【摘要】：研究背景和目的:前列腺癌嚴(yán)重影響著世界范圍內(nèi)的男性健康,該疾病在男性癌癥中的患病率已位居第二位,前列腺癌在中老年男性中的發(fā)病率隨年齡的增長快速上升,該疾病難以治療并且致死率也非常高,對(duì)人類的健康產(chǎn)生嚴(yán)重的威脅。所以目前依據(jù)前列腺癌的發(fā)病機(jī)制尋找該疾病的預(yù)防以及治療靶點(diǎn)和新方法迫在眉睫。近年來的報(bào)道顯示,前列腺癌的發(fā)病機(jī)制主要有雄性激素依賴性前列腺癌以及包括原發(fā)性和繼發(fā)性的激素非依賴性前列腺癌,并且指出雄性激素依賴性前列腺癌在該疾病的發(fā)生發(fā)展過程中占據(jù)重要位置。近來的研究發(fā)現(xiàn)微小RNA-103(microRNA-103,miR-103)調(diào)控程序性細(xì)胞死亡因子10(programmed cell death factor 10,PDCD10)在前列腺細(xì)胞中的表達(dá)對(duì)前列腺癌的發(fā)生發(fā)展具有較大影響,并且在前列腺癌發(fā)病機(jī)制中具有非常關(guān)鍵的作用。PDCD10能夠調(diào)控機(jī)體細(xì)胞的凋亡過程,對(duì)維持機(jī)體細(xì)胞的健康存活具有非常重要的作用,當(dāng)PDCD10在前列腺細(xì)胞中的表達(dá)失調(diào)會(huì)引起前列腺病變,甚至最終能夠引起前列腺癌變,同時(shí)PDCD10在人類細(xì)胞中的分布非常廣泛,能夠參與人體多種疾病的生理病理程序,目前已經(jīng)發(fā)現(xiàn)PDCD10在多種癌癥中發(fā)揮非常重要的調(diào)控作用。此外miR-103是一類miRNA,目前已發(fā)現(xiàn)其在直結(jié)腸癌等多種癌細(xì)胞中調(diào)控癌癥的發(fā)生發(fā)展過程。miRNAs一般通過調(diào)控與癌癥相關(guān)基因的表達(dá),發(fā)揮致癌或者抑癌功能。目前研究發(fā)現(xiàn)miR-103可能通過與靶基因的非編碼區(qū)結(jié)合調(diào)控目的基因在細(xì)胞中的表達(dá),進(jìn)而影響該基因功能的發(fā)揮,最終對(duì)多種癌癥的發(fā)病過程進(jìn)行調(diào)控。采用生物信息學(xué)方法(使用miRanda、TargetScan、PicTar等軟件)對(duì)miR-103和PDCD10基因的靶向匹配關(guān)系進(jìn)行預(yù)測,發(fā)現(xiàn)PDCD10可能是miR-103的潛在靶基因。綜上,miR-103對(duì)PDCD10在前列腺細(xì)胞中表達(dá)的調(diào)控與前列腺癌的發(fā)生發(fā)展可能具有緊密聯(lián)系,但是目前對(duì)于mi R-103和PDCD10與前列腺癌之間的聯(lián)系以及兩者之間的調(diào)控模式尚未有研究,所以相關(guān)的重要科學(xué)問題急需深入了解及研究。因此,本課題擬研究miR-103及PDCD10在前列腺癌患者癌細(xì)胞中的表達(dá)變化,并且以兩者之間的表達(dá)變化模式為切入點(diǎn),闡明miR-103及PDCD10在前列腺癌發(fā)病機(jī)制中作用,有助于從新的位點(diǎn)了解前列腺癌的發(fā)病機(jī)制,為前列腺癌的預(yù)防和治療提供新的靶點(diǎn)及理論基礎(chǔ)。研究方法:1.利用Real-time PCR方法檢測miR-103在前列腺癌患者組織和前列腺癌細(xì)胞系中的表達(dá)變化規(guī)律,并對(duì)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。2.運(yùn)用Western blotting以及Real-time PCR方法檢測PDCD10在前列腺癌患者組織和前列腺細(xì)胞系中的表達(dá)變化規(guī)律,并對(duì)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。3.體外實(shí)驗(yàn)中,利用MTT實(shí)驗(yàn)、流式細(xì)胞實(shí)驗(yàn)、細(xì)胞計(jì)數(shù)以及細(xì)胞轉(zhuǎn)染等方法檢測miR-103以及PDCD10對(duì)前列腺癌細(xì)胞的增殖、凋亡以及細(xì)胞周期的影響。另外利用酵母雙雜實(shí)驗(yàn)檢測miR-103對(duì)PDCD10與MST4互作的影響,利用Western blotting以及Real-time PCR方法檢測miR-103對(duì)ERK蛋白磷酸化的影響,利用熒光素酶報(bào)告系統(tǒng)分析miR-103與PDCD10的靶向關(guān)系。研究結(jié)果:1.miR-103在前列腺癌患者組織和前列腺癌系中表達(dá)顯著低于正常對(duì)照。2.體外實(shí)驗(yàn)中,miR-103在前列腺癌細(xì)胞中的表達(dá)變化與前列腺癌細(xì)胞的增殖率、侵襲呈負(fù)相關(guān)性,與前列腺癌細(xì)胞的凋亡率呈正相關(guān)性;miR-103在前列腺癌細(xì)胞中超表達(dá)時(shí),能夠抑制癌細(xì)胞由G1期向S期轉(zhuǎn)變。3.在前列腺癌患者中,PDCD10在前列腺癌組織和細(xì)胞系中的表達(dá)量顯著高于正常前列腺內(nèi)皮細(xì)胞。4.在體外實(shí)驗(yàn)中,PDCD10在前列腺癌細(xì)胞中的表達(dá)變化與前列腺癌細(xì)胞的凋亡呈負(fù)相關(guān),與前列腺癌細(xì)胞的增殖率、侵襲呈正相關(guān);在前列腺細(xì)胞中PDCD10蛋白能夠與MST4蛋白互作,并且兩者能夠協(xié)同表達(dá),PDCD10表達(dá)上調(diào)與ERK蛋白的磷酸化呈現(xiàn)正相關(guān)性,同時(shí)能夠增加前列腺癌細(xì)胞抵御氧化應(yīng)激對(duì)細(xì)胞的傷害。5.PDCD10的表達(dá)可以受miR-103直接調(diào)控,miR-103在前列腺癌細(xì)胞中超表達(dá)時(shí)會(huì)抑制PDCD10的表達(dá),并且引起MST4蛋白在前列腺癌細(xì)胞中的表達(dá)下調(diào),ERK蛋白的磷酸化水平也下降,導(dǎo)致前列腺癌抵御氧化應(yīng)激的能力下降。研究結(jié)論:1.在前列腺癌患者癌細(xì)胞中miR-103表達(dá)下降,PDCD10的表達(dá)水平上升,miR-103表達(dá)下降以及PDCD10表達(dá)上調(diào)會(huì)引起前列腺癌細(xì)胞持續(xù)增殖,阻礙細(xì)胞凋亡。2.在前列腺癌細(xì)胞中,miR-103能夠通過直接調(diào)控靶基因PDCD10的表達(dá)抑制前列腺癌細(xì)胞的增殖、侵襲,同時(shí)抑制前列腺癌細(xì)胞周期由G1期向S期轉(zhuǎn)變。3.miR-103能夠通過抑制抑制PDCD10的表達(dá),使得MST4蛋白表達(dá)下降以及ERK磷酸化水平下降,最終引起前列腺癌細(xì)胞抵抗氧化應(yīng)激刺激的能力下降。4.miR-103靶向調(diào)控PDCD10抑制前列腺癌的發(fā)生和進(jìn)展,為前列腺的治療提供了新的理論依據(jù)和新的靶點(diǎn)。
