本文選題：miR-1284 + 胃癌��； 參考：《廣西醫(yī)科大學(xué)》2016年博士論文

【摘要】：1背景與目的胃癌是我國(guó)最常見的消化道惡性腫瘤,年患病率和死亡率均為世界平均水平的2倍以上。目前臨床上主要利用手術(shù)、化療和放療等綜合手段治療胃癌,但胃癌總體生存率并沒有明顯改善,特別是進(jìn)展期胃癌出現(xiàn)侵襲及轉(zhuǎn)移后不僅加重機(jī)體損害而且疾病預(yù)后惡化。目前,傳統(tǒng)的腫瘤藥物治療一般是指化學(xué)治療,具有不分?jǐn)澄�、毒性較大的特點(diǎn),分子靶向治療比傳統(tǒng)的化療特異性強(qiáng)、毒副反應(yīng)小。以EFGR和ALK基因突變的非小細(xì)胞肺癌治療為例,過去傳統(tǒng)的化療有效率始終徘徊在20%到30%左右,生存率在9~10個(gè)月,采用抑制此類基因突變的分子靶向藥物,治療有效率能翻一番,超過50%~60%,生存率可延長(zhǎng)至18個(gè)月。因此,靶向治療可能成為未來腫瘤的個(gè)體化治療的關(guān)鍵,探尋細(xì)胞調(diào)控的關(guān)鍵分子靶點(diǎn)已成為當(dāng)前腫瘤治療研究的熱點(diǎn)。微小RNA((micro RNA,mi RNA))作為內(nèi)源性非編碼單鏈小RNA,其具有高效調(diào)控癌基因或抑癌基因的生物學(xué)特性,與腫瘤細(xì)胞的生長(zhǎng)、侵襲和凋亡等密切相關(guān),被認(rèn)為是高效靶向調(diào)控細(xì)胞途徑之一。最新的胃癌mi RNA表達(dá)譜芯片篩選研究報(bào)道了與胃癌相關(guān)的多個(gè)mi RNA,其中報(bào)道了mi R-1284在胃癌淋巴結(jié)轉(zhuǎn)移陽性組織比原胃癌組織表達(dá)少,然而,對(duì)mi R-1284在胃癌發(fā)生發(fā)展中的功能和作用機(jī)制并未見有深入研究和探討。為了驗(yàn)證芯片結(jié)果的準(zhǔn)確性,本研究擬通過熒光定量PCR方法檢測(cè)mi R-1284在胃正常細(xì)胞株、胃癌細(xì)胞系、胃癌組織和癌旁正常組織的表達(dá)情況,明確其在胃癌中的表達(dá),并分析其與患者年齡、組織學(xué)類型、淋巴結(jié)轉(zhuǎn)移、遠(yuǎn)處轉(zhuǎn)移、病理分期等的相關(guān)性,明確其在胃癌臨床診斷和預(yù)后中的意義。同時(shí)構(gòu)建mi R-1284慢病毒載體,觀察目的mi R-1284在體內(nèi)外對(duì)胃癌腫瘤細(xì)胞的功能作用,利用基因芯片、雙熒光素酶報(bào)告基因?qū)嶒?yàn)、實(shí)時(shí)定量PCR和Western Blot蛋白印跡實(shí)驗(yàn)檢測(cè)靶基因。明確目的mi R-1284與其靶基因在胃癌中的關(guān)系,闡明mi R-1284在胃癌發(fā)生發(fā)展中的功能和作用機(jī)制。2方法2.1應(yīng)用Trizol試劑提取胃癌組織和細(xì)胞的總RNA,應(yīng)用熒光定量PCR方法檢測(cè)mi R-1284在胃正常細(xì)胞株、胃癌細(xì)胞系、胃癌組織和癌旁正常組織的表達(dá)情況,并分析其與患者年齡、組織學(xué)類型、淋巴結(jié)轉(zhuǎn)移、遠(yuǎn)處轉(zhuǎn)移、病理分期等的相關(guān)性。2.2構(gòu)建包含目的mi R-1284的熒光慢病毒載體,陽性重組克隆進(jìn)行測(cè)序驗(yàn)證,收集重組過表達(dá)目的mi R-1284的病毒顆粒;Transwell法鑒定不同胃癌細(xì)胞的遷移和侵襲能力;采用目的mi R-1284慢病毒顆粒和陰性對(duì)照病毒載體分別轉(zhuǎn)染胃癌細(xì)胞MGC-803和SGC-7901,嘌呤霉素篩選過表達(dá)mi R-1284胃癌穩(wěn)定細(xì)胞株,熒光定量PCR檢測(cè)轉(zhuǎn)染細(xì)胞株mi R-1284的表達(dá);Transwell法和劃痕實(shí)驗(yàn)檢測(cè)mi R-1284對(duì)胃癌細(xì)胞遷移侵襲能力的影響;CCK-8法檢測(cè)mi R-1284對(duì)胃癌細(xì)胞增殖能力的影響;流式細(xì)胞術(shù)檢測(cè)mi R-1284對(duì)胃癌細(xì)胞周期和凋亡的影響;Hoechst染色法和AO-EB雙染色法檢測(cè)mi R-1284對(duì)胃癌細(xì)胞凋亡的影響。2.3將轉(zhuǎn)染后穩(wěn)定細(xì)胞株的細(xì)胞懸液200μl(6×106個(gè)/ml)每只,在裸鼠左側(cè)腋窩皮下注射構(gòu)建人胃癌裸鼠皮下移植瘤模型,隔2天1次,用游標(biāo)卡尺測(cè)量腫瘤體長(zhǎng)徑(a)和短徑(b),計(jì)算相對(duì)瘤體體積(RTV)并繪制腫瘤生長(zhǎng)曲線,21天后脫頸椎法處死裸鼠,完整切取腫瘤組織并用HE和TUNEL染色確認(rèn)腫瘤組織和檢測(cè)組織細(xì)胞凋亡情況;將轉(zhuǎn)染后穩(wěn)定細(xì)胞株的細(xì)胞懸液200μl(4×105個(gè)/ml)每只,注射入裸鼠尾靜脈構(gòu)建胃癌裸鼠轉(zhuǎn)移模型,第36天后脫頸椎法處死裸鼠,完整切取裸鼠肺部和肝臟組織,并用HE染色檢測(cè)胃癌肝肺轉(zhuǎn)移情況。2.4采用全基因表達(dá)譜芯片檢測(cè)mi R-1284過表達(dá)慢病毒轉(zhuǎn)染胃癌細(xì)胞顯著差異表達(dá)的基因;生物信息學(xué)預(yù)測(cè)軟件預(yù)測(cè)目的mi R-1284的靶基因,利用雙熒光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證靶基因;應(yīng)用實(shí)時(shí)定量PCR和Western Blot蛋白印跡實(shí)驗(yàn)檢測(cè)靶基因EIF4A1及其下游基因cMyc、MMP12、JUN、CDH1的m RNA及蛋白表達(dá),應(yīng)用免疫組化法檢測(cè)胃癌標(biāo)本靶基因EIF4A1蛋白表達(dá)。3結(jié)果3.1 q RT-PCR結(jié)果顯示,74例胃癌患者中,26例胃癌患者的胃癌組織mi R-1284表達(dá)量高于癌旁正常組織,占35.14%;48例胃癌患者的胃癌組織mi R-1284表達(dá)量低于癌旁正常組織,占64.86%,mi R-1284在胃癌組織中的表達(dá)量明顯低于胃癌旁的正常組織(P㩳0.05),mi R-1284在胃癌細(xì)胞株AGS、HGC-27、MGC-803和SGC-7901的表達(dá)量低于在胃上皮細(xì)胞株GES-1的表達(dá)量(P㩳0.05)。