肝癌中星形细胞上调基因1作用于microRNA-885-5p/基质金属蛋白酶9信号通路促进肝癌转移的机制研究

摘要

Objective To investigate the relationship between astrocyte elevated gene-l (AEG-1) and microRNA-885-5p,observe the influence of microRNA-885-5p on MHCC-97H migration and invasion,identify the target gene of microRNA-885-5p and uncover the mechanisms of AEG-1 promoting cancer metastasis.Methods The experimental study was adopted.The AEG-1 over-expressed plasmid constructed and the siRNA of silent AEG-1 synthesized were transiently transfected into MHCC-97H cells,respectively.For plasmid transfection,MHCC-97H cells tranfected with empty Vector NC were divided into group A as the control,and MHCC-97H cells tranfected with AEG-1 over-expressed plasmid were divided into group B.For AEG-1 siRNA transfection,MHCC-97H cells tranfected with negative siRNA were divided into group C as the control,and MHCC-97H cells tranfected with AEG-1 siRNA were divided into group D.The relative expressions of microRNA885-5p in MHCC-97H cells of group A,B,C,D were measured by fluorescent quantitative polymerase chain reaction(PCR).For microRNA-885-5p simulator transfection,MHCC-97H cells tranfected with negative microRNA simulator were divided into group E as the control,and tranfected with microRNA-885-5p simulator were divided into group F.Transwell assay was used to evaluate MHCC-97H migration and invasion.Targetscan software was used to predict the target gene of microRNA-885-5p.MHCC-97H cells co-transfected with reporter plasmid of target gene and negative control siRNA were divided into group G as the control,and MHCC-97H cells co-transfected with reporter plasmid of target gene and microRNA-885-5p simulator were divided into group H.MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and negative control siRNA were divided into group Ⅰ as the control,MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and microRNA-885-5p simulator were divided into group J.The luciferase activities of MHCC-97H cells in group G,H,I,J were tested.MHCC-97H cells transfected with negative control siRNA were divided into group K,and tranfectcd with microRNA-885-5p simulator were divided into group L.Western blot test was used to detect the relative expression of target gene of microRNA-885-5p.MHCC-97H cells transfected with siRNA of target gene of microRNA-885-5p were divided into group M,and transfected with negative control siRNA were divided into group N.Transwell assay was used to evaluate MHCC-97H migration and invasion in group M and group N.Measurement data with normal distribution were presented as x ± s and comparison between groups was done using the t test.Results The relative expressions of microRNA-885-5p in MHCC-97H cells of group A and group B were (10.68 ± 1.32) × 10-4 and (5.02 ± 0.20) × 10-4,respectively,showing significant difference between the 2 groups (t =7.357,P ＜ 0.05).The relative expressions of microRNA-885-5p in MHCC-97H cells of group C and group D were (11.04 ± 0.97) × 10-4 and (24.15 ± 3.71) × 10-4,respectively,showing significant difference between the 2 groups (t =5.920,P ＜ 0.05).The results of MHCC-97H migration showed that the numbers of MHCC-97H cells in lower chambers of transwell plate of group E and group F were 1 452 ± 212 and 778 ± 95,showing significant difference between the 2 groups (t =5.018,P ＜ 0.05).The results of MHCC-97H invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate of group E and group F were 1 237 ± 238 and 470 ± 70,showing significant difference between the 2 groups (t =5.353,P ＜ 0.05).MMP9 was predicted as a candidate for target gene.The luciferase activity of MHCC-97H cells was 0.73 ± 0.03 in group G and 0.46 ± 0.04 in group H,showing significant difference between the 2 groups (t =8.623,P ＜ 0.05).The reporter plasmid of binding region mutation mutMMP9 was detected.The luciferase activity of MHCC-97H cells was 0.69 ± 0.03 in group Ⅰ and 0.64 ± 0.08 in group J,showing no significant difference between the 2 groups (t =0.934,P ＞ 0.05).The relative expressions of MMP9 in MHCC-97H cells of group K and group L were 0.75 ± 0.03 and 0.25 ± 0.03,respectively,showing significant difference between the 2 groups (t =19.086,P ＜ 0.05).The results of MHCC-97H migration and invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate were 1 210 ± 163 and 1 192 ± 170 in group M,537 ± 16 and 374 ± 55 in group N,showing significant differences between the 2 groups (t =7.111,7.916,P ＜ 0.05).Conclusions AEG-1 downregulates the expression of microRNA-885-5p,the target gene of which is MMP9.MicroRNA-885-5p inhabits the migration and invasion of hepatoma cells by negative regulation of MMP9.%目的 探讨星形细胞上调基因-1(AEG-1)与microRNA-885-5p的关系,观察microRNA-885-5p对肝癌细胞MHCC-97H迁移和侵袭能力的影响,鉴定microRNA-885-5p的靶基因,揭示AEG-1促进肝癌转移的机制.方法 采用实验研究方法.构建AEG-1过表达质粒,合成沉默AEG-1的siRNA,分别瞬时转染进MHCC-97H细胞.对于转染质粒,以转染空载体Vector NC的MHCC-97H细胞作对照,设为A组;转染AEG-1过表达质粒的MHCC-97H细胞设为B组.对于转染AEG-1 siRNA,以转染阴性对照siRNA的MHCC-97H细胞作对照,设为C组;转染AEG-1 siRNA的MHCC-97H细胞设为D组.采用荧光定量PCR分别检测A、B、C、D组MHCC-97H细胞中microRNA-885-5p相对表达量.对于转染microRNA-885-5p模拟体,以转染阴性对照microRNA模拟体的MHCC-97H细胞作对照,设为E组;转染microRNA-885-5p模拟体的MHCC-97H细胞设为F组.采用Transwell小室法检测E、F组MHCC-97H细胞迁移和侵袭能力.采用Targetscan软件,预测microRNA-885-5p的靶基因.将靶基因报告质粒和阴性对照siRNA共转染进MHCC-97H细胞作对照,设为G组;将靶基因报告质粒和microRNA-885-5p模拟体共转染进MHCC-97H细胞,设为H组.将结合位点突变的靶基因报告质粒和阴性对照siRNA共转染进MHCC-97H细胞作对照,设为Ⅰ组;将结合位点突变的靶基因报告质粒和microRNA-885-5p模拟体共转染进MHCC-97H细胞,设为J组.检测G、H、I、J组细胞荧光素酶活性.在MHCC-97H细胞中转染阴性对照siRNA,设为K组;转染microRNA-885-5p模拟体,设为L组,采用Western blot检测各组中microRNA-885-5p靶基因蛋白相对表达量.将microRNA-885-5p靶基因siRNA转染进MHCC-97H细胞,设为M组;将阴性对照siRNA转染进MHCC-97H细胞,设为N组.采用Transwell小室法检测M、N组MHCC-97H细胞迁移和侵袭能力.符合正态分布的计量资料以x±s表示,两组间比较采用t检验.结果 A组MHCC-97H细胞中microRNA-885-5p的相对表达量为(10.68±1.32)×10-4,B组为(5.02±0.20)×10-4,两组比较,差异有统计学意义(t=7.357,P＜0.05).C组MHCC-97H细胞中microRNA-885-5p的相对表达量为(11.04±0.97) ×10-4,D组为(24.15 ±3.71) ×10-4,两组比较,差异有统计学意义(t =5.920,P ＜0.05).MHCC-97H细胞迁移实验结果:E组Transwell小室下层MHCC-97H细胞数目为(1 452±212)个,F组为(778±95)个,两组比较,差异有统计学意义(t=5.018,P＜0.05).MHCC-97H细胞侵袭实验结果:E组Transwell小室下层MHCC-97H细胞数目为(1 237±238)个,F组为(470 ±70)个,两组比较,差异有统计学意义(=5.353,P＜0.05).预测到一条靶基因候选者为MMP9.G组MHCC-97H细胞荧光素酶活性为0.73±0.03,H组为0.46±0.04,两组比较,差异有统计学意义(t=8.623,P＜0.05).检测到结合位点突变报告质粒mutMMP9.Ⅰ组MHCC-97H细胞荧光素酶活性为0.69±0.03,J组为0.64±0.08,两组比较,差异无统计学意义(t=0.934,P＞0.05).K组MHCC-97H细胞中MMP9蛋白相对表达量为0.75±0.03,L组为0.25±0.03,两组比较,差异有统计学意义(t=19.086,P＜0.05).MHCC-97H细胞迁移实验结果:M组Transwell小室下层MHCC-97H细胞数目为(1 210±163)个,N组为(537±16)个,两组比较,差异有统计学意义(t=7.111,P＜0.05).MHCC-97H细胞侵袭实验结果:M组Transwell小室下层MHCC-97H细胞数目为(1 192±170)个,N组为(374-±55)个,两组比较,差异有统计学意义(t=7.916,P＜0.05).结论 AEG-1负调控microRNA-885-5p的表达,microRNA-885-5p的靶基因为MMP9,micro-RNA-885-5p通过负调控MMP9,起到抑制肝癌细胞迁移和侵袭的作用.