Dickkopf-1基因在人胰腺癌生长侵袭中的作用机制探讨

发布时间：2017-12-27 23:26

本文关键词：Dickkopf-1基因在人胰腺癌生长侵袭中的作用机制探讨　出处：《蚌埠医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的:1.检测人胰腺癌细胞系PANC-1中DKK-1的表达情况,观察DKK-1 siRNA靶向抑制PANC-1中DKK-1表达前后癌细胞生物学特性的变化;2.检测DKK-1 siRNA干扰PANC-1细胞中DKK-1表达前后癌细胞c-Myc、cyclinD1及TGF-β1基因表达情况变化,并对DKK-1与c-Myc、cyclinD1及TGF-β1的相关性进行分析,探讨DKK-1基因在PANC-1细胞生长侵袭作用中与c-Myc、cyclinD1及TGF-β1基因的关系。方法:1.设计合成3对DKK-1干扰片段(siDKK-1-1、siDKK-1-2、siDKK-1-3),应用RT-PCR方法检测siDKK-1-1组、siDKK-1-2组、siDKK-1-3组PANC-1细胞中DKK-1 mRNA的表达,从siDKK-1-1、siDKK-1-2、siDKK-1-3中筛选出干扰效果最显著的目标片段;并从如下三个方面检测干扰DKK-1表达后PANC-1细胞生物学特性的变化:MTT法检测转染目标片段前后PANC-1细胞增殖能力变化,流式细胞仪检测转染目标片段前后PANC-1细胞凋亡能力变化,划痕实验检测转染目标片段前后PANC-1细胞运动侵袭能力变化,通过比较三个指标在各组之间的差异,进一步探讨抑制DKK-1表达对PANC-1细胞生物学特性的影响。2.应用RT-PCR法检测DKK-1 siRNA转染PANC-1细胞前后细胞中DKK-1、c-Myc、cyclinD1及TGF-β1基因m RNA的表达;Western-blot法检测DKK-1 siRNA转染PANC-1细胞前后细胞中DKK-1、c-Myc、cyclinD1及TGF-β1蛋白的表达,利用统计软件对转染前后各基因表达的变化进行相关性分析,进一步探讨DKK-1在促进人胰腺癌生长侵袭中与c-Myc、cyclinD1及TGF-β1的关系。结果:1.RT-PCR结果显示转染siDKK-1-1后PANC-1细胞内DKK-1 mRNA表达降低最明显。2.转染si DKK-1-1后PANC-1细胞增殖能力下降、凋亡增加及迁移能力降低,以上实验结果与空白组及NC组比较,差异有统计学意义(P均0.05)。3.RT-PCR检测结果显示转染siDKK-1-1后PANC-1细胞内c-Myc、cyclinD1及TGF-β1 mRNA表达明显降低(P均0.05);Western-blot检测结果显示转染si DKK-1-1后PANC-1细胞内c-Myc、cyclinD1及TGF-β1蛋白表达明显降低(P均0.05)。对上述实验结果进行相关性分析发现DKK-1、c-Myc、cyclinD1及TGF-β1表达两两之间均呈正相关。结论:1.靶向DKK-1 siRNA阻断人胰腺癌细胞PANC-1中DKK-1的表达,可有效抑制PANC-1细胞的增殖、侵袭能力,并诱导细胞凋亡。2.利用DKK-1 siRNA抑制胰腺癌细胞PANC-1中DKK-1的表达,可使c-Myc、cyclinD1及TGF-β1表达下调。3.DKK-1通过影响TGF-β1的表达来调控下游效应基因c-Myc、cyclinD1,从而促进胰腺癌细胞的增殖与迁移,并抑制其凋亡。
[Abstract]:Objective: to detect expression of DKK-1 in 1. human pancreatic cancer cell line PANC-1, observe DKK-1 siRNA targeted inhibition of DKK-1 expression and biological characteristics of cancer cells in PANC-1; 2. DKK-1 siRNA interference in PANC-1 cells to detect expression of DKK-1, cyclinD1 and c-Myc before and after cancer cell TGF- beta 1 gene expression changes, and the correlation between DKK-1 and c-Myc, cyclinD1 and TGF- beta 1 were analyzed to investigate the relationship between growth and c-Myc, cyclinD1 and DKK-1 in the invasion TGF- beta 1 gene expression in PANC-1 cells. Methods: 1. design and synthesis of 3 DKK-1 interference fragment (siDKK-1-1, siDKK-1-2, siDKK-1-3), the expression of RT-PCR was used to detect the siDKK-1-1 group, siDKK-1-2 group, siDKK-1-3 group, PANC-1 cells DKK-1 mRNA, screened out the interference effect from siDKK-1-1, siDKK-1-2, siDKK-1-3 of the most significant target fragment; and from the biological characteristics of PANC-1 cell three the detection of interference DKK-1 expression changes after changes in proliferation ability of PANC-1 cells before and after transfection were detected by MTT method to detect the changes of target fragments, the apoptosis of PANC-1 cells before and after transfection ability of target fragments by flow cytometry, ability of invasion of PANC-1 cells before and after transfection of target motion fragment detection scratch test, by comparing the three indicators among the groups, further to investigate the inhibitory effect of DKK-1 expression on the biological characteristics of PANC-1 cell. Expression of DKK-1, c-Myc, cyclinD1 and TGF- beta 1 gene m RNA DKK-1 siRNA detected before and after transfection of PANC-1 cells 2. by RT-PCR; expression of DKK-1 in cells, c-Myc, cyclinD1 and TGF- beta 1 protein were detected before and after DKK-1 siRNA was transfected into PANC-1 cells by Western-blot method, to analyze the correlation between the changes of expression of each gene transfected by the statistical software, further explore the relationship of DKK-1 in invasion and promote the growth of human pancreatic cancer with c-Myc, cyclinD1 and TGF- beta 1. Results: the results of 1.RT-PCR showed that the expression of DKK-1 mRNA in PANC-1 cells decreased most obviously after siDKK-1-1 transfection. 2. after transfection of Si DKK-1-1, the proliferation ability of PANC-1 cells decreased, the apoptosis increased and the migration ability decreased. The difference between the above experimental results and the blank group and NC group was statistically significant (P 0.05). 3.RT-PCR detection showed that the expression of c-Myc, cyclinD1 and TGF- beta 1 mRNA in PANC-1 cells decreased significantly after transfection of siDKK-1-1 (P 0.05), and Western-blot test showed that the expression of PANC-1 and PANC-1 in the cells of the Si cells decreased significantly after transfection Si DKK-1-1 (all 0.05). The correlation analysis of the experimental results showed that there was a positive correlation between DKK-1, c-Myc, cyclinD1 and TGF- beta 1 expression 22. Conclusion: 1. targeting DKK-1 siRNA can inhibit the expression of DKK-1 in human pancreatic cancer cell PANC-1, which can effectively inhibit the proliferation and invasion ability of PANC-1 cells, and induce cell apoptosis. 2. the expression of DKK-1 in pancreatic cancer cell PANC-1 was inhibited by DKK-1 siRNA, and the expression of c-Myc, cyclinD1 and TGF- beta 1 was down regulated. 3.DKK-1 regulates the downstream effect gene c-Myc and cyclinD1 by affecting the expression of TGF- beta 1, which promotes the proliferation and migration of pancreatic cancer cells and inhibits its apoptosis.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.9

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