LSD1在前列腺癌雄激素非依赖性表型转化中的作用及机制研究

发布时间：2018-03-07 13:47

  本文选题：前列腺癌　切入点：赖氨酸特异性去甲基化酶　出处：《华中科技大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分过表达和干扰LSD1的慢病毒载体构建和鉴定 目的构建和鉴定过表达和干扰LSD1慢病毒载体,为进一步的实验研究奠定基础。 方法1.采用PCR技术从LSD1cDNA克隆质粒中钓取LSD1基因,并将该基因克隆到慢病毒载体表达质粒GV166中,构建慢病毒载体表达质粒GV166-LSD1,通过PCR鉴定、测序比对目的基因LSD1,用GV166-LSD1转染293T细胞后Western Blot检测LSD1蛋白的表达,将GV166-LSD1质粒和包装质粒pHelperl.0、 pHelper2.0共转染293T细胞,包装和生产携带LSD1基因的重组慢病毒LV-LSD1,测定病毒滴度。LV-LSD1感染LNCaP细胞后,Real-time定量PCR和Western Blot检测LSD1基因mRNA和蛋白的表达水平。2.LV-LSD1-感染LNCaP细胞后,通过puromycin抗性压力筛选过表达LSD1的单克隆稳定株LNCaP-LSD1细胞,定量PCR和Western Blot检测LNCaP-LSD1细胞LSD1基因mRNA和蛋白的表达水平。3.根据权威文献验证过的LSD1基因siRNA干扰靶点序列,构建shRNA慢病毒(Lentivirus, LV)表达质粒,并进行PCR鉴定和测序比对,将GV307-LSD1-shRNA质粒载体及包装质粒pHelperl.0、pHelper2.0共转染293T细胞,进行病毒包装成LV-LSD1-shRNA,并进行滴度测定；LV-LSD1-shRNA感染LNCaP-AI细胞后,Real-time定量PCR和Western Blot检测LSD1基因mRNA和蛋白的表达水平。 结果1.成功构建过表达LSD1慢病毒载体LV-LSD1,病毒滴度为2.0E+8TU/ml, LV-LSD1感染LNCaP细胞后,其LSD1mRNA和蛋白的表达水平明显升高。2.筛选出过表达LSD1的单克隆稳定株LNCaP-LSD1细胞,其LSD1mRNA和蛋白的表达水平升高。3.构建出干扰LSD1表达慢病毒载体LV-LSD1-shRNA,病毒滴度为5.0E+7TU/ml, LV-LSD1-shRNA感染LNCaP-AI细胞后,其LSD1mRNA和蛋白的表达显著降低。 结论成功构建过表达LSD1慢病毒载体LV-LSD1;成功筛选获得过表达LSD1的单克隆稳定株LNCaP-LSD1细胞,并在mRNA和蛋白表达水平验证了LSD1过表达；成功构建干扰LSD1的慢病毒载体LV-LSD1-shRNA,并在LNCaP-AI细胞产生了预期的干扰效应。上述成果为进一步研究LSD1在前列腺癌雄激素非依赖性表型转化中的作用及机制奠定实验基础。 第二部分LSD1在前列腺癌雄激素非依赖性表型转化中的作用及可能机制 目的探讨LSD1在前列腺癌雄激素非依赖性表型转化中的作用及可能机制。 方法1.CCK-8法检测LNCaP-LSD1与LNCaP细胞在Bicalutamide处理后细胞增殖水平的差异,以及LSD1-siRNA、con-siRNA、Pargyline、Tranylcypromine处理后LNCaP和LNCaP-AI细胞的细胞活力的变化,以及以上处理联合R1881刺激后细胞数量的改变。2.定量PCR和Western blot检测LNCaP细胞和LNCaP-LSD1细胞AR mRNA和蛋白表达水平的差异；ELISA法检测LNCaP细胞与LNCaP-LSD1细胞在不同浓度Bicalutamide处理后PSA分泌水平的差异,并检测LSD1-siRNA、con-siRNA单独或与Bicalutamide联合处理后LNCaP-AI细胞PSA分泌水平的变化。3.FCM亚G1峰(sub G1)法检测Etoposide单独或与LSD1-siRNA、con-siRNA联合处理后LNCaP-AI细胞凋亡的差异；定量PCR和Western Blot检测LNCaP和LNCaP-LSD1细胞p53、p21和Bax mRNA和蛋白表达水平的差异,观察LSD1-siRNA处理后LNCaP-AI细胞p21和HDM2mRNA和蛋白的变化；Western Blot检测Con-siRNA和LSD1-siRNA处理后LNCaP-AI细胞的Bax. PUMA和survivin蛋白表达水平的差异。 结果1. LNCaP细胞在在去雄激素环境或Bicalutamide阻断雄激素受体后增殖能力受到明显抑制,而LNCaP-LSD1细胞仍能持续增殖；LSD1-siRNA、Pargyline和Tranylcypromine处理后,LNCaP和LNCaP-AI细胞活力均明显降低；此外,LSD1-siRNA和Tranylcypromine抑制了R1881对LNCaP和LNCaP-AI细胞的促增殖效应。2. LNCaP细胞和LNCaP-LSD1细胞ARmRNA和蛋白表达水平的无明显差异；LNCaP-LSD1细胞PSA分泌水平显著高于LNCaP细胞,10μmol/L Bicalutamide显著降低LNCaP细胞PSA分泌水平,但对LNCaP-LSD1细胞PSA分泌水平无影响；Bicalutamide对LNCaP-AI细胞PSA分泌无影响,LSD1-siRNA处理使Bicalutamide重新获得了对LNCaP-AI细胞PSA分泌的抑制效应。3.LSD1-siRNA促进了Etoposide诱导的LNCaP-AI细胞凋亡,LNCaP-LSD1细胞p53mRNA和蛋白表达量与LNCaP细胞无明显差别,p21、Bax mRNA和蛋白表达量较LNCaP细胞明显减少;LSD1-siRNA增加了LNCaP-AI细胞p21和HDM2mRNA表达水平,LSD1-siRNA上调了LNCaP-AI细胞p21、HDM2、Bax和PUMA蛋白表达,下调了survivin蛋白的表达。 结论LSD1可能在前列腺癌AI转化中扮演了重要角色。前列腺癌在雄激素剥夺环境中上调LSD1基因表达,LSD1通过活化AR信号通路,抑制p53依赖性凋亡通路,从而诱导前列腺癌获得AI表型。
[Abstract]:Construction and identification of lentivirus vectors overexpressing and interfering LSD1
Objective to construct and identify the expression and interference of LSD1 lentivirus vector, and to lay the foundation for further experimental research.
