轉錄因子KLF9在前列腺癌發(fā)生發(fā)展中的功能研究
發(fā)布時間：2018-10-16 15:30
【摘要】：[目的]：以前列腺癌細胞LNCaP、PC3、DU145為研究對象,并建立前列腺癌細胞小鼠荷瘤模型,檢測KLF9基因在前列腺癌細胞系中的表達情況及其對前列腺癌細胞生物學效應的影響,并初步探討其影響前列腺癌發(fā)生發(fā)展的可能機制。[方法]：在研究中,我們用實時熒光定量PCR(RT-PCR)和蛋白質印跡法(Western Blot)檢測KLF9基因在抗雄藥氟他胺(flutamide)在去雄誘導LNCaP細胞凋亡過程中的變化,以及KLF9在不同前列腺(癌)細胞系中的表達情況。我們構建了攜帶有四環(huán)素可調控(tetracycline-on, Tet-on)基因表達系統(tǒng)的真核表達載體pTRIPZ-KLF9,用慢病毒感染的方法導入人前列腺癌細胞系PC3和DU145中,并在強力霉素(doxycycline, Dox)的誘導下過表達KLF9,分別采用MTT法、克隆形成實驗、FACS法、TUNEL染色及免疫組化(IHC)檢測KLF9過表達對前列腺癌細胞增殖、凋亡和細胞周期的影響；用構建的pTRIPZ-KLF9和陰性對照質粒轉染的PC3或LNCaP細胞接種于小鼠皮下,觀察轉染了各質粒的PC3或LNCaP細胞在小鼠體內成瘤率、瘤體生長速度以及平均腫瘤體積等方面的變化。并且用Western Blot檢測了凋亡相關蛋白的變化情況以及可能的機制的變化情況,并對涉及機制的可能蛋白做了恢復實驗。[結果]：在氟他胺抗雄誘導LNCaP細胞發(fā)生凋亡的過程中,可誘導KLF9的表達,且具有隨時間變化的遞增關系;敲除內源性的KLF9基因可以部分降低氟他胺對LNCaP細胞造成的生長抑制。同時發(fā)現(xiàn),與非腫瘤細胞(RWPE-1、BPH1)相比,KLF9在前列腺癌細胞系中是低表達的,尤其是在惡性程度更高的雄激素非依賴性細胞系(PC3、DU145、C4-2B)中表達水平更低。過表達KLF9能顯著抑制前列腺癌PC3和DU145細胞的增殖及克隆形成能力(P0.001),并能明顯的誘導細胞凋亡(P0.001);且使細胞周期被阻滯在G 2/M期。在KLF9過表達抑制細胞增殖并且誘導凋亡的過程中,Western Blot及IHC實驗結果顯示,抗凋亡相關蛋白BCL2、Bcl-xl的表達水平降低(P0.01),凋亡相關蛋白Cleaved Casspase3、Cleaved PARP的表達水平明顯升高。在體內實驗中,接種pTRIPZ-KLF9組的小鼠腫瘤成瘤率下降(P0.01),小鼠瘤體生長速度明顯慢于對照組(P0.001),實驗終止時,腫瘤平均體積明顯小于對照組(P0.001)。在機制方面,KLF9過表達可抑制AKT的活化,同時也抑制AKT下游蛋白mTOR的活化;AKT過表達之后,可以部分恢復由KLF9誘導引起的對前列腺癌細胞PC3、DU145的生長抑制及細胞凋亡。[結論]：1)轉錄因子KLF9在前列腺癌細胞系中的表達低于其在相應的非腫瘤細胞系中的表達量；2)在抗雄藥物氟他胺誘導LNCaP細胞發(fā)生凋亡的過程中,KLF9基因的表達上調；3)在體外及體內實驗中,KLF9基因過表達后可顯著地誘導前列腺癌細胞發(fā)生凋亡,抑制前列腺癌的生長及體內成瘤；其可能為前列腺癌發(fā)病的一個抑癌基因;4)AKT信號通路的抑制參與了轉錄因子KLF9抑制前列腺癌細胞生長的過程。
[Abstract]:[objective]: to investigate the expression of KLF9 gene in prostate cancer cell line LNCaP,PC3,DU145 and its effect on the biological effect of prostate cancer cell line. The possible mechanism of its influence on the occurrence and development of prostate cancer was discussed. [methods]: in our study, we used real-time fluorescent quantitative PCR (RT-PCR) and Western blot (Western Blot) to detect the changes of KLF9 gene in the process of deandrogen-induced LNCaP cell apoptosis induced by androgenic flutamide (flutamide). And the expression of KLF9 in different prostate (cancer) cell lines. We constructed an eukaryotic expression vector pTRIPZ-KLF9, carrying tetracycline regulated (tetracycline-on, Tet-on) gene expression system into human prostate cancer cell lines PC3 and DU145 by lentiviral infection, and overexpressed KLF9, by MTT method under the induction of doxycycline (doxycycline, Dox). The effects of overexpression of KLF9 on the proliferation, apoptosis and cell cycle of prostate cancer cells were detected by FACS assay, TUNEL staining and immunohistochemical (IHC). The PC3 or LNCaP cells transfected with constructed pTRIPZ-KLF9 and negative control plasmids were inoculated subcutaneously in mice. The tumorigenic rate, tumor growth rate and mean tumor volume of PC3 or LNCaP cells transfected with each plasmid in mice were observed. Western Blot was used to detect the changes of apoptosis-related proteins and possible mechanisms. [results]: in the course of anti-androgen-induced apoptosis of LNCaP cells, flutamide could induce the expression of KLF9 and increase with