本文選題：二烯丙基二硫 + 人胃癌細胞�。� 參考：《南華大學(xué)》2015年博士論文

【摘要】：胃癌是最常見的惡性腫瘤之一,嚴重危害人類的健康和生命。隨著社會經(jīng)濟發(fā)展與生活水平的提高,在歐美等發(fā)達國家其發(fā)病率和死亡率呈明顯下降趨勢,然而在亞洲、拉丁美洲仍然高發(fā)。由于其發(fā)病機制非常復(fù)雜,其預(yù)后仍不容樂觀。二烯丙基二硫(diallyl disulfide,DADS)是大蒜中的脂溶性有效成分,可抑制多種腫瘤發(fā)生發(fā)展。我們前期研究證實,DADS能在體內(nèi)外抑制人胃癌細胞增殖,其機制與G2/M期阻滯、p38激活、ERK抑制及其他多條信號轉(zhuǎn)導(dǎo)通路異常有關(guān)。MicroRNAs是一類小分子非編碼RNA,近年研究表明,其與胃癌的關(guān)系密切,參與胃癌的發(fā)生發(fā)展,在胃癌的增殖、侵襲和轉(zhuǎn)移等過程中發(fā)揮了重要作用。miR-7是23個核苷酸miRNA,在多種腫瘤中低表達,具有潛在的抑瘤功能。XIAP(X-linked inhibitor of apoptosis)作為凋亡抑制因子家族(inhibitor of apoptosis protein,IAP)成員之一,在腫瘤發(fā)生發(fā)展中起著癌基因作用,與腫瘤發(fā)生、發(fā)展、凋亡、遷移、侵襲和轉(zhuǎn)移密切相關(guān)。我們前期采用mirna芯片技術(shù)篩選dads處理人胃癌細胞的差異mirna表達,發(fā)現(xiàn)dads處理后人胃癌細胞mir-7明顯上調(diào)。同時,qrt-pcr驗證mir-7在胃癌組織與胃癌細胞株中顯著下調(diào)。蛋白質(zhì)組學(xué)發(fā)現(xiàn)xiap等24種差異表達的蛋白質(zhì),并驗證顯示,多株人胃癌細胞xiap高表達,而dads處理胃癌細胞后,xiap明顯下調(diào)。本實驗分三部分探討mir-7和xiap對人胃癌細胞增殖侵襲的影響及二者相互關(guān)系。第一部分:dads上調(diào)mir-7抑制人胃癌細胞增殖和侵襲目的:探討dads和mir-7對人胃癌細胞增殖和侵襲的影響。方法:構(gòu)建mir-7高表達和沉默病毒載體,通過細胞轉(zhuǎn)染,建立mir-7穩(wěn)定高表達和沉默的sgc-7901和hgc-27細胞系,同時各組細胞經(jīng)30mg/ldads處理,然后采用qrt-pcr、cck-8、平板克隆、流式細胞術(shù)、劃痕實驗、遷移實驗、侵襲實驗和裸鼠移植等分析dads和mir-7對胃癌細胞增殖和侵襲的作用。結(jié)果:qrt-pcr證實成功構(gòu)建mir-7穩(wěn)定高表達和沉默的sgc-7901和hgc-27細胞系,且dads能上調(diào)各組細胞mir-7的表達。dads和mir-7高表達分別可顯著抑制sgc-7901和hgc-27細胞增殖,誘導(dǎo)sgc-7901細胞凋亡和細胞周期阻滯,抑制sgc-7901細胞克隆形成。劃痕實驗和transwell遷移和侵襲實驗表明,dads和mir-7高表達分別可顯著抑制sgc-7901和hgc-27細胞遷移和侵襲能力。裸鼠成瘤實驗也表明dads組和mir-7高表達組腫瘤生長較慢,瘤體較小,較對照組有顯著性差異(p0.05)。而mir-7沉默后表現(xiàn)出與mir-7高表達相反的結(jié)果。各組實驗均表明,mir-7高表達可協(xié)同dads抑制細胞增殖、遷移、侵襲、凋亡、移植瘤生長,誘導(dǎo)細胞凋亡和周期阻滯,反之,mir-7沉默可拮抗dads的作用。第二部分:dads下調(diào)xiap抑制人胃癌細胞增殖和侵襲目的:探討dads和xiap對人胃癌細胞增殖和侵襲的影響。方法:免疫組織化學(xué)檢測組織芯片中胃癌組織xiap的表達情況,構(gòu)建xiap高表達病毒載體和沉默質(zhì)粒,通過細胞轉(zhuǎn)染,建立xiap穩(wěn)定高表達和沉默的sgc-7901和hgc-27細胞系,同時各組細胞分別經(jīng)30mg/ldads處理,然后采用qrt-pcr、westernblot、細胞免疫熒光、cck-8、平板克隆、流式細胞術(shù)、劃痕實驗、遷移實驗、侵襲實驗以及裸鼠移植等分析dads和xiap對人胃癌細胞增殖和侵襲的作用。結(jié)果:xiap在胃癌中呈強陽性表達,陽性部位主要在細胞質(zhì)中,而正常上皮及不典型增生上皮呈陰性或部分陽性表達,陽性表達率差異具有統(tǒng)計學(xué)意義(p0.05)。qrt-pcr證實成功構(gòu)建xiap穩(wěn)定高表達和沉默的sgc-7901和hgc-27細胞系,且dads能下調(diào)各組細胞xiap的表達。westernblot實驗也表明xiap沉默的sgc-7901和hgc-27細胞xiap表達下降,而xiap高表達組則xiap表達上升,dads可使各組xiap表達均下降。細胞免疫熒光亦發(fā)現(xiàn),xiap陽性表達主要在細胞質(zhì)中,xiap表達水平與westernblot實驗一致。dads和xiap沉默分別可顯著抑制sgc-7901和hgc-27細胞增殖,誘導(dǎo)sgc-7901細胞凋亡和細胞周期阻滯,抑制sgc-7901細胞克隆形成。劃痕實驗和transwell遷移和侵襲實驗表明,dads和xiap沉默可分別顯著抑制sgc-7901和hgc-27細胞遷移和侵襲能力。裸鼠成瘤實驗也表明dads組和xiap沉默組腫瘤生長較慢,腫瘤瘤體較小,較對照組有顯著性差異(p0.05)。這些實驗結(jié)果與mir-7高表達一致,而xiap高表達后表現(xiàn)出與xiap沉默相反的結(jié)果。而且,各組實驗均表明,xiap沉默可協(xié)同dads抑制細胞增殖、遷移、侵襲、凋亡和細胞周期阻滯以及抑制移植瘤生長的作用,同時,亦可拮抗xiap過表達的作用。第三部分:mir-7靶向負性調(diào)控xiap的表達目的:探討mir-7和xiap的靶向關(guān)系。方法:從mirnabase數(shù)據(jù)庫查找成熟mir-7序列,同時從genbank查詢xiap3’utr序列;利用在線數(shù)據(jù)庫diana-microt-cds、targetscan和miranda進行靶基因預(yù)測,然后構(gòu)建包含xiap-3’utr-wt和xiap-3’utr-mt雙熒光素酶報告基因系統(tǒng),與mir-7高表達載體共轉(zhuǎn)染sgc-7901細胞,檢測熒光素酶報告基因活性;最后通過qrt-pcr和westernblot檢查mir-7穩(wěn)定高表達和沉默的sgc-7901和hgc-27細胞xiapmrna和蛋白的表達,最終確定mir-7與xiap的靶向關(guān)系。結(jié)果:在獲取miR-7成熟序列和XIAP 3’UTR序列基礎(chǔ)上,利用生物信息學(xué)分析發(fā)現(xiàn)miR-7與XIAP 3’UTR存在多個結(jié)合位點,三個在線軟件均預(yù)測XIAP為miR-7的靶基因之一;雙熒光素酶報告基因系統(tǒng)證實miR-7與XIAP 3’UTR野生型共轉(zhuǎn)染SGC-7901細胞,其熒光值與對照組比較,明顯下降;miR-7穩(wěn)定高表達的SGC-7901和HGC-27細胞XIAP mRNA和蛋白表達水平均下調(diào),反向?qū)嶒烌炞CmiR-7穩(wěn)定沉默的SGC-7901和HGC-27細胞XIAP mRNA和蛋白水平均上調(diào)。結(jié)論:1.DADS可上調(diào)miR-7抑制人胃癌細胞增殖、遷移侵襲與移植瘤生長和誘導(dǎo)凋亡與周期阻滯。2.DADS可下調(diào)XIAP抑制人胃癌細胞增殖、遷移侵襲與移植瘤生長和誘導(dǎo)凋亡與周期阻滯。3.miR-7靶向負性調(diào)控XIAP mRNA和蛋白的表達。