mi R-1284表達(dá)量與胃癌患者的年齡、性別、組織學(xué)分級(jí)、TNM分期和淋巴結(jié)轉(zhuǎn)移的相關(guān)無統(tǒng)計(jì)學(xué)顯著意義(P㧐0.05),但mi R-1284表達(dá)量與胃癌的腫瘤大小及遠(yuǎn)處轉(zhuǎn)移的相關(guān)有統(tǒng)計(jì)學(xué)顯著意義(P㩳0.05)。3.2陽性重組克隆測(cè)序結(jié)果顯示mi R-1284過表達(dá)載體重組序列無誤;Transwell實(shí)驗(yàn)檢測(cè)結(jié)果顯示,與胃癌細(xì)胞株AGS、HGC-27相比,胃癌細(xì)胞株MGC-803和SGC-7901的遷移和侵襲細(xì)胞數(shù)較多(P㩳0.05);熒光定量PCR檢測(cè)結(jié)果顯示,mi R-1284在胃癌細(xì)胞株MGC-803和SGC-7901中實(shí)驗(yàn)組的表達(dá)量高于陰性對(duì)照組的表達(dá)量(P㩳0.05);Transwell實(shí)驗(yàn)檢測(cè)結(jié)果顯示,與陰性對(duì)照組相比,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株MGC-803和SGC-7901的遷移和侵襲細(xì)胞數(shù)減少(P㩳0.05);劃痕實(shí)驗(yàn)結(jié)果顯示,胃癌細(xì)胞株SGC-7901在48小時(shí)時(shí)mi R-1284組細(xì)胞遷移間距為(312.42±16.87)μm較NC(陰性對(duì)照)組為(123.63±17.69)μm遷移細(xì)胞間距大,胃癌細(xì)胞株MGC-803在48小時(shí)時(shí)mi R-1284組細(xì)胞遷移間距為(112.46±11.18)μm較NC(陰性對(duì)照)組為(28.74±8.62)μm遷移細(xì)胞間距大,差異均有統(tǒng)計(jì)學(xué)顯著意義(P㩳0.05);CCK-8實(shí)驗(yàn)檢測(cè)結(jié)果顯示與陰性對(duì)照組相比,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株MGC-803和SGC-7901的增殖能力降低(P㩳0.05);流式細(xì)胞儀檢測(cè)結(jié)果顯示,與陰性對(duì)照組相比,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株MGC-803和SGC-7901的細(xì)胞周期阻滯在G0/G1期,S期細(xì)胞比例減少,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株MGC-803和SGC-7901的細(xì)胞凋亡率增加,差異均有統(tǒng)計(jì)學(xué)顯著意義(P0.05);Hoechst和AO-EB雙染檢測(cè)結(jié)果顯示,與陰性對(duì)照組相比,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株MGC-803和SGC-7901的細(xì)胞凋亡率增加,差異均有統(tǒng)計(jì)學(xué)顯著意義(P0.05)。3.3裸鼠皮下移植瘤模型腫瘤體積測(cè)量的結(jié)果顯示,與陰性對(duì)照組相比,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株SGC-7901、MGC-803的裸鼠皮下移植瘤模型腫瘤體積較小(P㩳0.05),HE染色確認(rèn)皮下移植瘤組織為惡性胃癌組織,TUNEL檢測(cè)結(jié)果顯示mi R-1284過表達(dá)的實(shí)驗(yàn)組SGC-7901、MGC-803的裸鼠皮下移植瘤凋亡率增加(P㩳0.05);轉(zhuǎn)移瘤模型的肝肺組織病理檢查結(jié)果顯示,mi R-1284過表達(dá)的實(shí)驗(yàn)組的肺轉(zhuǎn)移率為10.00%,與陰性對(duì)照組的肺轉(zhuǎn)移率70.00%相比,mi R-1284過表達(dá)的實(shí)驗(yàn)組胃癌細(xì)胞株MGC-803的細(xì)胞肺轉(zhuǎn)移率降低(P㩳0.05)。3.4全基因表達(dá)譜芯片檢測(cè)結(jié)果顯示,按差異基因的篩選條件設(shè)定為log2"#Flod change"#"g1.5且P㩳0.05,共有143個(gè)基因?yàn)椴町愋员磉_(dá),上調(diào)基因數(shù)97個(gè),下調(diào)基因數(shù)46個(gè);Mi Randa、Target Scan、micro RNA軟件預(yù)測(cè)mi R-1284的靶基因交集為138個(gè)基因;芯片表達(dá)差異基因和生物信息學(xué)網(wǎng)站數(shù)據(jù)庫預(yù)測(cè)靶基因的交集為EIF4A1、KLF10、C8orf4和SUMO1基因;雙熒光素酶報(bào)告實(shí)驗(yàn)結(jié)果顯示:EIF4A1 3'UTR-NC+mi RNA組和EIF4A1 3'UTR+mi RNA組的熒光素酶活性相比,差異有統(tǒng)計(jì)學(xué)顯著意義(P0.05);EIF4A1 3'UTR-NC+mi RNA組和EIF4A1 3'UTR-MU+mi RNA組的熒光素酶活性相比,差異無統(tǒng)計(jì)學(xué)顯著意義(P0.05);EIF4A13'UTR+mi RNA組和EIF4A1 3'UTR-MU+mi RNA組熒光素酶活性相比,EIF4A1 3'UTR-MU+mi RNA組的熒光素酶活性明顯增加,差異有統(tǒng)計(jì)學(xué)顯著意義(P0.05);熒光定量PCR和Western blotting實(shí)驗(yàn)結(jié)果顯示:與陰性對(duì)照組比較,上調(diào)胃癌細(xì)胞株MGC-803、SGC-7901的mi R-1284表達(dá)后,實(shí)驗(yàn)組靶基因EIF4A1和c-Myc、MMP12、JUN的m RNA和蛋白表達(dá)降低,CDH1的m RNA和蛋白表達(dá)明顯增加,差異均有統(tǒng)計(jì)學(xué)顯著意義(P0.05);免疫組化結(jié)果顯示,人胃癌組織中EIF4A1的蛋白表達(dá)量較匹配的癌旁正常組織低,差異有統(tǒng)計(jì)學(xué)顯著意義(P0.05)。4結(jié)論4.1 mi R-1284在胃癌細(xì)胞株的表達(dá)水平低于正常胃上皮細(xì)胞株GES-1,在胃癌組織中的表達(dá)量明顯低于胃癌組織,且mi R-1284的表達(dá)量與胃癌的腫瘤大小及遠(yuǎn)處轉(zhuǎn)移呈負(fù)相關(guān)。