time, and knockout of endogenous KLF9 gene could partly reduce the growth inhibition of LNCaP cells induced by flutamide. It was also found that KLF9 expression was lower in prostate cancer cell lines than in non-tumor cells (RWPE-1,BPH1), especially in androgen independent cell lines (PC3,DU145,C4-2B) with higher malignancy. Overexpression of KLF9 could significantly inhibit the proliferation and clone formation of prostate cancer PC3 and DU145 cells (P0.001), induce apoptosis (P0.001), and block the cell cycle in G _ 2 / M phase. The results of, Western Blot and IHC experiments showed that the expression of anti-apoptosis-related protein (BCL2,Bcl-xl) was decreased (P0.01) and the expression of apoptosis-related protein (Cleaved Casspase3,Cleaved PARP) was significantly increased during the process of over-expression of KLF9 inhibiting cell proliferation and inducing apoptosis. In vivo, the tumorigenesis rate of mice inoculated with pTRIPZ-KLF9 decreased (P0.01), and the tumor growth rate of mice was significantly slower than that of control group (P0.001). At the end of the experiment, the average tumor volume was significantly smaller than that of the control group (P0.001). In terms of mechanism, overexpression of KLF9 could inhibit the activation of AKT and the activation of mTOR, the downstream protein of AKT, and AKT overexpression could partially restore the growth inhibition and apoptosis of prostate cancer cell line PC3,DU145 induced by KLF9. [conclusion]: 1) the expression of transcription factor KLF9 in prostate cancer cell line is lower than that in the corresponding non-tumor cell line, 2) the expression of KLF9 gene is up-regulated during the apoptosis of LNCaP cells induced by androgenic flutamide. 3) in vitro and in vivo experiments, overexpression of KLF9 gene could significantly induce apoptosis of prostate cancer cells, inhibit the growth of prostate cancer and tumorigenesis in vivo. It may be a tumor suppressor gene in the pathogenesis of prostate cancer. 4) the inhibition of AKT signaling pathway is involved in the inhibition of the growth of prostate cancer cells by the transcription factor KLF9.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25
本文編號：2274801
[Abstract]:[objective]: to investigate the expression of KLF9 gene in prostate cancer cell line LNCaP,PC3,DU145 and its effect on the biological effect of prostate cancer cell line. The possible mechanism of its influence on the occurrence and development of prostate cancer was discussed. [methods]: in our study, we used real-time fluorescent quantitative PCR (RT-PCR) and Western blot (Western Blot) to detect the changes of KLF9 gene in the process of deandrogen-induced LNCaP cell apoptosis induced by androgenic flutamide (flutamide). And the expression of KLF9 in different prostate (cancer) cell lines. We constructed an eukaryotic expression vector pTRIPZ-KLF9, carrying tetracycline regulated (tetracycline-on, Tet-on) gene expression system into human prostate cancer cell lines PC3 and DU145 by lentiviral infection, and overexpressed KLF9, by MTT method under