三氧化二砷对前列腺癌DU145细胞中KEAP1基因表达作用研究

发布时间：2018-05-18 16:19

本文选题：三氧化二砷 + 前列腺癌　；参考：《郑州大学》2014年硕士论文

【摘要】：背景和目的前列腺癌是在男性泌尿生殖系统中最常见的恶性肿瘤之一。目前治疗前列腺癌的主要方法有手术，激素拮抗治疗，化疗和放疗。虽然大多数前列腺癌患者最初的雄激素去势有效，但他们通常会在数月至数年后出现在雄激素非依赖性。雄激素非依赖性的前列腺癌的预后比较差，且多对化疗和放射治疗不敏感。目前激素非依赖型前列腺癌对放、化疗抵抗的机制目前还不明确。因此了解肿瘤细胞如何产生耐药及如何才能逆转耐药是急需解决的重要问题。近几年来已有多项试验证实KEAP1-NRF2-ARE信号通路与多重耐药机制相关联[1,2]，目前已经在NSCL、乳腺癌、结肠癌等肿瘤组织中发现KEAP1基因的突变，使表达相关蛋白下降且NRF2相关蛋白高表达[3,4]，已有实验证实在非小细胞肺癌，结肠癌，前列腺癌DU145细胞中KEAP1基因启动子区CpG岛高甲基化而低表达[5,6]，前列腺癌PC3细胞中KEAP1基因高表达，所以本实验选择PC3细胞做为阳性对照[7]。我国学者将三氧化二砷用于治疗急性早幼粒细胞白血病取得显著疗效，引起了普遍关注；随后证实其对实体瘤也能获得同样效果，同时三氧化二砷的去甲基化作用也有报道。本研究通过用不同浓度As2O3溶液对前列腺癌DU145细胞进行处理，检测药物对细胞增殖抑制影响及对细胞中KEAP1基因表达的影响，初步探索As2O3对KEAP1基因可能的作用机制，从而为激素非依赖性前列腺癌的治疗提供新的治疗药物。材料和方法 1．雄激素非依赖性前列腺癌DU145细胞，用10%灭活胎牛血清含双抗的新鲜DMEM-H培养液在细胞培养箱中（T：37℃,5%CO2）常规培养；PC-3细胞用含10%灭活胎牛血清的新鲜RPMI-1640培养液培养。 2．应用MTT法检测不同浓度(0、0.5、1.0、2.0、4.0、6.0、8.0μmol/L)的As2O3不同作用时间(24、48、72h)对DU145细胞的生长抑制作用。 3.应用RT-PCR方法检测不同浓度（0、0.5、1.0、2.0、4.0μmol/L）As2O3作用于各组DU145细胞72h后KEAP1基因mRNA表达情况。 4.应用Western blot方法检测不同浓度组(0.0、0.5、1.0、2.0、4.0μmol/L）As2O3的作用激素非依赖性前列腺癌DU145细胞株72h后KEAP1蛋白表达的情况，PC3细胞组作为对照组。 5．统计学处理：实验数据用SPSS17.0统计软件进行处理，各组数据间采用非参数检验、t检验及方差分析进行差异比较，检验水准α=0.05。结果 1．MTT结果显示：随着As203浓度的升高对DU145细胞抑制作用越明显，在8．0μmol/L时抑制率最高；根据不同时间点对抑制率的测定在72h时抑制率最高。因此As203对DU145细胞的抑制作用具有剂量依赖性和时间依赖性。 2．RT-PCR结果显示：在不含As2O3的处理组中，前列腺癌DU145细胞的KEAP1基因mRNA表达不明显，经As2O3处理72h后，KEAP1基因表达逐渐增强，在2.0μmol/L时条带亮度最大，表达最强。 3．Western blot结果显示：阴性对照组keap1蛋白未表达；不同浓度As2O3处理人前列腺癌DU145细胞72h后，各观察组KEAP1蛋白均出现表达；2.0μmol/L As2O3处理组与阳性对照PC-3组显示的KEAP1蛋白条带最深。结论 1.As2O3对前列腺癌DU145细胞的增殖具有抑制作用，，并且随着剂量的增大或时间的增长抑制作用而逐渐增强，呈剂量依赖性和时间依赖性。 2.As2O3可以诱导KEAP1基因的mRNA和KEAP蛋白表达增加。
[Abstract]:Background and purpose
Prostate cancer is one of the most common malignant tumors in the male genitourinary system. The main methods for treating prostate cancer are surgery, hormone antagonism, chemotherapy and radiotherapy. Although the initial androgen deprivation of most prostate cancer patients is effective, they usually appear in androgens for months to years. The prognosis of hormone non dependent prostate cancer is poor, and it is not sensitive to chemotherapy and radiation therapy. The mechanism of hormone non dependent prostate cancer therapy is not clear at present. Therefore, it is an important problem to understand how cancer cells produce resistance and how to reverse the drug resistance.
In recent years, many experiments have proved that KEAP1-NRF2-ARE signaling pathway is associated with multidrug resistance mechanism [1,2]. The mutation of KEAP1 gene has been found in NSCL, breast cancer, colon cancer and other tumor tissues. The expression related protein is decreased and the NRF2 related protein is highly expressed [3,4]. It has been proved to be in the non small cell lung cancer, colon cancer, and the front row. The CpG island of KEAP1 gene promoter region of adenocarcinoma DU145 cells is highly methylation and low expression of [5,6], and the KEAP1 gene in PC3 cells of prostate cancer is highly expressed. Therefore, this experiment selected PC3 cells as a positive control [7]. in China to use arsenic trioxide to treat acute promyelocytic