摘要
目的探讨微小RNA-488(miR-488)对高迁移率族核小体结合域5(HMGN5)的靶向调控作用及非小细胞肺癌(NSCLC)细胞A549侵袭迁移的影响。方法采用实时定量PCR(QPCR)检测正常肺上皮细胞系(BEAS-2B)和A549细胞的miR-488水平。脂质体法向A549细胞转染miR-488模拟物(过表达组)或阴性对照序列(NC组)并设未转染的细胞为对照组。MTT比色法、Transwell实验和划痕愈合实验体外测定A549细胞的增殖、侵袭和迁移能力的变化,QPCR和Western blotting检测HMGN5、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-488对HMGN5的靶向调控作用。结果A549细胞的miR-488水平为0.256±0.028,低于BEAS-2B细胞的1.019±0.124(P<0.05);过表达组的miR-488水平为11.229±2.164,高于对照组的1.051±0.162和NC组的1.029±0.116(P<0.05)。与NC组和对照组相比,过表达组A549细胞转染48、72 h的增殖能力下降(P<0.05);过表达组的划痕愈合率和穿膜细胞数目分别为(25.53±3.75)%和(79.66±9.23)个,低于NC组的(58.56±4.76)%和(172.34±14.67)个及对照组的(54.34±5.65)%和(165.22±11.53)个(P<0.05);miR-488模拟物能降低HMGN5基因3’端非翻译区野生型的荧光素酶活性(P<0.05),但对3’端非翻译区突变型无影响(P>0.05)。与其余两组相比,过表达组的HMGN5、MMP-9和MMP-2水平降低(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)。结论miR-488在NSCLC细胞中低表达,上调该miRNA水平可抑制增殖和迁移侵袭能力并发挥类似抑癌基因的作用,可能与靶向调控HMGN5及抑制MMP-2/MMP-9通路有关,在NSCLC靶向中有一定参考价值。
Objective To investigate the effect of microRNA-488(miR-488)on the targeting regulation of high-mobility group nucleosome binding domain 5(HMGN5)and the invasion and migration of non-small cell lung cancer(NSCLC)A549 cells.Methods Real-time quantitative PCR(QPCR)was used to detect the miR-488 level in normal pulmonary epithelial cell line(BEAS-2 B)and A549 cells.A549 cells were transfected with miR-488 mimics(Overexpression group)or negative control sequence(NC group),and the untransfected cells were used as Control group.The proliferation,invasion and migration ability of A549 cells were measured by MTT,Transwell and scratch healing.Levels of HMGN5,matrix metalloproteinase(MMP)-2 and MMP-9 were detected by QPCR and Western blotting.The double luciferase reporter gene experiment was applied to verify the targeted regulation of miR-488 on HMGN5.Results The miR-488 level of A549 cells was 0.256±0.028,lower than 1.019±0.124 of BEAS-2 B cells(P<0.05).The miR-488 level in Overexpression group was 11.229±2.164,higher than 1.051±0.162 in Control group and 1.029±0.116 in NC group(P<0.05).Compared with the Control group and NC group,the cell proliferation ability of the Overexpression group decreased after 48 and 72 h-transfection(P<0.05).The scratch healing rate and number of membrane piercing cells in the Overexpression group were(25.53±3.75)%and 79.66±9.23,lower than(54.34±5.65)%and 165.22±11.53 in Control group and(58.56±4.76)%and 172.34±14.67 of NC group(P<0.05).MiR-488 could reduce the luciferase activity of the wild type of 3’untranslated region of HMGN5 gene(P<0.05),but had no effect on the mutant type of 3’untranslated region(P>0.05).Compared with other two groups,the levels of HMGN5,MMP-2 and MMP-9 in Overexpression group decreased(P<0.05).No significant difference was observed on the above indices between Control group and NC group(P>0.05).Conclusion MiR-488 is lowly expressed in NSCLC cells.Up-regulation of miR-488 can inhibit proliferation,migration and invasion,and play a role similar to tumor suppressor gene.It may be related to targeted regulation of HMGN5 and inhibition of MMP-2/MMP-9 pathway,which has certain reference value in NSCLC targeting therapy.
作者
孙伟
丁静
SUN Wei;DING Jing(Department of Respiratory,Huaibei People's Hospital,Huaibei 235000,China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2019年第11期973-977,共5页
Chinese Clinical Oncology