1. methods of using PCR technology to fish LSD1 gene from plasmid LSD1cDNA, then the gene was cloned into the lentiviral vector plasmid GV166, construct a lentiviral vector expression plasmid GV166-LSD1, identified by PCR, sequencing of LSD1 gene, used to detect the expression of LSD1 protein Western Blot GV166-LSD1 in 293T cells transfected with GV166-LSD1 plasmid. And the packaging plasmid pHelperl.0, pHelper2.0 were transfected into 293T cells, packaging and production of recombinant lentiviral LV-LSD1 carrying LSD1 gene,.LV-LSD1 cells infected with LNCaP virus titer was measured after Real-time PCR and Western Blot quantitative detection of LSD1 gene mRNA and protein expression level of.2.LV-LSD1- in LNCaP cells infected by puromycin resistance screening over expression of monoclonal pressure stable strain LNCaP-LSD1 LSD1 cells, the expression level of.3. PCR and Western Blot root quantitative detection of LNCaP-LSD1 cell LSD1 gene mRNA and protein According to the LSD1 gene siRNA interference target sequence verified the authoritative literature, construct shRNA lentiviral expression plasmids (Lentivirus, LV), and identified by PCR and sequencing the GV307-LSD1-shRNA plasmid and packaging plasmid pHelperl.0, pHelper2.0 were transfected into 293T cells. The virus was packaged into LV-LSD1-shRNA, and the titer of LV-LSD1-shRNA infected LNCaP-AI cells; later, the expression level of Real-time PCR and Western Blot quantitative detection of LSD1 gene and mRNA protein.
Results of the 1. successful construction of over expression of LSD1 LV-LSD1 lentivirus vector, the virus titer was 2.0E+8TU/ml, LV-LSD1 infected LNCaP cells, the expression of LSD1mRNA and protein were significantly increased..2. screened over expression of monoclonal LNCaP-LSD1 cells stable LSD1, increased the expression level of the protein LSD1mRNA and.3. constructed LSD1 interference Lentivirus Expression Vector LV-LSD1-shRNA. The virus titer was 5.0E+7TU/ml, LV-LSD1-shRNA infected LNCaP-AI cells, the expression of LSD1mRNA and protein decreased significantly.
Conclusion the successful construction of a lentiviral LSD1 expression vector LV-LSD1 successfully screened; overexpression of monoclonal LNCaP-LSD1 cells stable LSD1 expression level, and verified the expression of LSD1 in mRNA and protein; lentivirus vector LV-LSD1-shRNA was successfully constructed by LSD1 interference, and the interference effect in LNCaP-AI cells. The expected results for the further study of LSD1 in prostate cancer effects and mechanism of phenotypic transformation in the experimental basis.
The role of the second part of LSD1 in the androgen independent phenotype transformation of prostate cancer and its possible mechanism
Objective to investigate the role of LSD1 in the androgen independent phenotype transformation of prostate cancer and its possible mechanism.