[Abstract]:Research background and purpose: prostate cancer seriously affects the health of men worldwide. The incidence of the disease is the second in male cancer. The incidence of prostate cancer in middle-aged and old men is rising rapidly with age. The disease is difficult to treat and has a high mortality rate, which produces serious health to human health. So it is imminent to find the prevention and treatment targets and new methods of the disease according to the pathogenesis of prostate cancer. Recent reports have shown that the main pathogenesis of prostate cancer is androgen dependent prostate cancer and primary and secondary hormone non dependent prostate cancer, and the male irritable disease is pointed out. Recent studies have found that the expression of RNA-103 (microRNA-103, miR-103) regulatory programmed cell death factor 10 (programmed cell death factor 10, PDCD10) in prostate cells has a great influence on the development of prostate cancer. The key role of the pathogenesis of prostate cancer is that.PDCD10 can regulate the apoptosis process of the body cells, which plays a very important role in maintaining the healthy survival of the body cells. When the expression of PDCD10 in the prostate cells is dysfunctional, it can cause prostate disease, and even eventually lead to prostate cancer, while PDCD10 is very thin in human beings. The distribution of the cell is very extensive and can participate in the physiological and pathological procedures of various diseases. PDCD10 has been found to play a very important regulatory role in many kinds of cancer. In addition, miR-103 is a kind of miRNA. At present, it has been found that.MiRNAs regulates and regulates the progression of cancer in a variety of cancer cells such as colon cancer. The expression of cancer related genes exerts carcinogenic or tumor suppressor functions. At present, it is found that miR-103 may regulate the expression of target genes in cells by combining the non coding regions of the target genes, and then affect the function of the gene, and ultimately regulate the pathogenesis of various cancers. Bioinformatics (using miRanda, Ta) The target matching relationship between miR-103 and PDCD10 genes is predicted by rgetScan, PicTar and other software. It is found that PDCD10 may be a potential target gene for miR-103. To sum up, the regulation of miR-103 on the expression of PDCD10 in prostate cells may be closely related to the development of prostate cancer, but it is currently available for MI R-103 and PDCD10 and prostate cancer. The relationship between the two and the mode of regulation between the two has not been studied, so the important scientific questions need to be deeply understood and studied. Therefore, this topic intends to study the changes in the expression of miR-103 and PDCD10 in the cancer cells of the patients with prostate cancer, and to clarify that miR-103 and PDCD10 are in the forefront. The role of adenocarcinoma pathogenesis is helpful to understand the pathogenesis of prostate cancer from new sites and provide new targets and theoretical basis for the prevention and treatment of prostate cancer. Research methods: 1. the expression of miR-103 in the tissues of prostate cancer patients and prostate cancer cell lines was detected by Real-time PCR method, and the results were added to the results. Statistical analysis.2. used Western blotting and Real-time PCR to detect the changes in the expression of PDCD10 in the tissues and the prostate cell lines of the prostate