【摘要】：目的：MicroRNAs(miRNAs)是一類(lèi)長(zhǎng)約20～22個(gè)核苷酸的非編碼單鏈RNA分子,可通過(guò)與其相關(guān)的mRNA分子3’端非編碼區(qū)(untranslated region, UR)互補(bǔ)配對(duì),參與基因表達(dá)的轉(zhuǎn)錄后調(diào)控。大量證據(jù)證明miRNA在腫瘤的發(fā)展,分化,凋亡及增殖過(guò)程中發(fā)揮著關(guān)鍵作用。在前列腺癌中,miR-154的表達(dá)及具體功能目前研究甚少,本課題擬驗(yàn)證miR-154在前列腺癌中的表達(dá)趨勢(shì)及研究其對(duì)前列腺癌細(xì)胞生物學(xué)功能的影響。 材料和方法：采用qRT-PCR方法檢測(cè)27例前列腺癌組織和9例前列腺正常組織的樣本中miR-154的表達(dá)水平。運(yùn)用細(xì)胞增殖實(shí)驗(yàn)、集落形成實(shí)驗(yàn)、細(xì)胞周期實(shí)驗(yàn)及Western blot分析評(píng)估m(xù)iR-154和CCND2的表達(dá)變化對(duì)前列腺細(xì)胞系PC-3和DU145的影響。通過(guò)雙熒光素酶報(bào)告基因?qū)嶒?yàn)確定miR-154是否通過(guò)CCND2的3'-UTR區(qū)域調(diào)控CCND2。 結(jié)果：與癌旁前列腺正常組織相比,miR-154在前列腺癌組織中的表達(dá)水平明顯下調(diào)。而與Gleason評(píng)分、T分級(jí)相關(guān)無(wú)明顯相關(guān)性。體外實(shí)驗(yàn)中(CCK-8、集落形成實(shí)驗(yàn)、細(xì)胞周期分析)上調(diào)miR-154的表達(dá)水平和下調(diào)CCND2表達(dá)水平可以明顯抑制前列腺癌細(xì)胞的增殖能力。這種關(guān)系表明,在腫瘤的發(fā)生發(fā)展過(guò)程中miR-154與CCND2起協(xié)同作用。雙熒比素酶報(bào)告實(shí)驗(yàn)證明CCND2為miR-154直接調(diào)控的靶基因。轉(zhuǎn)染miR-154mimics后72小時(shí)后,細(xì)胞內(nèi)CCND2蛋白表達(dá)水平顯著降低。 結(jié)論：在前列腺癌中miR-154可作為一種腫瘤抑制因子,直接在轉(zhuǎn)錄后水平調(diào)控CCND2的表達(dá)。上調(diào)miR-154或下調(diào)CCND2的表達(dá)水平均可影響前列腺癌細(xì)胞的遷移和侵襲能力。miR-154有希望成為診斷和治療前列腺癌的分子標(biāo)記和靶點(diǎn)。
[Abstract]:Objective: microRNAs (miRNAs) are a class of non-coding single-stranded RNAs with a length of about 20 ~ 22 nucleotides. They may be involved in the posttranscriptional regulation of gene expression through complementary pairing of the 3 '-terminal noncoding region (untranslated region, UR of their related mRNAs. A great deal of evidence shows that miRNA plays a key role in tumor development, differentiation, apoptosis and proliferation. The expression and specific function of miR-154 in prostate cancer are rarely studied. This study aims to verify the expression trend of miR-154 in prostate cancer and its effect on the biological function of prostate cancer cells. Materials and methods: the expression of miR-154 was detected by qRT-PCR in 27 prostate cancer tissues and 9 normal prostate tissues. The effects of miR-154 and CCND2 expression on prostate cell line PC-3 and DU145 were evaluated by cell proliferation assay, colony formation assay, cell cycle assay and Western blot analysis. Whether miR-154 regulates CCND2 through CCND2 3- UTR region is determined by double luciferase reporter gene experiment. Results: the expression of miR-154 in prostate cancer tissues was significantly down-regulated compared with the adjacent normal prostate tissues. There was no significant correlation between Gleason score and T grade. In vitro (CCK-8, colony forming assay, cell cycle analysis), upregulation of miR-154 expression and down-regulation of CCND2 expression could significantly inhibit the proliferation of prostate cancer cells. This relationship suggests that miR-154 and CCND2 play a synergistic role in tumorigenesis and development. The double fluorinase report showed that CCND2 was the target gene directly regulated by miR-154. After 72 hours of miR-154 mimics transfection, the expression of CCND2 protein decreased significantly. Conclusion: miR-154 may act as a tumor suppressor in prostate cancer and regulate the expression of CCND2 directly at posttranscriptional level. Upregulation of miR-154 or down-regulation of CCND2 expression may affect the migration and invasion of prostate cancer cells. MiR-154 may become a molecular marker and target for the diagnosis and treatment of prostate cancer.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.25

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