IER2通过ITGB1介导的信号通路调控肝癌细胞的迁移和侵袭

发布时间：2018-04-20 11:58

  本文选题：人即刻早期应答 + 2　； 参考：《扬州大学》2017年硕士论文

【摘要】：目的:肝癌危害生命主要原因在于转移。而肝癌转移是多基因参与的、复杂的过程,它涉及大量基因异常表达以及相关信号通路的异常。人即刻早期应答2基因(immediate early response 2,IER2)的表达产物可能是一种核蛋白,作为一种早期转录因子或转录调控因子发挥作用。目前虽有研究表明,IER2在胰腺癌及乳腺癌中表达可以促进肿瘤细胞迁移和侵袭。然而,IER2在肝癌细胞中的生物功能和潜在调控机制仍然是未知的。因此,本研究的目的是探讨IER2对肝癌细胞迁移、侵袭以及与细胞外基质(ECM)黏附和铺展影响,初步阐明IER2调控肝癌细胞迁移、侵袭及与ECM黏附作用的机制。方法:以肝癌细胞为实验对象,构建IER2过表达和RNA干扰表达的重组慢病毒,感染细胞后通过嘌呤霉素筛选稳定表达细胞系,探讨IER2对肝癌细胞迁移、侵袭及与ECM黏附的影响。(1)分别采用RT-qPCR及Western blot实验方法检测IER2等在四种不同迁移潜能肝癌细胞中的表达。(2)采用CCK-8试剂盒检测IER2过表达及RNA干扰表达对肝癌细胞SMMC-7721和MHCC97H细胞活力的影响。(3)通过Transwell细胞迁移和侵袭实验研究IER2过表达和RNA干扰表达对SMMC-7721和MHCC97H细胞迁移和侵袭的影响。(4)利用肝癌细胞与ECM(细胞外基质)黏附实验以及细胞铺展实验研究IER2过表达和RNA干扰表达对SMMC-7721和MHCC97H细胞在不同基质上黏附以及在Fibronectin上的铺展的影响。(5)利用RT-qPCR及Western blot实验研究IER2过表达和RNA干扰表达对SMMC-7721和MHCC97H细胞中ITGB1和ITGB5表达水平的影响;利用荧光素酶报告基因实验检测IER2是否通过转录调控ITGB1的表达;进一步采用染色质免疫共沉淀(ChIP)实验验证IER2是否可以直接与ITGB1的启动子结合调控其转录;利用RNAi敲低过表达IER2的细胞中的ITGB1,采用Transwell细胞迁移、侵袭以及细胞铺展实验进一步验证IER2是否通过ITGB1影响肝癌细胞的迁移、侵袭和铺展。(6)利用Western blot实验研究IER2过表达和RNA干扰表达对SMMC-7721和MHCC97H细胞中ITGB1介导的相关信号分子表达及其活性的影响。结果:在成功构建慢病毒介导的IER2稳定过表达和RNA稳定干扰表达的SMMC-7721和MHCC97H细胞系的基础上,我们通过如下实验,系统地对IER2在肝癌细胞迁移、侵袭及与ECM黏附等指标中的地位和作用进行了初步探索。主要结果如下:(1)RT-qPCR和Western blot实验检测IER2在四种不同迁移潜能肝癌细胞中的表达,结果显示,具有低迁移能力的HepG2和SMMC-7721细胞中IER2的表达相对较低,而具有高迁移能力的MHCC97H和HCCLM3细胞中IER2的表达相对较高。(2)CCK-8细胞活力实验结果表明,IER2过表达和RNA干扰表达对SMMC-7721和MHCC97H细胞的细胞活力无显著性影响。(3)Transwell细胞迁移和Transwell侵袭实验结果均显示,IER2过表达可以显著促进SMMC-7721和MHCC97H细胞的迁移和侵袭(p0.05),而RNA干扰表达能明显抑制这两种细胞的迁移和侵袭过程(p0.05)。(4)细胞与ECM黏附实验结果表明,IER2过表达可以显著促进SMMC-7721和MHCC97H细胞与Fibronectin的黏附,而RNA干扰表达能明显抑制这两种细胞与Fibronectin的黏附(p0.05);无论是IER2过表达还是RNA干扰表达均不影响SMMC-7721 和 MHCC97H 细胞与 Collagen type Ⅰ以及 Matrigel 的黏附。(5)细胞铺展实验结果显示,IER2过表达显著促进SMMC-7721和MHCC97H细胞在Fibronectin上的铺展(p0.05),而IER2干扰表达则可以明显抑制这两种细胞在Fibronectin基质上铺展(p0.05)。(6)RT-qPCR和Western blot实验结果表明,IER2过表达和RNA干扰表达可以分别上调和下调SMMC-7721和MHCC97H细胞中ITGB1的mRNA水平及其蛋白水平,但对ITGB5的表达无显著影响;荧光素酶报告基因实验检测结果显示,与空白对照组和对照空载病毒组相比,IER2过表达可以显著提高ITGB1启动子的活性(p0.05),而IER2敲低表达可以明显降低ITGB1启动子的活性(p0.05);ChIP实验结果显示,IER2可以直接与ITGB启动子上相应序列结合进行转录调控;RNAi敲低过表达IER2的SMMC-7721和MHCC97H细胞中的ITGB1后,可以显著抑制IER2诱导的肝癌细胞的迁移、侵袭和铺展。(7)Western blot实验结果显示,与空白对照组及对照空载病毒组相比,IER2过表达可以显著上调SMMC-7721和MHCC97H细胞中p-Y397FAK、p-Y407FAK、p-Y576/Y577FAK、p-Y861FAK、p-Y925FAK、p-S910FAK、p-Y419Src 和 p-Y118 paxillin(p0.05),下调 p-Y527Src(p0.05);而 RNA 干扰 IER2 表达则可以下调这两种细胞中的p-Y527Src,上调细胞中p-Y419Src;无论是IER2过表达还是RNA干扰表达对细胞中的FAK、Src和paxillin的表达均无显著影响。结论:(1)IER2的表达与肝癌细胞的迁移能力相关。(2)IER2表达可以促进肝癌细胞的迁移、侵袭以及在Fibronectin基质上的黏附和铺展过程。(3)IER2通过对ITGB1的转录调控,促进ITGB1的表达,激活ITGB1-FAK-Src-paxillin信号通路来影响肝癌细胞与Fibronectin基质黏附、铺展、迁移及侵袭。