The difference, 1.CCK-8 method for detection of LNCaP-LSD1 and LNCaP on the cell proliferation level after treatment with Bicalutamide and LSD1-siRNA, con-siRNA, Pargyline, LNCaP and LNCaP-AI changes in cell viability of Tranylcypromine cells after treatment, and the combined treatment of R1881 cells after stimulation with more than a change in the quantity of.2. and Western blot PCR quantitative detection of LNCaP cells and LNCaP-LSD1 cells AR and mRNA protein expression level difference; ELISA method to detect LNCaP cells and LNCaP-LSD1 cells in different concentrations of Bicalutamide difference after PSA secretion, and to detect LSD1-siRNA,.3.FCM changes con-siRNA alone or in combination with Bicalutamide treated LNCaP-AI cells PSA secretion level of sub G1 peak (sub G1) method for the detection of Etoposide alone or with LSD1-siRNA, the difference of LNCaP-AI cell the apoptosis of con-siRNA after the treatment of PCR and Western Blot; quantitative detection of LNCaP and LNCaP-LSD1 The difference of cell p53, p21 and Bax mRNA and protein expression level was observed. The changes of p21 and HDM2mRNA and protein in LNCaP-AI cells after LSD1-siRNA treatment were observed. Western Blot was used to detect the difference of the expression level of PG and protein in the cells of Con-siRNA cells and Western treated cells.
Results 1. LNCaP cells in androgen environment or Bicalutamide blocking proliferation was markedly inhibited after androgen receptor, whereas LNCaP-LSD1 cells can continue proliferation; LSD1-siRNA, Pargyline and Tranylcypromine, LNCaP and LNCaP-AI cell activity were significantly decreased; in addition, there was no significant difference between LSD1-siRNA and Tranylcypromine inhibited the R1881 of LNCaP and LNCaP-AI cells promote the proliferation effect of.2. LNCaP cells and LNCaP-LSD1 cells in ARmRNA and protein expression level of LNCaP-LSD1 cells; the secretion of PSA was significantly higher than that of LNCaP cells, 10 mol/L Bicalutamide significantly reduced LNCaP cell PSA secretion of LNCaP-LSD1 cells, but the secretion level of PSA had no effect on LNCaP-AI secretion of PSA cells; Bicalutamide effect, LSD1-siRNA processing to enable Bicalutamide to obtain.3.LSD1-siRNA the inhibitory effect on the secretion of LNCaP-AI in PSA cells promoted Etopo LNCaP-AI induced apoptosis of side cells, LNCaP-LSD1 cells and p53mRNA protein expression had no significant difference, and the amount of LNCaP cell p21, Bax mRNA and protein expression of LNCaP cells is significantly decreased; LSD1-siRNA increased LNCaP-AI p21 cells and the expression of HDM2mRNA, LSD1-siRNA upregulation of LNCaP-AI cells p21, HDM2, Bax and PUMA protein expression, reduced the expression of survivin protein.
Conclusion LSD1 may play an important role in the AI transformation of prostate cancer. Prostate cancer upregulates LSD1 gene expression in androgen deprivation environment. LSD1 inhibits p53 dependent apoptosis pathway through activating AR signaling pathway, and induces prostate cancer to get AI phenotype.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

【参考文献】

相关期刊论文 前2条

1 孔德玉;杨冬梅;陈艳媛;王宇;张金桥;高萌;方芳;;雄激素非依赖性前列腺癌LNCaP-AI细胞亚系模型的建立[J];吉林医药学院学报;2012年06期

2 韩苏军;张思维;陈万青;李长岭;;中国前列腺癌发病现状和流行趋势分析[J];临床肿瘤学杂志;2013年04期

，

本文编号：1579544