miR-29b在前列腺癌中的表达及其作用和机制的初步探讨
发布时间：2019-03-17 10:40
【摘要】：第一部分miR-29b在前列腺癌组织中的表达及临床意义 目的：探讨miR-29b在前列腺癌组织中的表达及其临床意义。 方法：用实时定量PCR技术检测10例前列腺癌(PCa)及癌旁非肿瘤组织中miR-29b的表达情况,研究其与PCa病理分期、分级之间的关系。 结果：10例前列腺癌组织(prostate cancer, PCa)与配对的癌旁非肿瘤组织相比,miR-29b在PCa中的表达明显下调,有统计学差异(p0.05)。根据TNM病理分期,其中Tl-T2患者7例和T3b患者3例,两组的miR-29b无统计学差异(p0.05)；根据Gleason评分进行病理分级,其中G1-G2患者4例,G3-G4患者6例,两组miR-29b表达相比无统计学差异(p0.05)。 结论：(1)miR-29b在前列腺癌组织中的表达明显低于癌旁组织。(2) miR-29b的表达与PCa病理分期及病理分级没有明显相关性。 第二部分miR-29b在不同前列腺癌细胞株中的表达 目的：探讨miR-29b在不同前列腺癌细胞株中的表达及其与细胞恶性程度和雄激素依赖性的相关性,选择miR-29b表达相对较低的细胞株进行下一步实验。 方法：选取PC3、DU145、LNCap、LNCap-AI四种细胞系,其中PC3、DU145、LNCap为来源不同的细胞系。PC3和DU145具有恶性程度高且雄激素非依赖生长的特点,LNCap恶性程度低且雄激素依赖生长,而LNCap和LNCap-AI来源相同,LNCap-AI为LNCap在逐步去雄激素环境下培养出的雄激素非依赖细胞。用实时定量PCR技术检测这四种前列腺癌细胞株中miR-29b的表达情况。 结果：四种前列腺癌细胞系中表达水平由高到低分别为DU-145、PC-3、LNCap、LNCap-AI,四组间具有统计学差异(P0.05)。其中DU-145、PC-3中的rniR-29b表达水平明显高于LNCap,而LNCap细胞中rniR-29b表达水平高于LNCap-AI细胞。 结论：(1) miR-29b在不同的前列腺癌细胞系中有不同的表达水平,且与雄激素非依赖性转化过程有相关性。(2) LNCap细胞株中miR-29b表达相对较低,选择LNCap细胞株进行下一步实验。 第三部分miR-29b对前列腺癌细胞生物学行为的影响 目的：探讨]miR-29b对前列腺癌细胞化疗药物敏感性、细胞周期、增殖、凋亡及侵袭能力等细胞生物学行为的影响。 方法：构建miR-29b模拟物片段及干预序列,并转染到LNCap细胞中。在荧光镜下计算细胞转染效率并用实时定量PCR检测LNCap转染细胞株中1miR-29b的表达水平。分别用MTT法检测上调miR-29b对顺铂化疗作用的影响,观察细胞的存活率的变化,用流式细胞术检测细胞周期变化,AV/PI双标法FCM检测细胞的凋亡情况,Transwell细胞侵袭实验观察细胞浸袭能力。 结果：成功构建miR-29b模拟物片段及干预序列,并转染到LNCap细胞中,细胞的转染效率达到80%以上。与阴性对照组相比：miR-29b模拟物转染组癌细胞细胞存活率明显下降,而1niR-29b模拟物加顺铂组细胞存活率进一步下降,rniR-29b可以增加癌细胞对顺铂的化疗敏感性；1miR-29b模拟物转染组亚G0/G1期明显增加,G0/G1期、S期、G2/M期比例均减少,癌细胞增殖能力下降；AV/PI双标法FCM试验显示1miR-29b模拟物转染组细胞总凋亡率明显下降；Transwell细胞侵袭实验显示miR-29b模拟物转染组细胞侵袭数量明显减少。 结论：miR-29b高表达可以抑制前列腺癌细胞的增殖和侵袭能力,促进癌细胞凋亡并且增加癌细胞对化疗药物敏感性。 第四部分前列腺癌中miR-29b作用靶基因的初步筛查 目的：初步探讨前列腺癌中miR-29b可能的作用靶基因。 方法：生物信息学技术及查阅文献的方法预测miR-29b可能的靶基因,Western-blot检测LNCap细胞株中各预测靶基因蛋白的表达情况。结果：用生物信息学技术预测出8个miR-29b可能作用的靶基因：SOCS-1、Mcl-1、DNMT3b、MMP2、IFN-γ、SMAD3、SOCS5、AKT3、WB检测rniR-29b表达变化后8种可能靶基因的蛋白变化,其中AKT3、Mcl-1和DNMT3b的蛋白表达在miR-29b模拟物转染组有明显下降,在miR-29b抑制物转染组有明显上升(P0.05) 结论：AKT3、Mcl-1和DNMT3b是miR-29b在前列腺癌中三种可能作用的靶基因,miR-29b可能通过靶向作用于此三种基因而发挥抑癌功能。
[Abstract]:Expression of the first part of miR-29b in prostate cancer and its clinical significance Objective: To study the expression of miR-29b in prostate cancer and its clinical significance. Methods: The expression of miR-29b in 10 cases of prostate cancer (PCa) and non-tumor tissues was detected by real-time quantitative PCR. Results: The expression of miR-29b in PCa was significantly reduced in 10 cases of prostate cancer (PCa) compared with the paired non-tumor tissues, and there was a statistical difference (p 0.05). According to TNM pathological stage, there were 3 cases of T1-T2 and 3 cases of T3b, and there was no statistical difference between the two groups (p0.05); according to Gleason score, there were 4 cases of G1-G2 and 6 in G3-G4, and there was no statistical difference in the expression of miR-29b in the two groups (p Conclusion: (1) The expression of miR-29b in prostate cancer tissue The expression of miR-29b and the pathological staging and pathological grade of PCa were significantly lower than that in the adjacent tissues. There was no significant correlation. The second part of miR-29b was before Objective: To study the expression of miR-29b in different prostate cancer cell lines and its correlation with the degree of cell malignancy and androgen dependence, and to select the expression of miR-29b is relatively low. The method comprises the following steps of: selecting four cell lines of PC3, DU145, LNCap and LNCap-AI, wherein PC3, DU145 and L NCap is a source of different cell lines. PC3 and DU145 are characterized by a high degree of malignancy and non-dependent growth of androgen, with the same degree of malignancy and androgen-dependent growth of LNCap and the same source of LNCap and LNCap-AI, and LNCap-AI is the progressive deandrogen environment for LNCap. The four prostate cancer cell lines were detected by real-time quantitative PCR (RT-PCR). Results: The expression of miR-29b in four prostate cancer cell lines was DU-145, PC-3, LNCap, LNCap-AI and four groups, respectively. There was a statistical difference (P0.05). The expression level of the rniR-29b in the DU-145 and PC-3 was significantly