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miR-711通過CD44調(diào)控胃癌細(xì)胞EMT

發(fā)布時(shí)間:2018-01-07 22:03

  本文關(guān)鍵詞:miR-711通過CD44調(diào)控胃癌細(xì)胞EMT 出處:《南華大學(xué)》2016年碩士論文 論文類型:學(xué)位論文


  更多相關(guān)文章: miR-711 CD44 EMT 胃癌細(xì)胞


【摘要】:目的:在課題組前期研究的基礎(chǔ)上,進(jìn)一步研究miR-711對(duì)CD44的調(diào)控作用及其與胃癌細(xì)胞發(fā)生EMT的相關(guān)性,初步揭示miR-711對(duì)胃癌侵襲轉(zhuǎn)移的調(diào)節(jié)機(jī)制。方法:1)使用TargetScan靶基因預(yù)測(cè)數(shù)據(jù)庫(kù)檢索miR-711與CD44 3'UTR的結(jié)合位點(diǎn),mi R-711 mimics與CD44 3'UTR質(zhì)粒共轉(zhuǎn)染293T細(xì)胞,熒光素酶報(bào)告基因?qū)嶒?yàn)檢測(cè)熒光素酶活性,驗(yàn)證miR-711對(duì)CD44的靶向調(diào)控作用。2)通過脂質(zhì)體轉(zhuǎn)染法將miR-711過表達(dá)(miR-711 mimics)、miR-711抑制表達(dá)(miR-711 inhibitors)、miRNA空質(zhì)粒(miRNA-NC)分別轉(zhuǎn)染人胃癌細(xì)胞株MGC-803及SGC-7901,以miRNA空質(zhì)粒轉(zhuǎn)染組作為陰性對(duì)照組,以空白轉(zhuǎn)染組作為空白對(duì)照組,轉(zhuǎn)染48小時(shí)后在熒光顯微鏡下觀察轉(zhuǎn)染效率,Western Blot實(shí)驗(yàn)檢測(cè)各組CD44蛋白的表達(dá)量。3)通過脂質(zhì)體轉(zhuǎn)染將CD44干擾質(zhì)粒(沉默組)、CD44空質(zhì)粒轉(zhuǎn)染人胃癌細(xì)胞株MGC-803及SGC-7901,以CD44空質(zhì)粒作為陰性對(duì)照組,以空白轉(zhuǎn)染組作為空白對(duì)照組,轉(zhuǎn)染48小時(shí)后在熒光顯微鏡下觀察轉(zhuǎn)染效率,Western Blot實(shí)驗(yàn)檢測(cè)各組CD44蛋白的表達(dá)量及EMT相關(guān)分子標(biāo)志蛋白E-cadherin、Vimentin的表達(dá)量。結(jié)果:1)生物信息學(xué)分析結(jié)果顯示miR-711與CD44 3'UTR之間存在結(jié)合位點(diǎn);熒光素酶報(bào)告基因?qū)嶒?yàn)結(jié)果顯示,與對(duì)照組相比,miR-711 mimics與CD44 3'UTR熒光素酶報(bào)告質(zhì)粒共轉(zhuǎn)染細(xì)胞后,相對(duì)熒光素酶活性減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。2)人胃癌細(xì)胞株MGC-803、SGC-7901瞬時(shí)轉(zhuǎn)染miR-711過表達(dá)及抑制表達(dá)質(zhì)粒后,Western Blot結(jié)果均顯示,與miR-711 inhibitors組、陰性對(duì)照組及空白對(duì)照組相比,miR-711 mimics組CD44蛋白表達(dá)量減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05);與陰性對(duì)照組及空白對(duì)照組相比,miR-711inhibitors組CD44蛋白表達(dá)量增多,差異有統(tǒng)計(jì)學(xué)意義(p0.05);陰性對(duì)照組與空白對(duì)照組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。3)人胃癌細(xì)胞株MGC-803、SGC-7901瞬時(shí)轉(zhuǎn)染CD44干擾質(zhì)粒后,Western Blot結(jié)果均顯示,與陰性組及空白組相比,沉默組CD44蛋白表達(dá)量減少,間質(zhì)標(biāo)志物Vimentin蛋白表達(dá)量減少,上皮標(biāo)志物E-cadherin蛋白表達(dá)量增多,差異有統(tǒng)計(jì)學(xué)意義(p0.05),陰性組與空白組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論:1、CD44為miR-711的靶基因之一;2、miR-711可通過下調(diào)CD44的表達(dá)抑制人胃癌細(xì)胞株MGC-803、SGC-7901發(fā)生EMT。
[Abstract]:Objective: Based on previous studies, further study the relationship between the role of miR-711 in regulation of CD44 and EMT in gastric cancer cells, revealed miR-711 regulating the invasion mechanism of metastasis of gastric cancer. Methods: 1) using the TargetScan target genes predicted binding sites of miR-711 and CD44 3'UTR database, MI R-711 mimics and CD44 3'UTR plasmids were transfected into 293T cells. Luciferase reporter assay of luciferase activity, verification of miR-711 targeted regulation of CD44.2) by liposome transfection over expression of miR-711 (miR-711 mimics), miR-711 (miR-711 inhibitors), inhibit the expression of miRNA empty plasmid (miRNA-NC) were transfected into human gastric cancer cell lines MGC-803 and SGC-7901, to miRNA empty plasmid group as negative control group, the blank transfection group as the control group, after 48 hours of transfection efficiency of transfection was observed under fluorescence microscopy, The expression of.3 protein was detected by CD44 Western Blot in each experiment) by liposome transfection of CD44 plasmid CD44 (silent group), empty plasmid were transfected into human gastric cancer cell lines MGC-803 and SGC-7901, with CD44 empty plasmid as a negative control group, the blank transfection group as the control group, after 48 hours of transfection efficiency of transfection was observed under fluorescence under the microscope, each symbol protein E-cadherin detection of CD44 protein expression and Western Blot experiment of EMT related molecules, the expression of Vimentin. Results: 1) analysis showed the presence of binding sites between miR-711 and CD44 3'UTR bioinformatics; luciferase reporter assay results showed that compared with the control group, miR-711 mimics and CD44 3'UTR luciferase reporter plasmid co transfection, luciferase activity decreased, the difference was statistically significant (P0.05).2) in human gastric cancer cell line MGC-803, SGC-7901 transfected miR-711 Overexpression and inhibition of the expression plasmid, Western Blot showed that, miR-711 and inhibitors group, compared with the negative control group and blank control group, miR-711 mimics group, CD44 protein expression decreased, the difference was statistically significant (P0.05); compared with the negative control group and blank control group, miR-711inhibitors group increased the protein expression of CD44, a statistically significant difference (P0.05); compared with the negative control group and blank control group, the difference was not statistically significant (P0.05).3) in human gastric cancer cell line MGC-803, SGC-7901 transient transfection of CD44 plasmid, Western Blot showed that, compared with the negative group and blank group, silent group CD44 protein expression decreased, mesenchymal marker Vimentin protein expression decreased epithelial marker expression of E-cadherin protein increased, the difference was statistically significant (P0.05), compared with the negative group and blank control group, the difference was not statistically significant (P0.05). Conclusion: 1 CD44 is one of the target genes of miR-711; 2, miR-711 can inhibit the human gastric cancer cell line MGC-803 by downregulating the expression of CD44, and SGC-7901 can occur EMT.

【學(xué)位授予單位】:南華大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2016
【分類號(hào)】:R735.2

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