circ_0003159对胃癌细胞增殖、侵袭和迁移能力的影响及机制研究

Effect of circ_0003159 on proliferation,invasion and migration of gastric cancer cells and related mechanism

目的探讨circ_0003159对胃癌细胞增殖、侵袭和迁移能力的影响及其作用机制。方法选取2015年6月至2018年12月嘉兴市第二医院肿瘤外科经手术切除的30例患者的胃癌组织和配对的癌旁组织。采用qRT-PCR法检测胃癌组织和癌旁组织中circ_0003159、miR-32-5p和第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)的表达水平;以及胃癌细胞株(BGC823、AGS、MKN45)和正常胃黏膜上皮细胞株GES-1中circ_0003159的表达水平。选取circ_0003159表达水平最低的胃癌BGC823细胞系,建立circ_0003159超表达模型,设立circ_0003159超表达组(转染circ_0003159模拟物)和阴性对照组(转染空载体阴性对照物)。采用CCK-8法和Transwell小室实验检测circ_0003159超表达对BGC823细胞增殖、侵袭和迁移的影响;生物信息学方法对circ_0003159或miR-32-5p进行靶基因预测,并通过双荧光素酶报告基因验证miR-32-5p与circ_0003159或PTEN的靶向关系;采用Western blot法检测过表达miR-32-5p对PTEN蛋白表达的影响。结果胃癌组织中circ_0003159和PTEN的相对表达水平均明显低于癌旁组织(均P<0.05),但胃癌组织中miR-32-5p的相对表达水平明显高于癌旁组织(P<0.05)。胃癌细胞系BGC823、AGS、MKN45中circ_0003159的相对表达水平均明显低于正常胃黏膜上皮细胞GES-1(均P<0.05),且BGC823细胞中circ_0003159的相对表达水平最低。相比于阴性对照组,circ_0003159超表达组在48、72 h时OD450表达水平均明显下调(均P<0.05)。与阴性对照组比较,circ_0003159超表达组中细胞侵袭数和迁移数均明显减少(均P<0.05)。预测结果可知miR-32-5p为circ_0003159潜在的靶基因,PTEN是miR-32-5p潜在的靶基因。荧光素酶报告基因实验结果显示,与对照组比较,过表达miR-32-5p组中circ_0003159-MUT和PTEN-MUT相对活力值比较差异均无统计学意义(均P>0.05),而circ_0003159-WT和PTEN-WT相对活力值比较均明显下调(均P<0.05)。与对照组相比,过表达miR-32-5p组中PTEN蛋白相对表达水平明显下降(P<0.05)。结论circ_0003159超表达显著抑制了胃癌细胞增殖、侵袭和迁移能力,其机制可能是通过靶向下调miR-32-5p对PTEN的抑制作用。

Objective To explore the effect of circ_0003159 on proliferation,invasion,and migration of gastric cancer cells,and its underlying mechanism.Methods Real-time quantitative RT-PCR(qRT-PCR)was performed to detect the expr-ession levels of circ_0003159,miR-32-5p and gene of phosphate and tension homology deleted on chromosome ten(PTEN)in gastric cancer tissues and matched adjacent tissues,as well as the expression of circ_0003159 in gastric cancer cell lines BGC823,AGS,MKN45 and normal gastric mucosa cell line GSE-1.BGC823 cell line with the lowest circ_0003159 expression was selected for further study.BGC823 cells were transfected with pcDNA3.1-circ_0003159(circ_0003159 overexpression group)or pcDNA3.1-NC(negative control group).The effect of circ_0003159 overexpression on proliferation,invasion,and migration abilities of BGC823 cells were determined by CCK-8 assay and Transwell assay.Bioinformatics analysis for target gene prediction of circ_0003159 or miR-32-5p was performed,and target relationship between miR-32-5p and circ_0003159 or PTEN was validated by dual-luciferase reporter gene assay.The effect of miR-32-5p overexpression on the expression of PTEN protein was determined by Western blot.Results The circ_0003159 and PTEN was lowly-expressed in gastric cancer tissues compared with matched adjacent tissues(both P<0.05),whereas the expression of miR-32-5p was higher in gastric cancer tissues than that in matched adjacent tissues(P<0.05).Meanwhile,the expression of circ_0003159 in gastric cancer cell lines BG-C823,AGS,MKN45 was lower than that in normal gastric mucosa cell line GSE-1.Compared with negative control group,overexpression of circ_0003159 significantly suppressed BGC823 cell proliferation,invasion,and migration(all P<0.05).The dual-luciferase reporter gene assay showed that compared with the control group(mimic-NC),the differences in circ_0003159-MUT and PTEN-MUT expression of the overexpression miR-32-5p group(transfected with miR-32-5p mimic)were not statistically significant(both P>0.05),while both circ_0003159-WT and PTEN-WT expressions were significantly downregulated(both P<0.05).The expression of PTEN protein was significantly downregulated in the miR-32-5p overexpression group compared with the control group(P<0.05).Conclusion The elevated circ_0003159 can suppress the proliferation,invasion and migration of gastric cancer cells via targeting inhibitory effect of miR-32-5p on PTEN expression.