MicroRNA-124对喉鳞状细胞癌细胞自噬、侵袭和迁移的影响

Effect of microRNA-124 on autophagy,invasion and migration of laryngeal squamous cell carcinoma cells

目的探讨microRNA-124(miR-124)在人喉鳞状细胞癌(LSCC)组织中的表达及其对LSCC细胞自噬、侵袭和迁移的影响。方法选取2016年1月至2018年9月新乡医学院第一附属医院手术切除的LSCC组织及癌旁组织(距离肿瘤边缘1~2 cm)标本各12例,采用实时定量聚合酶链反应(qRT-PCR)检测LSCC组织和癌旁组织中miR-124表达;体外培养人Hep-2细胞,将其分为miR-124类似物(miR-124 mimics)组、miR-124抑制剂(miR-124 inhibitor)组、类似物阴性对照(mimics NC)组、抑制剂阴性对照(inhibitor NC)组、mimics+西罗莫司组和inhibitor+3-甲基腺嘌呤(3-MA)组,进行细胞转染;采用qRT-PCR检测miR-124 mimics组、miR-124 inhibitor组、mimics NC组、inhibitor NC组细胞中miR-124的表达水平,Western blot法检测LSCC组织、癌旁组织和mimics NC组、miR-124 mimics组、inhibitor NC组、miR-124 inhibitor组细胞中自噬相关蛋白LC3B-Ⅱ的表达,Transwell小室法检测miR-124 mimics组、miR-124 inhibitor组、mimics NC组、inhibitor NC组、mimics+西罗莫司组和inhibitor+3-MA组细胞的侵袭能力,细胞划痕实验检测miR-124 mimics组、miR-124 inhibitor组、mimics NC组、inhibitor NC组、mimics+西罗莫司组和inhibitor+3-MA组细胞的迁移能力。结果LSCC组织中miR-124相对表达量显著低于癌旁组织(P<0.05)。LSCC组织中LC3B-Ⅱ蛋白表达显著高于癌旁组织(P<0.05)。miR-124 mimics组细胞中miR-124相对表达量显著高于mimics NC组(P<0.05),miR-124 inhibitor组细胞中miR-124相对表达量显著低于inhibitor NC组(P<0.05)。miR-124 mimics组细胞LC3B-Ⅱ蛋白相对表达量显著低于mimics NC组(P<0.05),miR-124 inhibitor组细胞LC3B-Ⅱ蛋白相对表达量显著高于inhibitor NC组(P<0.05)。miR-124 mimics组侵袭细胞数显著少于mimics NC组和mimics+西罗莫司组(P<0.05),miR-124 inhibitor组侵袭细胞数显著多于inhibitor NC组和inhibitor+3-MA组(P<0.05)。划痕培养72 h后,inhibitor NC组和inhibitor+3-MA组细胞的划痕愈合率低于miR-124 inhibitor组(P<0.05);培养96 h后,mimics NC组和mimics+西罗莫司组细胞的划痕愈合率高于miR-124 mimics组(P<0.05)。结论人LSCC组织中miR-124表达显著下调,人LSCC组织和miR-124过表达细胞中自噬相关蛋白LC3B-Ⅱ表达显著上调,miR-124可能通过抑制自噬而抑制LSCC细胞的侵袭和迁移能力。

Objective To investigate the expression of microRNA-124(miR-124)in human laryngeal squamous cell carcinoma(LSCC)and its effect on autophagy,invasion and migration of LSCC cells.Method Twelve LSCC tissue specimens and twelve paracancerous tissue specimens(1-2 cm away from the edge of the tumor)were collected from the First Affiliated Hospital of Xinxiang Medical University from January 2016 to September 2018.The expression of miR-124 in LSCC and paracancerous tissues was detected by real-time quantitative polymerase chain reaction(qRT-PCR).Human LSCC Hep-2 cells were cultured in vitro and divided into miR-124 mimics group,miR-124 inhibitor group,mimics negative control(mimics NC)group,inhibitor negative control(inhibitor NC)group,mimics+sirolimus group and inhibitor+3-methyladenine(3-MA)group.After cell transfection,the expression of miR-124 in Hep-2 cells of in the miR-124 mimics group,miR-124 inhibitor group,mimics NC group and inhibitor NC group was detected by qRT-PCR.The expression of autophagy-related protein LC3B-Ⅱin LSCC tissues,paracancerous tissues and cells in the miR-124 mimics group,miR-124 inhibitor group,mimics NC group and inhibitor NC group were detected by Western blot method.The cell invasion ability and cell migration ability in the miR-124 mimics group,miR-124 inhibitor group,mimics NC group,inhibitor NC group,mimics+sirolimus group and inhibitor+3-MA group was detected by Transwell method and would healing assay,respectively.Result The relative expression of miR-124 in LSCC tissues was significantly lower than that in paracancerous tissues(P<0.05).The relative expression of autophagy-related protein LC3B-Ⅱin LSCC tissues was obviously higher than that in paracancerous tissues(P<0.05).The relative expression of miR-124 in the miR-124 mimics group was significantly higher than that in the mimics NC group(P<0.05).The relative expression of miR-124 in the miR-124 inhibitor group was significantly lower than that in the inhibitor NC group(P<0.05).The relative expression of LC3B-Ⅱprotein in the miR-124 mimics group was obviously lower than that in the mimics NC group(P<0.05).The relative expression of LC3B-Ⅱprotein in the miR-124 inhibitor group was significantly higher than that in the inhibitor NC group(P<0.05).The number of invasion cells in the miR-124 mimics group were significantly fewer than that in the mimics NC group and mimics+sirolimus group(P<0.05).The number of invasion cells in the miR-124 inhibitor group was significantly more than that in the inhibitor NC group and inhibitor+3-MA group(P<0.05).The healing rate in the inhibitor NC group and inhibitor+3-MA group was significantly lower than that in the miR-124 inhibitor group at 72 hours after scratch culturing(P<0.05).The healing rate in the mimics NC and mimics+sirolimus group was significantly higher than that in the miR-124 mimics group at 96 hours after scratch culturing(P<0.05).Conclusion The expression of miR-124 is significantly down-regulated in LSCC tissues.The expression of autophagy-related protein LC3B-Ⅱis obviously up-regulated in LSCC tissues and miR-124-overexpressed cells.MiR-124 may inhibit the invasion and migration ability of LSCC cells by inhibiting autophagy.