lncRNA RARB-AS2对口腔鳞状细胞癌细胞增殖、细胞周期和侵袭的影响及分子机制

Effects of lncRNA RARB-AS2 on proliferation,cell cycle and invasion of oral squamous cell carcinoma cells and its molecular mechanism

目的 观察长链非编码RNA(lncRNA)RARB-AS2在口腔鳞状细胞癌组织中的表达,探讨RARB-AS2对口腔鳞状细胞癌细胞增殖、细胞周期和侵袭的影响及分子机制。方法 癌症基因组图谱(TCGA)数据库分析RARB-AS2在口腔鳞状细胞癌组织和癌旁组织中表达水平。实时荧光定量PCR(qPCR)检测RARB-AS2在4种口腔鳞状细胞癌细胞SCC-9、CAL-27、HSC-3、SCC-25以及口腔上皮角质细胞HOK中表达水平。通过脂质体转染法在SCC-25细胞中分别转染RARB-AS2高表达质粒和对照质粒,定义为RARB-AS2组和对照组。qPCR检测SCC-25细胞中RARB-AS2表达水平。平板克隆实验、流式细胞术和Transwell实验分别检测SCC-25细胞增殖、细胞周期和侵袭能力。双荧光素酶报告实验验证RARB-AS2和miR-455-3p的靶向关系。TCGA数据库分析口腔鳞状细胞癌组织中RARB-AS2和miR-455-3p表达的相关性。qPCR检测SCC-25细胞中miR-455-3p表达水平。Western blot法检测SCC-25细胞中PI3K/AKT信号通路蛋白p-PI3K、PIP3、PDK1、p-AKT表达。结果 口腔鳞状细胞癌组织中RARB-AS2表达显著低于癌旁组织(P<0.01)。与口腔上皮角质细胞HOK比较,口腔鳞状细胞癌细胞系SCC-9、CAL-27、HSC-3、SCC-25中RARB-AS2呈低表达(P<0.01),在SCC-25细胞中RARB-AS2表达最低(P<0.01)。与对照组比较,RARB-AS2组SCC-25细胞的增殖能力显著降低(P<0.01),SCC-25细胞位于G0/G1期比例显著升高(P<0.01),S期比例显著下降(P<0.05),G2/M期比例显著下降(P<0.05),SCC-25细胞的侵袭能力显著降低(P<0.01)。RARB-AS2与miR-455-3p间存在互补关系(P<0.01)。口腔鳞状细胞癌组织中RARB-AS2与miR-455-3p表达呈负相关(P<0.01)。与对照组比较,RARB-AS2组SCC-25细胞中miR-455-3p表达显著降低(P<0.01),PI3K/AKT信号通路蛋白p-PI3K、PIP3、PDK1、p-AKT表达减少(P<0.01)。结论 口腔鳞状细胞癌组织和细胞系中RARB-AS2呈低表达,RARB-AS2可能通过调控miR-455-3p/PI3K/AKT信号通路,抑制口腔鳞状细胞癌SCC-25细胞的增殖、侵袭。

Objective To investigate the expression of long non-coding RNA(lncRNA)RARB-AS2 in oral squamous cell carcinoma tissues,and analyze the molecular mechanism of RARB-AS2 inhibiting the proliferation,the cell cycle and the invasion of oral squamous cell carcinoma cells.Methods The Cancer Genome Atlas(TCGA)database was used to analyze the expression level of RARB-AS2 in oral squamous cell carcinoma tissues and adjacent tissues.Real-time fluorescent quantitative PCR(qPCR)was used to detect the expression level of RARB-AS2 in four oral squamous cell carcinoma cells SCC-9,CAL-27,HSC-3,SCC-25 and oral epithelial keratinocytes HOK.SCC-25 cells were transfected with RARB-AS2 high-expression plasmid and control plasmid by lipofection method,which were defined as RARB-AS2 group and control group.The expression level of RARB-AS2 in transfected SCC-25 cells was detected by qPCR.Plate cloning assay,flow cytometry(FCM)and Transwell assay were used to detect the proliferation,the cell cycle and the invasion ability of SCC-25 cells,respectively.The dual-luciferase reporter assay was used to verify the targeting relationship between RARB-AS2 and miR-455-3p.The TCGA database was used to analyze the correlation between the expression of RARB-AS2 and miR-455-3p in oral squamous cell carcinoma tissues.The expression level of miR-455-3p in SCC-25 cells was detected by qPCR.The expression of PI3K/AKT signaling pathway proteins p-PI3K,PIP3,PDK1,p-AKT in SCC-25 cells was detected by Western blotting.Results The expression of RARB-AS2 in oral squamous cell carcinoma tissues was significantly lower than that in adjacent tissues(P<0.01).Compared with oral epithelial keratinocytes HOK cells,the expression of RARB-AS2 in oral squamous cell carcinoma cell lines SCC-9,CAL-27,HSC-3,SCC-25 was decreased(P<0.01),and the expression of RARB-AS2 in SCC-25 cells was the lowest(P<0.01).Compared with control group,the proliferation ability of SCC-25 cells was significantly reduced in RARB-AS2 group(P<0.01),the proportion of SCC-25 cells in G0/G1 phase was significantly increased(P<0.01),the proportion of S phase cells was decreased significantly(P<0.05),the proportion of G2/M phase cells was decreased significantly(P<0.05),and the invasion ability of SCC-25 cells was decreased significantly(P<0.01).There was a complementary relationship between RARB-AS2 and miR-455-3p(P<0.01).The expression levels of RARB-AS2 and miR-455-3p in oral squamous cell carcinoma tissues were negatively correlated(P<0.01).Compared with control group,the expression of miR-455-3p in SCC-25 cells was significantly decreased in RARB-AS2 group(P<0.01),and the expression of PI3K/AKT signaling pathway proteins p-PI3K,PIP3,PDK1,p-AKT was decreased(P<0.01).Conclusion The expression of RARB-AS2 in oral squamous cell carcinoma tissues and cell lines is low,and RARB-AS2 may inhibit the proliferation and invasion abilities of oral squamous cell carcinoma SCC-25 cells by regulating the miR-455-3p/PI3K/AKT signaling pathway.