[Abstract]:Objective: the main cause of liver cancer is metastasis, and liver cancer metastasis is a multi gene involved, complex process, which involves a large number of abnormal expression of genes and abnormal signal pathways. The expression of the 2 gene (immediate early response 2, IER2) may be a nucleoprotein, as an early transcriptional cause. Some studies have shown that the expression of IER2 in pancreatic and breast cancer can promote the migration and invasion of tumor cells. However, the biological functions and potential regulatory mechanisms of IER2 in the hepatoma cells are still unknown. Therefore, the purpose of this study is to explore the migration, invasion and effect of IER2 on hepatoma cells. The effect of extracellular matrix (ECM) adhesion and spreading, preliminarily clarifies the mechanism of IER2 regulation of liver cancer cell migration, invasion and adhesion to ECM. Methods: the recombinant lentivirus expressed by IER2 overexpression and RNA interference was constructed with hepatoma cells as the experimental object. After infected cells, the cell lines were screened by purinomycin, and IER2 was used to investigate the effect of IER2 on liver cancer. The effects of cell migration, invasion and adhesion to ECM. (1) RT-qPCR and Western blot were used to detect the expression of IER2 in four different migration potential hepatoma cells. (2) the effect of IER2 overexpression and RNA interference expression on the viability of SMMC-7721 and MHCC97H cells in hepatocellular carcinoma cells was detected by CCK-8 Kit. (3) through Transwell cell migration The effect of IER2 overexpression and RNA interference expression on the migration and invasion of SMMC-7721 and MHCC97H cells. (4) the adhesion of IER2 overexpression and RNA interference expression to the adhesion of SMMC-7721 and MHCC97H cells on the different substrates and in Fibronectin by the adhesion experiment of hepatoma cells with ECM (extracellular matrix) and the cell spreading experiment. (5) the effects of IER2 overexpression and RNA interference on the expression of ITGB1 and ITGB5 in SMMC-7721 and MHCC97H cells were investigated by RT-qPCR and Western blot, and the use of luciferase reporter gene test to detect the ITGB1 expression of IER2 through transcriptional regulation; further using chromatin immunoprecipitation (ChIP) experiment Verify whether IER2 can directly regulate its transcription with the promoter of ITGB1; use RNAi to knock down ITGB1 in IER2 cells, use Transwell cell migration, invasion and cell spreading experiments to further verify whether IER2 can affect the migration, invasion and spread of hepatoma cells through ITGB1. (6) IER2 over the use of Western blot. The effect of expression and RNA interference expression on the expression and activity of ITGB1 mediated signaling molecules in SMMC-7721 and MHCC97H cells. Results: on the basis of successful construction of SMMC-7721 and MHCC97H cell lines expressed by slow virus mediated IER2 stable overexpression and RNA stable interference, we systematically studied IER2 in liver cancer cells. The status and role of migration, invasion and ECM adhesion were preliminarily explored. The main results were as follows: (1) the expression of IER2 in four different migration potential hepatoma cells was detected by RT-qPCR and Western blot. The results showed that the expression of IER2 in HepG2 and SMMC-7721 cells with low migration ability was relatively low and had high migration. The expression of IER2 in MHCC97H