circ_0007059/miR-421对皮肤鳞状细胞癌发生及发展的影响

The Influence of circ_007059/miR-421 on the Occurrence and Development of Cutaneous Squamous Cell Carcinoma

目的探讨circ_0007059/miR-421对皮肤鳞状细胞癌(简称皮肤鳞癌)细胞生物学行为的影响及其对PDCD4的调控作用。方法采用qRT-PCR法检测皮肤鳞癌组织、癌旁组织中circ_0007059与miR-421的表达量;采用免疫组织化学法检测皮肤鳞癌组织中PDCD4蛋白阳性率;体外培养皮肤鳞癌细胞SCC-13,随机分为以下几组:pcDNA组、pcDNA-circ_0007059组、si-NC组、si-circ_0007059组、anti-miR-NC组、anti-miR-421组、pcDNA-circ_0007059+miR-NC组、pcDNA-circ_0007059+miR-421组。采用qRT-PCR法与Western blot实验分别检测细胞中circ_0007059、miR-421、PDCD4的表达量;采用MTT实验、平板克隆形成实验、流式细胞术、划痕实验、Transwell小室实验分别检测增殖、克隆形成、凋亡、迁移及侵袭;双荧光素酶报告基因实验检测circ_0007059与miR-421的靶向关系,以及miR-421与PDCD4的靶向关系。结果与癌旁组织比较,皮肤鳞癌组织中circ_0007059的表达水平降低,miR-421的表达水平升高,PDCD4蛋白阳性率降低,差异均有统计学意义(P<0.05);与pcDNA组比较,pcDNA-circ_0007059组miR-421的表达水平降低,PDCD4蛋白水平升高,细胞活力降低,集落形成数与侵袭细胞数减少,迁移距离减小,凋亡率升高,差异均有统计学意义(P<0.05);与si-NC组比较,si-circ_0007059组miR-421的表达量升高,PDCD4蛋白水平降低,细胞活力升高,集落形成数和侵袭细胞数增多,细胞凋亡率降低,迁移距离增加,差异均有统计学意义(P<0.05);circ_0007059可靶向结合miR-421,PDCD4是miR-421的靶基因;与anti-miR-NC组比较,anti-miR-421组PDCD4蛋白水平升高,细胞活力降低,集落形成数与侵袭细胞数减少,迁移距离减小,凋亡率升高,差异均有统计学意义(P<0.05);与pcDNA-circ_0007059+miR-NC组比较,pcDNA-circ_0007059+miR-421组PDCD4蛋白水平降低,细胞活力升高,集落形成数与侵袭细胞数增多,迁移距离增长,凋亡率降低,差异均有统计学意义(P<0.05)。结论circ_0007059过表达可调控miR-421/PDCD4分子轴而抑制皮肤鳞癌细胞增殖、克隆形成、迁移、侵袭及诱导细胞凋亡。

Objective To explore the effect of circ_0007059/miR-421 on the biological manifestation of cutaneous squamous cell carcinoma and its regulation on PDCD4.Methods The qRT-PCR method was used to detect the expression of circ_0007059 and miR-421 in cutaneous squamous cell carcinoma tissues and adjacent tissues.Immunohistochemical method was used to detect the positive rate of PDCD4 protein in cutaneous squamous cell carcinoma tissue.Skin squamous cell carcinoma cells SCC-13 were cultured in vitro and randomly divided into the following groups:pcDNA group,pcDNA-circ_0007059 group,si-NC group,si-circ_0007059 group,anti-miR-NC group,anti-miR-421 group,pcDNA-circ_0007059+miR-NC group,pcDNA-circ_0007059+miR-421 group.The expressions of circ_0007059,miR-421 and PDCD4 in the cells were detected by qRT-PCR and Western blot respectively.MTT experiment,plate clone formation experiment,flow cytometry,scratch experiment,and Transwell chamber experiment were used to detect proliferation,clone formation,apoptosis,migration and invasion,respectively.The dual luciferase reporter gene experiment was used to detect the targeting relationship between circ_0007059 and miR-421,and the targeting relationship between miR-421 and PDCD4.Results Compared with adjacent tissues,the expression level of circ_0007059 in cutaneous squamous cell carcinoma tissue was decreased,the expression level of miR-421 was increased,and the positive rate of PDCD4 protein was decreased,the difference was statistically significant(P<0.05).Compared with the pcDNA group,the expression level of miR-421 in the pcDNA-circ_0007059 group was decreased,but the protein level of PDCD4 was increased,and cell viability was decreased,the number of colonies formed and the number of invasive cells were decreased,the migration distance was decreased,but the apoptosis rate was increased,the difference was statistically significant(P<0.05).Compared with the si-NC group,the expression of miR-421 in the si-circ_0007059 group was increased,the protein level of PDCD4 was decreased,cell viability was increased,the number of colonies formed and the number of invasive cells were increased,the rate of cell apoptosis was decreased,and the migration distance was increased,the difference was statistically significant(P<0.05).circ_0007059 could target miR-421,PDCD4 was the target gene of miR-421.Compared with the anti-miR-NC group,the protein level of PDCD4 in the anti-miR-421 group was increased,but cell viability was decreased,and the number of colonies formed and the number of invasive cells was decreased,and migration distance was decreased,while the apoptosis rate was increased.Compared with the pcDNA-circ_0007059+miR-NC group,the protein level of PDCD4 of the pcDNA-circ_0007059+miR-421 group was decreased,but cell viability was increased,and the number of colonies formed and the number of invasive cells was increased,and the migration distance was increased,but the apoptosis rate was decreased,the difference was statistically significant(P<0.05).Conclusion Overexpression of circ_0007059 could regulate the molecular axis of miR-421/PDCD4 and inhibit cell proliferation,clone formation,migration,invasion and induce cell apoptosis in cutaneous squamous cell carcinoma.