circEPSTI1通过靶向调控miR-216b-5p调节胃癌细胞顺铂耐药性

CircEPSTI1 Regulates Cisplatin Resistance of Gastric Cancer Cells by Targeting miR-216b-5p

该研究的目的是为了观察circEPSTI1是否通过靶向调控miR-216b-5p对胃癌细胞顺铂耐药性产生影响,并探讨其可能的作用机制。采用实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)检测胃癌组织、癌旁组织、人胃黏膜细胞GES-1、人胃癌细胞HGC27与胃癌耐药性细胞HGC27/DDP中circEPSTI1与miR-216b-5p的表达情况;使用双荧光素酶报告实验验证circEPSTI1与miR-216b-5p的靶向调控关系;以HGC27/DDP细胞为研究对象,实验分组:si-NC组、si-circEPSTI1组、si-circEPSTI1+anti-miR-NC组、si-circEPSTI1+anti-miR-216b-5p组;采用CCK-8法、平板克隆形成实验、流式细胞术与Transwell实验分别检测细胞增殖、克隆形成、凋亡、迁移及侵袭;Western blot检测Cleavea-caspase 3、Cleavea-caspase 9、E-cadherin、N-cadherin蛋白表达情况。结果显示,与癌旁组织相比,胃癌组织中的circEPSTI1表达上调(P<0.05), miR-216b-5p表达下调(P<0.05);与GES-1细胞相比,HGC27细胞、HGC27/DDP细胞的circEPSTI1表达上调(P<0.05), miR-216b-5p表达下调(P<0.05);与HGC27细胞相比, HGC27/DDP细胞的circEPSTI1表达上调(P<0.05), miR-216b-5p表达下调(P<0.05);miR-216b-5p过表达可降低wt-circEPSTI1的荧光素酶活性(P<0.05),未影响mut-circEPSTI1的荧光素酶活性;与si-NC组相比, si-circEPSTI1组细胞增殖抑制率、凋亡率和Cleavea-caspase 3、Cleavea-caspase 9、E-cadherin蛋白表达上调(P<0.05),克隆形成数、迁移及侵袭细胞数减少(P<0.05), N-cadherin蛋白表达下调(P<0.05);与si-circEPSTI1+antimiR-NC组相比, si-circEPSTI1+anti-miR-216b-5p组细胞增殖抑制率、凋亡率和Cleavea-caspase 3、Cleavea-caspase 9、E-cadherin蛋白表达下调(P<0.05),克隆形成数、迁移及侵袭细胞数增加(P<0.05),N-cadherin蛋白表达上调(P<0.05)。结果表明, circEPSTI1通过靶向抑制miR-216b-5p表达促进胃癌耐药性细胞增殖、克隆形成、迁移及侵袭,抑制胃癌耐药性细胞凋亡,从而增强胃癌细胞对DDP的耐药性,该机制可能与改变凋亡相关蛋白表达情况和逆转上皮–间质转化过程有关。

The objective was to observe the effect of circEPSTI1 on cisplatin resistance of gastric cancer cells by targeting miR-216b-5p and to explore its possible mechanism.The qRT-PCR(quantitative real-time polymerase chain reaction)was used to detect the expression of circEPSTI1 and miR-216b-5p in gastric cancer tissues,adjacent tissues,human gastric mucosal cells GES-1,human gastric cancer cells HGC27 and gastric cancer drug-resistant cells HGC27/DDP.The dual luciferase reporting assay was used to verify the targeted regulatory relationship between circEPSTI1 and miR-216b-5p.HGC27/DDP cells were selected as the research object and divided into four groups:the si-NC group,the si-circEPSTI1 group,the si-circEPSTI1+anti-miR-NC group,and the si-circEPSTI1+anti-miR-216b-5p group.Cell proliferation,clonal formation,apoptosis,migration,and invasion were detected by CCK-8 assay,plate clonal formation assay,flow cytometry,and Transwell assay,respectively.Western blot was used to detect the protein expression of Cleavea-caspase 3,Cleavea-caspase 9,E-cadherin,and N-cadherin.Compared with adjacent tissues,the results showed that circEPSTI1 expression in gastric cancer tissues was up-regulated(P<0.05)and miR-216b-5p expression was down-regulated(P<0.05).Compared with GES-1 cells,circEPSTI1 expression in HGC27 cells and HGC27/DDP cells was up-regulated(P<0.05),while miR-216b-5p expression was down-regulated(P<0.05).Compared with HGC27 cells,circEPSTI1 expression in HGC27/DDP cells was up-regulated(P<0.05)and miR-216b-5p expression was down-regulated(P<0.05).Overexpression of miR-216b-5p could reduce the luciferase activity of wt-circEPSTI1(P<0.05),but did not affect the luciferase activity of mut-circEPSTI1.Compared with the si-NC group,cell proliferation inhibition rate,apoptosis rate,Cleavea-caspase 3,Cleavea-caspase 9,and E-cadherin protein expression in the si-circEPSTI1 group were up-regulated(P<0.05).The clone formation numbers,migration,and invasion cells were decreased(P<0.05),and the expression of N-cadherin protein was down-regulated(P<0.05).Compared with the si-circEPSTI1+anti-miRNC group,cell proliferation inhibition rate,apoptosis rate,Cleavea-caspase 3,Cleavea-caspase 9,and E-cadherin expression were down-regulated in the si-circEPSTI1+anti-miR-216b-5p group(P<0.05).The number of clone formation,migration,and invasion cells increased(P<0.05),and the expression of N-cadherin protein was upregulated(P<0.05).The results showed that circEPSTI1 promoted the proliferation,clone formation,migration,and invasion,and inhibited the apoptosis in drug-resistant gastric cancer cells by targeting the expression of miR216b-5p,thus promoting the drug resistance of gastric cancer cells to DDP.This mechanism may be related to the change of apoptosis-related protein expression and the reversal of the epithelial-mesenchymal transformation process.