长链非编码RNA肌动蛋白纤维相关蛋白1-反义RNA1在口腔鳞状细胞癌中的表达及其相关功能

Expression and functions of long non-coding RNA actin filament-associated protein 1-antisense RNA1 in oral squamous cell carcinoma

目的分析长链非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(AFAP1-AS1)在口腔鳞状细胞癌(OSCC)中的表达及其对OSCC细胞生物行为学的影响,初步探讨其可能的作用机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测OSCC患者(55例)癌组织、癌旁正常黏膜组织及人口腔鳞状细胞癌SCC25、人正常口腔角质细胞株(NOK)细胞中lncRNA AFAP1-AS1的表达,分析AFAP1-AS1与OSCC患者病理特征的相关性,通过Kaplan-Meier生存曲线分析AFAP1-AS1与患者预后的关系。AFAP1-AS1 siRNA转染SCC25细胞,细胞计数(CCK-8)及Transwell实验分别检测细胞增殖、迁移和侵袭的变化,蛋白质印迹(Western blot)检测侵袭相关蛋白、肌动蛋白纤维相关蛋白1(AFAP1)及Rho GTP酶家族成员蛋白的表达情况,免疫荧光染色检测细胞骨架肌动蛋白微丝结构的变化。结果OSCC组织中AFAP1-AS1的表达高于癌旁正常黏膜组织,SCC25细胞中AFAP1-AS1的表达高于NOK细胞(P<0.001)。AFAP1-AS1的表达与OSCC的分化程度、TNM分期及淋巴结转移密切相关(P<0.05),AFAP1-AS1高表达患者的生存率低于AFAP1-AS1低表达者(P<0.05)。AFAP1-AS1 siRNA转染后AFAP1-AS1的表达水平下调,SCC25细胞的增殖、迁移和侵袭能力降低,AFAP1、RhoA、Rac2、Rab10、RhoGDI和Pfn1的表达上调,RhoC的表达下调,细胞骨架中应力纤维丝减少,肌动蛋白完整性丢失。结论lncRNA AFAP1-AS1在OSCC组织及SCC25细胞中高表达,与OSCC的发生进展及预后相关。下调AFAP1-AS1能够抑制OSCC的增殖、迁移和侵袭能力,其可能是通过调控肌动蛋白纤维丝的完整性实现的。

Objective To analyze the expression and clinical significance of long non-coding RNA(lncRNA)actin filamentassociated protein 1-antisense RNA1(AFAP1-AS1)in oral squamous cell carcinoma(OSCC)and its effect on the biobehavior of OSCC cells.Methods Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of lncRNA AFAP1-AS1 in the tumor tissue and matching adjacent normal tissue of OSCC patients(n=55),SCC25 cells,and normal oral keratinocyte lines(NOK)cells.The correlation between AFAP1-AS1 expression and the clinicopathological characteristics of OSCC patients was analyzed.The relationship between AFAP1-AS1 and prognosis was also studied with the Kaplan-Meier method.AFAP1-AS1 siRNA was transfected into the SCC25 cells.Cell counting kit-8(CCK-8)and Transwell were used to detect cell proliferation,invasion,and migration.The expression of the invasion-associated protein,AFAP1,and Rho GTPase family members,was detected by Western blot.In addition,the immunofluorescence of the cytoskeletal actin filament was observed.Results The expression of AFAP1-AS1 was higher in the OSCC tissues than in the NOK cells,and the relative expression of AFAP1-AS1 was higher in the SCC25 cells than in the NOK cells(P<0.001).AFAP1-AS1 expression was associated with the degree of differentiation,TNM stage,lymphatic metastasis,and poor prognosis of OSCC(P<0.05).Patients with a high expression of AFAP1-AS1 had lower survival rates than those with a low expression of AFAP1-AS1(P<0.05).After transfected by AFAP1-AS1 siRNA,the expression of AFAP1-AS1 was downregulated.The inhibition of AFAP1-AS1 expression consequently suppressed the proliferation,invasion,and migration of SCC25.Moreover,AFAP1-AS1 siRNA upregulated the expression levels of AFAP1,RhoA,Rac2,Rab10,RhoGDI,and Pfn1 but downregulated the expression of RhoC.Immunofluorescence showed that AFAP1-AS1 also reduced the formation of stress filaments in the cytoskeleton and affected the integrity of the actin filament.Conclusion The expression of AFAP1-AS1 was high in the OSCC tissues and SCC25 cells and is associated with the development and prognosis of OSCC.The knockdown of AFAP1-AS1 might inhibit the proliferation and invasion of OSCC cells by regulating the integrity of the actin filament.