靶向TAK1基因的siRNA干扰前列腺癌DU145细胞的实验研究

发布时间：2018-05-28 13:46

本文选题：转化生长因子-β激活激酶 + 前列腺癌　；参考：《兰州大学》2014年硕士论文

【摘要】：研究背景局限性前列腺癌的五年生存率为100%,但是进展期的前列腺癌的五年生存率大约为31%。大多数患者采用内分泌治疗24月内,。绝大多数患者晚期前列腺癌又会发生骨转移,且骨转移事件一旦发生将会直接危害患者生活质量和生命。目前,前列腺癌唯一可以治愈的治疗手段为前列腺癌根治术。而不能行前列腺癌根治术的患者,目前多会全雄激素阻断治疗,继而大约70%的患者会出现骨转移现象。因此急需开发一种新的有效的治疗方法,来增加CRPC患者的生存时间、降低骨转移相关并发症对的发生率。前列腺癌的发生、发展和基因突变及多种细胞因子的失衡有关。进展期前列腺癌可以高表达多种和细胞生长、侵袭、迁移及成瘤性等相关的生长因子,TGF-岱是其中的一种。TGF-β在前列腺癌的发展中表现为双重作用,在肿瘤细胞的早期主要起到抑制肿瘤细胞生长的作用,但是在进展期肿瘤中TGF-β的作用转变为促进肿瘤细胞的侵袭性和至瘤性,高表达的TGF-β可以诱导溶骨细胞介导的骨质破坏。经典的TGF-β信号通路为smad信号通路,但是non-smad信号通路在肿瘤骨转移中的研究的也比较多。Non-samd信号通路中的转化生长因子β蛋白激活激酶1(TGF-β activated protein kinase1, TAK1)对乳腺癌的骨转移起着重要作用。因此本研究通过siRNA沉默人前列腺癌DU145细胞系TAK1基因的表达,初步研究敲除TAK1基因对人前列腺癌DU145细胞增殖、侵袭、迁移、凋亡及药物敏感性性的变化及作用机制。在前列腺癌发展、转移、耐药等方面,为深入研究TAK1基因的作用机制奠定基础。研究目的研究TAK1siRNA对目标基因表达的干扰作用,检测沉默TAK1表达对前列腺癌DU145细胞增殖、侵袭、迁移、凋亡及药物敏感性的影响及相关蛋白的表达情况,并初步探讨其作用机制。研究方法 1.应用siRNA靶向沉默前列腺癌DU145细胞的TAK1基因的表达,qRT-PCR检测TAK1mRNA的表达情况,计算TAK1siRNA的基因敲除效率。 2.应用蛋白免疫印迹方法检测TAK1siRNA转染前后TAK1蛋白表达的变化 3.成功沉默TAK1基因表达后,利用绘制的生长曲线检测沉默TAK1基因对前列腺癌细胞增殖能力影响。 4.应用Transwell小室和划痕实验来分析TAK1siRNA转染前后前列腺癌细胞DU145侵袭和迁徙能力的变化。 5.利用骨髓诱导细胞外基质(BM-ECM)和transwell小室(8.0μm和0.4μm孔径)构建体外骨转移模型,观察BM-ECM对前列腺癌细胞的生长促进作用和趋化作用。 6.在TAK1siRNA转染前后,利用流式细胞仪检测人前列腺癌细胞凋亡情况的变化。 7.用MTT法检测TAK1siRNA转染前后前列腺癌DU145细胞对三种化疗药物(多西紫杉醇(docetaxel)、奥沙利铂(L-OHP)及5-氟尿嘧啶(5-FU))的药物敏感性的变化。 8.在TAK1siRNA转染前后,通过实时荧光定量PCR和Western blot检测前列腺癌细胞表皮生长因子受体(EGFR)、凋亡抑制基因(Bcl-2)、环氧合酶-2(COX-2、β-连环蛋白(β-catenin)、MMP-2、9(基质金属蛋白酶-2、9)及氨基末端激酶(JNK)等基因在转录和翻译水平的变化。研究结果 1.利用脂质体转染TAK1siRNA可以高效的敲除TAK1基因的表达,RT-PCR和western blot分析提示,TAK1siRNA可以显著抑制TAK1基因的转录与翻译,差异有统计学意义(P0.05)。 2.细胞生长曲线提示利用TAK1siRNA敲除TAKl基因后,前列腺癌DU145细胞的增殖能力被减弱,从第四天开始差异有统计学意义(P0.05) 3.利用transwell和划痕实验检测侵袭和迁移能力,TAK1siRNA转染后前列腺癌DU145细胞的侵袭和迁移能力均明显下降,差异有统计学意义(P0.05)。 4.流式细胞仪检测转染前后前列腺癌细胞凋亡率分别为4.13%士0.11%和13.21%士1.41%,敲除TAKl基因表达可以明显增加前列腺癌细胞的凋亡率,两组之间比较,差异有统计学意义(P0.05)。 5.骨髓诱导细胞外基质(BM-ECM)对前列腺癌DU145细胞具有促进生长、侵袭和迁移作用,差异有统计学意义(P0.05)。 6.转染TAK1siRNA后,前列腺癌DU145对多西他赛紫杉醇、L-OHP、5-FU的IC50显著低于阴性对照组,TAK1基因沉默后前列腺癌DU145细胞对化疗药物的敏感性明显增加,差异有统计学意义(P0.05)；对照组之间三种药物的IC50之间IC50接近,差异没有统计学意义(P0.05)。 7.转染48h时,siRNA-TAK1组的COX-2、Bcl-2、JNK、β-Catenin, MMP2及MMP9的mRNA和蛋白相对表达量均低于negative control组及control组的表达量,差异有统计学意义(P0.05),而control组与negative control组之间,前列腺癌细胞中上述基因表达量差异无统计学意义P0.05) 结论：沉默前列腺癌DU145细胞TAK1基因的表达可以降低凋亡相关蛋白Bcl-2、JNK,耐药基因环氧合酶-2蛋白,表皮生长因子受体等的表达,从而促进细胞凋亡,抑制细胞生长,增加对化疗药物的敏感性；同时可以下调MMP2,MMP9表达可以降低前列腺癌DU145细胞的侵袭及迁移能力。TAKl基因可能是前列腺癌细胞信号转导网络中的一个关键性靶点。
[Abstract]:Research background
