【摘要】 目的探讨circ₀003159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circ₀003159、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circ₀003159的表达水平。选取circ₀003159表达水平最低的胃癌BGC823细胞系,建立circ₀003159超表达模型,设立circ₀003159超表达组（转染circ₀003159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circ₀003159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circ₀003159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circ₀003159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circ₀003159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circ₀003159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circ₀003159的相对表达水平最低。相比于阴性对照组,circ₀003159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,circ₀003159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circ₀003159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circ₀003159-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circ₀003159-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 circ₀003159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。更多还原

【Abstract】 Objective To explore the effect of circ₀003159 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 expression levels of circ₀003159, 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 circ₀003159 in gastric cancer cell lines BGC823, AGS, MKN45 and normal gastric mucosa cell line GSE-1. BGC823 cell line with the lowest circ₀003159 expression was selected for further study. BGC823 cells were transfected with pc DNA3.1-circ₀003159（circ₀003159 overexpression group）or pc DNA3.1-NC（negative control group）. The effect of circ₀003159 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 circ₀003159 or miR-32-5p was performed, and target relationship between miR-32-5p and circ₀003159 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 circ₀003159 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 circ₀003159 in gastric cancer cell lines BGC823, AGS, MKN45 was lower than that in normal gastric mucosa cell line GSE-1. Compared with negative control group, overexpression of circ₀003159 significantly suppressed BGC823 cell proliferation, invasion, and migration（all P<0.05）. The dualluciferase reporter gene assay showed that compared with the control group（mimic-NC）, the differences in circ₀003159-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 circ₀003159-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 circ₀003159 can suppress the proliferation, invasion and migration of gastric cancer cells via targeting inhibitory effect of miR-32-5p on PTEN expression.更多还原