miR-29a-3p通过调节血红素加氧酶1的表达在前列腺癌中的作用及机制研究

发布时间：2018-06-26 09:50

本文选题：前列腺癌 + MiR-29a-3p　；参考：《河北医科大学》2017年硕士论文

【摘要】：最新癌症数据统计显示在发达国家,前列腺癌仍然是男性癌症死亡的主要原因之一。而且,近年来在我国前列腺癌的患病率和死亡率也呈现上升趋势。然而,前列腺癌的基本机制仍然在很大程度上是未知的。因此,阐明前列腺癌进展的生物学机制是非常重要的。miR-29a-3p作为一种小的非编码RNA,参与细胞增殖,凋亡,分化等生理生化过程。miR-29a-3p在肿瘤的发展中也起到重要作用。最近,有研究显示,miR-29a-3p在前列腺癌组织中是异常表达的并且与细胞的侵袭、增殖等相关,但其在协调氧化应激,凋亡和增殖中的作用机制至今尚未报道。血红素加氧酶-1(HO-1)是一种细胞保护酶,具有抗氧化应激的作用。此外,其在抗炎和抗凋亡中也起关键作用。并且研究表明内源性HO-1通常在肿瘤组织中要比其在周围正常组织中表达要高。然而在前列腺癌中,HO-1和microRNA之间的作用关系尚没有研究。Bach2为CNC家族中B细胞特异性转录因子,有研究显示Bach2可通过抑制HO-1诱导细胞凋亡来应对氧化应激。并且Bach2在血液系统恶性肿瘤中发挥着重要作用,然而关于Bach2在实体瘤中的机制的报道却很少。目前在前列腺癌中关于Bach2调控氧化应激的作用尚未报道。因此在本研究中,我们旨在探讨miR-29a-3p/Bach2/HO-1在前列腺癌中作用及其分子机制。目的:探索miR-29a-3p对前列腺癌细胞增殖和凋亡的影响及其在前列腺癌进展中的作用。方法:1对在临床收取的20组人类前列腺癌组织和前列腺增生组织,HO-1联合miR-29a-3p荧光探针原位杂交定位miR-29a-3p的表达。2实时定量PCR(qRT-PCR)检测miR-29a-3p、Bach2在前列腺癌和前列腺增生组织中的表达。3免疫组织化学染色观察前列腺增生和前列腺癌组织中ho-1的表达量。4通过miranda和rnahybrid两网站对细胞凋亡和增殖相关基因进行预测。5合成靶基因3'非编码序列(utr)及其突变体并连接到pmir-glo双荧光报告质粒中,转染pc3细胞并进行双荧光报告基因活性检测。6pc3细胞中过表达或敲低mir-29a-3p,westernblot检测ho-1、bach2、pcna、caspase3和cyclind1的表达。7对pc3细胞进行敲低mir-29a-3p同时给予doc刺激,进行ho-1和bach2免疫双荧光染色。8在pc3细胞中过表达或敲低ho-1的同时给予doc刺激,tunel法检测细胞凋亡情况。9在pc3细胞中敲低mir-29a-3p的同时给予doc刺激,染色质免疫共沉淀技术(chip)检测bach2与ho-1启动子之间的关系。结果:1mir-29a-3p在临床前列腺癌组织和前列腺癌细胞系中是上调的荧光定量pcr结果显示,与bph组织相比,mir-29a-3p在前列腺癌组织中的表达显著增加(p0.01)。mir-29a-3p荧光探针免疫染色的原位杂交结果显示,mir-29a-3p在前列腺癌组织中显著表达,而在bph组织中表达很少。此外,荧光定量pcr结果显示,与正常前列腺上皮细胞相比,mir-29a-3p在所有lncap、pc3、du-145三种前列腺癌细胞系中的表达均显著上调。以上结果表明,mir-29a-3p在前列腺癌组织中的表达上调。2体外实验中mir-29a-3p促进前列腺癌细胞的迁移和增殖体外培养的pc3细胞分别过表达和敲低mir-29a-3p。细胞划痕实验结果显示,与对照组相比,过表达mir-29a-3p促进细胞的迁移。相反,敲低mir-29a-3p后,抑制细胞的迁移。同时,westernblot实验结果显示,过表达mir-29a-3p后,pcna、cyclind1的蛋白表达量增高,而caspase3的蛋白表达量降低。敲低mir-29a-3p后,pcna、cyclind1的蛋白表达量明显降低,caspase3的蛋白表达量显著升高。同时,在lncap细胞中,过表达和敲低mir-29a-3p,westernblot显示类似的结果。以上结果表明,在体外,mir-29a-3p能够促进前列腺癌细胞的迁移和增殖。3mir-29a-3p通过增加ho-1的表达来促进前列腺癌细胞的生长免疫组织化学染色结果显示,在前列腺癌不同阶段样本中,临床分期,psa水平以及gleason等级越高,ho-1的表达量也越高。转染pc3细胞24小时后,westernblot结果显示,较mimicctl组,mimicmir-29a-3p组ho-1蛋白的表达水平是升高的。而较mimicmir-29a-3p+anti-ctl组,mimicmir-29a-3p+anti-mir-29a-3p组ho-1蛋白表达水平则被抑制。mir-29a-3p荧光探针结合ho-1免疫染色的原位杂交结果显示,mir-29a-3p和ho-1在前列腺癌组织中的表达要比在bph组织中的表达明显增高。不同浓度doc处理pc3细胞24小时后,qrt-pcr结果显示,mir-29a-3p的表达呈多西紫杉醇浓度依赖性降低。转染pc3细胞,过表达和敲低mir-29a-3p,同时给予doc刺激,westernblot结果显示,较对照组,doc刺激组的ho-1蛋白表达水平降低,caspase3蛋白水平升高。较其他组,anti-mir-29a-3p+doc(10nm)组ho-1蛋白表达水平显著降低,而caspase3蛋白水平显著升高。过表达和敲低ho-1,并给与doc处理细胞,流式细胞术结果显示,较其他组,doc+ho-1敲低组显著增加细胞凋亡。tunel荧光染色分析结果显示类似的结果。以上结果显示mir-29a-3p增加ho-1的表达从而促进前列腺癌细胞的生长。4在前列腺癌中bach2是mir-29a-3p的直接靶标选取的mir-29a-3p的潜在靶基因进行荧光报告基因检测,结果显示,与对照组相比,含有wtbach23'utr的细胞可被mir-29a-3p模拟物抑制。westernblot实验显示,与对照组相比,mir-29a-3p过表达组bach2的蛋白水平显著降低,而敲低组bach2蛋白表达水平显著升高。qrt-pcr结果显示,与临床bph组织相比,前列腺癌组织中bach2的表达水平较低。随后进行bach2和mir-29a-3p表达的相关性分析,结果显示,bach2和mir-29a-3p在mrna水平上具有显著的统计学负相关性。5补救实验确认mir-29a-3p/bach2/ho-1轴调控前列腺癌发展补救实验,westernblot结果显示,在doc处理的条件下,与对照组相比,mir-29a-3p过表达可增加ho-1蛋白的表达,降低bach2蛋白的表达。相反,mir-29a-3p敲低可降低ho-1蛋白的表达,增加bach2蛋白的表达。当共转染ad-mir-29a-3p和si-bach2时,结果显示,与其他组相比,Ad-miR-29a-3p+si-Bach2组Bach2蛋白水平显著降低,而HO-1蛋白水平显著升高。6 Bach2通过结合HO-1启动子来调节HO-1的表达HO-1和Bach2免疫双荧光染色结果示,与对照组相比,miR-29a-3p敲低组的HO-1表达水平降低,Bach2的表达水平升高。当给予DOC刺激后,与对照组相比,miR-29a-3p敲低组的HO-1表达水平显著降低,Bach2的表达水平显著升高。荧光报告基因检测示,与对照组相比,敲低Bach2显著增加HO-1启动子活性。染色质免疫共沉淀(CHIP)实验结果显示,miR-29a-3p敲低组增加了Bach2与HO-1启动子的结合。以上结果表明Bach2可通过结合HO-1启动子来调控HO-1的表达。7在体内miR-29a-3p可促进前列腺癌的发生发展裸鼠皮下接种稳定转染的PC3细胞荷瘤。定期观察发现,稳定转染了LV-Anti-miR-29a-3p的PC3细胞的异种移植瘤组的肿瘤体积明显比对照组的小。上述所有结果表明mi R-29a-3p在前列腺癌进展中起到重要作用。结论:1 miR-29a-3p在前列腺癌组织和前列腺癌细胞中是高表达的。2 miR-29a-3p在体外和体内均可促进前列腺癌细胞的增殖。3 miR-29a-3p通过增加HO-1的表达,从而起到抗凋亡,促增殖作用。4在前列腺癌细胞中Bach2是mi R-29a-3p的直接靶基因。5 miR-29a-3p通过靶向抑制Bach2促进与氧化应激相关基因HO-1的表达,进而导致前列腺癌细胞的发展。