4.2成功構(gòu)建人mi R-1284過表達(dá)慢病毒載體和mi R-1284過表達(dá)的胃癌細(xì)胞株MGC-803、SGC-7901,mi R-1284過表達(dá)抑制胃癌細(xì)胞MGC-803、SGC-7901的遷移、侵襲、增殖能力,促進(jìn)胃癌細(xì)胞MGC-803、SGC-7901的凋亡,使MGC-803、SGC-7901的細(xì)胞周期阻滯在G0/G1期和S期細(xì)胞比例減少。4.3 mi R-1284過表達(dá)抑制裸鼠皮下移植瘤模型腫瘤的生長(zhǎng)速度,促進(jìn)腫瘤組織的細(xì)胞凋亡,抑制裸鼠轉(zhuǎn)移瘤模型的腫瘤細(xì)胞轉(zhuǎn)移能力。4.4 mi R-1284過表達(dá)對(duì)胃癌細(xì)胞基因譜表達(dá)有影響,EIF4A1是mi R-1284的直接靶基因,mi R-1284過表達(dá)可使胃癌細(xì)胞株MGC-803、SGC-7901的EIF4A1和c-Myc、MMP12、JUN基因的m RNA和蛋白表達(dá)降低,CDH1的m RNA和蛋白表達(dá)增加。4.5在胃癌患者中EIF4A1的蛋白表達(dá)與mi R-1284表達(dá)量呈負(fù)相關(guān),推測(cè)mi R-1284過表達(dá)極有可能直接通過EIF4A1/CDH1/JUN/MMP12和EIF4A1/c-Myc信號(hào)通路抑制胃癌的進(jìn)展。創(chuàng)新點(diǎn)1體外雙熒光素酶報(bào)告實(shí)驗(yàn)是研究micro RNA和靶基因間相互作用的有效手段,本研究采用了此技術(shù)驗(yàn)證mi R-1284和EIF4A1的相互作用,揭示了mi R-1284直接靶向調(diào)控EIF4A1的客觀存在性。2 micro RNA是機(jī)體內(nèi)調(diào)控基因表達(dá)的重要分子,與腫瘤的發(fā)生發(fā)展、侵襲轉(zhuǎn)移密切相關(guān),本課題從mi R-1284在胃癌中的表達(dá)入手,通過體外和體內(nèi)兩個(gè)方面進(jìn)行研究,揭示和闡明了mi R-1284靶向調(diào)控EIF4A1對(duì)胃癌進(jìn)展的影響其分子機(jī)制。
[Abstract]:1 background and objective gastric cancer is the most common malignant tumor in the digestive tract in China. The annual prevalence rate and mortality rate are more than 2 times more than the world average. At present, the main clinical use of surgery, chemotherapy and radiotherapy for the treatment of gastric cancer, but the overall survival rate of gastric cancer is not significantly improved, especially in the progression of gastric cancer after the invasion and metastasis. It not only aggravates the damage of the body but also worsens the prognosis of the disease. At present, the traditional treatment of tumor drugs generally refers to the chemical treatment, which has the characteristics of not dividing the enemy from the enemy, having a large toxicity, and the molecular targeting therapy is stronger than the traditional chemotherapy, and the toxic side effects are small. The traditional chemotherapy has been used as an example with the treatment of EFGR and ALK gene mutations in non small cell lung cancer. The efficiency is always around 20% to 30%, the survival rate is in 9~10 months. The effective rate can be doubled, the survival rate can be more than 50%~60%, and the survival rate can be extended to 18 months. Therefore, the target therapy may be the key to the individualized treatment of the tumor in the future, and the key molecular targets for cell regulation are explored. RNA (micro RNA (MI RNA)), as an endogenous non coding single strand RNA, has a close relationship with the growth, invasion and apoptosis of tumor cells, which is considered to be one of the highly effective targeting cell pathways. The latest gastric cancer mi RNA table is considered as an endogenous non coding single strand RNA. A number of MI RNA related to gastric cancer were reported by the spectrum chip screening. It was reported that the expression of MI R-1284 in gastric cancer lymph node metastasis was less than that of the original gastric carcinoma. However, the function and mechanism of MI R-1284 in the development of gastric cancer had not been further studied and