miR-509通过调控二磷酸腺苷-核糖基化因子6对人舌鳞状细胞癌SCC15细胞生物学行为的影响

Effect of miR-509 on the biological behaviors of SCC15 cells by regulating ARF6

目的探讨miR-509对人舌鳞状细胞癌SCC15细胞生物学行为的影响及对二磷酸腺苷-核糖基化因子6(adenosine diphosphate-ribosylation factor 6, ARF6)信号通路的调控作用。方法取对数生长期SCC15细胞,随机分为miR-509 mimic组(转染miR-509 mimic)、miR-509 inhibitor组(转染miR-509 inhibitor)、mimic NC组(转染mimic NC)、inhibitor NC组(转染inhibitor NC)、对照组(仅转染脂质体)。转染24 h, 5组采用实时荧光定量PCR法检测miR-509相对表达量,采用流式细胞术检测细胞凋亡,采用Transwell小室试验检测细胞侵袭能力,采用Western blot法检测ARF6蛋白相对表达量。采用双荧光素酶报告基因实验验证miR-509与ARF6的靶向关系。结果 miR-509 mimic组miR-509相对表达量(6.17±0.25)、细胞凋亡率[(12.46±0.37)%]均高于对照组[4.28±0.36、(3.08±0.30)%]和mimic NC组[4.25±0.34、(2.98±0.41)%](P<0.05),侵袭细胞数[(8.82±2.07)个]少于对照组[(27.40±3.26)个]和mimic NC组[(28.36±3.14)个](P<0.05),ARF6蛋白相对表达量(0.27±0.05)低于对照组(0.62±0.05)和mimic NC组(0.64±0.04)(P<0.05);miR-509 inhibitor组miR-509相对表达量(2.50±0.28)、细胞凋亡率[(2.16±0.45)%]均低于对照组和inhibitor NC组[4.26±0.33、(3.06±0.24)%](P<0.05),侵袭细胞数[(125.26±3.36)个]多于对照组和inhibitor NC组[(28.93±2.85)个](P<0.05),ARF6蛋白相对表达量(0.86±0.06)高于对照组和inhibitor NC组(0.68±0.06)(P<0.05);miR-509 inhibitor组miR-509相对表达量、细胞凋亡率均低于miR-509 mimic组(P<0.05),侵袭细胞数多于miR-509 mimic组(P<0.05),ARF6蛋白相对表达量高于miR-509 mimic组(P<0.05);对照组、mimic NC组和inhibitor NC组miR-509相对表达量、细胞凋亡率、侵袭细胞数、ARF6蛋白相对表达量比较差异均无统计学意义(P>0.05)。ARF6 WT+miR-509 mimic组荧光素酶活性(0.52±0.17)低于ARF6 WT+mimic NC组(1.55±0.22)(t=6.417,P<0.001);ARF6 MUT+miR-509 mimic组荧光素酶活性(1.53±0.26)与ARF6 MUT+mimic NC组(1.56±0.25)比较差异无统计学意义(t=0.144,P=0.892)。结论 miR-509上调可促进人舌鳞状细胞癌SCC15细胞凋亡、抑制其侵袭,可能通过靶向抑制ARF6蛋白表达发挥作用。

Objective To investigate the effect of miR-509 on the biological behaviors of human tongue squamous cell carcinoma SCC15 cells and its role in regulating adenosine diphosphate-ribosylation factor 6(ARF6) signaling pathway. Methods The SCC15 cells in logarithmic growth phase were randomly divided into miR-509 inhibitor group(transfected with miR-509 inhibitor), miR-509 mimic group(transfected with miR-509 mimic), mimic NC group(transfected with mimic NC), inhibitor NC group(transfected with inhibitor NC), and control group(transfected with liposome). After transfection for 24 h, the relative expression of miR-509 was detected by real-time fluorescence quantitative PCR, the cell apoptosis rate was detected by flow cytometry, the cell invasiveness was detected by Transwell assay, the relative expression of ARF6 protein was detected by Western blot, and the targeting relationship between miR-509 and ARF6 was detected by double luciferase reporter gene assay. Results The relative expression of miR-509 and apoptosis rate were higher in miR-509 mimic group [6.17±0.25,(12.46±0.37)%] than those in control group [4.28±0.36,(3.08±0.30)%] and mimic NC group [4.25±0.34,(2.98±0.41)%](P<0.05), while the invasive cell number was less and the relative expression of ARF6 protein was lower in miR-509 mimic group(8.82±2.07,0.27±0.05)than that in control group(27.40±3.26,0.62±0.05)and mimic NC group(28.36±3.14,0.64±0.04)(P<0.05).The relative expression of miR-509 and cell apoptosis rate were lower in miR-509 inhibitor group[2.50±0.28,(2.16±0.45)%]than those in inhibitor NC group[4.26±0.33,(3.06±0.24)%]and control group(P<0.05),the invasive cell number was more in miR-509 inhibitor group(125.26±3.36)than that in inhibitor NC group(28.93±2.85)and control group(P<0.05),and the relative expression of ARF6 protein was higher in miR-509 inhibitor group(0.86±0.06)than that in inhibitor NC group(0.68±0.06)and control group(P<0.05).The relative expression of miR-509 and cell apoptosis rate were lower in miR-509 inhibitor group that those in miR-509 mimic group(P<0.05),the invasive cell number was more than that in miR-509 mimic group(P<0.05),and the relative expression of ARF6 protein was higher than that in miR-509 mimic group(P<0.05).There were no significant differences in the relative expression of miR-509,cell apoptosis rate,invasive cell number and relative expression of ARF6 protein among control group,mimic NC group and inhibitor NC group(P>0.05).The luciferase activity was lower in ARF6 WT+miR-509 mimic group(0.52±0.17)than that in ARF6 WT+mimic NC group(1.55±0.22)(t=6.417,P<0.001),and showed no significant difference between ARF6 MUT+ miR-509 mimic group(1.53±0.26)and ARF6 MUT+ mimic NC group(1.56±0.25)(t=0.144,P=0.892).Conclusion The up-regulation of miR-509 promotes the apoptosis and decrease the invasion of SCC15,possibly by targetedly inhibiting the expression of ARF6 protein.