ING4通过逆转上皮间充质转化（EMT）抑制乳腺癌细胞的迁移和侵袭

发布时间：2018-08-22 10:37

【摘要】：目的:探讨慢病毒介导的生长抑制因子ING4过表达对乳腺癌细胞迁移、侵袭的作用及其机制。方法:(1)以携带人源化ING4编码序列(CDS)的pAdTrack-CMV/ING4质粒为模板,利用ING4特异性引物(ING4-F:5'-gaa gct agc gcc acc atg gct gct ggg atg tat ttg-3';ING4-R:5'-ata ggc gcg ccc tat ttc ttc ttc cgt tct tg-3')通过PCR扩增获得ING4 CDS片段。(2)在GFP标记的p Lenti6.3/IRES/GFP慢病毒质粒的NheI和Sgs I限制性酶切位点之间插入ING4目的片段构建ING4重组慢病毒质粒pLenti6.3/ING4/IRES/GFP,并通过PCR、双酶切、DNA测序鉴定。(3)用脂质体Lipofectamine2000将构建正确的pLenti6.3/ING4/IRES/GFP及其对照pLenti6.3/IRES/GFP慢病毒质粒分别与辅助包装质粒pLP1、p LP2、VSVG共转染293T人胚肾细胞包装GFP标记的ING4重组慢病毒(LV-ING4)及其对照空病毒(LV),并将慢病毒培养上清高速离心浓缩后,用有限稀释法根据GFP的表达进行滴度测定。(4)LV-ING4、LV分别用10MOI的最佳感染剂量感染MDA-MB-468三阴性乳腺癌细胞,通过10μg/ml的灭瘟素(BSD)筛选后获得MDA-MB-468-LV-ING4(简写为MDA-MB-468-ING4)、MDA-MB-468-LV(简写为MDA-MB-468-Mock)。荧光显微镜、流式细胞仪检测GFP分析转基因效率,及RT-PCR、Western blot检测慢病毒介导ING4在MDA-MB-468细胞中的表达。(5)采用Transwell迁移、侵袭实验检测MDA-MB-468-ING4、MDA-MB-468-Mock乳腺癌细胞的迁移、侵袭能力,分析慢病毒介导的ING4过表达对MDA-MB-468三阴性乳腺癌细胞迁移、侵袭的作用。(6)利用Western blot检测MDA-MB-468-ING4、MDA-MB-468-Mock乳腺癌细胞上皮间充质转化(EMT)相关蛋白(E-cadherin、N-cadherin、Snail1、Snail2、Zeb1、Zeb2、Twist1、Twist2)的表达,分析慢病毒介导的ING4过表达抑制MDA-MB-468三阴性乳腺癌细胞迁移、侵袭的分子机制。结果:(1)成功制备了表达人源化ING4的GFP标记重组慢病毒(LV-ING4)。(2)成功构建了慢病毒介导的ING4转基因MDA-MB-468三阴性乳腺癌细胞。(3)慢病毒介导的ING4过表达能够明显抑制MDA-MB-468三阴性乳腺癌细胞的迁移、侵袭能力,MDA-MB-468-ING4较MDA-MB-468-Mock的相对迁移、侵袭力分别为45.7%、36.8%。(4)ING4能够明显下调MDA-MB-468三阴性乳腺癌细胞Snail1、Snail2 EMT诱导转录因子(EMT-TFs)和N-cadherin间充质标志物及上调E-cadherin上皮标志物,而对Zeb1、Zeb2、Twist1、Twist2 EMT-TFs几乎无影响。结论:慢病毒介导的ING4过表达能够下调Snail1、Snail2 EMT-TFs逆转EMT的方式抑制乳腺癌转移。
[Abstract]:Aim: to investigate the effect of lentivirus-mediated growth suppressor ING4 overexpression on the migration and invasion of breast cancer cells and its mechanism. Methods: (1) pAdTrack-CMV/ING4 plasmid carrying humanized ING4 coding sequence (CDS) was used as template. ING4 CDS fragments were obtained by PCR amplification using ING4 specific primers (ING4-F:5'-gaa gct agc gcc acc atg gct ggg atg tat ttg-3 (ING4-F:5'-gaa gct agc gcc acc atg gct ggg atg tat ttg-3) ING4-R: 5a ggc gcg ccc tat ttc cgt tct tg-3'). (2) inserted between NheI and Sgs I restriction endonuclease sites of GFP labeled pLenti6.3/IRES/GFP lentivirus plasmids. The ING4 recombinant lentivirus plasmid pLenti6.3 / ING4 / IRES- / GFP was constructed by ING4 target fragment, and identified by PCR and double enzyme digestion. (3) the constructed lentivirus plasmid and its control pLenti6.3/IRES/GFP lentivirus plasmid were cotransfected into 293T human embryo kidney by liposome Lipofectamine2000 and co-transfected with pLP1pLP2VSVG, respectively. Cell packaging GFP labeled ING4 recombinant lentivirus (LV-ING4) and its control empty virus (LV), and culturing lentivirus supernatant with high speed centrifugation and concentration. The titer of LV-ING4LV was determined by limited dilution method according to GFP expression. (4) LV-ING4LV was infected with MDA-MB-468 tri-negative breast cancer cells with the best infection dose of 10MOI, and MDA-MB-468-LV-ING4 (MDA-MB-468-ING4) MDA-MB-468-LV (MDA-MB-468-Mock) was obtained by 10 渭 g/ml (BSD) screening. Fluorescence microscopy, flow cytometry and RT-PCR Western blot were used to detect the expression of ING4 mediated by lentivirus in MDA-MB-468 cells. (5) Transwell migration and invasion assay were used to detect the migration and invasion ability of MDA-MB-468-MNG4MDA-MB-468-Mock breast cancer cells. To analyze the effect of lentivirus-mediated overexpression of ING4 on the migration and invasion of MDA-MB-468 tri-negative breast cancer cells. (6) Western blot was used to detect the expression of E-cadherin N-cadherin Snail2Zeb2Zeb2Zeb2Zeb2Zeb2Twist1 Twist2 protein in MDA-MB-468-Mock breast cancer cells. To analyze the molecular mechanism of lentivirus-mediated overexpression of ING4 inhibiting the migration and invasion of MDA-MB-468 tri-negative breast cancer cells. Results: (1) GFP labeled lentivirus (LV-ING4). (2) expressing human ING4 was successfully prepared to construct ING4 transgene MDA-MB-468 triple negative breast cancer cells mediated by lentivirus. (3) Lentivirus mediated ING4 overexpression could significantly inhibit MDA-MB-468 triple negative breast cancer cells. The migration of adenocarcinoma cells, The invasion ability of MDA-MB-468-ING4 was 45.7 and 36.8than that of MDA-MB-468-Mock, respectively. (4) ING4 could significantly down-regulate EMT-TFs and N-cadherin mesenchymal markers and up-regulate E-cadherin epithelial markers in MDA-MB-468 tri-negative breast cancer cells, but had little effect on Zeb1Twist1Twist2 EMT-TFs. Conclusion: lentivirus-mediated overexpression of ING4 can inhibit the metastasis of breast cancer by down-regulating Snail1 and Snail2 EMT-TFs reversing EMT.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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