【摘要】 目的 观察微小RNA-216a(miR-216a)和钙蛋白酶6(CAPN6)基因过表达的人宫颈癌细胞系HeLa增殖、迁移、侵袭变化及靶向关系，探讨miR-216a对HeLa细胞增殖迁移侵袭影响的作用机制。方法 取对数生长期HeLa细胞，分为一、二、三、四、五组，一组转染miR-216a mimic，二组转染pcDNA3.1-CAPN6质粒，三组顺序转染miR-216a mimic、pcDNA3.1-CAPN6，四组转染pcDNA3.1，五组转染miR-216a mimic NC，培养48 h时分别采用CCK-8法、划痕愈合实验、Transwell试验观察各组细胞的增殖迁移侵袭情况，培养24 h时采用qRT-PCR法检测各组细胞miR-216a、采用Western Blotting法检测各组细胞CAPN6及信号转导子与激活子3(STAT3)蛋白。取对数生长期HeLa细胞分为四组：A组细胞顺序转染miR-216a mimic、 pGL3-CAPN6-WT质粒，B组细胞顺序转染miR-216a mimic、pGL3-CAPN6-MUT质粒，C组细胞顺序转染miR-216a mimic NC、pGL3-CAPN6-WT,D组细胞顺序转染miR-216a mimic NC、pGL3-CAPN6-MUT，培养36 h时收集各组细胞，采用双荧光素酶报告基因检测试剂盒测算各组细胞相对荧光素酶活性。结果 与五组相比，培养48 h时一组细胞增殖活性及划痕前缘迁移距离百分比低、侵袭细胞数少（P均<0.01）；与四组相比，二组细胞增殖活性及划痕前缘迁移距离百分比高、侵袭细胞数多（P均<0.01）；与一组相比，三组细胞增殖活性及划痕前缘迁移距离百分比高、侵袭细胞数多（P均<0.01）；与二组相比，三组细胞增殖活性及划痕前缘迁移距离百分比低、侵袭细胞数少（P均<0.01）。与五组相比，一组细胞miR-216a相对表达量高（P<0.01）。与四组相比，培养24 h时二组细胞CAPN6蛋白、p-STAT3/STAT3相对表达量高，三组细胞表达CAPN6蛋白、p-STAT3/STAT3相对表达量低；与五组相比，一组细胞CAPN6蛋白、p-STAT3/STAT3相对表达量低；与一组相比，三组细胞CAPN6蛋白、p-STAT3/STAT3相对表达量高。与B组相比，A组细胞荧光素酶活性低（P<0.05）。结论 miR-216a过表达可抑制HeLa细胞的增殖侵袭和迁移。HeLa细胞中miR-216a与CAPN6基因存在靶向关系。miR-216a可能通过调控CAPN6基因表达，抑制HeLa的增殖、迁移及侵袭。更多还原

【Abstract】 Objective To observe the changes in proliferation, migration, and invasion of human cervical cancer cell line HeLa with overexpression of microRNA-216a(miR-216a)/Calpain 6(CAPN6) gene and their targeting relationships, and to explore the effects of miR-216a on the proliferation, migration, and invasion of HeLa cells. Methods HeLa cells in the logarithmic growth phase were divided into five groups: groups Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ, with 6 well plates in each group. Cells in the group Ⅰ were transfected with miR-216a mimic, cells in the group Ⅱ were transfected with pcDNA3.1-CAPN6 plasmid, cells in the group Ⅲ were sequentially transfected with miR-216a mimic and pcDNA3.1-CAPN6, cells in the group Ⅳ were transfected with pcDNA3.1, and cells in the group Ⅴ were transfected with miR-216a mimic NC. At 48 h of cultivation, the proliferation, migration, and invasion of cells in each group were observed by CCK-8 method, Scratch healing test and Transwell test. At 24 h of cultivation, miR-216a in each group was detected by qRT-PCR, and CAPN6 and signal transducer and activator of transcription 3(STAT3) proteins in each group were detected by Western blotting. In addition, HeLa cells in the logarithmic growth phase were divided into four groups: cells in the group A were sequentially transfected with miR-216a mimic and pGL3-CAPN6-WT plasmids, cells in the group B were sequentially transfected with miR-216a mimic and pGL3-CAPN6-MUT plasmids, cells in the group C were sequentially transfected with miR-216a mimic NC and pGL3-CAPN6-WT, and cells in the group D were sequentially transfected with miR-216a mimic NC and pGL3-CAPN6-MUT plasmids. At 36 h of cultivation, cells from each group were collected and the relative luciferase activity was calculated by the dual-luciferase reporter gene assay kit. Results Compared with the group Ⅴ, group Ⅰ had lower cell proliferation activity and percentage of scratch front migration distance, and fewer invasive cells at 48 h of cultivation(all P<0. 01). Compared with the group Ⅳ, group Ⅱ had higher cell proliferation activity and percentage of scratch front migration distance, and more invasive cells(all P<0. 01). Compared with the group Ⅰ, group Ⅲ had higher cell proliferation activity and percentage of scratch front migration distance, and more invasive cells(all P<0. 01). Compared with the group Ⅱ, group Ⅲ had lower cell proliferation activity and percentage of scratch front migration distance, and fewer invasive cells(all P<0. 01). Compared with group Ⅴ, the relative expression level of miR-216a of cells in the group Ⅰ was much higher(P<0. 01). Compared with the group Ⅳ, the relative expression levels of CAPN6 protein and p-STAT3/STAT3 in the group Ⅱ were much higher at 24 h of cultivation, while the relative expression levels of CAPN6 protein and p-STAT3/STAT3 were lower in the group Ⅲ. Compared with the group Ⅴ, the relative expression levels of CAPN6 protein and pSTAT3/STAT3 in the group Ⅰ were lower. Compared with group Ⅰ, the relative expression levels of CAPN6 protein and pSTAT3/STAT3 in the group Ⅲ were higher. Compared with the group B, group A had lower cell luciferase activity(P<0. 05). Conclusions Over-expression of miR-216a may inhibit the proliferation, invasion, and migration of Hela cells.There is a targeted relationship between miR-216a and CAPN6 gene in HeLa cells. MiR-216a may inhibit the proliferation, migration, and invasion of HeLa by regulating the expression of CAPN6 gene.更多还原