miR-711通过CD44调控胃癌细胞EMT

发布时间：2018-01-07 22:03

本文关键词：miR-711通过CD44调控胃癌细胞EMT　出处：《南华大学》2016年硕士论文　论文类型：学位论文

更多相关文章： miR-711 CD44 EMT 胃癌细胞

【摘要】：目的:在课题组前期研究的基础上,进一步研究miR-711对CD44的调控作用及其与胃癌细胞发生EMT的相关性,初步揭示miR-711对胃癌侵袭转移的调节机制。方法:1)使用TargetScan靶基因预测数据库检索miR-711与CD44 3'UTR的结合位点,mi R-711 mimics与CD44 3'UTR质粒共转染293T细胞,荧光素酶报告基因实验检测荧光素酶活性,验证miR-711对CD44的靶向调控作用。2)通过脂质体转染法将miR-711过表达(miR-711 mimics)、miR-711抑制表达(miR-711 inhibitors)、miRNA空质粒(miRNA-NC)分别转染人胃癌细胞株MGC-803及SGC-7901,以miRNA空质粒转染组作为阴性对照组,以空白转染组作为空白对照组,转染48小时后在荧光显微镜下观察转染效率,Western Blot实验检测各组CD44蛋白的表达量。3)通过脂质体转染将CD44干扰质粒(沉默组)、CD44空质粒转染人胃癌细胞株MGC-803及SGC-7901,以CD44空质粒作为阴性对照组,以空白转染组作为空白对照组,转染48小时后在荧光显微镜下观察转染效率,Western Blot实验检测各组CD44蛋白的表达量及EMT相关分子标志蛋白E-cadherin、Vimentin的表达量。结果:1)生物信息学分析结果显示miR-711与CD44 3'UTR之间存在结合位点;荧光素酶报告基因实验结果显示,与对照组相比,miR-711 mimics与CD44 3'UTR荧光素酶报告质粒共转染细胞后,相对荧光素酶活性减少,差异有统计学意义(p0.05)。2)人胃癌细胞株MGC-803、SGC-7901瞬时转染miR-711过表达及抑制表达质粒后,Western Blot结果均显示,与miR-711 inhibitors组、阴性对照组及空白对照组相比,miR-711 mimics组CD44蛋白表达量减少,差异有统计学意义(p0.05);与阴性对照组及空白对照组相比,miR-711inhibitors组CD44蛋白表达量增多,差异有统计学意义(p0.05);阴性对照组与空白对照组相比,差异无统计学意义(p0.05)。3)人胃癌细胞株MGC-803、SGC-7901瞬时转染CD44干扰质粒后,Western Blot结果均显示,与阴性组及空白组相比,沉默组CD44蛋白表达量减少,间质标志物Vimentin蛋白表达量减少,上皮标志物E-cadherin蛋白表达量增多,差异有统计学意义(p0.05),阴性组与空白组相比,差异无统计学意义(p0.05)。结论:1、CD44为miR-711的靶基因之一;2、miR-711可通过下调CD44的表达抑制人胃癌细胞株MGC-803、SGC-7901发生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.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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