higher than that of the LNCap, whereas the rniR-29b table in the LNCap cell Conclusion: (1) miR-29b has different expression levels in different prostate cancer cell lines, and the expression of the miR-29b in the LNCap cell line is relatively low, The LNCap cell line was selected for the next experiment. The third part mi Objective: To investigate the effect of R-29b on the biological behavior of prostate cancer cells: to study the sensitivity and cell cycle of the miR-29b to the chemotherapy of prostate cancer cells. Effects of proliferation, apoptosis, and invasive capacity on the biological behavior of cells. Methods: Construction of miR-29b The cell transfection efficiency was calculated under the fluorescence microscope and the L was detected by real-time quantitative PCR. The expression level of 1-miR-29b in NCap-transfected cell line was detected by MTT method. The effect of up-regulation of miR-29b on the chemotherapy of cisplatin was detected by MTT method, and the cell cycle change was detected by flow cytometry, and the apoptosis of the cells was detected by the method of flow cytometry. Transwell cell invasion experiment to observe the ability of cell immersion. Results: The miR-29b mimetic fragment and the intervention sequence were successfully constructed and transferred. In LNCap cells, the transfection efficiency of the cells reached more than 80%. Compared with the negative control group, the survival rate of the cells in the transfected group of the miR-29b analog was significantly decreased, while the survival rate of the 1niR-29b analog in the cisplatin group decreased further, and the rniR-29b can increase the chemotherapy sensitivity of the cancer cells to the cisplatin, and the miR-29b simulation There was a significant increase in the subG0/ G1 phase, the G0/ G1 phase, the S phase, the G2/ M phase, and the proliferation ability of the cancer cells. Conclusion: The high expression of miR-29b can inhibit the proliferation of prostate cancer cells. The ability to attack, to promote the apoptosis of cancer cells and to increase the sensitivity of cancer cells to chemotherapy A preliminary study on the effect of miR-29b on the target gene in the fourth part of prostate cancer Objective: To study the possible target genes of miR-29b in prostate cancer. Methods: The possible target genes of miR-29b were predicted by bioinformatics and the methods of reference. The expression of each of the predicted target genes in the LNCap cell line was detected by western-blot. Results: The possible target genes for miR-29b were predicted by bioinformatics technique: SOCS-1, Mcl-1, DNMT3b, MMP2, IFN-1, SMAD3, SOCS5, AKT3, and WB. Protein changes of 8 possible target genes after the expression of the rniR-29b expression, in which the protein expression of AKT3, Mcl-1 and DNMT3b is transferred to the miR-29b analog Conclusion: AKT3, Mcl-1 and DNMT3b are the three possible genes of miR-29b in the treatment of prostate cancer.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2442217
[Abstract]:Expression of the first part of miR-29b in prostate cancer and its clinical significance Objective: To study the expression of miR-29b in prostate cancer and its clinical significance. Methods: The expression of miR-29b in 10 cases of prostate cancer (PCa) and non-tumor tissues was detected by real-time quantitative PCR. Results: The expression of miR-29b in PCa was significantly reduced in 10 cases of prostate cancer (PCa) compared with the paired non-tumor tissues, and there was a statistical difference (p 0.05). According to TNM pathological stage, there were 3 cases of T1-T2 and 3 cases of T3b, and there was no statistical difference between the two groups (p0.05); according to Gleason score, there were 4 cases of G1-G2 and 6 in G3-G4, and there was no statistical difference in the expression of miR-29b in the two groups (p Conclusion: (1) The expression of miR-29b in prostate cancer tissue The expression of miR-29b and the pathological staging and pathological grade of PCa were significantly lower than that in the adjacent tissues. There was no significant correlation. The