discussed. The expression of MI R-1284 in normal gastric cell line, gastric cancer cell line, gastric cancer tissue and normal tissue were detected by fluorescence quantitative PCR, and its expression in gastric cancer was determined. The correlation with age, histological type, lymph node metastasis, distant metastasis and disease stage was analyzed, and the clinical diagnosis and diagnosis of gastric cancer were also made. The significance of MI R-1284 lentivirus vector was also constructed to observe the function of MI R-1284 on gastric cancer cells in vivo and in vivo and in vivo and in vivo, using gene chip, double luciferase reporter gene test, real-time quantitative PCR and Western Blot blot test to detect target genes. The purpose of this study was to determine the purpose of MI R-1284 and its target gene in gastric cancer. To elucidate the function and mechanism of MI R-1284 in the development of gastric cancer.2 method 2.1 using Trizol reagent to extract the total RNA of gastric cancer tissues and cells, and to detect the expression of MI R-1284 in normal gastric cell lines, gastric cancer cell lines, gastric cancer tissues and normal tissues by Trizol reagent, and to analyze the age and histology of the patients with the gastric cancer cell line, gastric cancer tissue and the carcinoma adjacent to the cancer. Type, lymph node metastasis, distant metastasis, pathological staging, and other correlation.2.2 constructs a fluorescent lentivirus carrier containing target mi R-1284, positive recombinant clones are sequenced and verified, and the recombinant virus particles that express mi R-1284 are collected; the migration and invasion ability of different gastric cancer cells are identified by Transwell method, and the target mi R-1284 lentivirus is used. MGC-803 and SGC-7901 were transfected by granular and negative control virus vectors respectively. The expression of MI R-1284 gastric cancer cell line was screened by purinomycin, the expression of MI R-1284 in the transfected cell line was detected by fluorescence quantitative PCR; the influence of MI R-1284 on the invasion ability of gastric cancer cells was detected by Transwell and scratch test; CCK-8 method was used to detect mi The effect of MI R-1284 on the cell cycle and apoptosis of gastric cancer cells by flow cytometry; the effect of Hoechst staining and AO-EB double staining on the apoptosis of gastric cancer cell line.2.3,.2.3 will be used for 200 mu (6 x 106 /ml) per mouse of stable cell suspension after transfection and subcutaneous injections in the left armpit of nude mice The subcutaneous tumor model of nude mice was built. 