GNMT的表达及调控对人前列腺癌细胞系LNCap和PC3生物学特性的影响

发布时间：2018-06-23 03:46

本文选题：GNMT + 前列腺癌细胞系　；参考：《复旦大学》2014年博士论文

【摘要】：前列腺癌的发病率在世界范围内及我国均呈逐年上升趋势,众多的分子生物学及遗传学机制主导着这一疾病的进程。Sreekumar等通过代谢组学的研究方法,发现肌氨酸与前列腺癌的发生及进展密切相关。它在前列腺癌尤其是侵袭性前列腺癌组织中的浓度明显高于良性前列腺增生组织中的浓度,并且进一步的研究发现在良性前列腺增生细胞系中加入肌氨酸后可以使细胞具有侵袭性。组织细胞中的肌氨酸浓度受到甘氨酸N-甲基转移酶(GNMT)的调节而发生变化。最新研究发现,前列腺癌细胞比正常前列腺上皮细胞表达更多的IGNMT,并且敲除GNMT基因可以抑制前列腺癌细胞增殖,并诱导其凋亡。因此,调节GNMT的表达或功能,有望为前列腺癌的发生及进展机制和治疗方法的研究提供一种全新的思路。GNMT作为一种具有多种功能的蛋白质,其甲基转移酶的活性可以被其催化产物S-腺苷高半胱氨酸(SAH)所抑制,这种作用在体外及体内均可观察到,并且SAH还可以在转录水平上抑制GNMT的表达。维甲酸(retinoic acid, RA)可以显著增加小鼠体内GNMT的活性,并可在转录水平上促进其表达。因此,我们可以尝试利用SAH和RA来实现体外和体内对GNMT表达及活性的调控,从而进行进一步的研究。目前尚无研究证实,外源性调控GNMT是否能够改变前列腺癌细胞的生物学特性,而这正是我们这一研究试图回答的问题。我们旨在通过对GNMT的体外及体内调控,观察其对人前列腺癌细胞系LNCap和PC3的分化、凋亡、增殖、侵袭性以及成瘤能力的影响,从而对前列腺癌的发生机制及治疗途径进行新的探索。第一部分 GNMT在人前列腺癌细胞系LNCap和PC3中的表达目的：研究不同侵袭性的人前列腺癌细胞系LNCap和PC3中GNMT的表达差异,分析GNMT与前列腺癌细胞侵袭性的关系。方法：RT-PCR检测LNCap和PC3细胞内GNMTmRNA的表达,Western Blot检测两种细胞内GNMT蛋白的表达。结果：LNCap细胞中GNMTmRNA的相对表达量为(1.000±0.331),PC3细胞中GNMT mRNA的相对表达量为(2.579±0.863),二者的差异有统计学意义,p0.05。LNCap细胞中GNMT蛋白的相对表达量为(0.341±0.072),PC3细胞中3NMT蛋白的相对表达量为(0.841±0.116),二者的差异有显著统计学意义p0.01。结论：GNMT的表达量和前列腺癌细胞系的侵袭性相关,侵袭性较高的PC3细胞中表达较高,侵袭性较低的LNCap细胞中表达较低。第二部分 GNMT的体外调控对人前列腺癌细胞系LNCap和PC3生物学特性的影响目的：研究GNMT的体外调控对人前列腺癌细胞系LNCap和PC3的分化、凋亡、增殖和侵袭性等各项生物学特性的影响。方法：细胞培养液中分别加入GNMT (10μg/ml)和SAH (10ng/ml)来上调和下调LNCap和PC3细胞内GNMT的活性。在12h、24h、36h和48h这4个时间点,光镜下观察细胞分化,TUNEL和流式细胞法检测细胞凋亡,MTT法检测细胞增殖,Transwell实验检测细胞侵袭性。结果：①光镜观察第48h,GNMT和SAH处理过的LNCap和PC3细胞形态在200倍光镜下观察,均没有发生明显变化,延长培养时间至5d后结果仍是一样。②TUNEL两种细胞中凋亡率,GNMT组均低于对照组,SAH组均高于对照组。其差异在LNCap细胞中GNMT组和对照组之间,第48h无统计学意义,p0.05,第12h、24h和36h有统计学意义,p0.05; SAH组和对照组之间,4个时间点均有统计学意义,p0.05。在PC3细胞中GNMT组和对照组之间,第12h和48h无统计学意义,p0.05,第24h和36h有统计学意义,p0.05; SAH组和对照组之间,第12h无统计学意义,p0.05,第24h、36h和48h有统计学意义,p0.05。③流式细胞两种细胞中凋亡率,GNMT组均低于对照组,SAH组均高于对照组。其差异在LNCap细胞中3组之间两两比较,4个时间点均有显著统计学意义,p0.01。在PC3细胞中GNMT组和对照组之间,第48h无统计学意义,p0.05,第12h、24h和36h有统计学意义,p0.05; SAH组和对照组之间,4个时间点均有显著统计学意义,p0.01。 ④MTT两种细胞中吸光度值,GNMT组均高于对照组,SAH组均低于对照组。其差异只在PC3细胞中的48h时间点SAH组与对照组之间有统计学意义,p0.05。⑤Transwell两种细胞中穿膜细胞数,GNMT组均高于对照组,SAH组均低于对照组。其差异在两种细胞中均在36h和48h时间点才有统计学意义,p0.05。结论：在细胞培养液中分别加入GNMT和SAH,可以实现对GNMT的体外调控,可以改变前列腺癌细胞系LNCap和PC3除细胞分化以外的各项生物学特性。GNMT活性上调表现出的作用：可以抑制LNCap和PC3细胞凋亡,并且程度与细胞的雄激素依赖特性无关；对LNCap和PC3细胞的增殖没有明显影响；可以增加前列腺癌细胞,尤其是PC3细胞的侵袭性。GNMT活性下调表现出的作用：可以促进LNCap和PC3细胞凋亡,并且程度与细胞的雄激素依赖特性无关；可以抑制PC3细胞增殖,但是不能抑制LNCap细胞增殖；可以减弱LNCap和PC3细胞的侵袭性,并且程度与细胞的雄激素依赖特性无关。第三部分 GNMT的体内调控对人前列腺癌细胞系PC3成瘤作用的影响目的：研究GNMT的体内调控对PC3细胞在裸鼠体内成瘤能力的影响。方法：将人前列腺癌细胞系PC3注入裸鼠皮下成瘤。随机分为3组,2个实验组每天分别喂食顺式维甲酸(CRA,30μmol/kg)口S-腺苷高半胱氨酸(SAH, 10g/kg),对照组正常饮食,连续6周,每周测量肿瘤的大小,绘制肿瘤生长曲线,处死后免疫组化检测瘤体内GNMT的表达。结果：6周后,肿瘤体积CRA组比对照组明显增大,差异有统计学意义,p0.05；SAH组比对照组明显缩小,差异有统计学意义,p0.05。瘤体内GNMT表达CRA组比对照组明显增加,差异有统计学意义(阳性细胞率差异,p0.01；累积光密度差异,p0.05)；SAH组比对照组明显减少,差异有统计学意义(阳性细胞率差异,p0.01；累积光密度差异,p0.05)。结论：通过饮食摄入CRA和SAH可以改变荷瘤裸鼠PC3细胞所致人前列腺癌肿瘤的生长。这一影响可能是通过调控肿瘤内GNMT的活性和表达实现的,摄入CRA可以上调GNMT从而促进肿瘤生长；摄入SAH可以下调GNMT从而抑制肿瘤生长。
[Abstract]:The incidence of prostate cancer is increasing worldwide and in our country. Many molecular biological and genetic mechanisms dominate the process of the disease, such as.Sreekumar, and so on. By metabonomics, it is found that muscle ammonia is closely related to the development and progression of prostate cancer. It is especially an aggressive prostatic cancer in prostate cancer. The concentration in the adenocarcinoma tissue is significantly higher than that in the benign prostatic hyperplasia tissue, and further studies have found that the addition of mylysine to the benign prostatic hyperplasia cell line can make the cells invasive. The concentration of creatine in the tissue cells is changed by the regulation of glycine N- methyltransferase (GNMT). The latest study It is found that prostate cancer cells express more IGNMT than normal prostatic epithelial cells, and knockout GNMT gene can inhibit the proliferation of prostate cancer cells and induce apoptosis. Therefore, the