本文選題：肝細胞肝癌 + miR-150��； 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：【背景】肝細胞肝癌是全世界最常見的惡性腫瘤之一,是世界第三位、我國第二位的腫瘤相關(guān)性死亡原因。近年來,盡管外科手術(shù)技術(shù)不斷提高、現(xiàn)代影像技術(shù)和非手術(shù)局部治療技術(shù)不斷進步,大大提高了肝癌的治療效果,然而,肝癌患者的預(yù)后并沒有因此得到很大的改善。主要原因是肝癌早期診斷困難、容易發(fā)生復(fù)發(fā)和轉(zhuǎn)移等。因此,深入研究肝癌發(fā)生、發(fā)展的分子機制,以期尋找能夠用于肝癌早期診斷的分子標(biāo)志物以及尋找有效治療肝癌的新靶點是一項亟待解決的問題。micro RNA是一類非編碼小RNA,主要通過與靶基因m RNA 3’非編碼區(qū)相互結(jié)合進而負向調(diào)控靶基因的表達,從而參與多種細胞學(xué)功能,包括細胞增殖、凋亡、分化、代謝和調(diào)節(jié)內(nèi)分泌系統(tǒng)等。mi RNA表達異常和包括腫瘤在內(nèi)的多種疾病相關(guān)。在腫瘤的研究中發(fā)現(xiàn),mi RNA參與了腫瘤細胞增殖、凋亡、分化、耐藥和侵襲轉(zhuǎn)移等多個方面的調(diào)控,其中,mi R-150在腫瘤的發(fā)生發(fā)展過程中也發(fā)揮了重要作用。在結(jié)腸癌、胰腺癌、卵巢癌中mi R-150表達下調(diào),發(fā)揮抑癌作用,而在胃癌、乳腺癌中mi R-150高表達,促進腫瘤的進展。因此,mi R-150在不同的腫瘤類型中發(fā)揮著不同的作用。有研究表明,通過基因芯片分析CD133+和CD133-的肝癌細胞中micro RNA的表達差異發(fā)現(xiàn),mi R-150在CD133+的肝癌干細胞中表達下調(diào)。另有一篇文獻報道,通過比較肝癌轉(zhuǎn)移灶和原位肝癌中的micro RNA表達差異,發(fā)現(xiàn)mi R-150在轉(zhuǎn)移灶中表達下調(diào),推測mi R-150可能參與了肝癌的侵襲轉(zhuǎn)移過程。然而,mi R-150是否參與調(diào)控了肝癌的發(fā)生、發(fā)展過程?其在肝癌中的具體作用及機制是什么?至今尚不明確,仍待進一步研究�！灸康摹�1、檢測肝癌組織和對應(yīng)癌旁組織中mi R-150的表達,分析mi R-150表達水平與患者臨床病理特征及患者預(yù)后之間的關(guān)系。2、研究mi R-150對肝癌細胞增殖和侵襲轉(zhuǎn)移能力的影響。3、探討mi R-150調(diào)控肝癌細胞惡性表型的分子機制。【方法】1、通過熒光實時定量PCR方法檢測mi R-150在84對肝癌組織和對應(yīng)癌旁組織中的表達情況,分析mi R-150的表達和肝癌患者臨床病理特征以及患者預(yù)后之間的關(guān)系。2、構(gòu)建mi R-150過表達慢病毒載體,感染mi R-150表達相對較低的肝癌細胞系,建立穩(wěn)定表達mi R-150和對照NC的肝癌細胞。通過CCK-8增殖實驗、平板克隆形成實驗和裸鼠皮下成瘤實驗檢測mi R-150對肝癌細胞增殖能力的影響;通過Transwell侵襲、遷移實驗和裸鼠肺轉(zhuǎn)移模型實驗檢測mi R-150對肝癌細胞侵襲、轉(zhuǎn)移能力的影響。3、通過生物信息學(xué)軟件預(yù)測mi R-150的下游靶基因,經(jīng)綜合分析,篩選GAB1可能是mi R-150的直接靶基因之一。通過雙熒光素酶報告基因?qū)嶒�、熒光實時定量PCR和western blot實驗進行驗證。檢測肝癌組織中GAB1的表達,分析其與mi R-150表達水平的相關(guān)性。采用RNAi技術(shù)沉默肝癌細胞中GAB1的表達,觀察其對肝癌細胞增殖、侵襲、遷移能力的影響,過表達GAB1后觀察肝癌細胞的功能恢復(fù)情況。進一步通過western blot和免疫組織化學(xué)染色方法檢測mi R-150對GAB1表達水平、ERK1/2磷酸化水平和EMT標(biāo)志物蛋白表達水平的影響,深入研究其中的分子機制�！窘Y(jié)果】1、mi R-150在肝癌組織中的表達水平顯著低于對應(yīng)癌旁組織,結(jié)合肝癌患者臨床病理資料分析,mi R-150低表達與腫瘤大小、靜脈侵犯和轉(zhuǎn)移密切相關(guān)。進一步研究發(fā)現(xiàn),mi R-150低表達組患者的總體生存時間顯著低于mi R-150高表達組患者。低mi R-150表達能夠作為肝癌患者預(yù)后較差的獨立指標(biāo)。2、mi R-150在肝癌細胞系中的表達水平顯著低于永生化人肝細胞系HL-7702,選用mi R-150表達相對較低的SMMC-7721和MHCC97-H細胞用于后續(xù)實驗。成功構(gòu)建mi R-150過表達慢病毒載體后,感染細胞,構(gòu)建過表達mi R-150的肝癌細胞系。CCK-8實驗、平板克隆實驗、裸鼠皮下成瘤實驗結(jié)果顯示,過表達mi R-150能夠顯著抑制肝癌細胞的增殖能力、克隆形成能力和裸鼠皮下成瘤能力。3、Transwell實驗證實,過表達mi R-150能夠顯著抑制肝癌細胞的侵襲、遷移能力。裸鼠體內(nèi)實驗結(jié)果顯示,過表達mi R-150能夠顯著抑制肝癌細胞的肺轉(zhuǎn)移能力。4、通過生物信息學(xué)軟件預(yù)測,GAB1可能是mi R-150的靶基因。雙熒光素酶報告基因?qū)嶒烇@示,野生型GAB1-WT-3’UTR報告基因載體與mi R-150 mimic共轉(zhuǎn)染可使熒光素酶活性顯著降低,而mi R-150 mimic與突變型GAB1-MUT-3’UTR共轉(zhuǎn)染后熒光素酶活性無明顯改變。進一步實驗證實,過表達mi R-150能夠顯著降低GAB1m RNA和蛋白表達水平。在肝癌組織中GAB1 m RNA表達上調(diào),通過分析發(fā)現(xiàn)肝癌組織中mi R-150表達和GAB1 m RNA表達呈負相關(guān)關(guān)系。表明GAB1是mi R-150的直接靶基因。5、GAB1-si RNA下調(diào)GAB1表達能夠抑制肝癌細胞的增殖、侵襲和遷移能力,模擬了mi R-150過表達對肝癌細胞的抑制作用,而恢復(fù)GAB1的表達后,部分逆轉(zhuǎn)了mi R-150的抑制作用。6、western blot實驗表明,過表達mi R-150抑制肝癌細胞GAB1表達的同時還抑制了ERK1/2的磷酸化,并且上調(diào)上皮樣細胞分子標(biāo)記物E-cadherin蛋白的表達,下調(diào)間質(zhì)樣細胞分子標(biāo)記物N-cadherin和Vimentin蛋白的表達,提示過表達mi R-150能夠抑制肝癌細胞的EMT過程。最后在裸鼠移植瘤組織水平驗證了過表達mi R-150能夠抑制GAB1的表達和ERK1/2的磷酸化。【結(jié)論】1、mi R-150在肝癌組織中低表達,并且mi R-150低表達和肝癌的惡性表型以及患者預(yù)后密切相關(guān)。2、過表達mi R-150能夠在體外和體內(nèi)水平抑制肝癌細胞的增殖、侵襲和轉(zhuǎn)移能力。3、GAB1是mi R-150的直接靶基因,mi R-150通過負性調(diào)節(jié)GAB1-ERK1/2軸抑制肝癌細胞的增殖、侵襲、轉(zhuǎn)移能力。臨床標(biāo)本進一步證實mi R-150的表達水平與GAB1 m RNA表達水平呈負相關(guān)關(guān)系。因此,mi R-150-GAB1-ERK1/2軸有可能成為肝癌治療的新靶點。
