长链非编码RNA PAPAS对口腔鳞状细胞癌增殖、侵袭和上皮-间充质转化的影响

Effects of long non-coding RNA PAPAS on proliferation, invasion and epithelial mesenchymal transformation of oral squamous cell carcinoma

目的观察长链非编码RNA(lncRNA)PAPAS对口腔鳞状细胞癌增殖、侵袭和上皮-间充质转化的影响。方法选取河南大学淮河医院2015年10月至2019年10月收集的58例口腔鳞状细胞癌及其癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)分析lncRNA PAPAS表达水平;人口腔鳞状细胞HSC-4分为lncRNA对照组和短发卡RNA(shRNA)PAPAS组。采用细胞计数试剂(CCK-8)和克隆形成实验分析两组细胞增殖;采用Transwell分析分析两组细胞侵袭;采用蛋白质免疫印迹(Western blot)分析间质标志物神经性钙黏蛋白(N-cadhenrin)和波形蛋白(Vimentin)及上皮性标志物上皮性钙黏蛋白(E-cadherin)表达水平。组间计量资料比较采用t检验。结果癌旁组织中lncRNA PAPAS表达水平(1.24±0.21)明显低于口腔鳞状细胞癌组织(3.09±0.31),差异有统计学意义(t=3.018,P<0.05)。lncRNA对照组细胞48 h吸光度值(2.17±0.28)明显高于shRNA PAPAS组细胞(1.52±0.19),差异有统计学意义(t=3.819,P<0.05)。克隆形成实验结果显示,lncRNA对照组细胞克隆形成率[(85.28±6.99)%]明显高于shRNA PAPAS组细胞[(38.19±3.61)%],差异有统计学意义(t=5.882,P<0.05)。lncRNA对照组细胞迁移数量[(152.37±10.29)个]明显高于shRNA PAPAS组细胞[(87.33±7.49)个],差异有统计学意义(t=4.398,P<0.05)。lncRNA对照组细胞上皮细胞标志物E-cadherin表达水平(1.09±0.14)明显低于shRNA PAPAS组细胞(2.58±0.20),差异有统计学意义(t=3.609,P<0.05)。lncRNA对照组细胞间质细胞标志物N-cadhenrin和Vimentin表达水平(1.54±0.13;1.29±0.16)明显低于shRNA PAPAS组细胞(0.63±0.11;0.72±0.17),差异有统计学意义(t=3.018,P<0.05;t=3.237,P<0.05)。生物信息学和双荧光素酶报告基因显示lncRNA PAPAS和miR-34a存在碱基互补,通过海绵吸附调控miR-34a水平。结论lncRNA PAPAS在口腔鳞状细胞癌表达显著上调,通过海绵吸附miR-34a参与口腔鳞状细胞癌的增殖、侵袭和上皮-间充质转化过程。

Objective To investigate the effects of long non-coding RNA(lncRNA)PAPAS on proliferation,invasion and epithelial mesenchymal transformation of oral squamous cell carcinoma.Methods 58 cases of oral squamous cell carcinoma and their adjacent tissues collected in our hospital from October 2015 to October 2019 were selected as the research objects.The expression level of lncRNA PAPAS was analyzed by fluorescence quantitative polymerase chain reaction(PCR).Human oral squamous cell CAL27 was divided into lncRNA control group and short hairpin RNA(shRNA)PAPAS group.The cell proliferation of the two groups was analyzed by cell counting reagent(CCK-8)and clone formation experiment.The cell invasion of the two groups was analyzed by Transwell.The expression levels of interstitial markers N-cadherin,vimentin and epithelial markers E-cadherin were analyzed by Western blotting.The measurement data between groups were compared by t-test.Results The expression level of lncRNA PAPAS in adjacent tissues(1.24±0.21)was significantly lower than that in oral squamous cell carcinoma(3.09±0.31,t=3.018,P<0.05).The 48 h light absorption value of cells in lncRNA control group(2.17±0.28)was significantly higher than that in shRNA papas group(1.52±0.19,t=3.819,P<0.05).The results of clone formation experiment showed that the clone formation rate of lncRNA control group[(85.28±6.99)%]was significantly higher than that of shRNA PAPAS Group[(38.19±3.61)%,t=5.882,P<0.05].The migration number of cells in lncRNA control group[(152.37±10.29)cells]was significantly higher than that in shRNA PAPAS group[(87.33±7.49)cells,t=4.398,P<0.05].The expression level of E-cadherin in lncRNA control group(1.09±0.14)was significantly lower than that in shRNA PAPAS group(2.58±0.20,t=3.609,P<0.05).The expression levels of interstitial cell markers N-Cadhenrin and Vimentin in lncRNA control group(1.54±0.13;1.29±0.16)were significantly lower than those in shRNA PAPAS group(0.63±0.11;0.72±0.17,t=3.018,P<0.05;t=3.237,P<0.05).Bioinformatics and double luciferase reporter gene showed that lncRNA PAPAS and miR-34a were base complementary,and the level of miR-34a was regulated by sponge adsorption.Conclusion The expression of lncRNA PAPAS is significantly up-regulated in oral squamous cell carcinoma.LncRNA PAPAS as sponge of miR-34a is involved in the proliferation,invasion and epithelial mesenchymal transformation of oral squamous cell carcinoma.