leukemia. It is confirmed that it can also achieve the same effect on solid tumor, and the demethylation of arsenic trioxide is also reported. This study was conducted by using different concentrations of As2O3 solution to treat DU145 cells in prostate cancer, to detect the effect of drugs on cell proliferation inhibition and to the expression of KEAP1 gene in cells, and to explore the possibility of As2O3 to the KEAP1 gene. Therefore, it provides a new treatment for hormone independent prostate cancer.
Materials and methods
1. the androgen independent prostate cancer DU145 cells were cultured in the cell culture box (T:37, 5%CO2) with 10% inactivated fetal bovine serum containing fresh DMEM-H culture in the cell culture box (T:37, 5%CO2), and the PC-3 cells were cultured with fresh RPMI-1640 culture containing 10% inactivated fetal bovine serum.
2. MTT method was used to detect the growth inhibition of DU145 cells with different concentrations (0,0.5,1.0,2.0,4.0,6.0,8.0 mu mol/L) of As2O3 at different time of action (24,48,72h).
3. RT-PCR method was used to detect mRNA expression of KEAP1 gene after different concentrations (0,0.5,1.0,2.0,4.0 mol/L) As2O3 acting on 72h cells of each group.
4. the Western blot method was used to detect the expression of KEAP1 protein in the non dependent prostate cancer DU145 cell line 72h, and the PC3 cell group was used as the control group. The Western blot method was used to detect the different concentration group (0.0,0.5,1.0,2.0,4.0 mu mol/L).
5. statistical processing: the experimental data were processed by SPSS17.0 statistical software. The data between each group were tested by nonparametric test, t test and variance analysis were compared, and the level of alpha =0.05. was tested.
Result
1.MTT results showed that the inhibition of DU145 cells was more obvious with the increase of As203 concentration and the highest inhibition rate at 8 mol/L; the inhibition rate was highest at 72h at different time points. Therefore, the inhibitory effect of As203 on DU145 cells was dose-dependent and time dependent.
2.RT-PCR results showed that in the treatment group without As2O3, the expression of KEAP1 gene mRNA in DU145 cells of prostate cancer was not obvious. After As2O3 treatment 72h, the expression of KEAP1 gene was increased gradually, and the band brightness was the greatest and the expression was the strongest at 2 u.
The results of 3.Western blot showed that the Keap1 protein in the negative control group was not expressed, and the KEAP1 protein was expressed in all the observation groups after 72h of As2O3 treated DU145 cells of human prostate cancer, and the KEAP1 protein bands in the 2 u mol/L As2O3 treatment group and the positive control PC-3 group were the deepest.
conclusion
1.As2O3 has a inhibitory effect on the proliferation of DU145 cells in prostate cancer and gradually increases with the dose increase or time growth inhibition, which is dose-dependent and time dependent.
2.As2O3 can induce the expression of mRNA and KEAP protein in KEAP1 gene.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

【参考文献】