[Abstract]:[background] hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. It is the third world in the world and the cause of tumor related death in the second of our country. In recent years, although the surgical technique has been improved, modern imaging technology and non-surgical local treatment technology have been progressing, the treatment effect of liver cancer has been greatly improved. However, the patients with liver cancer have been greatly improved. The main reason is that the early diagnosis of liver cancer is difficult, and the recurrence and metastasis are easy to occur. Therefore, it is urgent to study the molecular mechanism of the development of liver cancer and to find the molecular markers that can be used in the early diagnosis of liver cancer and to find new targets for effective treatment of liver cancer. Problem.Micro RNA is a class of non coding small RNA, which is mainly mediated by the non coding region of the target gene m RNA 3 'and then negatively regulates the expression of target genes, thus participating in a variety of cytological functions, including cell proliferation, apoptosis, differentiation, metabolism and regulation of the.Mi RNA expression, such as the endocrine system, and a variety of diseases, including tumors. In the study of tumor, MI RNA has been involved in the regulation of tumor cell proliferation, apoptosis, differentiation, resistance and invasion and metastasis. Among them, MI R-150 also plays an important role in the development of tumor. The expression of MI R-150 in colon, pancreatic and ovarian cancer is downregulated, and MI R in gastric cancer and breast cancer. -150 is highly expressed and promotes the progression of tumors. Therefore, MI R-150 plays a different role in different tumor types. Studies have shown that the expression of micro RNA in CD133+ and CD133- cells by gene chip analysis showed that MI R-150 was downregulated in the liver cancer stem cells of CD133+. The difference in the expression of micro RNA in cancer metastasis and in situ liver cancer showed that MI R-150 was down regulated in the metastasis, and that MI R-150 may be involved in the invasion and metastasis of liver cancer. However, whether mi R-150 participates in the regulation of the occurrence and development of liver cancer, what is its specific role and mechanism in the liver cancer? It is still not clear and still needs to be advanced. [Objective] [Objective] [Objective] 1 to detect the expression of MI R-150 in liver cancer tissues and adjacent tissues, to analyze the relationship between the expression of MI R-150 and the clinicopathological features of the patients and the prognosis of the patients.2, and to study the effect of MI R-150 