本文選題：前列腺癌 + MiR-29a-3p��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：最新癌癥數(shù)據(jù)統(tǒng)計(jì)顯示在發(fā)達(dá)國(guó)家,前列腺癌仍然是男性癌癥死亡的主要原因之一。而且,近年來在我國(guó)前列腺癌的患病率和死亡率也呈現(xiàn)上升趨勢(shì)。然而,前列腺癌的基本機(jī)制仍然在很大程度上是未知的。因此,闡明前列腺癌進(jìn)展的生物學(xué)機(jī)制是非常重要的。miR-29a-3p作為一種小的非編碼RNA,參與細(xì)胞增殖,凋亡,分化等生理生化過程。miR-29a-3p在腫瘤的發(fā)展中也起到重要作用。最近,有研究顯示,miR-29a-3p在前列腺癌組織中是異常表達(dá)的并且與細(xì)胞的侵襲、增殖等相關(guān),但其在協(xié)調(diào)氧化應(yīng)激,凋亡和增殖中的作用機(jī)制至今尚未報(bào)道。血紅素加氧酶-1(HO-1)是一種細(xì)胞保護(hù)酶,具有抗氧化應(yīng)激的作用。此外,其在抗炎和抗凋亡中也起關(guān)鍵作用。并且研究表明內(nèi)源性HO-1通常在腫瘤組織中要比其在周圍正常組織中表達(dá)要高。然而在前列腺癌中,HO-1和microRNA之間的作用關(guān)系尚沒有研究。Bach2為CNC家族中B細(xì)胞特異性轉(zhuǎn)錄因子,有研究顯示Bach2可通過抑制HO-1誘導(dǎo)細(xì)胞凋亡來應(yīng)對(duì)氧化應(yīng)激。并且Bach2在血液系統(tǒng)惡性腫瘤中發(fā)揮著重要作用,然而關(guān)于Bach2在實(shí)體瘤中的機(jī)制的報(bào)道卻很少。目前在前列腺癌中關(guān)于Bach2調(diào)控氧化應(yīng)激的作用尚未報(bào)道。因此在本研究中,我們旨在探討miR-29a-3p/Bach2/HO-1在前列腺癌中作用及其分子機(jī)制。目的:探索miR-29a-3p對(duì)前列腺癌細(xì)胞增殖和凋亡的影響及其在前列腺癌進(jìn)展中的作用。方法:1對(duì)在臨床收取的20組人類前列腺癌組織和前列腺增生組織,HO-1聯(lián)合miR-29a-3p熒光探針原位雜交定位miR-29a-3p的表達(dá)。2實(shí)時(shí)定量PCR(qRT-PCR)檢測(cè)miR-29a-3p、Bach2在前列腺癌和前列腺增生組織中的表達(dá)。3免疫組織化學(xué)染色觀察前列腺增生和前列腺癌組織中ho-1的表達(dá)量。4通過miranda和rnahybrid兩網(wǎng)站對(duì)細(xì)胞凋亡和增殖相關(guān)基因進(jìn)行預(yù)測(cè)。5合成靶基因3'非編碼序列(utr)及其突變體并連接到pmir-glo雙熒光報(bào)告質(zhì)粒中,轉(zhuǎn)染pc3細(xì)胞并進(jìn)行雙熒光報(bào)告基因活性檢測(cè)。6pc3細(xì)胞中過表達(dá)或敲低mir-29a-3p,westernblot檢測(cè)ho-1、bach2、pcna、caspase3和cyclind1的表達(dá)。7對(duì)pc3細(xì)胞進(jìn)行敲低mir-29a-3p同時(shí)給予doc刺激,進(jìn)行ho-1和bach2免疫雙熒光染色。8在pc3細(xì)胞中過表達(dá)或敲低ho-1的同時(shí)給予doc刺激,tunel法檢測(cè)細(xì)胞凋亡情況。9在pc3細(xì)胞中敲低mir-29a-3p的同時(shí)給予doc刺激,染色質(zhì)免疫共沉淀技術(shù)(chip)檢測(cè)bach2與ho-1啟動(dòng)子之間的關(guān)系。結(jié)果:1mir-29a-3p在臨床前列腺癌組織和前列腺癌細(xì)胞系中是上調(diào)的熒光定量pcr結(jié)果顯示,與bph組織相比,mir-29a-3p在前列腺癌組織中的表達(dá)顯著增加(p0.01)。