2 days 1 times, the tumor body length (a) and short diameter (b) were measured with the vernier caliper. The relative tumor volume (RTV) was calculated and the tumor growth curve was plotted. After 21 days, the nude mice were killed by deactivation of the cervical vertebra. The tumor tissue was stained with HE and TUNEL, and the apoptosis of the tissue cells was confirmed. The cell suspension of the stable cell line was 200 L (4 x 105 /ml) each, and the nude mice were injected into the tail vein of the nude mice to construct the metastatic model of gastric cancer in nude mice. After thirty-sixth days, the nude mice were killed by the deactivation of the cervical vertebra. The lung and liver tissues of the nude mice were completely cut out, and the liver and lung metastasis of the gastric cancer was detected by HE staining. The full gene expression spectrum chip was used to detect the slow expression of MI R-1284. The gene was transfected by the virus, and the target gene of MI R-1284 was predicted by the bioinformatics prediction software, and the target gene was verified by the double luciferase reporter gene experiment. The target gene EIF4A1 and its downstream gene cMyc, MMP12, JUN, CDH1 m RNA and protein were detected by real-time quantitative PCR and Western Blot blotting. The results of the expression of the target gene EIF4A1 protein expression of.3 3.1 Q RT-PCR by immunohistochemistry showed that in 74 cases of gastric cancer, the expression of MI R-1284 in gastric cancer tissues of 26 cases of gastric cancer was higher than that of normal tissue, accounting for 35.14%, and the expression of MI R-1284 in gastric cancer tissues of 48 cases of gastric cancer was lower than that of normal tissue adjacent to cancer, accounting for 64.86%, MI. The expression of R-1284 in gastric cancer tissues was significantly lower than that of normal tissue adjacent to gastric cancer (P? 0.05). The expression of MI R-1284 in gastric cancer cell lines AGS, HGC-27, MGC-803 and SGC-7901 was lower than that in gastric epithelial cell strain GES-1 (P? 0.05).Mi R-1284 expression and age, sex, histological grading, staging and lymph node metastasis of gastric cancer patients. The correlation was not statistically significant (P? 0.05), but the expression of MI R-1284 was significantly correlated with the tumor size and distant metastasis of gastric cancer (P? 0.05).3.2 positive recombinant cloning sequencing results showed that MI R-1284 overexpressed vector recombinant sequence was unmistakable; Transwell experimental results showed that gastric cancer was compared with AGS, HGC-27, gastric cancer. The number of cell migration and invasion of cell lines MGC-803 and SGC-7901 was more (P? 0.05), and the results of fluorescence quantitative PCR detection showed that the expression of MI R-1284 in the gastric cancer cell line MGC-803 and SGC-7901 was higher than that of the negative control group (P? 0.05). The Transwell test results showed that MI R-1284 was over expressed in comparison with the negative control group. The number of migration and invasion cells of gastric cancer cell lines MGC-803 and SGC-7901 decreased (P? 0.05), and the results of scratch test showed that the cell migration interval of the MI R-1284 group was (312.42 + 16.87) mu m at 48 hours and (123.63 + 17.69) mu m migrating cell spacing in the mi R-1284 group, and MGC-803 of the