[Abstract]:The latest statistics of cancer data show that prostate cancer is still one of the main causes of male cancer death in developed countries. Moreover, the prevalence and mortality rate of prostate cancer in China are also rising in recent years. However, the basic mechanism of prostate cancer is still largely unknown. The physical mechanism is a very important.MiR-29a-3p as a small non coding RNA, and it plays an important role in the development of tumor, such as cell proliferation, apoptosis, differentiation and other physiological and biochemical processes. Recently, studies have shown that miR-29a-3p is abnormal expression in the prostate cancer tissue and is associated with cell invasion and proliferation. The mechanism of its role in coordinating oxidative stress, apoptosis and proliferation has not yet been reported. Heme oxygenase -1 (HO-1) is a kind of cytoprotective enzyme that has the effect of antioxidant stress. In addition, it also plays a key role in anti-inflammatory and anti apoptosis. And the study shows that endogenous HO-1 is usually in tumor tissue than it is in normal tissue. However, in prostate cancer, the relationship between HO-1 and microRNA has not yet been studied by.Bach2 as a B cell specific transcription factor in the CNC family. Studies have shown that Bach2 can respond to oxidative stress by inhibiting HO-1 induced apoptosis. And Bach2 plays an important role in the malignant tumor of the blood system, but on Bac The mechanism of H2 in solid tumors is rarely reported. The role of Bach2 in the regulation of oxidative stress in prostate cancer has not been reported. Therefore, in this study, we aim to explore the role and molecular mechanism of miR-29a-3p/Bach2/HO-1 in prostate cancer. The role in the progress of prostate cancer. Methods: 1 pairs of human prostate cancer tissues and prostatic hyperplasia in 20 groups of clinical cases, HO-1 combined with miR-29a-3p fluorescence probe in situ hybridization localization of miR-29a-3p,.2 real-time quantitative PCR (qRT-PCR) detection miR-29a-3p, Bach2 in prostatic adenocarcinoma and prostatic hyperplasia in the expression of.3 immune tissue The expression of HO-1 in prostate hyperplasia and prostate cancer tissue was observed by chemical staining.4 through the Miranda and rnahybrid two sites to predict the.5 synthetic target gene 3'non coding sequence (UTR) and its mutant and connect to the pmir-glo double fluorescent report substance, and transfect the PC3 cell and carry out the double fluorescent report base. The expression of HO-1, Bach2, PCNA, Caspase3 and CyclinD1 in.6pc3 cells expressed or knocked down by the activity detection of mir-29a-3p, and the expression of.7 on PC3 cells was low mir-29a-3p at the same time. Apoptotic condition.9 was stimulated by Doc at the same time in PC3 cells with low mir-29a-3p knockout, and chromatin immunoprecipitation (chip) was used to detect the relationship between Bach2 and HO-1 promoter. Results: 1mir-29a-3p in the clinical prostate cancer and prostate cancer cell lines was up-regulated and the results showed that the mir-29a-3p was compared with BPH tissue. The expression in the adenocarcinoma tissue was significantly increased (P0.01).Mir-29a-3p fluorescence probe