cancer patients, and the results were statistically analyzed in.3. in vitro, and miR-103 and P were detected by MTT experiment, flow cytometry, cell count and cell transfection. The effect of DCD10 on the proliferation, apoptosis and cell cycle of prostate cancer cells. In addition, the effect of miR-103 on the interaction between PDCD10 and MST4 was detected by yeast double heterozygosity. The effect of miR-103 on the phosphorylation of ERK protein was detected by Western blotting and Real-time PCR method. The target direction of miR-103 and PDCD10 was analyzed by the fluorescent enzyme report system. The results: the expression of 1.miR-103 in the tissue and prostate cancer of the prostate cancer patients was significantly lower than that in the normal control.2. in vitro. The expression of miR-103 in the prostate cancer cells was negatively correlated with the proliferation rate and invasion of the prostate cancer cells, and was positively correlated with the apoptosis rate of the prostate cancer cells; miR-103 was in the prostate cancer. The expression of PDCD10 in prostate cancer patients was significantly higher than that of normal prostate cancer cells and cell lines. The expression of PDCD10 in prostate cancer tissues and cell lines was significantly higher than that of normal prostate endothelial cells (.4.) in vitro. The expression of PDCD10 in prostate cancer cells was negatively correlated with the apoptosis of prostate cancer cells, and the expression of.3. in prostate cancer cells was negatively correlated with the apoptosis of prostate cancer cells. The proliferation rate and invasion of prostate cancer cells are positively correlated. In prostate cells, PDCD10 protein can interact with MST4 protein, and both can be co expressed. The up regulation of PDCD10 expression is positively correlated with the phosphorylation of ERK protein, and can also increase the expression of.5.PDCD10 in prostate cancer cells against oxidative stress. Under the direct regulation of miR-103, the overexpression of miR-103 in prostate cancer cells inhibits the expression of PDCD10, and causes the expression of MST4 protein in the prostate cancer cells to decrease, and the level of phosphorylation of ERK protein also decreases, which leads to the decline in the ability of prostate cancer to resist oxidative stress. The conclusion: 1. in the cancer cells of prostate cancer patients, miR-103 The expression level of PDCD10 increased, the expression level of miR-103 and the up-regulated expression of PDCD10 may cause the proliferation of prostate cancer cells and inhibit the apoptosis of.2. in prostate cancer cells. MiR-103 can inhibit the proliferation, invasion and inhibition of prostate cancer cell cycle by direct regulation of the expression of target gene PDCD10. The transition from G1 to S phase.3.miR-103 can inhibit the expression of PDCD10, decrease the expression of MST4 protein and decrease the level of ERK phosphorylation, and eventually lead to the decline in the ability of prostate cancer cells to resist oxidative stress stimulation..4.miR-103 targeted regulation of PDCD10 to inhibit the occurrence and development of prostate cancer and provide the prostate treatment. New theoretical basis and new targets.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25
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本文編號(hào)：2165656