gastric cancer cell line was mi R at 48 hours. The cell migration distance of group -1284 was (112.46 + 11.18) mu m than that of NC (28.74 + 8.62) mu m (28.74 + 8.62). The difference was significant (P? 0.05). The results of CCK-8 test showed that the proliferation ability of gastric cancer cell line MGC-803 and SGC-7901 was lower than that of negative control group (P? 0.05). The results of flow cytometry showed that compared with the negative control group, the cell cycle block of gastric cancer cell line MGC-803 and SGC-7901 in the MI R-1284 overexpressed experimental group was in G0/G1 stage, and the proportion of S phase cells decreased, and the apoptosis rate of MGC-803 and SGC-7901 increased in the experimental group of MI R-1284, and the difference was statistically significant. (P0.05), the results of Hoechst and AO-EB double staining showed that compared with the negative control group, the apoptosis rate of MGC-803 and SGC-7901 in the experimental group of gastric cancer cells with overexpression of MI R-1284 increased, and the difference was statistically significant (P0.05) in.3.3 nude mice, the results of the tumor volume measurement of the subcutaneous transplanted tumor model showed that the MI R-128 compared with the negative control group. 4 over expression of the experimental group of gastric cancer cell line SGC-7901, MGC-803 nude mice subcutaneous tumor model tumor volume smaller (P? 0.05), HE staining confirmed that subcutaneous transplantation tumor tissue was malignant gastric cancer tissue, TUNEL test results showed that MI R-1284 overexpressed experimental group SGC-7901, MGC-803 nude mice subcutaneous transplantation tumor apoptosis rate increased (P? 0.05); metastatic tumor model The pathological examination of liver and lung tissue showed that the lung metastasis rate of the MI R-1284 overexpressed experimental group was 10%, compared with the lung metastasis rate of the negative control group 70%. The lung metastasis rate of the gastric cancer cell line MGC-803 in the MI R-1284 overexpressed group was reduced (P? 0.05).3.4 whole gene expression spectrum chip detection results showed that the differential gene was screened by the differential gene screening. The condition was set as log2 "#Flod change", "g1.5 and P? 0.05, there were 143 genes of differential expression, 97 up-regulated genes and 46 down regulated genes; Mi Randa, Target Scan, micro RNA software predicted the target gene of MI to be 138 genes, and the intersection of chip expression differentially gene and bioinformatics website database predicted target gene. F4A1, KLF10, C8orf4 and SUMO1 genes, and the results of the double luciferase reporter experiment showed that the difference of luciferase activity of EIF4A1 3'UTR-NC+mi RNA group and EIF4A1 3'UTR+mi RNA group was statistically significant (P0.05), and there was no significant difference in the activity of luciferase activity. (P0.05); the luciferase activity of the EIF4A1 3'UTR-MU+mi RNA group was significantly increased