immunostaining in situ hybridization results showed that mir-29a-3p was significantly expressed in the prostate cancer tissue, but the expression in the BPH tissue was few. In addition, the fluorescence quantitative PCR results showed that mir-29a-3p was in all LNCaP, PC3, DU-145 three compared with normal prostatic skin cells. The expression in the prostate cancer cell line was significantly up-regulated. The above results showed that the expression of mir-29a-3p in the prostate cancer tissue was up regulated in.2 in vitro and mir-29a-3p promoted the migration of prostate cancer cells and the proliferation of PC3 cells in vitro, and the results of low mir-29a-3p. cell scratch test showed that compared with the control group, The overexpression of mir-29a-3p promoted cell migration. On the contrary, after knocking down the mir-29a-3p, the cell migration was inhibited. At the same time, the Westernblot experiment showed that after overexpression of mir-29a-3p, the protein expression of PCNA, CyclinD1 was increased and the protein expression of Caspase3 decreased. After knocking down mir-29a-3p, the protein expression of PCNA, CyclinD1 decreased significantly, Caspase3. The expression of protein was significantly increased. At the same time, in LNCaP cells, over expression and knock down mir-29a-3p, Westernblot showed similar results. The above results suggest that in vitro, mir-29a-3p can promote the migration and proliferation of prostate cancer cells and.3mir-29a-3p can promote the growth of prostate cancer cells by increasing the expression of HO-1. The results showed that in the samples of different stages of prostate cancer, the higher the clinical stage, the PSA level and the Gleason level, the higher the expression of HO-1. After 24 hours transfection of PC3 cells, Westernblot results showed that the expression level of HO-1 protein in group mimicmir-29a-3p was higher than that of group mimicctl, and compared with group mimicmir-29a-3p+anti-ctl, mimicmir-29a-3p+ant. The expression level of HO-1 protein in i-mir-29a-3p group was inhibited by in situ hybridization of.Mir-29a-3p fluorescence probe combined with HO-1 immunostaining. The expression of mir-29a-3p and HO-1 in prostate cancer tissues was significantly higher than that in BPH tissues. After 24 hours of DOC treated PC3 cells at different concentrations, qRT-PCR results showed that mir-29a-3p expression was present. The concentration dependence of docetaxel decreased. Transfection of PC3 cells, overexpression and knock down mir-29a-3p, and DOC stimulation. Westernblot results showed that the expression level of HO-1 protein in the doc stimulation group decreased and the level of Caspase3 protein increased. The expression level of HO-1 protein in anti-mir-29a-3p+ doc (10nm) group was significantly lower than that of the other groups, and Caspase3 (10nm) was significantly lower than those in the other groups. The protein level was increased significantly. HO-1 was overexpressed and knocked down, and DOC treated cells were given. Flow cytometry showed that compared with other groups, the results of apoptotic.Tunel staining analysis in doc+ho-1 knockout groups showed similar results. The above results showed that mir-29a-3p increased the expression of HO-1 and thus promoted the growth of prostate cancer cells in.4. In prostate cancer, Bach2 is the potential target gene of the direct target of mir-29a-3p for the detection