RPL34基因敲减抑制黑素瘤细胞侵袭与生长研究

RPL34 gene knockdown inhibits the invasion and growth of melanoma cells

目的:研究核糖体蛋白60SL34(RPL34)对黑素瘤细胞增殖、迁移和侵袭能力的影响及其相关机制。方法:比较20例皮肤黑素瘤和正常皮肤组织内RPL34蛋白表达,采用慢病毒感染法构建人黑素瘤细胞A375 RPL34稳定干扰的细胞株。实时荧光定量PCR(RT-PCR)及免疫印迹法检测细胞中RPL34基因的干扰效率。用细胞增殖和克隆形成试验检测RPL34干扰对A375细胞增殖和克隆形成能力的影响。细胞迁移、侵袭和划痕试验观察RPL34干扰对A375细胞迁移运动和侵袭能力影响。结果:免疫组化显示,患者黑素瘤组织中RPL34的表达高于正常组织。在A375细胞中干扰RPL34后,细胞增殖(F=25.362,P<0.05)和克隆形成的能力(t=23.171,P=0.00065)明显减弱,细胞迁移运动能力(t=6.99,P=0.00185),侵袭能力(t=7.576,P=0.00156)亦显著减弱。A375细胞中干扰RPL34后,TWIST1基因、波形蛋白(vimentin)、组织抑制因子(TIMP)2、基质金属蛋白酶(MMP)7、MMP9和MMP13的表达均低于对照细胞(P<0.05)。结论:在黑素瘤中降低RPL34表达抑制了细胞增殖及克隆形成的能力,影响细胞上皮间质化的进程,从而抑制黑素瘤细胞的迁移、侵袭。

Objective:To explore the effect of 60 S ribosomal protein L34(RPL34)on proliferation,migration and invasion of melanoma cells and the related mechanisms.Methods:Immunohistochemistry(IHC)staining was performed to investigate the expression of RPL34 in 20 pairs of melanoma tissue and corresponding normal skin tissue.Human melanoma cell line A375 stably expressing shNC(negative control)or shRPL34 was constructed using lentivirus.The knockdown efficiency of RPL34 was confirmed using RT-PCR and Western blot.Proliferation and colony formation assays were performed to detect cell proliferation and clone formation ability.Cell motility was assessed by wound healing assay,and the migration and invasion abilities of these cells were detected using transwell assay.Results:IHC staining suggested higher RPL34 expression in melanoma tissue than in corresponding normal skin tissue.The knockdown of RPL34 inhibited A375 cell proliferation(F=25.362,P<0.05)and clone formation(t=23.171,P=0.00065),as well as migration(t=6.99,P=0.00185)motility and invasion(t=7.576,P=0.00156).Downregulation of TWIST1,vimentin,TIMP2,MMP7,MMP9,and MMP13 expression was found in RPL34 knock-down A375 cells(P<0.05).Conclusion:The knockdown of RPL34 in melanoma suppresses cell proliferation,colony formation,and epithelial-mesenchymal transition,thus inhibiting migration and invasion of melanoma cells.