[Abstract]:Gastric cancer is one of the most common malignant tumors and seriously endangers human health and life. With the development of social economy and the improvement of living standard, the incidence and mortality of the developed countries in Europe and America are obviously decreasing. However, in Asia, Latin America is still high. The prognosis is still not optimistic because its pathogenesis is very complex. Two Allyl two (diallyl disulfide, DADS) is a fat soluble active ingredient in garlic, which inhibits a variety of tumor development. Our previous study confirmed that DADS could inhibit the proliferation of human gastric cancer cells in vivo and in vivo, and its mechanism was associated with G2/M phase block, p38 activation, ERK inhibition, and other multiple signal transduction pathways associated with.MicroRNAs as a class of small fractions. Subcoding RNA, which is closely related to gastric cancer in recent years, is involved in the development of gastric cancer and plays an important role in the proliferation, invasion and metastasis of gastric cancer..miR-7 is 23 nucleotides miRNA, low expression in a variety of tumors and potential inhibition of.XIAP (X-linked inhibitor of apoptosis) as a inhibition of apoptosis One of the members of inhibitor of apoptosis protein (IAP) plays a oncogene role in the development of tumor, which is closely related to the occurrence, development, apoptosis, migration, invasion and metastasis of tumor. We used miRNA chip technique to screen the differential miRNA expression of human gastric cancer cells in dads, and found miR-7 for the miR-7 of the human gastric cancer cells after dads treatment. At the same time, qRT-PCR showed that miR-7 was significantly down regulated in gastric cancer tissue and gastric cancer cell lines. The proteomics found 24 different proteins such as XIAP and other proteins, and demonstrated that XIAP was highly expressed in many human gastric cancer cells, and XIAP was obviously down regulated after dads treatment of gastric cancer cells. This experiment was divided into three parts to discuss miR-7 and XIAP to human gastric cancer cells. The effect of proliferation and invasion and the relationship between the two. Part one: Dads up-regulation miR-7 inhibits the proliferation and invasion of human gastric cancer cells: To explore the effect of dads and miR-7 on the proliferation and invasion of human gastric cancer cells. Methods: to construct miR-7 high expression and silent virus vector, and to establish miR-7 stable and silent SGC-7901 and hgc-27 through cell transfection. Cell lines were treated by 30mg/ldads, and then the effects