regulation of GNMT expression or function may provide a new idea for the study of the pathogenesis and progression of prostate cancer and the treatment methods,.GNMT as a new way of thinking. A protein with multiple functions, the activity of its methyltransferase can be inhibited by its catalytic product S- adenosine homocysteine (SAH), which can be observed in vitro and in vivo, and SAH can also inhibit the expression of GNMT at transcriptional level. Retinoic acid (RA) can significantly increase the activity of GNMT in mice. Therefore, we can try to use SAH and RA to regulate the expression and activity of GNMT in vitro and in vivo, so that we can do further research. There is no research on whether exogenous GNMT can change the biological characteristics of prostate cancer cells, and this is our study. To answer the question, we aim to observe the effects of GNMT on the differentiation, apoptosis, proliferation, invasiveness and tumorigenicity of human prostate cancer cell lines, LNCap and PC3 in vitro and in vivo, and to explore the mechanism and treatment of prostate cancer. The first part of GNMT is in the human prostate cancer cell line LNCap and in the first part of the human prostate cancer cell line LNCap PC3 expression objective: To study the difference of GNMT expression in LNCap and PC3 of different invasive human prostate cancer cell lines and to analyze the relationship between GNMT and the invasiveness of prostate cancer cells. Methods: RT-PCR was used to detect the expression of GNMTmRNA in LNCap and PC3 cells. Western Blot detected the expression of two kinds of intracellular GNMT proteins. The relative expression amount was (1 + 0.331), the relative expression of GNMT mRNA in PC3 cells was (2.579 + 0.863), the difference between the two was statistically significant, the relative expression of GNMT protein in p0.05.LNCap cells was (0.341 + 0.072), and the relative expression of 3NMT protein in PC3 cells was (0.841 + 0.116), and the differences of two were statistically significant p0.01. knots. The expression of GNMT is associated with the invasiveness of the prostate cancer cell lines, high expression of PC3 cells with higher invasiveness and low expression in the less invasive LNCap cells. The effect of the second part of GNMT on the biological characteristics of LNCap and PC3 in human prostate cancer cell lines: the study of GNMT in vitro regulation of human prostate cancer The effects of the differentiation, apoptosis, proliferation and invasiveness of cell lines LNCap and PC3. Methods: GNMT (10 mu g/ml) and SAH (10ng/ml) were added to the cell culture solution to up and down the activity of GNMT in LNCap and PC3 cells. At the 4 time points of 12h, 24h, 36h, and PC3, the cell differentiation was observed under light microscope, and the flow cytometry was observed. Detection of cell apoptosis, MTT assay to detect cell proliferation and Transwell test to detect cell invasiveness. Results: (1) the morphology of LNCap and PC3 cells treated by 48h, GNMT and SAH was observed under 200 times of light microscope, and no obvious changes were observed, and the same results were found after prolonging the incubation time to 5D. (2) the apoptotic rate in the TUNEL two cells and the GNMT group were all The difference between group SAH and control group was higher than that of control group. The difference between group GNMT and control group in LNCap cells was not statistically significant, P0.05, 12h, 24h and 36h were statistically significant, P0.05, between the SAH group and the control group, the 4 time points were statistically significant, p0.05. was between the PC3 cells and the control group, and there was no statistical difference. Significance, P0.05, 24h and 36h were statistically significant, P0.05, SAH group and control group, 12h had no statistical significance, P0.05, 24h, 36h and 48h were statistically significant, p0.05. 