on the proliferation and invasion and metastasis of hepatoma cells.3, and to explore the molecular mechanism of the MI R-150 regulation of the malignant phenotype of hepatoma cells. [method] 1, the expression of MI R-150 in 84 liver cancer tissues and adjacent tissues was detected by real time fluorescence quantitative PCR method, and the relationship between the expression of MI R-150 and the clinicopathological features of the patients with liver cancer and the prognosis of the patients was analyzed.2. The expression of MI R-150 by the overexpression of the lentivirus carrier and the infection of the low expression of the MI R-150 to the lower liver cancer cells The hepatoma cells that express mi R-150 and control NC were established. The effects of MI R-150 on the proliferation of hepatoma cells were detected by CCK-8 proliferation test, and the effect of MI R-150 on the proliferation of hepatoma cells. The invasion, migration test and lung metastasis model test of nude mice were used to detect the invasion and metastasis of MI R-150 to the liver cancer cells. Influence.3 and predict the downstream target gene of MI R-150 through bioinformatics software. Through comprehensive analysis, screening GAB1 may be one of the direct target genes of MI R-150. Through double luciferase reporter gene experiment, fluorescence real-time quantitative PCR and Western blot experiment were verified. The expression of GAB1 in liver cancer group was detected, and the expression level of MI R-150 was analyzed. RNAi technique was used to silence the expression of GAB1 in hepatoma cells, to observe the effect of its effect on the proliferation, invasion and migration of hepatoma cells. After overexpressing GAB1, the functional recovery of hepatoma cells was observed. The expression level of MI R-150 to GAB1, ERK1/2 phosphorylation level and E were further detected by western blot and immunohistochemical staining. The molecular mechanism of the expression level of MT marker protein was studied. [results] 1, the expression level of MI R-150 in the liver cancer tissues was significantly lower than that of the para cancerous tissue. The low expression of MI R-150 was closely related to the size of the tumor and the invasion and metastasis of the tumor. The further study found that MI R-150 was found. The overall survival time of the low expression group was significantly lower than that of the MI R-150 high expression group. The low MI R-150 expression could be used as an independent indicator of the poor prognosis of the liver cancer patients.2. The expression level of MI R-150 in the hepatocellular carcinoma cell lines was significantly lower than that of the immortalized human hepatocyte line HL-7702, and MI R-150 was selected to express relatively low SMMC-7721 and MHCC97-H cells. After the successful construction of the MI R-150 overexpressed lentivirus vector, the infected cells were infected and the hepatoma cell line expressing mi R-150 was constructed by.CCK-8 experiment. The experiment of flat clones and