miR-155-5p靶向ARID2对口腔鳞状细胞癌有促进作用

Promoting effect of miR-155-5p targeting ARID2 on oral squamous cell carcinoma

目的 探讨miR-155-5p对口腔鳞状细胞癌(OSCC)细胞增殖、迁移和侵袭的调控作用和机制。方法 收集人口腔癌细胞系CAL-27、HN4、HN6、SCC4、TCA8113和正常口腔上皮角质细胞系HOK,检测miR-155-5p表达。根据转染质粒的不同分为miR-NC组(转染miR-155-5p模拟物阴性对照)和miR-155-5p组(转染miR-155-5p模拟物)、anti-NC组(转染miR-155-5p抑制物阴性对照)、anti-miR-155-5p组(转染miR-155-5p抑制物)、Vector组(转染空质粒载体)、pc-ARID2组(转染ARID2过表达质粒)、miR-NC+Vector组(共转染阴性对照和空质粒载体)、miR-155-5p+Vector组(共转染miR-155-5p模拟物和空质粒载体)和miR-155-5p+pc-ARID2组(共转染miR-155-5p模拟物和ARID2过表达质粒)。采用CCK-8实验、克隆形成实验检测细胞增殖情况,分别采用划痕实验和侵袭实验检测CAL-27细胞、HN4细胞的迁移、侵袭情况,采用免疫印迹法检测ARID2蛋白的表达情况。采用双荧光素酶报告基因实验验证miR-155-5p与ARID2的靶向关系。结果 与正常口腔上皮角质细胞系HOK比较,OSCC细胞系(SCC4、HN6、HN4、CAL-27、TCA8113)miR-155-5p表达均显著上调(P<0.01)。与anti-NC组比较,anti-miR-155-5p组miR-155-5p相对表达量显著降低(P<0.01),增殖活性、克隆形成数、划痕闭合率和侵袭细胞数均显著降低(P<0.01)。与miR-NC组比较,miR-155-5p组ARID2蛋白表达显著下调(P<0.01),增殖活性、克隆形成数、划痕闭合率和侵袭细胞数均显著升高(P<0.01);anti-miR-155-5p组ARID2蛋白表达显著上调(P<0.01),增殖活性、克隆形成数、划痕闭合率和侵袭细胞数均显著降低(P<0.01)。双荧光素酶报告基因实验证实,与共转染miR-NC和pmiR-ARID2野生型质粒(pmiR-ARID2-wt)比较,共转染miR-155-5p mimic和pmiR-ARID2突变型质粒(pmiR-ARID2-wt)后荧光素酶活性显著降低(P<0.01)。与Vector组比较,pc-ARID2组ARID2 mRNA表达显著上调(P<0.01),增殖活性、克隆形成数、划痕闭合率和侵袭细胞数均显著降低(P<0.01)。与miR-NC+Vector组比较,miR-155-5p+Vector组增殖活性、克隆形成数、侵袭细胞数和划痕闭合率均显著升高(P<0.01);与miR-155-5p+Vector组比较,miR-155-5p+pc-ARID2组增殖活性、克隆形成数、侵袭细胞数和划痕闭合率均显著降低(P<0.01)。结论 OSCC细胞中miR-155-5p表达上调,并可通过靶向抑制ARID2表达促进OSCC细胞增殖、迁移和侵袭,进而发挥致癌基因作用。

Objective To investigate the regulation and mechanism of miR-155-5p on the proliferation,migration and invasion of oral squamous cell carcinoma(OSCC)cells.Methods Human OSCC cell lines CAL-27,HN4,HN6,SCC4,TCA8113 and normal oral epithelial keratinocyte line HOK were collected to determine the expression of miR-155-5p.According to the different transfection plasmids,they were classified into miR-NC group(transfected negative control with miR-155-5p mimics),miR-155-5p group(transfected with miR-155-5p mimics)and anti-NC group(transfected negative control with miR-155-5p inhibitor),anti-miR-155-5p group(transfected with miR-155-5p inhibitor),Vector group(transfected with empty plasmid vector),pc-ARID2 group(transfected with ARID2 overexpression plasmid),miR-NC+Vector group(co-transfected with negative control and empty plasmid vector),miR-155-5p+Vector group(co-transfected with miR-155-5p mimics and empty plasmid vector)and miR-155-5p+pc-ARID2 group(co-transfected with miR-155-5p mimics and ARID2 overexpression plasmid).The cell proliferation was determined by CCK-8 assay and clonal formation assay,the migration and invasion of CAL-27 cells and HN4 cells were determined by scratch assay and invasion assay,and the expression of ARID2 protein was determined by western blot.Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-155-5p and ARID2.Results Compared with oral epithelial keratinocyte line HOK,OSCC cell lines(SCC4,HN6,HN4,CAL-27,TCA8113)showed significantly up-regulated miR-155-5p expression(P<0.01).Compared with anti-NC group,the relative expression of anti-miR-155-5p inhibitor group was significantly decreased(P<0.01).The proliferation activity,clonal formation number,scratch closure rate and invasion cell number were decreased(P<0.01).Compared with miR-NC group,the expression of ARID2 in miR-155-5p group was down-regulated(P<0.01),and the proliferation activity,clonal formation number,scratch closure rate and invasion cell number were increased(P<0.01).The expression of ARID2 in anti-miR-155-5p inhibitor group was up-regulated(P<0.01),and the proliferation activity,clonal formation number,scratch closure rate and invasion cell number were decreased(P<0.01).Compared with co-transfected miR-NC and pmiR-ARID2 wild-type plasmid(pmiR-ARID2-wt),the luciferase activity was decreased after co-transfecting miR-155-5p mimic and pmiR-ARID2 mutant-type plasmid(pmiR-ARID2-wt)(P<0.01).Compared with Vector group,the expression of ARID2 mRNA in pc-ARID2 group was up-regulated(P<0.01),and the proliferation activity,clonal formation number,scratch closure rate and invasion cell number were decreased(P<0.01).Compared with miR-NC+Vector group,the proliferation activity,clonal formation number,scratch closure rate and invasion cell number in miR-155-5p+Vector group were increased(P<0.01).Compared with miR-155-5p+Vector group,the proliferation activity,clonal formation number,scratch closure rate and invasion cell number in miR-155-5p+pc-ARID2 group were decreased(P<0.01).Conclusions The expression of miR-155-5p is up-regulated in OSCC cells,and it can promote the proliferation,migration and invasion of OSCC cells through targeted inhibition of ARID2 expression,thus playing the role of oncogene.