猫白血病C亚类病毒受体1的反义RNA1靶向抑制微小RNA-515-5p表达对鼻咽癌细胞增殖、迁移和侵袭的影响

Feline leukemia virus subgroup C virus receptor 1 antisense RNA 1 promotes the proliferation,migration and invasion of nasopharyngeal carcinoma cells by targeting to inhibit the expression of microRNA-515-5p

目的探讨猫白血病C亚类病毒受体1的反义RNA1(feline leukemia virus subgroup C virus receptor 1 antisense RNA 1,FLVCR1-AS1)对鼻咽癌细胞增殖、迁移和侵袭的影响及其机制。方法以人永生化鼻咽上皮细胞NP69(简称NP69细胞)为对照,实时荧光定量PCR(realtime fluorescent quantitative PCR,qRT-PCR)法检测鼻咽癌细胞系(5-8F、C666-1、6-10B)中FLVCR1-AS1和微小RNA(microRNA,miR)-515-5p表达。将C666-1细胞分为si-NC组、si-FLVCR1-AS1组、miR-NC组、miR-515-5p组、si-FLVCR1-AS1+anti-miR-NC组和si-FLVCR1-AS1+anti-miR-515-5p组,CCK-8法和克隆形成实验检测细胞增殖,划痕实验和Transwell检测分别检测细胞迁移和侵袭。双荧光素酶报告基因实验验证FLVCR1-AS1和miR-515-5p调控关系。结果鼻咽癌细胞系(5-8F、C666-1、6-10B)中FLVCR1-AS1表达高于NP69细胞(2.26±0.11、4.19±0.24、3.54±0.18 vs 1.00±0.00,P<0.05),而miR-515-5p表达低于NP69细胞(0.86±0.09、0.20±0.03、0.47±0.05 vs 1.00±0.00,P<0.05)。与si-NC组比较,si-FLVCR1-AS1组C666-1细胞光密度(optical density,OD)值(0.59±0.03 vs 1.34±0.08)、克隆数[(55.67±1.70)个vs(114.67±3.30)个]、迁移距离[(75.34±3.09)μm vs(186.29±9.29)μm]、侵袭数[(69.33±3.68)个vs(149.67±6.80)个]降低(P均<0.05)。与miR-NC组比较,miR-515-5p组C666-1细胞OD值(0.51±0.04 vs 1.36±0.07)、克隆数[(46.67±1.25)个vs(115.67±4.11)个]、迁移距离[(65.89±1.96)μm vs(186.49±8.33)μm]、侵袭数[(58.33±2.05)个vs(150.00±6.98)个]降低(P均<0.05)。FLVCR1-AS1可靶向负调控miR-515-5p。与si-FLVCR1-AS1+anti-miR-NC组比较,si-FLVCR1-AS1+anti-miR-515-5p组C666-1细胞OD值(1.16±0.06 vs 0.59±0.04)、克隆数[(106.00±3.56)个vs(55.33±2.49)个]、迁移距离[(164.06±7.40)μm vs(75.48±2.62)μm]、侵袭数[(134.67±5.31)个vs(69.00±2.94)个]升高(P均<0.05)。结论 FLVCR1-AS1可能靶向下调miR-515-5p促进鼻咽癌细胞增殖、迁移和侵袭。

OBJECTIVE To explore the effect of feline leukemia virus subgroup C virus receptor 1 antisense RNA 1(FLVCR1-AS1) on the proliferation,migration and invasion of nasopharyngeal carcinoma cells and its mechanism.METHODS Using human immortalized nasopharyngeal epithelial cells NP69 as a control,realtime fluorescent quantitative PCR(qRT-PCR) was used to detect the expression of FLVCR1-AS1 and microRNA(miR)-515-5p in nasopharyngeal carcinoma cell lines(5-8F,C666-1,6-10B).C666-1 cells were divide into si-FLVCR1-AS1 group,si-NC group,miR-515-5p group,miR-NC group,si-FLVCR1-AS1+anti-miR-515-5p group and si-FLVCR1-AS1+anti-miR-NC group.And then CCK-8 method and clone formation test were used to detect cell proliferation,the scratch test was used to detect cell migration,and Transwell was used to detect cell invasion.The dual luciferase reporter gene experiment verified the regulatory relationship between FLVCR1-AS1 and miR-515-5p.RESULTS The expressionof FLVCR1-AS1 in nasopharyngeal carcinoma cell lines(5-8F,C666-1,6-10B) were higher than those in NP69 cells(2.26±0.11,4.19±0.24,3.54±0.18 vs 1.00±0.00,P<0.05),but the expression of miR-515-5p were lower than those in NP69 cells(0.86±0.09,0.20±0.03,0.47±0.05 vs 1.00±0.00,P<0.05).Compared with si-NC group,the OD value(0.59±0.03 vs 1.34±0.08),the number of colony formation(55.67±1.70 vs 114.67±3.30),migration distance[(75.34±3.09)μm vs (186.29±9.29)μm],the number of invasion(69.33±3.68 vs 149.67±6.80) of C666-1 cells in si-FLVCR1-AS1 group were decreased(P<0.05).Compared with miR-NC group,the OD value(0.51±0.04 vs 1.36±0.07),the number of colony formation(46.67±1.25 vs 115.67±4.11),migration distance[(65.89±1.96)μm vs (186.49±8.33)μm],the number of invasion(58.33±2.05 vs 150.00±6.98) of C666-1cells in miR-515-5p group were decreased(P<0.05).FLVCR1-AS1 could target and negatively regulate miR-515-5p.Compared with si-FLVCR1-AS1+anti-miR-NC group,the OD value(1.16±0.06 vs 0.59±0.04),the number of colony formation(106.00±3.56 vs 55.33±2.49),migration distance[(164.06±7.40) μm vs (75.48±2.62)μm],the number of invasion(134.67±5.31 vs 69.00±2.94) of C666-1 cells in si-FLVCR1-AS1+anti-515-5p group were decreased(P<0.05).CONCLUSION FLVCR1-AS1 may inhibit the proliferation,migration and invasion of nasopharyngeal carcinoma cells by targeting miR-515-5p.