mir-29a-3p熒光探針免疫染色的原位雜交結(jié)果顯示,mir-29a-3p在前列腺癌組織中顯著表達(dá),而在bph組織中表達(dá)很少。此外,熒光定量pcr結(jié)果顯示,與正常前列腺上皮細(xì)胞相比,mir-29a-3p在所有l(wèi)ncap、pc3、du-145三種前列腺癌細(xì)胞系中的表達(dá)均顯著上調(diào)。以上結(jié)果表明,mir-29a-3p在前列腺癌組織中的表達(dá)上調(diào)。2體外實(shí)驗(yàn)中mir-29a-3p促進(jìn)前列腺癌細(xì)胞的遷移和增殖體外培養(yǎng)的pc3細(xì)胞分別過表達(dá)和敲低mir-29a-3p。細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示,與對(duì)照組相比,過表達(dá)mir-29a-3p促進(jìn)細(xì)胞的遷移。相反,敲低mir-29a-3p后,抑制細(xì)胞的遷移。同時(shí),westernblot實(shí)驗(yàn)結(jié)果顯示,過表達(dá)mir-29a-3p后,pcna、cyclind1的蛋白表達(dá)量增高,而caspase3的蛋白表達(dá)量降低。敲低mir-29a-3p后,pcna、cyclind1的蛋白表達(dá)量明顯降低,caspase3的蛋白表達(dá)量顯著升高。同時(shí),在lncap細(xì)胞中,過表達(dá)和敲低mir-29a-3p,westernblot顯示類似的結(jié)果。以上結(jié)果表明,在體外,mir-29a-3p能夠促進(jìn)前列腺癌細(xì)胞的遷移和增殖。3mir-29a-3p通過增加ho-1的表達(dá)來促進(jìn)前列腺癌細(xì)胞的生長(zhǎng)免疫組織化學(xué)染色結(jié)果顯示,在前列腺癌不同階段樣本中,臨床分期,psa水平以及gleason等級(jí)越高,ho-1的表達(dá)量也越高。轉(zhuǎn)染pc3細(xì)胞24小時(shí)后,westernblot結(jié)果顯示,較mimicctl組,mimicmir-29a-3p組ho-1蛋白的表達(dá)水平是升高的。而較mimicmir-29a-3p+anti-ctl組,mimicmir-29a-3p+anti-mir-29a-3p組ho-1蛋白表達(dá)水平則被抑制。mir-29a-3p熒光探針結(jié)合ho-1免疫染色的原位雜交結(jié)果顯示,mir-29a-3p和ho-1在前列腺癌組織中的表達(dá)要比在bph組織中的表達(dá)明顯增高。不同濃度doc處理pc3細(xì)胞24小時(shí)后,qrt-pcr結(jié)果顯示,mir-29a-3p的表達(dá)呈多西紫杉醇濃度依賴性降低。轉(zhuǎn)染pc3細(xì)胞,過表達(dá)和敲低mir-29a-3p,同時(shí)給予doc刺激,westernblot結(jié)果顯示,較對(duì)照組,doc刺激組的ho-1蛋白表達(dá)水平降低,caspase3蛋白水平升高。較其他組,anti-mir-29a-3p+doc(10nm)組ho-1蛋白表達(dá)水平顯著降低,而caspase3蛋白水平顯著升高。過表達(dá)和敲低ho-1,并給與doc處理細(xì)胞,流式細(xì)胞術(shù)結(jié)果顯示,較其他組,doc+ho-1敲低組顯著增加細(xì)胞凋亡。tunel熒光染色分析結(jié)果顯示類似的結(jié)果。以上結(jié)果顯示mir-29a-3p增加ho-1的表達(dá)從而促進(jìn)前列腺癌細(xì)胞的生長(zhǎng)。4在前列腺癌中bach2是mir-29a-3p的直接靶標(biāo)選取的mir-29a-3p的潛在靶基因進(jìn)行熒光報(bào)告基因檢測(cè),結(jié)果顯示,與對(duì)照組相比,含有wtbach23'utr的細(xì)胞可被mir-29a-3p模擬物抑制。