The five year survival rate of localized prostate cancer is 100%, but the five year survival rate of advanced prostate cancer is about 31%. in most patients with endocrine therapy for 24 months. Most patients with advanced prostate cancer will have bone metastases, and the occurrence of bone metastases will directly harm the quality of life and life of the patients.
At present, the only cure for prostate cancer is radical prostatectomy. But patients who do not undergo radical prostatectomy have more androgen blockade, and then about 70% of the patients have bone metastases. Therefore, a new and effective treatment method is urgently needed to increase the survival time of CRPC patients. The incidence of complications associated with bone metastases.
The development of prostate cancer is related to gene mutation and the imbalance of various cytokines. Progressing stage prostate cancer can express a variety of growth factors, such as cell growth, invasion, migration and tumorigenicity. TGF- Dai is one of the.TGF- beta in the development of prostate cancer, which is a double role in the early stage of cancer cells. It plays a role in inhibiting the growth of tumor cells, but the role of TGF- beta in advanced tumors is to promote the invasiveness and tumorigenicity of tumor cells. The high expression of TGF- beta can induce osteoblast mediated bone destruction. The classic TGF- beta signaling pathway is the Smad signaling pathway, but it is the research of the non-Smad signaling pathway in the metastasis of tumor bone. In addition, the TGF beta protein activated kinase 1 (TGF- beta activated protein kinase1, TAK1) in multiple.Non-samd signaling pathways plays an important role in bone metastasis of breast cancer.
Therefore, through the expression of TAK1 gene in the DU145 cell line of siRNA silent human prostate cancer, this study preliminarily studies the changes in the proliferation, invasion, migration, apoptosis and drug sensitivity of the TAK1 gene in human prostate cancer cells, and the mechanism of the development, metastasis, and drug resistance of prostate cancer, which lays a foundation for the research of the mechanism of the TAK1 gene in the development of prostate cancer. Set the foundation.
research objective
To investigate the effect of TAK1siRNA on the expression of target gene, and to detect the effect of silent TAK1 expression on the proliferation, invasion, migration, apoptosis and drug sensitivity of DU145 cells in prostate cancer and the expression of related proteins, and to explore the mechanism of its action.
research method
1. siRNA was used to silence the expression of TAK1 gene in prostate cancer DU145 cells, qRT-PCR was used to detect the expression of TAK1mRNA, and the gene knockout efficiency of TAK1siRNA was calculated.
