LncRNA-PANDAR介导EphA2、TRPM8对OSCC细胞侵袭、迁移及凋亡的作用机制

LncRNA-PANDAR mediated EphA2 and TRPM8 on invasion, migration and apoptosis of OSCC cells

目的探究LncRNA-PANDAR介导EphA2、TRPM8对OSCC细胞生物活性的作用。方法人OSCC细胞系(SCC6、SCC9、SCC25)和正常人口腔角质形成细胞(HOK)来自上海子实公司,于实验室保存,经检测后PANDAR在SCC25细胞中水平最高,因此后续选择SCC25细胞进行实验。将PANDAR转染SCC25细胞后分为口腔癌组(癌细胞未处理)、对照组(转染不相关序列组)、转染组(转染PANDAR组)。运用RT-PCR、Transwell法、流式细胞仪、免疫印记法检测PANDAR水平、SCC25细胞侵袭、迁移、凋亡及EphA2、TRPM8、FAK蛋白表达;双荧光素酶报告基因检测LncRNA-PANDAR与EphA2、TRPM8、FAK相关性。结果各口腔鳞癌细胞中PANDAR mRNA表达水平较HOK组细胞低(P<0.05),SCC25组中PANDAR表达水平高于其他口腔鳞癌细胞,组间比较显著差异(P<0.05),因此本文后续实验选用SCC25细胞进行。口腔癌组与对照组PANDAR、EphA2、TRPM8、FAK、侵袭数量、迁移数量、凋亡率差异无统计学意义(P>0.05)。双荧光素酶结果显示,转染LncRNA-PANDAR后EphA2、TRPM8及FAK野生型活性下降(P<0.05),对突变基因无影响(P>0.05),表明EphA2、TRPM8及FAK是LncRNA-PANDAR的靶基因。结论 LncRNA-PANDAR通过靶向降低EphA2、TRPM8活性从而抑制口腔鳞癌细胞的迁移、侵袭,促进其凋亡。

Objective To explore the effect of LncRNA-PANDAR-mediated EphA2 and TRPM8 on the biological activity of OSCC cells.Methods Human OSCC cell lines(SCC6,SCC9,SCC25) and normal human oral keratinocytes(HOK) were from Shanghai Zishi Company, stored in the laboratory.PANDAR had the highest level in SCC25 cells after testing.Therefore, SCC25 cells were subsequently selected for experiments.SCC25 cells were divided into oral cancer group(untreated cancer cells),control group(transfection irrelevant sequence group),and transfection group(transfection PANDAR group).RT-PCR,Transwell method, flow cytometry, immunoblotting method were used to detect PANDAR level, SCC25 cell invasion, migration, apoptosis and EphA2,TRPM8,FAK protein expression were measured;The dual luciferase reporter assay was used to detect the correlation between LncRNA-PANDAR and EphA2,TRPM8,and FAK.Results The expression level of PANDAR mRNA in oral squamous cell carcinoma cells was lower than that in the HOK group(P<0.05).The expression level of PANDAR in the SCC25 group was higher than other oral squamous cell carcinoma cells, and there were significant differences between the groups(P<0.05).Therefore, SCC25 cells are used for subsequent experiments The expression of PANDAR,EphA2,TRPM8 and FAK,the number of invasion, number of migration, and apoptosis rate of oral cancer group and control group were not different(P>0.05).Transfection with LncRNA-PANDAR in SCC25 cells significantly decreased these measures.The results of dual luciferase showed that EphA2,TRPM8 and FAK wild-type activities decreased after transfection of LncRNA-PANDAR,and the difference between the groups was significant(P<0.05).No effect on the mutant gene(P>0.05),indicating that EphA2,TRPM8 and FAK are the target genes of LncRNA-PANDAR.Conclusion LncRNA-PANDAR can inhibit the migration and invasion of oral squamous cell carcinoma cells and promote their apoptosis by targeting to reduce the activity of EphA2 and TRPM8.