miR-21-5p影響子宮內(nèi)膜癌細胞增殖、遷移及侵襲機制的研究
發(fā)布時間：2018-10-23 17:55
【摘要】：一、目的本實驗旨在探討miR-21-5p在不同子宮內(nèi)膜癌細胞Ishikawa及HEC-1A中的表達差異,上調(diào)或下調(diào)miR-21-5p對細胞增殖、遷移及侵襲的影響。探尋miR-21-5p的直接靶標分子,為子宮內(nèi)膜癌的診斷及治療提供新的靶點。二、方法1.Real-time PCR法檢測并分析不同子宮內(nèi)膜癌細胞Ishikawa及HEC-1A中miR-21-5p的表達差異;2.利用脂質(zhì)體轉(zhuǎn)染法,以miR-21-5p mimics(上調(diào)miR-21-5p)及其對照(Negative control)或miR-21-5p inhibitor(下調(diào)miR-21-5p)及其對照(Inhibitor NC),分別轉(zhuǎn)染Ishikawa及HEC-1A細胞,Real-time PCR法檢測并分析miR-21-5p表達變化;3.CCK8實驗檢測上調(diào)/下調(diào)miR-21-5p對Ishikawa及HEC-1A細胞增殖能力的影響;4.Transwell實驗及細胞劃痕實驗分別檢測上調(diào)/下調(diào)miR-21-5p對Ishikawa及HEC-1A細胞遷移及侵襲的影響;5.利用targetscan、mirdb等靶基因預(yù)測軟件,篩選與侵襲轉(zhuǎn)移相關(guān)的mir-21-5p靶基因;6.上調(diào)hec-1a細胞中mir-21-5p的表達,real-timepcr檢測靶基因(pitx2、vcl、jag1和fbxo11)mrna的變化;在ishikawa和hec-1a細胞中上調(diào)/下調(diào)mir-21-5p的表達,westernblot檢測pitx2及vcl蛋白表達變化;7.構(gòu)建mir-21-5p靶基因(pitx2、vcl、jag1和fbxo11)3’utr野生型的克隆載體和雙熒光素酶重組載體;分別用脂質(zhì)體瞬時轉(zhuǎn)染,雙熒光素酶(dual-luciferase)報告基因?qū)嶒灆z測各轉(zhuǎn)染組的雙熒光素酶活性。三、結(jié)果1.mir-21-5p在hec-1a細胞中的表達明顯高于ishikawa細胞;2.mir-21-5pmimics(對照組negativecontrol)轉(zhuǎn)染到ishikawa及hec-1a細胞后,mir-21-5p的表達升高(ishikawa細胞升高140倍,hec-1a升高15倍);mir-21-5pinhibitor(對照組inhibitornc)轉(zhuǎn)染到ishikawa及hec-1a細胞后,mir-21-5p的表達下降(ishikawa細胞降低2倍,hec-1a降低2.5倍);3.上調(diào)mir-21-5p能夠促進ishikawa和hec-1a細胞的增殖能力,差異有統(tǒng)計學(xué)意義(p0.05);下調(diào)mir-21-5p對ishikawa和hec-1a的增殖能力無顯著影響;4.上調(diào)mir-21-5p能夠促進ishikawa和hec-1a細胞的遷移及侵襲能力;下調(diào)mir-21-5p能抑制ishikawa和hec-1a的遷移及侵襲能力;差異具有統(tǒng)計學(xué)意義(p0.05);5.targetscanhuman7.1預(yù)測顯示mir-21-5p的“種子”區(qū)為“uauucga”,并篩選出與侵襲轉(zhuǎn)移相關(guān)的mir-21-5p的靶基因pitx2、vcl、jag1和fbxo11;6.上調(diào)hec-1a細胞mir-21-5p的表達,靶基因pitx2、vcl、jag1和fbxo11的mrna與對照組相比呈顯著降低趨勢;上調(diào)ishikawa、hec-1a細胞中mir-21-5p的表達pitx2及vcl蛋白的表達降低;相反,下調(diào)mir-21-5p的表達時,可促進pitx2和vcl蛋白的表達;7.靶基因3’utr區(qū)克隆載體、野生型及突變型重組載體測序結(jié)果與預(yù)想一致;雙熒光素酶結(jié)果顯示,pitx2、vcl、jag1和fbxo11是mir-21-5p的直接靶基因。四、結(jié)論1.miR-21-5p在HEC-1A中的表達顯著高于Ishikawa細胞。2.上調(diào)miR-21-5p能夠促進Ishikawa細胞及HEC-1A細胞的增殖、遷移及侵襲能力。3.本實驗證明了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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33
本文編號：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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33
【參考文獻】
相關(guān)期刊論文 前5條
1 Yu Yang;Jia-Xiang Guo;Zhi-Qiang Shao;;miR-21 targets and inhibits tumor suppressor gene PTEN to promote prostate cancer cell proliferation and invasion: An experimental study[J];Asian Pacific Journal of Tropical Medicine;2017年01期
2 唐開;崔玉蘭;;miRNA在子宮內(nèi)膜癌發(fā)病機制中的研究進展[J];現(xiàn)代腫瘤醫(yī)學(xué);2016年05期
3 王麗琴;;子宮內(nèi)膜癌臨床診治的研究進展[J];中國腫瘤臨床與康復(fù);2012年05期
4 郄明蓉;張竹;;子宮內(nèi)膜癌流行病學(xué)及發(fā)病因素[J];中國實用婦科與產(chǎn)科雜志;2011年11期
5 賈軍民;劉向芳;;子宮內(nèi)膜癌58例臨床病理分析[J];中國藥物與臨床;2010年04期
，本文編號：2290033
本文鏈接：http://sikaile.net/yixuelunwen/fuchankeerkelunwen/2290033.html
最近更新
教材專著