3 cell apoptosis rate in the two cells, GNMT groups were higher than those in the control group. The difference between the 3 groups was 22 and 4 times. There was significant statistical significance. There was no statistical significance in p0.01. between group GNMT and control group in PC3 cells. P0.05, 12h, 24h and 36h were statistically significant, and there were significant statistical significance in the 4 time points between the SAH group and the control group. The absorbance value of the p0.01. MTT two cells was higher than that of the control group, and the groups were all lower than those in the control group. In the control group, the difference was only statistically significant between the 48h time point SAH group and the control group in the PC3 cells. The number of membrane cells in the p0.05. Transwell two cells, the GNMT group were all higher than the control group, and the SAH group were all lower than the control group. The difference between the two cells was statistically significant at 36h and 48h time points, p0.05. conclusion: in cell culture The addition of GNMT and SAH, respectively, can be used to regulate and regulate GNMT in vitro, which can change the role of the biological properties of prostate cancer cell lines, LNCap and PC3, to inhibit the apoptosis of.GNMT and LNCap and PC3 cells, and the degree is not related to the androgen dependence of the cells; and LNCap and PC3 are fine. Cell proliferation has no obvious effect; it can increase the downregulation of invasive.GNMT activity of prostate cancer cells, especially PC3 cells: it can promote apoptosis of LNCap and PC3 cells, and is not related to the androgen dependence of cells; it can inhibit the proliferation of PC3 cells, but can not inhibit the proliferation of LNCap cells; can reduce the proliferation of cells; can reduce the proliferation of cells; The invasiveness of weak LNCap and PC3 cells and irrelevant to the androgen dependence of cells. Third the effect of part GNMT on human prostate cancer cell line PC3 tumorigenesis purpose: To study the effect of GNMT in vivo regulation of PC3 cells on the tumorigenicity of PC3 cells in nude mice. The mice were subcutaneously divided into 3 groups. The 2 experimental groups were fed with S- adenosine homocysteine (SAH, 10g/kg) at the mouth of CRA, 30 mu mol/kg. The control group had a normal diet for 6 weeks. The tumor size was measured every week and the tumor growth curve was plotted. After death, the expression of GNMT in the tumor was detected by immunohistochemistry. Results: 6 weeks after the tumor, the tumor body was detected. CRA group was significantly higher than the control group, the difference was statistically significant, P0.05, SAH group was significantly smaller than the control group, and the difference was statistically significant. The GNMT expression of CRA group in p0.05. tumor was significantly higher than the control group, the difference was statistically significant (the positive cell rate difference, P0.01; the cumulative light density difference, P0.05), and the SAH group was significantly less than the control group, and the difference was significantly lower than the control group. There is statistical significance (positive cell rate difference, P0.01; cumulative light density difference, P0.05). Conclusion: dietary intake of CRA and SAH can change the growth of human prostate cancer induced by PC3 cells in nude mice. This effect may be achieved by regulating the activity and expression of GNMT in the tumor. The intake of CRA can increase GNMT and thus promote the growth of the tumor. Tumor growth; ingestion of SAH can downregulate GNMT and inhibit tumor growth.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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