and HCCLM3 cells was relatively high. (2) the results of CCK-8 cell viability test showed that IER2 overexpression and RNA interference expression had no significant effect on the cell viability of SMMC-7721 and MHCC97H cells. (3) Transwell cell migration and Transwell invasion experiment results showed that IER2 overexpression could significantly promote SMMC-7721 and Transwell. The migration and invasion of MHCC97H cells (P0.05), and RNA interference expression can significantly inhibit the migration and invasion process of these two cells (P0.05). (4) the results of cell and ECM adhesion experiments show that IER2 overexpression can significantly promote the adhesion of SMMC-7721 and MHCC97H cells to Fibronectin, and RNA interference expression can significantly inhibit the two cells and Fibronect. The adhesion of in (P0.05), both IER2 overexpression and RNA interference expression did not affect the adhesion of SMMC-7721 and MHCC97H cells to Collagen type I and Matrigel. (5) cell spreading experimental results showed that the overexpression of IER2 significantly promoted the spread of SMMC-7721 and MHCC97H cells on the MHCC97H cells. The inhibition of these two cells on the Fibronectin matrix (P0.05). (6) RT-qPCR and Western blot experimental results showed that IER2 overexpression and RNA interference expression could up and down regulate mRNA level and protein level of ITGB1 in SMMC-7721 and MHCC97H cells, but have no significant influence on the expression of ITGB5, the luciferase reporter gene test test Compared with the blank control group and the control group, the overexpression of IER2 could significantly increase the activity of ITGB1 promoter (P0.05), while the low expression of IER2 knocked down the activity of ITGB1 promoter (P0.05). The results of ChIP experiment showed that IER2 could be regulated directly with the corresponding sequence on the ITGB promoter; RNAi knocking. After low overexpression of IER2 in SMMC-7721 and MHCC97H cells, ITGB1 could significantly inhibit the migration, invasion and spreading of IER2 induced hepatoma cells. (7) the results of Western blot test showed that the over expression of IER2 could significantly increase the p-Y397FAK in SMMC-7721 and MHCC97H cells compared with the blank control group and the control group. 77FAK, p-Y861FAK, p-Y925FAK, p-S910FAK, p-Y419Src and p-Y118 paxillin (P0.05), down regulated p-Y527Src (P0.05), while RNA interference IER2 expression can down regulate these two cells. Conclusion: (1) the expression of IER2 is related to the migration ability of hepatoma cells. (2) the expression of IER2 can promote the migration, invasion and adhesion and spreading process on the Fibronectin matrix. (3) IER2 can promote the expression of ITGB1 and activate the ITGB1-FAK-Src-paxillin signaling pathway to affect the hepatoma cells and Fibronecti through the transcription of ITGB1 N matrix adhesion, spreading, migration and invasion.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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本文编号：1777649