miR-29b在前列腺癌中的表達(dá)及其作用和機(jī)制的初步探討
發(fā)布時(shí)間：2019-03-17 10:40
【摘要】：第一部分miR-29b在前列腺癌組織中的表達(dá)及臨床意義 目的：探討miR-29b在前列腺癌組織中的表達(dá)及其臨床意義。 方法：用實(shí)時(shí)定量PCR技術(shù)檢測(cè)10例前列腺癌(PCa)及癌旁非腫瘤組織中miR-29b的表達(dá)情況,研究其與PCa病理分期、分級(jí)之間的關(guān)系。 結(jié)果：10例前列腺癌組織(prostate cancer, PCa)與配對(duì)的癌旁非腫瘤組織相比,miR-29b在PCa中的表達(dá)明顯下調(diào),有統(tǒng)計(jì)學(xué)差異(p0.05)。根據(jù)TNM病理分期,其中Tl-T2患者7例和T3b患者3例,兩組的miR-29b無(wú)統(tǒng)計(jì)學(xué)差異(p0.05)；根據(jù)Gleason評(píng)分進(jìn)行病理分級(jí),其中G1-G2患者4例,G3-G4患者6例,兩組miR-29b表達(dá)相比無(wú)統(tǒng)計(jì)學(xué)差異(p0.05)。 結(jié)論：(1)miR-29b在前列腺癌組織中的表達(dá)明顯低于癌旁組織。(2) miR-29b的表達(dá)與PCa病理分期及病理分級(jí)沒(méi)有明顯相關(guān)性。 第二部分miR-29b在不同前列腺癌細(xì)胞株中的表達(dá) 目的：探討miR-29b在不同前列腺癌細(xì)胞株中的表達(dá)及其與細(xì)胞惡性程度和雄激素依賴(lài)性的相關(guān)性,選擇miR-29b表達(dá)相對(duì)較低的細(xì)胞株進(jìn)行下一步實(shí)驗(yàn)。 方法：選取PC3、DU145、LNCap、LNCap-AI四種細(xì)胞系,其中PC3、DU145、LNCap為來(lái)源不同的細(xì)胞系。PC3和DU145具有惡性程度高且雄激素非依賴(lài)生長(zhǎng)的特點(diǎn),LNCap惡性程度低且雄激素依賴(lài)生長(zhǎng),而LNCap和LNCap-AI來(lái)源相同,LNCap-AI為L(zhǎng)NCap在逐步去雄激素環(huán)境下培養(yǎng)出的雄激素非依賴(lài)細(xì)胞。用實(shí)時(shí)定量PCR技術(shù)檢測(cè)這四種前列腺癌細(xì)胞株中miR-29b的表達(dá)情況。 結(jié)果：四種前列腺癌細(xì)胞系中表達(dá)水平由高到低分別為DU-145、PC-3、LNCap、LNCap-AI,四組間具有統(tǒng)計(jì)學(xué)差異(P0.05)。其中DU-145、PC-3中的rniR-29b表達(dá)水平明顯高于LNCap,而LNCap細(xì)胞中rniR-29b表達(dá)水平高于LNCap-AI細(xì)胞。 結(jié)論：(1) miR-29b在不同的前列腺癌細(xì)胞系中有不同的表達(dá)水平,且與雄激素非依賴(lài)性轉(zhuǎn)化過(guò)程有相關(guān)性。(2) LNCap細(xì)胞株中miR-29b表達(dá)相對(duì)較低,選擇LNCap細(xì)胞株進(jìn)行下一步實(shí)驗(yàn)。 第三部分miR-29b對(duì)前列腺癌細(xì)胞生物學(xué)行為的影響 目的：探討]miR-29b對(duì)前列腺癌細(xì)胞化療藥物敏感性、細(xì)胞周期、增殖、凋亡及侵襲能力等細(xì)胞生物學(xué)行為的影響。 方法：構(gòu)建miR-29b模擬物片段及干預(yù)序列,并轉(zhuǎn)染到LNCap細(xì)胞中。在熒光鏡下計(jì)算細(xì)胞轉(zhuǎn)染效率并用實(shí)時(shí)定量PCR檢測(cè)LNCap轉(zhuǎn)染細(xì)胞株中1miR-29b的表達(dá)水平。分別用MTT法檢測(cè)上調(diào)miR-29b對(duì)順鉑化療作用的影響,觀察細(xì)胞的存活率的變化,用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期變化,AV/PI雙標(biāo)法FCM檢測(cè)細(xì)胞的凋亡情況,Transwell細(xì)胞侵襲實(shí)驗(yàn)觀察細(xì)胞浸襲能力。 結(jié)果：成功構(gòu)建miR-29b模擬物片段及干預(yù)序列,并轉(zhuǎn)染到LNCap細(xì)胞中,細(xì)胞的轉(zhuǎn)染效率達(dá)到80%以上。與陰性對(duì)照組相比：miR-29b模擬物轉(zhuǎn)染組癌細(xì)胞細(xì)胞存活率明顯下降,而1niR-29b模擬物加順鉑組細(xì)胞存活率進(jìn)一步下降,rniR-29b可以增加癌細(xì)胞對(duì)順鉑的化療敏感性；1miR-29b模擬物轉(zhuǎn)染組亞G0/G1期明顯增加,G0/G1期、S期、G2/M期比例均減少,癌細(xì)胞增殖能力下降；AV/PI雙標(biāo)法FCM試驗(yàn)顯示1miR-29b模擬物轉(zhuǎn)染組細(xì)胞總凋亡率明顯下降；Transwell細(xì)胞侵襲實(shí)驗(yàn)顯示miR-29b模擬物轉(zhuǎn)染組細(xì)胞侵襲數(shù)量明顯減少。 結(jié)論：miR-29b高表達(dá)可以抑制前列腺癌細(xì)胞的增殖和侵襲能力,促進(jìn)癌細(xì)胞凋亡并且增加癌細(xì)胞對(duì)化療藥物敏感性。 第四部分前列腺癌中miR-29b作用靶基因的初步篩查 目的：初步探討前列腺癌中miR-29b可能的作用靶基因。 方法：生物信息學(xué)技術(shù)及查閱文獻(xiàn)的方法預(yù)測(cè)miR-29b可能的靶基因,Western-blot檢測(cè)LNCap細(xì)胞株中各預(yù)測(cè)靶基因蛋白的表達(dá)情況。結(jié)果：用生物信息學(xué)技術(shù)預(yù)測(cè)出8個(gè)miR-29b可能作用的靶基因：SOCS-1、Mcl-1、DNMT3b、MMP2、IFN-γ、SMAD3、SOCS5、AKT3、WB檢測(cè)rniR-29b表達(dá)變化后8種可能靶基因的蛋白變化,其中AKT3、Mcl-1和DNMT3b的蛋白表達(dá)在miR-29b模擬物轉(zhuǎn)染組有明顯下降,在miR-29b抑制物轉(zhuǎn)染組有明顯上升(P0.05) 結(jié)論：AKT3、Mcl-1和DNMT3b是miR-29b在前列腺癌中三種可能作用的靶基因,miR-29b可能通過(guò)靶向作用于此三種基因而發(fā)揮抑癌功能。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.25
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.25
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