發(fā)布時(shí)間：2018-04-20 11:58

  本文選題：人即刻早期應(yīng)答 + 2��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：目的:肝癌危害生命主要原因在于轉(zhuǎn)移。而肝癌轉(zhuǎn)移是多基因參與的、復(fù)雜的過(guò)程,它涉及大量基因異常表達(dá)以及相關(guān)信號(hào)通路的異常。人即刻早期應(yīng)答2基因(immediate early response 2,IER2)的表達(dá)產(chǎn)物可能是一種核蛋白,作為一種早期轉(zhuǎn)錄因子或轉(zhuǎn)錄調(diào)控因子發(fā)揮作用。目前雖有研究表明,IER2在胰腺癌及乳腺癌中表達(dá)可以促進(jìn)腫瘤細(xì)胞遷移和侵襲。然而,IER2在肝癌細(xì)胞中的生物功能和潛在調(diào)控機(jī)制仍然是未知的。因此,本研究的目的是探討IER2對(duì)肝癌細(xì)胞遷移、侵襲以及與細(xì)胞外基質(zhì)(ECM)黏附和鋪展影響,初步闡明IER2調(diào)控肝癌細(xì)胞遷移、侵襲及與ECM黏附作用的機(jī)制。方法:以肝癌細(xì)胞為實(shí)驗(yàn)對(duì)象,構(gòu)建IER2過(guò)表達(dá)和RNA干擾表達(dá)的重組慢病毒,感染細(xì)胞后通過(guò)嘌呤霉素篩選穩(wěn)定表達(dá)細(xì)胞系,探討IER2對(duì)肝癌細(xì)胞遷移、侵襲及與ECM黏附的影響。(1)分別采用RT-qPCR及Western blot實(shí)驗(yàn)方法檢測(cè)IER2等在四種不同遷移潛能肝癌細(xì)胞中的表達(dá)。(2)采用CCK-8試劑盒檢測(cè)IER2過(guò)表達(dá)及RNA干擾表達(dá)對(duì)肝癌細(xì)胞SMMC-7721和MHCC97H細(xì)胞活力的影響。(3)通過(guò)Transwell細(xì)胞遷移和侵襲實(shí)驗(yàn)研究IER2過(guò)表達(dá)和RNA干擾表達(dá)對(duì)SMMC-7721和MHCC97H細(xì)胞遷移和侵襲的影響。(4)利用肝癌細(xì)胞與ECM(細(xì)胞外基質(zhì))黏附實(shí)驗(yàn)以及細(xì)胞鋪展實(shí)驗(yàn)研究IER2過(guò)表達(dá)和RNA干擾表達(dá)對(duì)SMMC-7721和MHCC97H細(xì)胞在不同基質(zhì)上黏附以及在Fibronectin上的鋪展的影響。(5)利用RT-qPCR及Western blot實(shí)驗(yàn)研究IER2過(guò)表達(dá)和RNA干擾表達(dá)對(duì)SMMC-7721和MHCC97H細(xì)胞中ITGB1和ITGB5表達(dá)水平的影響;利用熒光素酶報(bào)告基因?qū)嶒?yàn)檢測(cè)IER2是否通過(guò)轉(zhuǎn)錄調(diào)控ITGB1的表達(dá);進(jìn)一步采用染色質(zhì)免疫共沉淀(ChIP)實(shí)驗(yàn)驗(yàn)證IER2是否可以直接與ITGB1的啟動(dòng)子結(jié)合調(diào)控其轉(zhuǎn)錄;利用RNAi敲低過(guò)表達(dá)IER2的細(xì)胞中的ITGB1,采用Transwell細(xì)胞遷移、侵襲以及細(xì)胞鋪展實(shí)驗(yàn)進(jìn)一步驗(yàn)證IER2是否通過(guò)ITGB1影響肝癌細(xì)胞的遷移、侵襲和鋪展。(6)利用Western blot實(shí)驗(yàn)研究IER2過(guò)表達(dá)和RNA干擾表達(dá)對(duì)SMMC-7721和MHCC97H細(xì)胞中ITGB1介導(dǎo)的相關(guān)信號(hào)分子表達(dá)及其活性的影響。結(jié)果:在成功構(gòu)建慢病毒介導(dǎo)的IER2穩(wěn)定過(guò)表達(dá)和RNA穩(wěn)定干擾表達(dá)的SMMC-7721和MHCC97H細(xì)胞系的基礎(chǔ)上,我們通過(guò)如下實(shí)驗(yàn),系統(tǒng)地對(duì)IER2在肝癌細(xì)胞遷移、侵襲及與ECM黏附等指標(biāo)中的地位和作用進(jìn)行了初步探索。主要結(jié)果如下:(1)RT-qPCR和Western blot實(shí)驗(yàn)檢測(cè)IER2在四種不同遷移潛能肝癌細(xì)胞中的表達(dá),結(jié)果顯示,具有低遷移能力的HepG2和SMMC-7721細(xì)胞中IER2的表達(dá)相對(duì)較低,而具有高遷移能力的MHCC97H和HCCLM3細(xì)胞中IER2的表達(dá)相對(duì)較高。