转录因子KLF9在前列腺癌发生发展中的功能研究
发布时间：2018-10-16 15:30
【摘要】：[目的]：以前列腺癌细胞LNCaP、PC3、DU145为研究对象,并建立前列腺癌细胞小鼠荷瘤模型,检测KLF9基因在前列腺癌细胞系中的表达情况及其对前列腺癌细胞生物学效应的影响,并初步探讨其影响前列腺癌发生发展的可能机制。[方法]：在研究中,我们用实时荧光定量PCR(RT-PCR)和蛋白质印迹法(Western Blot)检测KLF9基因在抗雄药氟他胺(flutamide)在去雄诱导LNCaP细胞凋亡过程中的变化,以及KLF9在不同前列腺(癌)细胞系中的表达情况。我们构建了携带有四环素可调控(tetracycline-on, Tet-on)基因表达系统的真核表达载体pTRIPZ-KLF9,用慢病毒感染的方法导入人前列腺癌细胞系PC3和DU145中,并在强力霉素(doxycycline, Dox)的诱导下过表达KLF9,分别采用MTT法、克隆形成实验、FACS法、TUNEL染色及免疫组化(IHC)检测KLF9过表达对前列腺癌细胞增殖、凋亡和细胞周期的影响；用构建的pTRIPZ-KLF9和阴性对照质粒转染的PC3或LNCaP细胞接种于小鼠皮下,观察转染了各质粒的PC3或LNCaP细胞在小鼠体内成瘤率、瘤体生长速度以及平均肿瘤体积等方面的变化。并且用Western Blot检测了凋亡相关蛋白的变化情况以及可能的机制的变化情况,并对涉及机制的可能蛋白做了恢复实验。[结果]：在氟他胺抗雄诱导LNCaP细胞发生凋亡的过程中,可诱导KLF9的表达,且具有随时间变化的递增关系;敲除内源性的KLF9基因可以部分降低氟他胺对LNCaP细胞造成的生长抑制。同时发现,与非肿瘤细胞(RWPE-1、BPH1)相比,KLF9在前列腺癌细胞系中是低表达的,尤其是在恶性程度更高的雄激素非依赖性细胞系(PC3、DU145、C4-2B)中表达水平更低。过表达KLF9能显著抑制前列腺癌PC3和DU145细胞的增殖及克隆形成能力(P0.001),并能明显的诱导细胞凋亡(P0.001);且使细胞周期被阻滞在G 2/M期。在KLF9过表达抑制细胞增殖并且诱导凋亡的过程中,Western Blot及IHC实验结果显示,抗凋亡相关蛋白BCL2、Bcl-xl的表达水平降低(P0.01),凋亡相关蛋白Cleaved Casspase3、Cleaved PARP的表达水平明显升高。在体内实验中,接种pTRIPZ-KLF9组的小鼠肿瘤成瘤率下降(P0.01),小鼠瘤体生长速度明显慢于对照组(P0.001),实验终止时,肿瘤平均体积明显小于对照组(P0.001)。在机制方面,KLF9过表达可抑制AKT的活化,同时也抑制AKT下游蛋白mTOR的活化;AKT过表达之后,可以部分恢复由KLF9诱导引起的对前列腺癌细胞PC3、DU145的生长抑制及细胞凋亡。[结论]：1)转录因子KLF9在前列腺癌细胞系中的表达低于其在相应的非肿瘤细胞系中的表达量；2)在抗雄药物氟他胺诱导LNCaP细胞发生凋亡的过程中,KLF9基因的表达上调；3)在体外及体内实验中,KLF9基因过表达后可显著地诱导前列腺癌细胞发生凋亡,抑制前列腺癌的生长及体内成瘤；其可能为前列腺癌发病的一个抑癌基因;4)AKT信号通路的抑制参与了转录因子KLF9抑制前列腺癌细胞生长的过程。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25
【共引文献】
相关博士学位论文 前1条
1 谭靖;i谌缢岫郧傲邢侔┫赴曛滤雷饔眉袄嗨瓶拱┮┪锷秆》椒ǖ难芯縖D];中南大学;2013年
，本文编号：2274801
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