本文關(guān)鍵詞：MicroRNA455-3p調(diào)節(jié)三陰性乳腺癌侵襲轉(zhuǎn)移的機制研究　出處：《山東大學》2016年碩士論文　論文類型：學位論文

  更多相關(guān)文章： miR-455-3p 三陰性乳腺癌 浸潤 轉(zhuǎn)移 EI24

【摘要】：研究背景乳腺癌是一種女性常見的惡性腫瘤,是當前威脅女性生命和健康的主要原因之一。近年來由于經(jīng)濟的飛速發(fā)展、生活環(huán)境和生活方式的改變以及多方面競爭造成的壓力等因素的影響,乳腺癌發(fā)病率呈逐年增高的趨勢。作為一種異質(zhì)性疾病,乳腺癌在形態(tài)、臨床表現(xiàn)及對治療的敏感性上均有所差異。三陰性乳腺癌(triple negative breast cancer, TNBC)是乳腺癌分子亞型分型中的一種特殊類型,因其分子表型缺乏雌激素受體(estrogen receptor, ER)、孕激素受體(progestrone receptor, PR)和人表皮生長因子受體-2(human epidermal growth factor 2, HER-2)的表達而得名。TNBC約占所有乳腺癌的15-20%,與其他類型的乳腺癌相比,好發(fā)于相對年輕的婦女,其細胞分化程度更差,侵襲性更強,更易出現(xiàn)早期復發(fā)和遠處轉(zhuǎn)移,因此臨床預后差。目前TNBC在臨床上的主要治療方法是外科手術(shù)和輔助性化療,但因缺乏激素受體及HER-2等治療靶點,尚無針對性的有效治療方案,因此TNBC患者的預后仍不理想。近年來TNBC發(fā)生的分子機制已成為眾多學者關(guān)注和研究的熱點。MicroRNA(簡稱miRNA)是一類含有18～22個核苷酸的內(nèi)源性的非編碼的RNA分子,它廣泛存在于真核生物中,是一類重要的基因表達調(diào)控因子,在細胞的增殖、發(fā)育、分化及凋亡中發(fā)揮著重要作用。miRNA可以通過和mRNA的31-UTR區(qū)特異性結(jié)合抑制靶基因的轉(zhuǎn)錄或誘導mRNA的降解,在生物體內(nèi)發(fā)揮癌基因或抑制癌基因的作用。根據(jù)生物信息學分析,人類基因的92%受miRNA的調(diào)節(jié),現(xiàn)有證據(jù)表明miRNA的異常調(diào)節(jié)參與乳腺癌的發(fā)生發(fā)展。例如miR-10b在轉(zhuǎn)移性乳腺癌中表達上調(diào),通過負性調(diào)控HOXD10促進乳腺癌的轉(zhuǎn)移和浸潤,乳腺癌中miR-106b的高表達,預示著病人復發(fā)的風險較大,而miR-206在Noth3抑制乳腺癌細胞的增殖、浸潤和轉(zhuǎn)移,但目前為止在TNBC中確認功能的miRNA卻少之又少。作為p53的一個反應性凋亡前體因子,E124在抑制細胞生長以及自噬激活的過程中起到了重要的作用。而且,EI24的低表達與EMT的誘導和腫瘤的進展存在密切的聯(lián)系：EI24的缺失引起的依托泊苷抵抗,表明其可能成為一個指示化療有效性的潛在因子。Jung-Min的研究表明在非小細胞肺癌中降低的EI24表達可以通過IGF-1R信號通路顯著提高癌細胞對吉非替尼的抵抗能力；而在食管鱗狀細胞癌中,miR-483-3p通過靶向結(jié)合EI24基因從而促進了腫瘤的進展,表明miR與EI24之間存在密切的關(guān)聯(lián)。但是,目前尚沒有EI24在TNBC中的研究報道。目的通過檢測TNBC組織及細胞中microRNA的差異性表達情況,確定候選microRNA;進一步驗證其作用及調(diào)控的靶基因,探討其影響TNBC侵襲轉(zhuǎn)移的機制。實驗方法(1) miRNA表達譜檢測：收集TNBC以及激素受體陽性浸潤性導管癌(ER、PR表達均為陽性,HER-2表達呈陰性)石蠟包埋組織各3例,運用miRCURY LNA Array系統(tǒng)對miRNA表達譜進行分析。miRNA表達譜的檢測委托聯(lián)川生物技術(shù)公司進行。(2)根據(jù)芯片檢測結(jié)果,篩選出3個在TNBC及對照組中具有差異性表達且有統(tǒng)計學意義的候選miRNA,通過qRT-PCR技術(shù)檢測117例TNBC和42例激素受體陽性浸潤性導管癌組織中3個候選miRNA的表達情況,選取差異性最顯著的miRNA進行后續(xù)實驗。(3)細胞功能學實驗,對三陰性乳腺癌細胞株MDA-MB-231和MDA-MB-468體外轉(zhuǎn)染miR-455-3p mimics、negative control、miR-455-3p inhibitor及inhibitor negative control,隨后進行MTS和Transwell實驗,觀察體外其對三陰性乳腺癌細胞增殖、浸潤和遷移的影響。(4)生物信息學預測miR-455-3p靶基因,結(jié)合芯片結(jié)果及相關(guān)文獻,選定LTBR, EI24, SMAD2作為miR-455-3p可能的靶基因。(5)通過雙熒光素酶報告基因?qū)嶒灪蚖estern blot實驗,驗證miR-455-3p調(diào)控的靶基因。結(jié)果(1)根據(jù)芯片結(jié)果分析,篩選出3個候選miRNAs,分別是miR-455-3p, miR-425-5p, miR-196a-5p,其中miR-455-3p和miR-196a-5p在TNBC中顯著上調(diào),而miR-425-5p表達呈下調(diào)。提取117例三陰性乳腺癌組織和42例浸潤性導管癌組織的miRNA,通過qRT-PCR實驗檢測,結(jié)果發(fā)現(xiàn)miR-455-3p在三陰性乳腺癌組織中表達上調(diào)10倍以上； 隨后在乳腺癌細胞MCF-7、MDA-MB-231和MDA-MB-468中進行驗證,結(jié)果顯示miR-455-3p在三陰性乳腺癌細胞中表達明顯上調(diào),差異具有統(tǒng)計學意義(P0.05)。(2) Transwell細胞實驗結(jié)果：細胞轉(zhuǎn)染miR-455-3p mimic后遷移和浸潤能力明顯增強；而轉(zhuǎn)染miR-455-3p inhibitor組的細胞遷移和浸潤能力比對照組明顯降低,這提示miR-455-3p可以促進三陰性乳腺癌細胞的遷移和浸潤。(3)MTS實驗結(jié)果顯示：轉(zhuǎn)染miR-455-3p mimic后提高了細胞的增殖能力；相反,轉(zhuǎn)染miR-455-3p inhibitor后抑制了細胞miR-455-3p的表達,與對照組相比(P0.05),細胞增殖能力減弱,說明miR-455-3p對三陰性乳腺癌細胞的生長和增殖有促進作用。