of dads and miR-7 on the proliferation and invasion of gastric cancer cells were analyzed by qRT-PCR, CCK-8, flat clones, flow cytometry, scratching experiments, migration experiments, invasive experiments and nude mice. Results: qRT-PCR confirmed the successful construction of miR-7 stable high expression and silent SGC-7901 and hgc-27. The proliferation of SGC-7901 and hgc-27 cells was significantly inhibited by dads, and the proliferation of SGC-7901 and hgc-27 cells could be inhibited by the higher expression of.Dads and miR-7 in each group of cells, inducing apoptosis and cell cycle arrest and inhibiting the formation of SGC-7901 cells. The scratch test and Transwell migration and invasion showed that the high expression of dads and miR-7 could significantly inhibit the expression of dads and miR-7. The proliferation and invasion ability of SGC-7901 and hgc-27 cells in nude mice showed that the tumor growth of dads group and miR-7 high expression group was slower, and the tumor body was smaller than that of the control group (P0.05). The miR-7 silence showed the contrary results with the high expression of miR-7. All the experiments showed that the high expression of miR-7 could inhibit the proliferation of the cells with dads. Migration, invasion, apoptosis, tumor growth, induction of cell apoptosis and cycle arrest, and conversely, miR-7 silencing can antagonize the effect of dads. The second part: Dads down XIAP inhibits the proliferation and invasion of human gastric cancer cells: explore the effects of dads and XIAP on the proliferation and invasion of human gastric cancer cells. Methods: immunohistochemical detection of gastric cancer group in tissue microarray The expression of XIAP, XIAP high expression virus vector and silent plasmid were constructed, SGC-7901 and hgc-27 cell lines were stable and highly expressed and silent through cell transfection, and the cells were treated by 30mg/ldads, and then qRT-PCR, Westernblot, cell immunofluorescence, CCK-8, flat clones, flow cytometry, scratching experiments, and migrated experiments were carried out. The effect of dads and XIAP on the proliferation and invasion of human gastric cancer cells was analyzed by migration experiments, invasive experiments and nude mice. Results: the positive expression of XIAP in gastric cancer was strongly positive, the positive parts were mainly in the cytoplasm, while the normal epithelium and atypical hyperplasia epithelium were negative or partial positive, and the difference of positive expression rate was statistically significant (P0.05). QRT-PCR confirmed the successful construction of XIAP and hgc-27 cell lines with high expression and silence, and dads could downregulate the expression of XIAP in each group of cells.Westernblot experiments also showed that XIAP silenced SGC-7901 and hgc-27 cells XIAP expression decreased, while XIAP high expression group increased expression. It was also found that the positive expression of XIAP was mainly in the cytoplasm. The expression level of XIAP