tongue squamous cell carcinoma cells through downregulating STAT3 phosphorylation

目的:探讨miR-98-5p对人舌鳞癌细胞SCC-25凋亡和肿瘤干样特性及裸鼠成瘤的影响。方法:将SCC-25细胞转染mimic mock、miR-98-5p mimic、inhibitor-NC、miR-98-5p inhibitor后分为对照(control)组、模拟物对照(mimic-mock)组、模拟物(mimic)组、抑制剂阴性对照(inhibitor-NC)组和抑制剂(inhibitor)组。逆转录-聚合酶链反应(RT-PCR)检测miR-98-5p表达;克隆形成法检测细胞增殖;流式细胞仪检测细胞凋亡;RT-PCR检测Ki67、增殖细胞核抗原(PCNA)、Bax、Bcl-2 mRNA水平;细胞成球实验检测细胞成球体积、成球数目;免疫印迹检测信号转导和转录活化因子3(STAT3)磷酸化情况;建立移植瘤裸鼠模型,慢病毒转染mimic,将裸鼠随机分为control组和mimic组,检测肿瘤质量和体积,免疫印迹检测STAT3磷酸化情况,免疫组织化学检测Ki67阳性表达率。结果:与control组相比较,mimic组miR-98-5p水平显著升高,克隆形成率显著降低,凋亡率显著升高,Ki67、PCNA mRNA水平显著降低,Bax/Bcl-2比值升高,成球体积、成球数目显著降低,p-STAT3/STAT3水平显著降低;inhibitor组miR-98-5p水平显著降低,克隆形成率显著升高,Ki67、PCNA mRNA水平显著升高,Bax/Bcl-2比值降低,成球体积、成球数目显著升高,p-STAT3/STAT3水平显著升高;mimic组肿瘤质量显著降低,肿瘤体积显著降低,Ki67阳性表达率显著降低,p-STAT3/STAT3水平显著降低。结论:miR-98-5p mimic可通过抑制STAT3诱导人舌鳞癌细胞SCC-25凋亡,抑制增殖和移植瘤生长。

Objective:To explore the effects of miR-98-5p on proliferation,apoptosis,and tumorigenesis of SCC-25 human tongue squamous cell carcinoma cells.Methods:SCC-25 cells respectively transfected with miR-98-5p mimic,mimic-mock,miR-98-5p inhibitor,miR-98-5p inhibitor-NC were submitted to detect expression of miR-98-5p by RT-PCR.And then,differently treated cell populations underwent in vitro detection of proliferation by clone formation and mRNA levels of Ki67 and PCNA by RT-PCR.Besides,role of miR-98-5p on apoptosis was evaluated in vitro using flow cytometry and by expression changes of Bax and Bcl-2.Carcinogenesis capability of differently treated cells were explored using sphere formation assay and cell derived xenograft.The expressions of STAT3 and pSTAT3 were analyzed by Western blotting in vitro and in vivo,and Ki67+cells of tumor tissues in xenograft mice was examined using immunohistochemistry.Results:Transfection of miR-98-5p mimic produced significant decrease in clone formation and mRNA levels of Ki67 and PCNA,and statistical increase in apoptotic rate and elevation of Bax/Bcl-2 mRNA ratio.Also,treatment of miR-98-5p mimic led to statistical decrease in volume and numbers of formed spheres in vitro,as well as in weight and volume of tumor in xenograft mice.Both in vitro and in vivo experiments showed that treatment of miR-98-5p mimic generated statistical decrease in pSTAT3/STAT3.Similar to in vitro experiment,transfection of miR-98-5p mimic resulted in significantly decrease in Ki67 expression in vivo.Conclusion:miR-98-5p could inhibit proliferation,apoptosis and carcinogenesis of SCC-25 human tongue squamous cell carcinoma cells through downregulating STAT3 phosphorylation.