in the group of EIF4A13'UTR+mi RNA and the EIF4A1 3'UTR-MU+mi RNA group, and the difference was statistically significant (P0.05). After the expression of R-1284, the target gene EIF4A1 and c-Myc, MMP12, JUN, m RNA and protein expression decreased, the m RNA and protein expression of CDH1 increased significantly, and the difference was statistically significant (P0.05). The immunohistochemical results showed that the expression of EIF4A1 protein in human gastric carcinoma tissue was lower than that of the normal tissue adjacent to the cancer, and the difference was statistically significant. The expression level of P0.05.4 conclusion 4.1 mi R-1284 in gastric cancer cell line is lower than that of normal gastric epithelial cell strain GES-1, and the expression in gastric cancer tissue is significantly lower than that of gastric cancer tissue, and the expression of MI R-1284 is negatively correlated with the size and distant metastasis of gastric cancer, which is a successful construction of the MI R-1284 overexpressed lentivirus carrier and MI R-1284. The expression of MGC-803, SGC-7901, and MI R-1284 overexpressed the migration, invasion, and proliferation of gastric cancer cell MGC-803, SGC-7901, and promoted the apoptosis of MGC-803 and SGC-7901 in gastric cancer cells, so that the cell cycle block in MGC-803 and SGC-7901 decreased in the G0/G1 phase and the S phase cells to inhibit the subcutaneous tumor model in nude mice The growth rate of tumor, promoting the apoptosis of tumor tissue, inhibiting the metastasis ability of tumor cells in the metastatic tumor model of nude mice,.4.4 mi R-1284 overexpression affects gene expression of gastric cancer cells. EIF4A1 is the direct target gene of MI R-1284. Mi R-1284 overexpression can make the gastric cancer cell strain MGC-803, SGC-7901 EIF4A1 and c-Myc The expression of M RNA and protein decreased, CDH1 m RNA and protein expression increased.4.5 expression in gastric cancer patients with MI R-1284 expression in negative correlation. It is presumed that MI R-1284 overexpression may inhibit the progression of gastric cancer directly through EIF4A1/CDH1/JUN/MMP12 and signaling pathway. Innovation point 1 in vitro double Luciferase Report The test is an effective means to study the interaction between micro RNA and target genes. This study uses this technique to verify the interaction between MI R-1284 and EIF4A1, and reveals that the objective existence.2 micro RNA of MI R-1284 targeting EIF4A1 is an important molecule in the regulation of gene expression in the body, which is closely related to the development of the tumor and the invasion and metastasis of the tumor. This topic begins with the expression of MI R-1284 in gastric cancer and through two aspects in vitro and in vivo, reveals and clarifies the molecular mechanism of the effect of MI R-1284 targeting EIF4A1 on the progression of gastric cancer.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.2

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