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沉默DJ-1对DADS抑制MGC803细胞增殖与迁移侵袭的影响

发布时间:2018-09-14 06:59
【摘要】:目的:研究沉默DJ-1对DADS抑制MGC803细胞增殖与迁移侵袭的影响。证实DJ-1是DADS抑制人胃癌细胞迁移与侵袭的作用靶点之一。方法:构建DJ-1沉默质粒,转染MGC803细胞,建立稳定DJ-1沉默MGC803细胞系。DADS处理MGC803细胞、空载体组细胞以及DJ-1沉默MGC803细胞后,采用MTT、平板克隆形成实验、划痕实验与Transwell侵袭实验、裸鼠成瘤实验检测DADS与DJ-1沉默对MGC803细胞增殖、迁移侵袭及裸鼠成瘤能力的影响。细胞免疫荧光、q RT-PCR、Western Blot检测DADS处理前后MGC803细胞DJ-1的表达。Western Blot检测DADS与DJ-1沉默对MGC803细胞PTEN、Akt、p-Akt表达的改变。结果:1.成功构建稳定DJ-1沉默MGC803细胞。将DJ-1干扰质粒及阴性对照质粒分别转染MGC803细胞,通过Western Blot、q RT-PCR验证质粒A对DJ-1的干扰效果最明显,转染后用Puromycin筛选,建立稳定DJ-1沉默MGC803细胞系。2.DJ-1沉默与DADS对MGC803细胞增殖与迁移侵袭能力的影响。MTT和平板克隆实验结果表明,DJ-1沉默与DADS均能抑制MGC803细胞的增殖能力,并且DJ-1沉默可增强DADS对MGC803细胞增殖抑制的作用。划痕实验与Transwell侵袭实验结果显示,DJ-1沉默和DADS处理后MGC803细胞的迁移与侵袭能力减弱(P0.05),并且DJ-1沉默可增强DADS抑制肿瘤迁移与侵袭的能力。3.DADS处理前后各组MGC803细胞DJ-1表达的改变。细胞免疫荧光、q RT-PCR、Western Blot实验结果显示,DJ-1主要分布在细胞浆中,DADS处理各组MGC803细胞后较未处理组DJ-1、p-Akt表达明显减少,PTEN的表达增加(P0.05)。4.DJ-1沉默与DADS对MGC803细胞裸鼠成瘤能力的影响裸鼠成瘤实验表明,DJ-1沉默和DADS能明显抑制MGC803细胞裸鼠体内成瘤能力。免疫组化结果显示,DJ-1沉默和DADS处理后,移植瘤组织中DJ-1阳性表达较对照组明显减少,而PTEN的表达较对照组明显增加。另外,DJ-1沉默及DADS处理后与对照组比较,移植瘤组织中Ki-67、CD-34、Vimentin阳性表达下降,而E-cadherin明显增强。结论:1.DADS可下调DJ-1通过PTEN/Akt通路抑制MGC803细胞增殖与迁移侵袭。2.DJ-1沉默可抑制MGC803细胞的增殖与迁移侵袭,增强DADS对MGC803细胞的增殖与迁移侵袭的抑制作用。3.DADS与沉默DJ-1均可抑制人胃癌MGC803细胞的成瘤能力,并且两者具有协同作用。
[Abstract]:Aim: to study the effect of silencing DJ-1 on the proliferation, migration and invasion of MGC803 cells inhibited by DADS. It is confirmed that DJ-1 is one of the targets of DADS in inhibiting migration and invasion of human gastric cancer cells. Methods: DJ-1 silencing plasmid was constructed and transfected into MGC803 cells. Stable DJ-1 silencing MGC803 cell line. Dads was established to treat MGC803 cells. After empty vector group and DJ-1 silencing MGC803 cells, MTT, flat clone formation test, scratch test and Transwell invasion test were used. The effects of DADS and DJ-1 silencing on the proliferation, migration and invasion of MGC803 cells and the tumorigenic ability of nude mice were detected by tumorigenic assay in nude mice. The expression of DJ-1 in MGC803 cells before and after DADS treatment. Western Blot was used to detect the changes of PTEN,Akt,p-Akt expression in MGC803 cells induced by DADS and DJ-1 silencing. The result is 1: 1. Stable DJ-1 silencing MGC803 cells were successfully constructed. DJ-1 interference plasmids and negative control plasmids were transfected into MGC803 cells respectively. Western Blot,q RT-PCR was used to verify the interference effect of plasmid A to DJ-1. After transfection, Puromycin was used to screen. Establishment of stable DJ-1 silencing MGC803 cell line. 2. The effects of DADS and DADS on the proliferation and invasion of MGC803 cells. The results showed that both DJ-1 silencing and DADS could inhibit the proliferation of MGC803 cells. DJ-1 silencing can enhance the inhibitory effect of DADS on the proliferation of MGC803 cells. The results of scratch test and Transwell invasion test showed that the migration and invasion ability of MGC803 cells was decreased after DJ-1 silencing and DADS treatment (P0.05), and DJ-1 silencing enhanced the ability of DADS to inhibit tumor migration and invasion. 3. The DJ-1 expression of MGC803 cells was increased before and after dads treatment. The results of cytoimmunofluorescence Q RT-PCR,Western Blot assay showed that DJ-1 mainly distributed in cytoplasm of MGC803 cells treated with dads significantly decreased the expression of DJ-1,p-Akt (P0.05) .4.The silencing of DJ-1 and the effect of DADS on the tumorigenic ability of MGC803 cells in nude mice Tumorigenic assay in nude mice showed that DJ-1 silencing and DADS could significantly inhibit the tumorigenesis of MGC803 cells in nude mice. The immunohistochemical results showed that the positive expression of DJ-1 was significantly decreased and the expression of PTEN was significantly increased in the transplanted tumor tissue after silencing and DADS treatment. In addition, compared with the control group, the positive expression of Ki-67,CD-34,Vimentin was decreased and E-cadherin was significantly increased after DJ-1 silencing and DADS treatment. Conclusion: 1. Dads can down-regulate the inhibition of MGC803 cell proliferation and migration invasion by DJ-1 via PTEN/Akt pathway. 2. DJ-1 silencing can inhibit the proliferation and migration of MGC803 cells. Enhancing the inhibitory effect of DADS on proliferation and migration and invasion of MGC803 cells. 3. Both dads and silencing DJ-1 can inhibit the tumorigenesis of MGC803 cells of human gastric cancer, and both of them have synergistic effects.
【学位授予单位】:南华大学
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:R735.2

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