IRF6通过激活MMP-12转录促进肺腺癌转移的实验研究

Experimental study on the promotion of lung adenocarcinoma metastasis by IRF6 activation of MMP-12 transcription

目的探讨干扰素调节因子6(interferon regulatory factor 6,IRF6)对肺腺癌(lung adenocarcinoma,LUAD)细胞转移的影响及可能调控机制。方法从GEPIA和Ualcan在线数据库获取IRF6在LUAD组织和正常肺组织中的表达情况。采用实时荧光定量PCR(qPCR)检测IRF6和基质金属蛋白酶12(matrix metallopeptidase 12,MMP-12)在LUAD细胞和正常人支气管上皮细胞中的表达情况。分别采用sh-NC和sh-IRF6质粒转染LUAD细胞,Transwell小室实验检测两组细胞的迁移能力。采用染色质免疫共沉淀(ChIP)实验检测MMP-12启动子富集情况;采用荧光素酶报告基因实验验证IRF6和MMP-12的靶向关系。结果在线数据库分析结果显示,LUAD组织中IRF6表达显著高于正常组织。与正常细胞(BEAS-2B)比较,A549和Calu3细胞中IRF6表达显著升高(5.82±0.92,7.11±1.31,P<0.01)。与sh-NC组比较,sh-IRF6转染组A549和Calu3细胞的迁移数减少(52±9.23和63±11.38 vs.21±4.24和33±6.11),差异有统计学意义(P<0.01);sh-IRF6转染组A549和Calu3细胞的MMP-12表达降低为0.31±0.06和0.45±0.11,差异有统计学意义(P<0.01)。ChIP实验结果表明,相对于阴性对照Anti-IgG组,在Anti-IRF6组A549和Calu3细胞的MMP-12启动子富集度升高了(31.24±5.78)倍和(26.27±4.11)倍,差异有统计学意义(P<0.01)。荧光素酶报告基因实验显示,干扰IRF6后,A549和Calu3细胞的野生型MMP-12转录活性显著降低为0.28±0.04和0.38±0.06(P<0.01)。干扰IRF6的A549和Calu3细胞中过表达MMP-12使细胞迁移数由25±4.21和36±7.28增加为48±8.89和60±11.17(P<0.01)。结论IRF6在LUAD细胞中高表达,敲低IRF6表达能够显著抑制LUAD细胞转移。

Objective To investigate the effect of interferon regulatory factor 6(IRF6)on lung adenocarcinoma(LUAD)cell metastasis and its possible mechanism.Methods The expression of IRF6 in LUAD tissues and normal lung tissues was obtained from GEPIA and Ualcan online databases.Real-time quantitative fluorescent PCR(qPCR)was used to detect the expression of IRF6 and matrix metallopeptidase 12(MMP-12)in LUAD cells and normal bronchial epithelial cells.LUAD cells were transfected with sh-NC and sh-IRF6 respectively,and the migration ability of the two groups of cells was detected by Transwell assay.MMP-12 promoter enrichment was detected by chromatin immunoprecipitation(ChIP)assay.The targeting relationship between IRF6 and MMP-12 was verified by luciferase reporter gene assay.Results The result of online database analysis showed that the expression of IRF6 in LUAD tissues was significantly higher than that in normal tissues.Compared with normal cells(BEAS-2B),the expression of IRF6 in A549 and Calu3 cells was significantly increased(5.82±0.92,7.11±1.31,P<0.01).Compared with sh-NC group,the migration numbers of A549 and Calu3 cells in sh-IRF6 transfection group were decreased(52±9.23 and 63±11.38 vs.21±4.24 and 33±6.11),and the difference was statistically significant(P<0.01).The expression of MMP-12 in A549 and Calu3 cells decreased to 0.31±0.06 and 0.45±0.11 in sh-IRF6 transfection group,and the difference was statistically significant(P<0.01).The ChIP test result showed that compared with the negative Anti-IgG group,the MMP-12 promoter enrichment of A549 and Calu3 cells in Anti-IRF6 group was increased by(31.24±5.78)fold and(26.27±4.11)fold,and the difference was statistically significant(P<0.01).After interference with IRF6,the transcriptional activity of MMP-12 in A549 and Calu3 cells was significantly reduced to 0.28±0.04 and 0.38±0.06(P<0.01).The overexpression of MMP-12 in IRF6-interfering A549 and Calu3 cells increased the cell migration numbers from 25±4.21 and 36±7.28 to 48±8.89 and 60±11.17(P<0.01).Conclusion IRF6 is highly expressed in LUAD cells,and knocking down IRF6 expression can significantly inhibit LUAD cell metastasis.