HPV16 E6通过影响外泌体中β⁃联蛋白和紧密连接蛋白1表达促进宫颈癌细胞增殖、迁移和侵袭

HPV16 E6 Promotes Proliferation,Migration and Invasion of Cervical Cancer Cells Through Affecting the Expression of β-Catenin and Claudin-1 in Exosomes

为探讨人乳头瘤病毒16(human papillomavirus 16,HPV16)E6癌蛋白对宫颈癌细胞的生物学行为及外泌体中β-联蛋白(β-catenin)和紧密连接蛋白(claudin-1)表达的影响,本研究利用RNA干扰技术建立HPV16 E 6敲低细胞模型(shE6组),通过CCK8试剂盒、流式细胞仪、划痕实验和Transwell实验对细胞增殖、细胞周期、迁移和侵袭特征进行检测,发现shE6相对于对照组(NC组),细胞增殖速率减慢、细胞周期阻滞在G_(0)/G_(1)期向S期的过渡阶段,细胞迁移能力显著降低,侵袭能力下降。同时提取细胞上清液中外泌体,利用Western印迹对β-联蛋白和紧密连接蛋白-1表达量进行检测,发现相对于NC组,shE6组细胞内β-联蛋白表达量减少,但外泌体中β-联蛋白量增加,紧密连接蛋白-1在细胞内和外泌体中均增加。上述结果提示,HPV16 E6促进细胞恶性表型可能与E6蛋白能够抑制β-联蛋白以外泌体形式释放,从而增加其在细胞内的积累,以及抑制紧密连接蛋白-1在细胞内和外泌体中的积累有关。

This study aimed to investigate the effect of human papillomavirus 16(HPV16)on theβ-catenin and claudin-1 protein expression and proliferation,migration and invasion of cervical cancer cells.RNA interfere technology was utilized to establish the cell model of HPV 16 E 6 knockdown(shE6 group),followed by the assays of CCK8 kit,flow cytometry,scratch and transwell tests to detect the cell proliferation,cell cycle,apoptosis,and migration.We found that in contrast with NC group,the cell cycle was arrested in the transition phase from G_(0)/G_(1)phase to the S phase,and the cellular ability of proliferation,migration and invasion was decreased significantly in shE6 group.The exosomes were extracted from cell lysate supernatant and the expression ofβ-catenin and claudin-1 was measured by Western blotting.We found that in shE6 group,the expression of cellularβ-catenin was lower,but the corresponding expression in exosomes was increased when compared to NC group.And the expression of claudin-1 protein was increased both in the cells and exosomes.Our results suggested that HPV16 E6 can promote the malignant phenotype of cells,which may be related to the fact that E6 protein inhibited the release ofβ-catenin via exosomes,thereby increased its cellular accumulation and inhibited the accumulation of claudin-1 in cells and exosomes.