(4)EI24是miR-455-3p的直接靶基因：將EI24熒光素酶載體和miR-455-3p mimic共轉(zhuǎn)染到MDA-MB-231和MDA-MB-468細胞中,miR-455-3p可以直接和EI24的3＇UTR區(qū)結(jié)合,在轉(zhuǎn)錄水平影響EI24的表達,熒光素酶報告基因分析顯示,EI24是miR-455-3p的直接靶基因；Western blot實驗結(jié)果顯示,在EI24低表達的三陰性乳腺癌細胞系MDA-MB-231和MDA-MB-468以及EI24高表達的乳腺癌細胞MCF-7中過表達miR-455-3p,EI24蛋白表達均明顯降低(P0.05)。進一步驗證了EI24是miR-455-3p調(diào)控的靶基因。(5)在三陰性乳腺癌細胞中干擾掉EI24后能促進細胞的遷移和浸潤,與miR-455-3p過表達對三陰性乳腺癌細胞的影響相似,這提示EI24受miR-455-3p的調(diào)控且miR-455-3p能促進三陰性乳腺癌細胞的遷移和浸潤。結(jié)論miR-455-3p在三陰性乳腺癌組織和三陰性乳腺癌細胞中均顯著性上調(diào),發(fā)揮著癌基因的作用,體外細胞學實驗證實miR-455-3p對三陰性乳腺癌細胞的增殖、浸潤和轉(zhuǎn)移起促進作用,其是通過調(diào)控EI24促進三陰性乳腺癌的發(fā)生發(fā)展。
[Abstract]:Background: breast cancer is a common malignant tumor in women, is one of the main causes of threat to women's life and health. In recent years due to the rapid development of economy, the living environment and lifestyle changes and many competitive pressures and other factors, the incidence of breast cancer is increasing year by year. A heterogeneous disease, breast cancer in morphology, clinical manifestations and sensitivity to treatment are different. Three negative breast cancer (triple negative breast cancer, TNBC) is a special type of breast cancer molecular subtypes in the molecular phenotype because of the lack of estrogen receptor (estrogen, receptor, ER) progesterone receptor (progestrone receptor, PR) and human epidermal growth factor receptor -2 (human epidermal growth factor 2, HER-2) expression named approximately.TNBC of all breast cancer 15-20%, and other types of Breast cancer compared to occur in relatively young women, the degree of cell differentiation is even worse, more aggressive, more prone to early recurrence and metastasis, so the prognosis is poor. The main treatment method in clinical TNBC is surgery and adjuvant chemotherapy, but due to lack of hormone receptors and HER-2 therapy there is no point, effective treatment for the patients with TNBC, so the prognosis is still not ideal. The molecular mechanism of TNBC in recent years has become the focus of attention of many scholars and research.MicroRNA (miRNA) is a class containing 18 to 22 nucleotides of endogenous non encoding RNA molecules, it widely exists in eukaryotic organisms, is one of the important factors to regulate the expression of genes in development, cell proliferation, differentiation and apoptosis plays an important role in the.MiRNA 31-UTR region specific through the combination of mRNA and transcription inhibition of target gene or inducing The degradation of mRNA, an oncogene or suppressor role in the organism. According to bioinformatics analysis, the regulation of human gene 92% by miRNA, the existing evidence suggests that abnormal regulation involved in the occurrence and development of breast cancer miRNA. For example the expression of miR-10b was up-regulated in metastatic breast cancer, through the negative regulation of HOXD10 to promote breast cancer metastasis and invasion, high expression of