2. protein immunoblotting was used to detect the expression of TAK1 protein before and after TAK1siRNA transfection.
3. after successful silencing of TAK1 gene expression, the growth curve was used to detect the effect of silencing TAK1 gene on the proliferation of prostate cancer cells.
4. Transwell chamber and scratch test were used to analyze the changes of invasion and migration ability of prostate cancer cell DU145 before and after TAK1siRNA transfection.
5. bone marrow induced extracellular matrix (BM-ECM) and Transwell compartment (8 m and 0.4 micron m aperture) were used to construct an in vitro bone metastasis model to observe the growth and chemotaxis effect of BM-ECM on the growth of prostate cancer cells.
6. before and after TAK1siRNA transfection, the apoptosis of human prostate cancer cells was detected by flow cytometry.
7. the changes in susceptibility to three chemotherapeutic drugs (docetaxel (docetaxel), Asha Leigh Per (L-OHP) and 5- fluorouracil (5-FU) were detected by MTT method before and after TAK1siRNA transfection.
8. before and after TAK1siRNA transfection, the expression of epidermal growth factor receptor (EGFR), apoptosis suppressor gene (Bcl-2), COX-2, beta catenin (beta -catenin), MMP-2,9 (-2,9) and amino terminal kinase (JNK) were detected by real-time fluorescence quantitative PCR and Western blot in the transcription and translation levels. Change.
Research results
1. transfection of TAK1siRNA with liposomes can efficiently knock out the expression of TAK1 gene. RT-PCR and Western blot analysis suggest that TAK1siRNA can significantly inhibit the transcription and translation of the TAK1 gene, and the difference is statistically significant (P0.05).
The 2. cell growth curve suggested that the proliferation ability of DU145 cells in prostate cancer was weakened after TAK1siRNA knockout TAKl gene, and the difference was statistically significant from the beginning of the fourth day (P0.05).
3. the invasion and migration ability was detected by Transwell and scratch test. The invasion and migration ability of DU145 cells in prostate cancer were significantly decreased after TAK1siRNA transfection, and the difference was statistically significant (P0.05).
4. flow cytometry was used to detect the apoptosis rate of prostate cancer cells before and after transfection. The apoptosis rate of prostate cancer cells was significantly increased by knockout TAKl gene expression, and the difference between the two groups was statistically significant (P0.05).
5. bone marrow induced extracellular matrix (BM-ECM) promoted the growth, invasion and migration of prostate cancer DU145 cells, and the difference was statistically significant (P0.05).
After 6. transfection of TAK1siRNA, the IC50 of prostate cancer DU145 to docetaxel paclitaxel, L-OHP, 5-FU was significantly lower than that in the negative control group. The sensitivity of DU145 cells to chemotherapeutic drugs was significantly increased after the TAK1 gene silencing, and the difference was statistically significant (P0.05); the IC50 of the three drugs between the control groups was close to the IC50, and the difference was not statistically significant. Meaning (P0.05).
The expression of mRNA and protein in COX-2, Bcl-2, JNK, beta -Catenin, MMP2 and MMP9 in group siRNA-TAK1 was lower than that of negative control and control groups when transfected with 48h, and there was a significant difference between the negative control group and the control group. There was no significant difference in the expression of the above gene expression in the Prost adenocarcinoma cells between the group and the group. 5)
Conclusion:
The expression of TAK1 gene of silent prostate cancer DU145 cells can reduce the expression of apoptosis related protein Bcl-2, JNK, drug resistant gene cyclooxygenase -2 protein, epidermal growth factor receptor and so on, thus promoting apoptosis, inhibiting cell growth, increasing sensitivity to chemotherapeutic drugs, decreasing MMP2, MMP9 expression can reduce the prostate cancer DU14. 5 the invasion and migration ability of.TAKl cells may be a key target in prostate cancer cell signal transduction network.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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