miR-21-5p影响子宫内膜癌细胞增殖、迁移及侵袭机制的研究
发布时间：2018-10-23 17:55
【摘要】：一、目的本实验旨在探讨miR-21-5p在不同子宫内膜癌细胞Ishikawa及HEC-1A中的表达差异,上调或下调miR-21-5p对细胞增殖、迁移及侵袭的影响。探寻miR-21-5p的直接靶标分子,为子宫内膜癌的诊断及治疗提供新的靶点。二、方法1.Real-time PCR法检测并分析不同子宫内膜癌细胞Ishikawa及HEC-1A中miR-21-5p的表达差异;2.利用脂质体转染法,以miR-21-5p mimics(上调miR-21-5p)及其对照(Negative control)或miR-21-5p inhibitor(下调miR-21-5p)及其对照(Inhibitor NC),分别转染Ishikawa及HEC-1A细胞,Real-time PCR法检测并分析miR-21-5p表达变化;3.CCK8实验检测上调/下调miR-21-5p对Ishikawa及HEC-1A细胞增殖能力的影响;4.Transwell实验及细胞划痕实验分别检测上调/下调miR-21-5p对Ishikawa及HEC-1A细胞迁移及侵袭的影响;5.利用targetscan、mirdb等靶基因预测软件,筛选与侵袭转移相关的mir-21-5p靶基因;6.上调hec-1a细胞中mir-21-5p的表达,real-timepcr检测靶基因(pitx2、vcl、jag1和fbxo11)mrna的变化;在ishikawa和hec-1a细胞中上调/下调mir-21-5p的表达,westernblot检测pitx2及vcl蛋白表达变化;7.构建mir-21-5p靶基因(pitx2、vcl、jag1和fbxo11)3’utr野生型的克隆载体和双荧光素酶重组载体;分别用脂质体瞬时转染,双荧光素酶(dual-luciferase)报告基因实验检测各转染组的双荧光素酶活性。三、结果1.mir-21-5p在hec-1a细胞中的表达明显高于ishikawa细胞;2.mir-21-5pmimics(对照组negativecontrol)转染到ishikawa及hec-1a细胞后,mir-21-5p的表达升高(ishikawa细胞升高140倍,hec-1a升高15倍);mir-21-5pinhibitor(对照组inhibitornc)转染到ishikawa及hec-1a细胞后,mir-21-5p的表达下降(ishikawa细胞降低2倍,hec-1a降低2.5倍);3.上调mir-21-5p能够促进ishikawa和hec-1a细胞的增殖能力,差异有统计学意义(p0.05);下调mir-21-5p对ishikawa和hec-1a的增殖能力无显著影响;4.上调mir-21-5p能够促进ishikawa和hec-1a细胞的迁移及侵袭能力;下调mir-21-5p能抑制ishikawa和hec-1a的迁移及侵袭能力;差异具有统计学意义(p0.05);5.targetscanhuman7.1预测显示mir-21-5p的“种子”区为“uauucga”,并筛选出与侵袭转移相关的mir-21-5p的靶基因pitx2、vcl、jag1和fbxo11;6.上调hec-1a细胞mir-21-5p的表达,靶基因pitx2、vcl、jag1和fbxo11的mrna与对照组相比呈显著降低趋势;上调ishikawa、hec-1a细胞中mir-21-5p的表达pitx2及vcl蛋白的表达降低;相反,下调mir-21-5p的表达时,可促进pitx2和vcl蛋白的表达;7.靶基因3’utr区克隆载体、野生型及突变型重组载体测序结果与预想一致;双荧光素酶结果显示,pitx2、vcl、jag1和fbxo11是mir-21-5p的直接靶基因。四、结论1.miR-21-5p在HEC-1A中的表达显著高于Ishikawa细胞。2.上调miR-21-5p能够促进Ishikawa细胞及HEC-1A细胞的增殖、迁移及侵袭能力。3.本实验证明了JAG1、FBX011、VCL和PITX2等基因是mi R-21-5p的靶基因。
[Abstract]:Objective: to investigate the expression of miR-21-5p in Ishikawa and HEC-1A of different endometrial cancer cells, and to up-regulate or down-regulate the effects of miR-21-5p on cell proliferation, migration and invasion. To explore the direct target molecules of miR-21-5p and to provide a new target for the diagnosis and treatment of endometrial carcinoma. Methods 1.Real-time PCR assay was used to detect and analyze the expression of miR-21-5p in Ishikawa and HEC-1A of different endometrial cancer cells. 2. Using liposome transfection, Ishikawa and HEC-1A cells were transfected with miR-21-5p mimics (up-regulated miR-21-5p), (Negative control) or miR-21-5p inhibitor (down-regulated miR-21-5p) and (Inhibitor NC), respectively, and miR-21-5p expression was detected and analyzed by Real-time PCR assay. Effect of miR-21-5p upregulation / down-regulation on proliferation of Ishikawa and HEC-1A cells by 3.CCK8 assay The effects of up-regulation and down-regulation of miR-21-5p on the migration and invasion of Ishikawa and HEC-1A cells were detected by 4.Transwell assay and cell scratch assay respectively. Targetscan,mirdb and other target gene prediction software was used to screen mir-21-5p target genes related to invasion and metastasis. The expression of mir-21-5p was up-regulated in hec-1a cells, the changes of target gene (pitx2,vcl,jag1 and fbxo11) mrna were detected by real-timepcr, mir-21-5p expression was upregulated / down-regulated in ishikawa and hec-1a cells, and pitx2 and vcl proteins were detected by westernblot. The wild-type clone vector and