miR-106b in breast cancer, indicates that the greater risk of relapse, miR-206 inhibited the proliferation of breast cancer cells in Noth3, invasion and metastasis, but so far in TNBC confirm the function of miRNA is less and less. As a p53 reaction of apoptosis before the body factor, E124 plays an important role in the process of cell growth inhibition and activation of autophagy. Moreover, there is a close relationship in low expression of EMT and EI24 induced by tumor: the loss of EI24 induced by etoposide In resistance, indicating that it might become a research indicating the effectiveness of chemotherapy potential factor.Jung-Min showed a decrease in non small cell lung cancer EI24 expression through IGF-1R signaling pathway significantly increased cancer cells to gefitinib resistance; in esophageal squamous cell carcinoma, miR-483-3p by targeting to EI24 gene to promote the progress of the tumor shows that there is close correlation between miR and EI24. However, there are no reports on EI24 in TNBC. Through the differential expression of microRNA TNBC detected the tissues and cells, to determine the candidate target gene microRNA; to further verify its effect and regulation, to explore the mechanism of the effect of TNBC the invasion and metastasis of experimental methods. (1) the expression of miRNA and TNBC spectrum detection: collect hormone receptor positive invasive ductal carcinoma (ER, PR expression was positive, HER-2 negative expression) Shi Labao Embedded tissues of the 3 cases, the use of miRCURY LNA Array expression spectrum analysis to detect the expression of.MiRNA by spectrum Lianchuan Biotech Corp of miRNA. (2) according to the result of the microarray, we selected 3 in TNBC and control group with different expression and there was statistical significance when miRNA expression by 3 the candidate miRNA qRT-PCR detection of 117 cases of TNBC and 42 cases of hormone receptor positive invasive ductal carcinoma, select the most significant difference miRNA for subsequent experiments. (3) experimental study on cell function, three negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 in vitro transfection of miR-455-3p mimics, negative control, miR-455-3p inhibitor and inhibitor negative control, followed by MTS and Transwell in vitro experiment, the proliferation of three negative breast cancer cells, effects of infiltration and migration. (4) bioinformatics prediction based on miR-455-3p target Because, according to the microarray results and related literature, selected LTBR, EI24, SMAD2 as the target gene of miR-455-3p. (5) by dual luciferase reporter assay and Western blot experiments, verify the target gene regulated by miR-455-3p. Results (1) according to the chip results, we selected 3 candidate miRNAs, respectively miR-455-3p, miR-425-5p miR-196a-5p, miR-455-3p and miR-196a-5p, which in TNBC was significantly increased, while the expression of