九香虫水提液通过miR-1287-5p/泛素特异性肽酶22通路调控非小细胞肺癌增殖、侵袭和迁移的分子机制

Molecular Mechanism of Jiuxiang Aqueous Extract Regulating Pproliferation,Invasion and Migration of Non-Small Cell Lung Cancer through miR-1287-5p/Ubiquitin Specific Peptidase 22 Pathway

将九香虫水提液用于非小细胞肺癌的治疗,探讨九香虫水提液对非小细胞肺癌细胞增殖、迁移和侵袭的调控机制。用九香虫水提取液(44、66、99 mg/L)处理A549细胞,筛选最适浓度99 mg/L。用脂质体法将miR-NC组(转染miR-NC)、miR-1287-5p组(转染miR-1287-5p mimics)、anti-miR-NC组(转染anti-miR-NC)、antimiR-1287-5p组(转染anti-miR-1287-5p)、Jiuxiang aqueous extract+anti-miR-NC组(转染anti-miR-NC用99 mg/L九香虫水提取液处理)、Jiuxiang aqueous extract+anti-miR-1287-5p组(转染anti-miR-1287-5p用99 mg/L九香虫水提取液处理)转染至A549细胞;噻唑蓝(MTT)法、迁移实验(Transwell)小室、免疫印迹(Western blot)实验、实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测细胞的抑制率、迁移侵袭、细胞周期蛋白依赖激酶(Cyclin D1)、细胞周期依赖性蛋白激酶抑制因子1A(p21)、基质金属蛋白酶(MMP) 2、MMP-9、干扰泛素特异肽酶(USP22)、miR-1287-5p的表达;双荧光素酶报告基因实验检测细胞的荧光活性。九香虫水提取液呈浓度依赖性显著提高A549细胞的抑制率,抑制细胞的迁移和侵袭,下调Cyclin D1、MMP-2、MMP-9,上调p21(P<0.05)。九香虫水提取液呈浓度依赖性显著上调miR-1287-5p的表达,下调USP22表达,并且过表达miR-1287-5p能够抑制肺癌细胞的增殖、迁移和侵袭,下调Cyclin D1、MMP-2、MMP-9,上调p21,而抑制miR-1287-5p可逆转九香虫水提取液对肺癌细胞增殖、迁移和侵袭的抑制作用,并上调Cyclin D1、MMP-2、MMP-9,下调p21。此外,miR-1287-5p明显的抑制野生型USP22的A549细胞荧光活性。九香虫水提取液可抑制非小细胞肺癌细胞的增殖、迁移和侵袭,其机制与上调miR-1287-5p进而靶向抑制USP22相关,可为九香虫水提取液治疗非小细胞肺癌提供支持。

To investigate the regulation mechanism of Jiuxiang aqueous extract on proliferation,migration and invasion of non-small cell lung cancer cells,A549 cells were treated with Jiuxiang aqueous extract(44,66,99 mg/L) and the optimum concentration was99 mg/L.The miR-NC group(transfected miR-NC),miR-1287-5 p group(transfected miR-1287-5 p mimics),anti-miR-NC group(transfected anti-miR-NC),anti-miR-1287-5 p group(transfected anti-miR-1287-5 p),Jiuxiang aqueous extract + anti-miR-NC group(transfected anti-miR-NC with 99 mg/L Jiuxiang aqueous extract treatment),Jiuxiang aqueous extract + anti-miR-1287-5 p group(transfected anti-miR-1287-5 p treated with 99 mg/L Jiuxiang aqueous extract) was transfected into A549 cells by liposome method.Methyl thiazolyl tetrazolium(MTT) assay,Transwell chamber,Western blotting(Western blot) assay,Real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR) assay were used to detect cell inhibition,migration and invasion,expression of Cyclin D1,Cell cycle-dependent protein kinase inhibitor 1 A(p21),matrix metalloproteinase(MMP)-2,MMP-9,ubiquitin specific peptidase(USP22),miR-1287-5 p.Dual luciferase assay genetic experiments were used to detect the fluorescence activity of cells.The Jiuxiang aqueous extract significantly increased the inhibition rate of A549 cells,inhibited the migration and invasion of cells,down-regulated Cyclin D1,MMP-2 and MMP-9,and up-regulated p21(P<0.05).The Jiuxiang aqueous extract significantly up-regulated the expression of miR-1287-5 p in a concentration-dependent manner,down-regulated the expression of USP22,and overexpression of miR-1287-5 p inhibited the proliferation,migration and invasion,down-regulated Cyclin D1,MMP-2 and MMP-9,and up-regulated p21 of lung cancer cells,while inhibiting miR-1287-5 pit can reverse the inhibitory effect of Jiuxiang aqueous extract on the proliferation,migration and invasion,up-regulated Cyclin D1,MMP-2 and MMP-9,and down-regulated p21 of lung cancer cells.In addition,miR-1287-5 p significantly inhibited the fluorescence activity of wild-type USP22 A549 cells.Jiuxiang aqueous extract can inhibit the proliferation,migration and invasion of non-small cell lung cancer cells.The mechanism is related to the up-regulation of miR-1287-5 p and targeted inhibition of USP22,which will provide support for the treatment of non-small cell lung cancer with Jiuxiang aqueous extract.