(2)CCK-8細(xì)胞活力實(shí)驗(yàn)結(jié)果表明,IER2過(guò)表達(dá)和RNA干擾表達(dá)對(duì)SMMC-7721和MHCC97H細(xì)胞的細(xì)胞活力無(wú)顯著性影響。(3)Transwell細(xì)胞遷移和Transwell侵襲實(shí)驗(yàn)結(jié)果均顯示,IER2過(guò)表達(dá)可以顯著促進(jìn)SMMC-7721和MHCC97H細(xì)胞的遷移和侵襲(p0.05),而RNA干擾表達(dá)能明顯抑制這兩種細(xì)胞的遷移和侵襲過(guò)程(p0.05)。(4)細(xì)胞與ECM黏附實(shí)驗(yàn)結(jié)果表明,IER2過(guò)表達(dá)可以顯著促進(jìn)SMMC-7721和MHCC97H細(xì)胞與Fibronectin的黏附,而RNA干擾表達(dá)能明顯抑制這兩種細(xì)胞與Fibronectin的黏附(p0.05);無(wú)論是IER2過(guò)表達(dá)還是RNA干擾表達(dá)均不影響SMMC-7721 和 MHCC97H 細(xì)胞與 Collagen type Ⅰ以及 Matrigel 的黏附。(5)細(xì)胞鋪展實(shí)驗(yàn)結(jié)果顯示,IER2過(guò)表達(dá)顯著促進(jìn)SMMC-7721和MHCC97H細(xì)胞在Fibronectin上的鋪展(p0.05),而IER2干擾表達(dá)則可以明顯抑制這兩種細(xì)胞在Fibronectin基質(zhì)上鋪展(p0.05)。(6)RT-qPCR和Western blot實(shí)驗(yàn)結(jié)果表明,IER2過(guò)表達(dá)和RNA干擾表達(dá)可以分別上調(diào)和下調(diào)SMMC-7721和MHCC97H細(xì)胞中ITGB1的mRNA水平及其蛋白水平,但對(duì)ITGB5的表達(dá)無(wú)顯著影響;熒光素酶報(bào)告基因?qū)嶒?yàn)檢測(cè)結(jié)果顯示,與空白對(duì)照組和對(duì)照空載病毒組相比,IER2過(guò)表達(dá)可以顯著提高ITGB1啟動(dòng)子的活性(p0.05),而IER2敲低表達(dá)可以明顯降低ITGB1啟動(dòng)子的活性(p0.05);ChIP實(shí)驗(yàn)結(jié)果顯示,IER2可以直接與ITGB啟動(dòng)子上相應(yīng)序列結(jié)合進(jìn)行轉(zhuǎn)錄調(diào)控;RNAi敲低過(guò)表達(dá)IER2的SMMC-7721和MHCC97H細(xì)胞中的ITGB1后,可以顯著抑制IER2誘導(dǎo)的肝癌細(xì)胞的遷移、侵襲和鋪展。(7)Western blot實(shí)驗(yàn)結(jié)果顯示,與空白對(duì)照組及對(duì)照空載病毒組相比,IER2過(guò)表達(dá)可以顯著上調(diào)SMMC-7721和MHCC97H細(xì)胞中p-Y397FAK、p-Y407FAK、p-Y576/Y577FAK、p-Y861FAK、p-Y925FAK、p-S910FAK、p-Y419Src 和 p-Y118 paxillin(p0.05),下調(diào) p-Y527Src(p0.05);而 RNA 干擾 IER2 表達(dá)則可以下調(diào)這兩種細(xì)胞中的p-Y527Src,上調(diào)細(xì)胞中p-Y419Src;無(wú)論是IER2過(guò)表達(dá)還是RNA干擾表達(dá)對(duì)細(xì)胞中的FAK、Src和paxillin的表達(dá)均無(wú)顯著影響。結(jié)論:(1)IER2的表達(dá)與肝癌細(xì)胞的遷移能力相關(guān)。(2)IER2表達(dá)可以促進(jìn)肝癌細(xì)胞的遷移、侵襲以及在Fibronectin基質(zhì)上的黏附和鋪展過(guò)程。(3)IER2通過(guò)對(duì)ITGB1的轉(zhuǎn)錄調(diào)控,促進(jìn)ITGB1的表達(dá),激活I(lǐng)TGB1-FAK-Src-paxillin信號(hào)通路來(lái)影響肝癌細(xì)胞與Fibronectin基質(zhì)黏附、鋪展、遷移及侵襲。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7
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本文編號(hào)：1777649