double luciferase recombinant vector of mir-21-5p target gene (pitx2,vcl,jag1 and fbxo11) 3'utr were constructed, and the double luciferase activity of each transfection group was detected by liposome transient transfection and double luciferase (dual-luciferase) reporter gene experiment. Three Results the expression of 1.mir-21-5p in hec-1a cells was significantly higher than that in ishikawa cells, the expression of mir-21-5p in ishikawa and hec-1a cells was increased after transfection of 2.mir-21-5pmimics (control negativecontrol) to ishikawa and hec-1a cells (140-fold increase in ishikawa cells and 15-fold increase in hec-1a), and the expression of mir-21-5p in ishikawa and hec-1a cells decreased after mir-21-5pinhibitor (control group inhibitornc) was transfected into ishikawa and hec-1a cells. (ishikawa cells decreased by 2 times, hec-1a decreased by 2. 5 times); Upregulation of mir-21-5p could promote the proliferation of ishikawa and hec-1a cells, the difference was statistically significant (p0.05), down-regulation of mir-21-5p had no significant effect on the proliferation of ishikawa and hec-1a. 4. Upregulation of mir-21-5p promoted the migration and invasion of ishikawa and hec-1a cells, down-regulation of mir-21-5p inhibited the migration and invasion of ishikawa and hec-1a, the difference was statistically significant (p0.05). 5.targetscanhuman7.1 predicted that the "seed" region of mir-21-5p was "uauucga". The target genes pitx2,vcl,jag1 and fbxo11;6. of mir-21-5p associated with invasion and metastasis were screened. When the expression of mir-21-5p was up-regulated, the mrna of target gene pitx2,vcl,jag1 and fbxo11 decreased significantly compared with the control group, the expression of mir-21-5p and vcl decreased in ishikawa,hec-1a cells, and the expression of pitx2 and vcl increased when the expression of mir-21-5p was down-regulated. 7. The sequencing results of target gene 3'utr region clone vector, wild type recombinant vector and mutant recombinant vector were consistent with expectations, and double luciferase analysis showed that pitx2,vcl,jag1 and fbxo11 were direct target genes of mir-21-5p. 4. Conclusion the expression of 1.miR-21-5p in HEC-1A is significantly higher than that in Ishikawa cells. 2. 2. Upregulation of miR-21-5p could promote proliferation, migration and invasion of Ishikawa and HEC-1A cells. It is proved that JAG1,FBX011,VCL and PITX2 are the target genes of mi R-21-5p.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
本文编号：2290033
[Abstract]:Objective: to investigate the expression of miR-21-5p in Ishikawa and HEC-1A of different endometrial cancer cells, and to up-regulate or down-regulate the effects of miR-21-5p on cell proliferation, migration and invasion. To explore the direct target molecules of miR-21-5p and to provide a new target for the diagnosis and treatment of endometrial carcinoma. Methods 1.Real-time PCR assay was used to detect and analyze the expression of miR-21-5p in Ishikawa and HEC-1A of different endometrial cancer cells. 