and the XIAP silence could significantly inhibit the proliferation of SGC-7901 and hgc-27 cells, induce apoptosis and cell cycle arrest of SGC-7901 cells and inhibit the formation of SGC-7901 cell clones. The scratch test and Transwell migration and invasion experiments showed that dads and XIAP were used. XIAP silencing could significantly inhibit the migration and invasion of SGC-7901 and hgc-27 cells. The tumorigenicity test in nude mice also showed that the tumor growth of the dads group and the XIAP silent group was slower, and the tumor tumor body was smaller than the control group (P0.05). These experimental results were consistent with the high expression of miR-7, and the high expression of XIAP showed the opposite knot with the XIAP silence. Moreover, all experiments showed that XIAP silencing could cooperate with dads to inhibit cell proliferation, migration, invasion, apoptosis and cell cycle arrest, and inhibit the growth of xenografts. At the same time, it can also antagonize the role of XIAP overexpression. The third part: miR-7 targeting the negative regulation of XIAP expression: explore the targeting relationship between miR-7 and XIAP. Method: mirn The abase database searches the mature miR-7 sequence and queries the xiap3 'UTR sequence from the GenBank, uses the online database diana-microt-cds, targetscan and Miranda to predict the target gene, then constructs the xiap-3' utr-wt and xiap-3 'utr-mt double luciferase reporter gene system, and CO transfects the cells with the high expression vector and detects the fluorescent cells. In the end, qRT-PCR and Westernblot were used to detect the expression of xiapmrna and protein in SGC-7901 and hgc-27 cells with high expression and silence by qRT-PCR and Westernblot. Finally, the target relationship between miR-7 and XIAP was determined. Results: on the basis of obtaining miR-7 mature sequence and XIAP 3 'UTR sequence, bioinformatics analysis found miR-7 and minerals. 3 'UTR had multiple binding sites and three online software predicted that XIAP was one of the target genes of miR-7, and the dual luciferase reporter gene system confirmed that miR-7 and XIAP 3' UTR wild-type co transfected SGC-7901 cells, and the fluorescence values were significantly lower than those of the control group; miR-7 stable and high apparent SGC-7901 and HGC-27 cells XIAP mRNA and protein expression water. The average down-regulation of SGC-7901 and HGC-27 cells XIAP mRNA and protein in HGC-27 cells all up. Conclusion: 1.DADS can up regulate the proliferation of human gastric cancer cells with miR-7 inhibition. Migration and invasion and growth of transplanted tumor and induction of apoptosis and cycle arrest.2.DADS can reduce the proliferation of XIAP suppressing human gastric cancer cells, migration and invasion and xenografts. Long and induced apoptosis and cycle arrest.3.miR-7 target negatively regulate XIAP mRNA and protein expression.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2

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