miR-425-5p was down regulated. From 117 cases of three breast cancer tissues and 42 cases of invasive ductal carcinoma of the miRNA, through the qRT-PCR test, the results showed that miR-455-3p up-regulated more than 10 times in the three breast cancer tissues; then in breast cancer cells MCF-7, confirmed by MDA-MB-231 and MDA-MB-468, the results showed that the expression of miR-455-3p is upregulated in three negative breast cancer cells, the difference was statistically significant (P0.05). (2) Transw The results of ell cell: cells transfected with miR-455-3p mimic significantly enhanced the ability of migration and invasion; migration of transfected miR-455-3p cells in the inhibitor group and the invasion ability was significantly lower than in the control group, suggesting that miR-455-3p can promote the migration of three negative breast cancer cells and infiltration of MTS. (3) experimental results showed that the transfection of miR-455-3p cells increased after mimic the proliferation ability; instead, after transfection of miR-455-3p inhibitor inhibited the expression of miR-455-3p cells, compared with the control group (P0.05), reduced cell proliferation and miR-455-3p on growth and proliferation of three negative breast cancer cells promoted. (4) EI24 is a direct target gene of miR-455-3p: the EI24 luciferase reporter vector and miR-455-3p mimic and MDA-MB-468 were transfected into MDA-MB-231 cells, with 3 'UTR miR-455-3p can be directly and EI24, at the transcriptional level affect the expression of EI24 Analysis and luciferase reporter gene showed that EI24 is a direct target gene of miR-455-3p Western blot; the experimental results show that miR-455-3p overexpression in breast cancer cells with high expression of MCF-7 in the low expression of EI24 three negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, EI24, EI24 protein expression decreased significantly (P0.05). EI24 is further verified the target genes of miR-455-3p. (5) in three negative breast cancer cells interfered after EI24 can promote cell migration and invasion, and the effect of miR-455-3p overexpression on three negative breast cancer cells, showed that EI24 regulated by miR-455-3p and miR-455-3p can promote the migration of three negative breast cancer cells and infiltration. Conclusion miR-455-3p in three breast cancer tissues and three negative breast cancer cells were significantly up-regulated, play the role of oncogenes, the in vitro experiments showed that miR-455-3p of three yin The proliferation, infiltration and metastasis of sexual breast cancer cells promote the development of three negative breast cancer by regulating EI24.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.9

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1 杜文英;乳腺癌分子亞型的臨床與病理特點[D];鄭州大學;2011年

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5 葛廣哲;樹，

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