本文選題：前列腺癌　切入點：賴氨酸特異性去甲基化酶　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分過表達和干擾LSD1的慢病毒載體構(gòu)建和鑒定 目的構(gòu)建和鑒定過表達和干擾LSD1慢病毒載體,為進一步的實驗研究奠定基礎(chǔ)。 方法1.采用PCR技術(shù)從LSD1cDNA克隆質(zhì)粒中釣取LSD1基因,并將該基因克隆到慢病毒載體表達質(zhì)粒GV166中,構(gòu)建慢病毒載體表達質(zhì)粒GV166-LSD1,通過PCR鑒定、測序比對目的基因LSD1,用GV166-LSD1轉(zhuǎn)染293T細胞后Western Blot檢測LSD1蛋白的表達,將GV166-LSD1質(zhì)粒和包裝質(zhì)粒pHelperl.0、 pHelper2.0共轉(zhuǎn)染293T細胞,包裝和生產(chǎn)攜帶LSD1基因的重組慢病毒LV-LSD1,測定病毒滴度。LV-LSD1感染LNCaP細胞后,Real-time定量PCR和Western Blot檢測LSD1基因mRNA和蛋白的表達水平。2.LV-LSD1-感染LNCaP細胞后,通過puromycin抗性壓力篩選過表達LSD1的單克隆穩(wěn)定株LNCaP-LSD1細胞,定量PCR和Western Blot檢測LNCaP-LSD1細胞LSD1基因mRNA和蛋白的表達水平。3.根據(jù)權(quán)威文獻驗證過的LSD1基因siRNA干擾靶點序列,構(gòu)建shRNA慢病毒(Lentivirus, LV)表達質(zhì)粒,并進行PCR鑒定和測序比對,將GV307-LSD1-shRNA質(zhì)粒載體及包裝質(zhì)粒pHelperl.0、pHelper2.0共轉(zhuǎn)染293T細胞,進行病毒包裝成LV-LSD1-shRNA,并進行滴度測定；LV-LSD1-shRNA感染LNCaP-AI細胞后,Real-time定量PCR和Western Blot檢測LSD1基因mRNA和蛋白的表達水平。 結(jié)果1.成功構(gòu)建過表達LSD1慢病毒載體LV-LSD1,病毒滴度為2.0E+8TU/ml, LV-LSD1感染LNCaP細胞后,其LSD1mRNA和蛋白的表達水平明顯升高。2.篩選出過表達LSD1的單克隆穩(wěn)定株LNCaP-LSD1細胞,其LSD1mRNA和蛋白的表達水平升高。3.構(gòu)建出干擾LSD1表達慢病毒載體LV-LSD1-shRNA,病毒滴度為5.0E+7TU/ml, LV-LSD1-shRNA感染LNCaP-AI細胞后,其LSD1mRNA和蛋白的表達顯著降低。 結(jié)論成功構(gòu)建過表達LSD1慢病毒載體LV-LSD1;成功篩選獲得過表達LSD1的單克隆穩(wěn)定株LNCaP-LSD1細胞,并在mRNA和蛋白表達水平驗證了LSD1過表達；成功構(gòu)建干擾LSD1的慢病毒載體LV-LSD1-shRNA,并在LNCaP-AI細胞產(chǎn)生了預(yù)期的干擾效應(yīng)。上述成果為進一步研究LSD1在前列腺癌雄激素非依賴性表型轉(zhuǎn)化中的作用及機制奠定實驗基礎(chǔ)。 第二部分LSD1在前列腺癌雄激素非依賴性表型轉(zhuǎn)化中的作用及可能機制 目的探討LSD1在前列腺癌雄激素非依賴性表型轉(zhuǎn)化中的作用及可能機制。 方法1.CCK-8法檢測LNCaP-LSD1與LNCaP細胞在Bicalutamide處理后細胞增殖水平的差異,以及LSD1-siRNA、con-siRNA、Pargyline、Tranylcypromine處理后LNCaP和LNCaP-AI細胞的細胞活力的變化,以及以上處理聯(lián)合R1881刺激后細胞數(shù)量的改變。2.定量PCR和Western blot檢測LNCaP細胞和LNCaP-LSD1細胞AR mRNA和蛋白表達水平的差異；ELISA法檢測LNCaP細胞與LNCaP-LSD1細胞在不同濃度Bicalutamide處理后PSA分泌水平的差異,并檢測LSD1-siRNA、con-siRNA單獨或與Bicalutamide聯(lián)合處理后LNCaP-AI細胞PSA分泌水平的變化。3.FCM亞G1峰(sub G1)法檢測Etoposide單獨或與LSD1-siRNA、con-siRNA聯(lián)合處理后LNCaP-AI細胞凋亡的差異；定量PCR和Western Blot檢測LNCaP和LNCaP-LSD1細胞p53、p21和Bax mRNA和蛋白表達水平的差異,觀察LSD1-siRNA處理后LNCaP-AI細胞p21和HDM2mRNA和蛋白的變化；Western Blot檢測Con-siRNA和LSD1-siRNA處理后LNCaP-AI細胞的Bax. PUMA和survivin蛋白表達水平的差異。 結(jié)果1. LNCaP細胞在在去雄激素環(huán)境或Bicalutamide阻斷雄激素受體后增殖能力受到明顯抑制,而LNCaP-LSD1細胞仍能持續(xù)增殖；LSD1-siRNA、Pargyline和Tranylcypromine處理后,LNCaP和LNCaP-AI細胞活力均明顯降低；此外,LSD1-siRNA和Tranylcypromine抑制了R1881對LNCaP和LNCaP-AI細胞的促增殖效應(yīng)。2. LNCaP細胞和LNCaP-LSD1細胞ARmRNA和蛋白表達水平的無明顯差異；LNCaP-LSD1細胞PSA分泌水平顯著高于LNCaP細胞,10μmol/L Bicalutamide顯著降低LNCaP細胞PSA分泌水平,但對LNCaP-LSD1細胞PSA分泌水平無影響；Bicalutamide對LNCaP-AI細胞PSA分泌無影響,LSD1-siRNA處理使Bicalutamide重新獲得了對LNCaP-AI細胞PSA分泌的抑制效應(yīng)。3.LSD1-siRNA促進了Etoposide誘導(dǎo)的LNCaP-AI細胞凋亡,LNCaP-LSD1細胞p53mRNA和蛋白表達量與LNCaP細胞無明顯差別,p21、Bax mRNA和蛋白表達量較LNCaP細胞明顯減少;LSD1-siRNA增加了LNCaP-AI細胞p21和HDM2mRNA表達水平,LSD1-siRNA上調(diào)了LNCaP-AI細胞p21、HDM2、Bax和PUMA蛋白表達,下調(diào)了survivin蛋白的表達。 結(jié)論LSD1可能在前列腺癌AI轉(zhuǎn)化中扮演了重要角色。前列腺癌在雄激素剝奪環(huán)境中上調(diào)LSD1基因表達,LSD1通過活化AR信號通路,抑制p53依賴性凋亡通路,從而誘導(dǎo)前列腺癌獲得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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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本文編號：1579544