of the potential target gene of mir-29a-3p. The results show that compared with the control group, the cells containing wtbach23'utr can be inhibited by the mir-29a-3p analogue to the.Westernblot experiment, compared with the control group, the protein level of the Bach2 in the mir-29a-3p overexpression group is significant. The expression level of Bach2 protein in the lower group was significantly higher than that in the lower group..qrt-pcr results showed that the expression of Bach2 in the prostate cancer tissue was lower compared with the clinical BPH tissue. Then the correlation analysis of Bach2 and mir-29a-3p expression was carried out. The results showed that Bach2 and mir-29a-3p have significant statistical negative correlation in mRNA water leveling.5 remedial reality. Mir-29a-3p/bach2/ho-1 axis regulation of prostate cancer development remedial experiment, Westernblot results show that under the condition of DOC treatment, compared with the control group, mir-29a-3p overexpression can increase the expression of HO-1 protein and reduce the expression of Bach2 protein. On the contrary, mir-29a-3p knockdown can lower the expression of HO-1 protein and increase the expression of Bach2 protein. When transfected with ad-mir-29a-3p and si-bach2, the results showed that the level of Bach2 protein in the Ad-miR-29a-3p+si-Bach2 group decreased significantly compared with the other groups, while the HO-1 protein level significantly increased.6 Bach2 by combining the HO-1 promoter to regulate the HO-1 expression HO-1 and Bach2 immunofluorescence staining results. Compared with the control group, the.6 Bach2 was compared with the control group. The level of Bach2 expression increased. When DOC was stimulated, the expression level of HO-1 in the miR-29a-3p knockout group was significantly lower than that of the control group, and the expression level of Bach2 increased significantly. The fluorescence report gene showed that the low Bach2 significantly increased the activity of HO-1 promoter compared with the control group. The chromatin immunoprecipitation (CHIP) experimental node was significantly increased. The results showed that the miR-29a-3p knockout group increased the combination of Bach2 and HO-1 promoter. The above results showed that Bach2 could regulate the expression of HO-1 by combining HO-1 promoter and.7 in the body miR-29a-3p promoting the development of prostate cancer in nude mice. The stable transfected PC3 cell tumor in nude mice. The stable transfection of LV-Anti-miR-29a-3 was found. The tumor volume in the xenograft group of P PC3 cells was significantly smaller than that in the control group. All of the above results suggest that MI R-29a-3p plays an important role in the progression of prostate cancer. Conclusion: 1 miR-29a-3p, the high expression of.2 miR-29a-3p in the prostate and prostate cancer cells, can promote the increase of prostate cancer cells in vitro and in vivo. Colonization of.3 miR-29a-3p, by increasing the expression of HO-1, plays a role in anti apoptosis and proliferation promoting effect of.4 in prostate cancer cells, Bach2 is a direct target gene for MI R-29a-3p.5 miR-29a-3p through targeting inhibition Bach2 to promote the expression of HO-1 on oxidative stress related genes, thus leading to the development of prostate cancer cells.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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