2. Using liposome transfection, Ishikawa and HEC-1A cells were transfected with miR-21-5p mimics (up-regulated miR-21-5p), (Negative control) or miR-21-5p inhibitor (down-regulated miR-21-5p) and (Inhibitor NC), respectively, and miR-21-5p expression was detected and analyzed by Real-time PCR assay. Effect of miR-21-5p upregulation / down-regulation on proliferation of Ishikawa and HEC-1A cells by 3.CCK8 assay The effects of up-regulation and down-regulation of miR-21-5p on the migration and invasion of Ishikawa and HEC-1A cells were detected by 4.Transwell assay and cell scratch assay respectively. Targetscan,mirdb and other target gene prediction software was used to screen mir-21-5p target genes related to invasion and metastasis. The expression of mir-21-5p was up-regulated in hec-1a cells, the changes of target gene (pitx2,vcl,jag1 and fbxo11) mrna were detected by real-timepcr, mir-21-5p expression was upregulated / down-regulated in ishikawa and hec-1a cells, and pitx2 and vcl proteins were detected by westernblot. The wild-type clone vector and double luciferase recombinant vector of mir-21-5p target gene (pitx2,vcl,jag1 and fbxo11) 3'utr were constructed, and the double luciferase activity of each transfection group was detected by liposome transient transfection and double luciferase (dual-luciferase) reporter gene experiment. Three Results the expression of 1.mir-21-5p in hec-1a cells was significantly higher than that in ishikawa cells, the expression of mir-21-5p in ishikawa and hec-1a cells was increased after transfection of 2.mir-21-5pmimics (control negativecontrol) to ishikawa and hec-1a cells (140-fold increase in ishikawa cells and 15-fold increase in hec-1a), and the expression of mir-21-5p in ishikawa and hec-1a cells decreased after mir-21-5pinhibitor (control group inhibitornc) was transfected into ishikawa and hec-1a cells. (ishikawa cells decreased by 2 times, hec-1a decreased by 2. 5 times); Upregulation of mir-21-5p could promote the proliferation of ishikawa and hec-1a cells, the difference was statistically significant (p0.05), down-regulation of mir-21-5p had no significant effect on the proliferation of ishikawa and hec-1a. 4. Upregulation of mir-21-5p promoted the migration and invasion of ishikawa and hec-1a cells, down-regulation of mir-21-5p inhibited the migration and invasion of ishikawa and hec-1a, the difference was statistically significant (p0.05). 5.targetscanhuman7.1 predicted that the "seed" region of mir-21-5p was "uauucga". The target genes pitx2,vcl,jag1 and fbxo11;6. of mir-21-5p associated with invasion and metastasis were screened. When the expression of mir-21-5p was up-regulated, the mrna of target gene pitx2,vcl,jag1 and fbxo11 decreased significantly compared with the control group, the expression of mir-21-5p and vcl decreased in ishikawa,hec-1a cells, and the expression of pitx2 and vcl increased when the expression of mir-21-5p was down-regulated. 7. The sequencing results of target gene 3'utr region clone vector, wild type recombinant vector and mutant recombinant vector were consistent with expectations, and double luciferase analysis showed that pitx2,vcl,jag1 and fbxo11 were direct target genes of mir-21-5p. 4. Conclusion the expression of 1.miR-21-5p in HEC-1A is significantly higher than that in Ishikawa cells. 2. 2. Upregulation of miR-21-5p could promote proliferation, migration and invasion of Ishikawa and HEC-1A cells. It is proved that JAG1,FBX011,VCL and PITX2 are the target genes of mi R-21-5p.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33
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