the induction of doxycycline (doxycycline, Dox). The effects of overexpression of KLF9 on the proliferation, apoptosis and cell cycle of prostate cancer cells were detected by FACS assay, TUNEL staining and immunohistochemical (IHC). The PC3 or LNCaP cells transfected with constructed pTRIPZ-KLF9 and negative control plasmids were inoculated subcutaneously in mice. The tumorigenic rate, tumor growth rate and mean tumor volume of PC3 or LNCaP cells transfected with each plasmid in mice were observed. Western Blot was used to detect the changes of apoptosis-related proteins and possible mechanisms. [results]: in the course of anti-androgen-induced apoptosis of LNCaP cells, flutamide could induce the expression of KLF9 and increase with time, and knockout of endogenous KLF9 gene could partly reduce the growth inhibition of LNCaP cells induced by flutamide. It was also found that KLF9 expression was lower in prostate cancer cell lines than in non-tumor cells (RWPE-1,BPH1), especially in androgen independent cell lines (PC3,DU145,C4-2B) with higher malignancy. Overexpression of KLF9 could significantly inhibit the proliferation and clone formation of prostate cancer PC3 and DU145 cells (P0.001), induce apoptosis (P0.001), and block the cell cycle in G _ 2 / M phase. The results of, Western Blot and IHC experiments showed that the expression of anti-apoptosis-related protein (BCL2,Bcl-xl) was decreased (P0.01) and the expression of apoptosis-related protein (Cleaved Casspase3,Cleaved PARP) was significantly increased during the process of over-expression of KLF9 inhibiting cell proliferation and inducing apoptosis. In vivo, the tumorigenesis rate of mice inoculated with pTRIPZ-KLF9 decreased (P0.01), and the tumor growth rate of mice was significantly slower than that of control group (P0.001). At the end of the experiment, the average tumor volume was significantly smaller than that of the control group (P0.001). In terms of mechanism, overexpression of KLF9 could inhibit the activation of AKT and the activation of mTOR, the downstream protein of AKT, and AKT overexpression could partially restore the growth inhibition and apoptosis of prostate cancer cell line PC3,DU145 induced by KLF9. [conclusion]: 1) the expression of transcription factor KLF9 in prostate cancer cell line is lower than that in the corresponding non-tumor cell line, 2) the expression of KLF9 gene is up-regulated during the apoptosis of LNCaP cells induced by androgenic flutamide. 3) in vitro and in vivo experiments, overexpression of KLF9 gene could significantly induce apoptosis of prostate cancer cells, inhibit the growth of prostate cancer and tumorigenesis in vivo. It may be a tumor suppressor gene in the pathogenesis of prostate cancer. 4) the inhibition of AKT signaling pathway is involved in the inhibition of the growth of prostate cancer cells by the transcription factor KLF9.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25
【共引文獻】
相關博士學位論文 前1條
1 譚靖;i諶縊岫鄖傲邢侔┫赴曛濾雷饔眉襖嗨瓶拱┮┪鍔稈》椒ǖ難芯縖D];中南大學;2013年
，本文編號：2274801
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