subcutaneous tumor formation in nude mice showed that overexpression of MI R-150 could significantly inhibit the proliferation of hepatoma cells, the cloning ability and the subcutaneous tumor formation ability of nude mice. 3, Transwell experiments confirmed that overexpression of MI R-150 could significantly inhibit the invasion and migration of hepatoma cells. The experimental results in nude mice showed that overexpression of MI R-150 could significantly inhibit the lung metastasis of hepatoma cells.4, and GAB1 might be the target gene for MI R-150 through bioinformatics software. The double luciferase reporter gene experiment showed that the expression of GAB1 could be the target gene. The results showed that the co transfection of the wild type GAB1-WT-3 'UTR reporter gene carrier and MI R-150 mimic could significantly reduce the luciferase activity, while the MI R-150 mimic and the mutant GAB1-MUT-3' UTR co transfected luciferase activity did not change significantly. Further experiments showed that the overexpression of MI R-150 could significantly reduce the level of the protein expression and protein expression. The expression of GAB1 m RNA was up-regulated in the tissue. It was found that the expression of MI R-150 in the liver cancer tissues was negatively correlated with the expression of GAB1 m RNA, indicating that GAB1 is the direct target gene.5 of MI R-150, which inhibits the proliferation, invasion and migration of hepatoma cells. When the expression of GAB1 was restored, the inhibitory effect of MI R-150 on.6 was partly reversed. The Western blot experiment showed that overexpression of MI R-150 inhibited the GAB1 expression of liver cancer cells and inhibited the phosphorylation of ERK1/2, and up regulated the expression of E-cadherin protein of the molecular markers of the epithelioid cells, and down regulated the molecular marker N-cadherin of interstitial like cells. The expression of tin protein suggests that over expression of MI R-150 can inhibit the EMT process of hepatoma cells. Finally, the expression of MI R-150 can inhibit the expression of GAB1 and the phosphorylation of ERK1/2 in nude mice. [Conclusion] 1, MI R-150 is low expression in liver cancer tissues, and MI R-150 is low expression and malignant phenotype of liver cancer and patients with liver cancer. The prognosis is closely related to.2, and overexpression of MI R-150 can inhibit the proliferation, invasion and metastasis of hepatoma cells in vitro and in vivo,.3, GAB1 is the direct target gene of MI R-150, MI R-150 can inhibit the proliferation, invasion and metastasis of hepatoma cells by negative regulation GAB1-ERK1/2 axis. The expression level of M RNA is negatively correlated. Therefore, MI R-150-GAB1-ERK1/2 axis may become a new target for treatment of HCC.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.7

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Current progress in epigenetic research for hepato-carcinomagenesis[J];Science in China(Series C:Life Sciences);2009年01期

，

本文編號：2081493