摘要
目的探讨微小RNA-224(miR-224)对口腔鳞状细胞癌(OSCC)细胞侵袭迁移和KLLN表达的影响。方法采用实时定量PCR(QPCR)检测正常口腔上皮细胞HOK和OSCC细胞(CAL-27、TSCCa和Tca8113)的miR-224水平,脂质体法向CAL-27细胞转染miR-224模拟物(Mimics组)或阴性对照序列(NC组),另以仅脂质体处理的细胞为对照组,活细胞计数(CCK-8)法、Transwell小室实验和划痕实验评估细胞增殖、侵袭和迁移能力,Western blotting检测KLLN、基质金属蛋白酶(MMP)-2和MMP-9的水平,双荧光素酶报告基因实验验证miR-224对KLLN的靶向调控作用。结果QPCR结果显示,OSCC细胞的miR-224水平较HOK细胞下调(P<0.05),其中CAL-27细胞的miR-224水平最低。Mimics组的miR-224水平为12.325±1.250,高于对照组的1.003±0.058和NC组的1.029±0.072(P<0.05);Mimics组转染24、48 h后的增殖活力低于对照组和NC组(P<0.05);Mimics组的划痕愈合率和穿膜细胞数分别为(23.161±3.609)%和(187.977±19.644)个,低于对照组的(47.372±4.717)%和(352.429±28.372)个及NC组的(45.094±5.651)%和(187.977±19.644)个,差异有统计学意义(P<0.05);Mimics组的KLLN、MMP-2和MMP-9水平低于对照组和NC组(P<0.05)。对照组和NC组上述指标的差异无统计学意义(P>0.05)。MiR-224模拟物对载有突变型KLLN细胞的荧光素酶活性不产生影响,而抑制了野生型KLLN细胞的荧光素酶活性(P<0.05)。结论OSCC细胞的miR-224水平降低且上调miR-224可能通过靶向KLLN来抑制OSCC细胞的迁移侵袭,该miRNA有望成为OSCC的预后生物标志物和治疗靶点。
Objective To investigate the effect of microRNA-224(miR-224)on the invasion,migration and KLLN expression of oral squamous cell carcinoma(OSCC)cells.Methods Real-time quantitative PCR(QPCR)was used to detect the miR-224 level in OSCC cells(CAL-27,TSCCa and Tca8113)and normal oral epithelial cell HOK.CAL-27 cells were transfected with miR-224 mimics(Mimics group)or negative control sequence(NC group)by liposome method,and cells treated only by liposome were used as Control group.The abilities of cell proliferation,invasion and migration were evaluated by Cell Counting Kit-8(CCK-8),Transwell chamber test and scratch test.Western blotting was used to detect the levels of KLLN,matrix metalloproteinase(MMP)-2 and MMP-9,and double luciferase reporter gene experiment was used to verify the targeted regulation of miR-224 on KLLN.Results QPCR showed that the miR-224 levels in OSCC cells were significantly lower than those in HOK cells(P<0.05)with the lowest level in CAL-27 cells.The miR-224 level of Mimics group was 12.325±1.250,higher than 1.003±0.058 of Control group and 1.029±0.072 of NC group(P<0.05).The proliferation activity of Mimics group after 24 and 48 h-transfection was lower than that in Control group and NC group(P<0.05).The scratch healing rate and number of transmembrane cells in Mimics group were(23.161±3.609)%and 187.977±19.644,lower than(47.372±4.717)%and 352.429±28.372 in Control group and(45.094±5.651)%and 187.977±19.644 in NC group(P<0.05).Levels of KLLN,MMP-2 and MMP-9 in Mimics group were lower than those in Control group and NC group(P<0.05).There was no statistically significant difference in terms of above indices between Control group and NC group(P>0.05).MiR-224 mimics had no effect on the luciferase activity of cells harboring mutated KLLN,but significantly inhibited the luciferase activity of cells harboring wild KLLN cells.Conclusion MiR-224 level in OSCC cells decreased and up-regulated its level can inhibit the migration and invasion of OSCC cells possibly by targeting KLLN,suggesting that miR-224 can be used as a prognosis biomarker and treatment target of OSCC.
作者
李启期
吕锡旌
周政
查小雨
李琦
LI Qiqi;LV Xijing;ZHOU Zheng;ZHA Xiaoyu;LI Qi(Department of Stomatology,the First Affiliated Hospital of Shihezi University School of Medicine,Shihezi 832008,China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2020年第2期127-132,共6页
Chinese Clinical Oncology
