miR-203靶向ΔNp63对口腔鳞状细胞癌细胞增殖、侵袭、凋亡和干细胞特性的影响

Effects of miR-203 targetingΔNp63 on proliferation,invasion,apoptosis and stem-cell characteristics of squamous cell carcinoma cells

目的:研究微小RNA-203(miR-203)靶向ΔNp63对口腔鳞状细胞癌(OSCC)细胞增殖、侵袭、凋亡和干细胞特性的影响。方法:TCGA数据库分析miR-203和ΔNp63在头颈鳞状细胞癌及其邻近正常组织中的表达。采用RT-qPCR法检测OSCC细胞系(Tca8113、CAL-27、SCC-9)和正常人口腔角质形成细胞(NHOK)中miR-203和ΔNp63 mRNA表达。将miR-NC、miR-203 mimic、pcDNA、pcDNA-ΔNp63分别转染及miR-203 mimic和pcDNA-ΔNp63共转染CAL-27细胞。Western blotting法检测ΔNp63、Bax、Bcl-2、Ki67、MMP-2、E-cadherin、N-cadherin、Lin28B、SOX2和OCT4蛋白表达。荧光素酶报告实验分析miR-203和ΔNp63的靶向关系。CCK8、Transwell、流式细胞术分别检测细胞活力、侵袭能力、凋亡和ALDH阳性细胞数目。构建裸鼠移植瘤模型,检测miR-203对体内肿瘤的作用。结果:miR-203在头颈鳞状细胞癌组织和OSCC细胞中表达下调,ΔNp63表达上调(P<0.05)。miR-203靶向负调控ΔNp63的表达。miR-203组ΔNp63、Ki67、MMP-2、N-cadherin、Bcl-2、Lin28B、SOX2和OCT4表达及CAL-27细胞活力降低,侵袭细胞数和ALDH阳性细胞数明显减少,细胞凋亡率及Bax和E-cadherin表达升高(均P<0.05)。pcDNA-ΔNp63组结果与之相反,且过表达miR-203可逆转ΔNp63对CAL-27细胞活力、侵袭和干细胞样特性的促进作用(P<0.05)。miR-203能抑制口腔鳞癌细胞CAL-27裸鼠移植瘤的生长,并下调肿瘤组织ΔNp63表达。结论:miR-203通过抑制ΔNp63表达在OSCC细胞中发挥抑癌作用。

Objective:To investigate the effect of miR-203 targetingΔNp63 on proliferation,invasion,apoptosis and stemcell characteristics of oral squamous cell carcinoma(OSCC)cells.Methods:TCGA database was used to analyze the expression of miR-203 andΔNp63 in head and neck squamous cell carcinoma and adjacent normal tissues.RT-qPCR was used to detect the expression of miR-203 andΔNp63 mRNA in OSCC cells(Tca8113,CAL-27,and SCC-9)and normal human oral keratinocytes(NHOK).The CAL-27 cells were transfected with miR-NC,miR-203 mimic,pcDNA,or pcDNA-ΔNp63,and co-transfected with miR-203 mimic and pcDNA-ΔNp63.The expressions ofΔNp63,Bax,Bcl-2,Ki67,MMP-2,E-cadherin,N-cadherin,Lin28B,SOX2 and OCT4 were detected by Western blotting.Luciferase reporter assay was used to analyze the targeting relationship between miR-203 andΔNp63.CCK8,Transwell,and flow cytometry were performed respectively to detect cell viability,invasion,apoptosis and the number of ALDH positive cells.A nude mouse xenograft tumor was constructed to detect the effect of miR-203 on tumors in vivo.Results:The expression of miR-203 was down-regulated while the expression ofΔNp63 was up-regulated in head and neck squamous cell carcinoma and OSCC cells(P<0.05).miR-203 targeted and negatively regulated the expression ofΔNp63.The expressions ofΔNp63,Ki67,MMP-2,N-cadherin,Bcl-2,Lin28B,SOX2 and OCT4,the viability of CAL-27 cells,the number of invasive cells and ALDH-positive cells were decreased significantly,while the apoptosis rate and the protein expression levels of Bax and E-cadherin were increased in miR-203 group(P<0.01).The results ofΔNp63 group were opposite.miR-203 mimic reversed the promotive effects of overexpressedΔNp63 on CAL-27 cell viability,invasion and stem cell-like properties(P<0.05).miR-203 inhibited tumor growth in nude mice translanted with OSCC cell line and downregulatedΔNp63 expression.Conclusion:miR-203 plays a tumor-suppressive role in OSCC by inhibiting the expression ofΔNp63.