westernblot實(shí)驗(yàn)顯示,與對(duì)照組相比,mir-29a-3p過表達(dá)組bach2的蛋白水平顯著降低,而敲低組bach2蛋白表達(dá)水平顯著升高。qrt-pcr結(jié)果顯示,與臨床bph組織相比,前列腺癌組織中bach2的表達(dá)水平較低。隨后進(jìn)行bach2和mir-29a-3p表達(dá)的相關(guān)性分析,結(jié)果顯示,bach2和mir-29a-3p在mrna水平上具有顯著的統(tǒng)計(jì)學(xué)負(fù)相關(guān)性。5補(bǔ)救實(shí)驗(yàn)確認(rèn)mir-29a-3p/bach2/ho-1軸調(diào)控前列腺癌發(fā)展補(bǔ)救實(shí)驗(yàn),westernblot結(jié)果顯示,在doc處理的條件下,與對(duì)照組相比,mir-29a-3p過表達(dá)可增加ho-1蛋白的表達(dá),降低bach2蛋白的表達(dá)。相反,mir-29a-3p敲低可降低ho-1蛋白的表達(dá),增加bach2蛋白的表達(dá)。當(dāng)共轉(zhuǎn)染ad-mir-29a-3p和si-bach2時(shí),結(jié)果顯示,與其他組相比,Ad-miR-29a-3p+si-Bach2組Bach2蛋白水平顯著降低,而HO-1蛋白水平顯著升高。6 Bach2通過結(jié)合HO-1啟動(dòng)子來調(diào)節(jié)HO-1的表達(dá)HO-1和Bach2免疫雙熒光染色結(jié)果示,與對(duì)照組相比,miR-29a-3p敲低組的HO-1表達(dá)水平降低,Bach2的表達(dá)水平升高。當(dāng)給予DOC刺激后,與對(duì)照組相比,miR-29a-3p敲低組的HO-1表達(dá)水平顯著降低,Bach2的表達(dá)水平顯著升高。熒光報(bào)告基因檢測(cè)示,與對(duì)照組相比,敲低Bach2顯著增加HO-1啟動(dòng)子活性。染色質(zhì)免疫共沉淀(CHIP)實(shí)驗(yàn)結(jié)果顯示,miR-29a-3p敲低組增加了Bach2與HO-1啟動(dòng)子的結(jié)合。以上結(jié)果表明Bach2可通過結(jié)合HO-1啟動(dòng)子來調(diào)控HO-1的表達(dá)。7在體內(nèi)miR-29a-3p可促進(jìn)前列腺癌的發(fā)生發(fā)展裸鼠皮下接種穩(wěn)定轉(zhuǎn)染的PC3細(xì)胞荷瘤。定期觀察發(fā)現(xiàn),穩(wěn)定轉(zhuǎn)染了LV-Anti-miR-29a-3p的PC3細(xì)胞的異種移植瘤組的腫瘤體積明顯比對(duì)照組的小。上述所有結(jié)果表明mi R-29a-3p在前列腺癌進(jìn)展中起到重要作用。結(jié)論:1 miR-29a-3p在前列腺癌組織和前列腺癌細(xì)胞中是高表達(dá)的。2 miR-29a-3p在體外和體內(nèi)均可促進(jìn)前列腺癌細(xì)胞的增殖。3 miR-29a-3p通過增加HO-1的表達(dá),從而起到抗凋亡,促增殖作用。4在前列腺癌細(xì)胞中Bach2是mi R-29a-3p的直接靶基因。5 miR-29a-3p通過靶向抑制Bach2促進(jìn)與氧化應(yīng)激相關(guān)基因HO-1的表達(dá),進(jìn)而導(dǎo)致前列腺癌細(xì)胞的發(fā)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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2 陳金鎖;miR-29a-3p通過調(diào)節(jié)血紅素加氧酶1的表達(dá)在前列腺癌中的作用及機(jī)制研究[D];河北醫(yī)科大學(xué);2017年

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