KRTAP5-AS1 负调控miR-335 对甲状腺癌细胞增殖、迁移及凋亡的影响

KRTAP5-AS1 negatively regulates effect of miR-335 on proliferation,migrationand apoptosis of thyroid cancer cells

目的:探究长链非编码RNA(lncRNA)KRTAP5-AS1是否通过负调控miR-335影响甲状腺癌细胞的增殖、迁移和凋亡。方法:qRT-PCR检测33例甲状腺癌组织中KRTAP5-AS1和miR-335的表达。甲状腺癌细胞SW579分为si-NC组、si-KRTAP5-AS1组、si-KRTAP5-AS1+miR-NC组和si-KRTAP5-AS1+miR-335 mimic组,CCK-8法、平板细胞克隆形成实验、细胞划痕实验、Transwell实验、流式细胞术与Western blot分别检测细胞活性、克隆形成、划痕愈合率、侵袭细胞数、凋亡和B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)表达。双荧光素酶报告实验测定KRTAP5-AS1和miR-335的靶向关系。结果:qRT-PCR结果显示,KRTAP5-AS1在甲状腺癌组织中呈高表达,miR-335呈低表达;si-KRTAP5-AS1组KRTAP5-AS1表达水平低于si-NC组,miR-335表达水平高于si-NC组,差异有统计学意义(P<0.05)。CCK-8结果表明,与si-NC组相比,si-KRTAP5-AS1组SW579细胞活性降低;与si-KRTAP5-AS1+miR-NC组相比,si-KRTAP5-AS1+miR-335 mimic组SW579细胞活性升高,差异有统计学意义(P<0.05)。平板细胞克隆形成实验结果发现,si-KRTAP5-AS1组克隆形成数少于si-NC组;si-KRTAP5-AS1+miR-335 mimic组克隆形成数多于si-KRTAP5-AS1+miR-NC组,差异有统计学意义(P<0.05)。细胞划痕实验和Transwell实验数据显示,与si-NC组相比,si-KRTAP5-AS1组划痕愈合率、侵袭细胞数减少;与si-KRTAP5-AS1+miR-NC组相比,si-KRTAP5-AS1+miR-335 mimic组划痕愈合率、侵袭细胞数增多,差异有统计学意义(P<0.05)。流式细胞术结果表明,si-KRTAP5-AS1组SW579细胞凋亡率高于si-NC组;si-KRTAP5-AS1+miR-335 mimic组SW579细胞凋亡率低于si-KRTAP5-AS1+miR-NC组,差异有统计学意义(P<0.05)。Western blot结果显示,与si-NC组相比,si-KRTAP5-AS1组SW579细胞中Bax、E-cadherin表达升高,Bcl-2、N-cadherin表达降低;与si-KRTAP5-AS1+miR-NC组相比,si-KRTAP5-AS1+miR-335 mimic组SW579细胞中Bax、E-cadherin表达降低,Bcl-2、N-cadherin表达升高,差异有统计学意义(P<0.05)。结论:KRTAP5-AS1通过负调控miR-335促进甲状腺癌细胞增殖、迁移,并抑制其凋亡。

Objective:To investigate whether long non-coding RNA(lncRNA)KRTAP5-AS1 negatively regulates miR-335 to affect proliferation,migration and apoptosis of thyroid cancer cells.Methods:qRT-PCR was used to detect expressions of KRTAP5-AS1 and miR-335 in 33 cases of thyroid cancer.Thyroid cancer cell SW579 were divided into si-NC group,si-KRTAP5-AS1 group,si-KRTAP5-AS1+miR-NC group and si-KRTAP5-AS1+miR-335 mimic group,CCK-8 method,plate cell clone formation experiment,cell scratch test,Transwell method,flow cytometry and Western blot to detect cell activity,clone formation,scratch healing rate,number of invasive cells,apoptosis and expressions of B cell lymphoma/leukemia-2(Bcl-2),Bcl-2 related X protein(Bax),E-cadherin and N-cadherin.Dual luciferase reporter experiment determined the targeting relationship between KRTAP5-AS1 and miR-335.Results:qRT-PCR results showed that KRTAP5-AS1 was highly expressed in thyroid cancer tissues,while miR-335 was low expression;expression level of KRTAP5-AS1 in si-KRTAP5-AS1 group was lower than that in si-NC group,while expression level of miR-335 was higher than that of si-NC group,the difference was statistically significant(P<0.05).CCK-8 results showed that compared with si-NC group,SW579 cell activity in si-KRTAP5-AS1 group was reduced;compared with si-KRTAP5-AS1+miR-NC group,SW579 cell activity of si-KRTAP5-AS1+miR-335 mimic group was increased,the difference was statistically significant(P<0.05).Results of plate cell clone formation experiment found that the number of clones formed in si-KRTAP5-AS1 group was less than that in si-NC group;the number of clones formed in si-KRTAP5-AS1+miR-335 mimic group was more than that in si-KRTAP5-AS1+miR-NC group,the difference was statistically significant(P<0.05).Cell scratch test data and Transwell method showed that compared with si-NC group,scratch healing rate and number of invasion cells in si-KRTAP5-AS1 group decreased;compared with si-KRTAP5-AS1+miR-NC group,scratch healing rate and number of invasion cells in si-KRTAP5-AS1+miR-335 mimic group increased,the difference was statistically significant(P<0.05).Flow cytometry results showed that apoptosis rate of SW579 cells in si-KRTAP5-AS1 group was higher than that in si-NC group;apoptosis rate of SW579 cells in si-KRTAP5-AS1+miR-335 mimic group was lower than that of si-KRTAP5-AS1+miR-NC group,the difference was statistically significant(P<0.05).Western blot results showed that compared with si-NC group,expressions of Bax and E-cadherin in SW579 cells of si-KRTAP5-AS1 group were increased,while expressions of Bcl-2 and N-cadherin were decreased;compared with si-KRTAP5-AS1+miR-NC group,expressions of Bax and E-cadherin in SW579 cells of si-KRTAP5-AS1+miR-335 mimic group were decreased,while expressions of Bcl-2 and N-cadherin were increased,the difference was statistically significant(P<0.05).Conclusion:KRTAP5-AS1 negatively regulates miR-335 to promote proliferation and migration of thyroid cancer cells and inhibit apoptosis.