second part of miR-29b was before Objective: To study the expression of miR-29b in different prostate cancer cell lines and its correlation with the degree of cell malignancy and androgen dependence, and to select the expression of miR-29b is relatively low. The method comprises the following steps of: selecting four cell lines of PC3, DU145, LNCap and LNCap-AI, wherein PC3, DU145 and L NCap is a source of different cell lines. PC3 and DU145 are characterized by a high degree of malignancy and non-dependent growth of androgen, with the same degree of malignancy and androgen-dependent growth of LNCap and the same source of LNCap and LNCap-AI, and LNCap-AI is the progressive deandrogen environment for LNCap. The four prostate cancer cell lines were detected by real-time quantitative PCR (RT-PCR). Results: The expression of miR-29b in four prostate cancer cell lines was DU-145, PC-3, LNCap, LNCap-AI and four groups, respectively. There was a statistical difference (P0.05). The expression level of the rniR-29b in the DU-145 and PC-3 was significantly higher than that of the LNCap, whereas the rniR-29b table in the LNCap cell Conclusion: (1) miR-29b has different expression levels in different prostate cancer cell lines, and the expression of the miR-29b in the LNCap cell line is relatively low, The LNCap cell line was selected for the next experiment. The third part mi Objective: To investigate the effect of R-29b on the biological behavior of prostate cancer cells: to study the sensitivity and cell cycle of the miR-29b to the chemotherapy of prostate cancer cells. Effects of proliferation, apoptosis, and invasive capacity on the biological behavior of cells. Methods: Construction of miR-29b The cell transfection efficiency was calculated under the fluorescence microscope and the L was detected by real-time quantitative PCR. The expression level of 1-miR-29b in NCap-transfected cell line was detected by MTT method. The effect of up-regulation of miR-29b on the chemotherapy of cisplatin was detected by MTT method, and the cell cycle change was detected by flow cytometry, and the apoptosis of the cells was detected by the method of flow cytometry. Transwell cell invasion experiment to observe the ability of cell immersion. Results: The miR-29b mimetic fragment and the intervention sequence were successfully constructed and transferred. In LNCap cells, the transfection efficiency of the cells reached more than 80%. Compared with the negative control group, the survival rate of the cells in the transfected group of the miR-29b analog was significantly decreased, while the survival rate of the 1niR-29b analog in the cisplatin group decreased further, and the rniR-29b can increase the chemotherapy sensitivity of the cancer cells to the cisplatin, and the miR-29b simulation There was a significant increase in the subG0/ G1 phase, the G0/ G1 phase, the S phase, the G2/ M phase, and the proliferation ability of the cancer cells. Conclusion: The high expression of miR-29b can inhibit the proliferation of prostate cancer cells. The ability to attack, to promote the apoptosis of cancer cells and to increase the sensitivity of cancer cells to chemotherapy A preliminary study on the effect of miR-29b on the target gene in the fourth part of prostate cancer Objective: To study the possible target genes of miR-29b in prostate cancer. Methods: The possible target genes of miR-29b were predicted by bioinformatics and the methods of reference. The expression of each of the predicted target genes in the LNCap cell line was detected by western-blot. Results: The possible target genes for miR-29b were predicted by bioinformatics technique: SOCS-1, Mcl-1, DNMT3b, MMP2, IFN-1, SMAD3, SOCS5, AKT3, and WB. Protein changes of 8 possible target genes after the expression of the rniR-29b expression, in which the protein expression of AKT3, Mcl-1 and DNMT3b is transferred to the miR-29b analog Conclusion: AKT3, Mcl-1 and DNMT3b are the three possible genes of miR-29b in the treatment of prostate cancer.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25
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