It was previously demonstrated that miR-199a was down-regulated in testicular germ cell tumor (TGCT) partly caused by hypermethylation of its promoter. More detailed analyses showed that miR-199a-5p, one of its two derivatives, suppressed TGCT invasiveness and proliferation via directing targeting PODXL and MAFB. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this project we identified DNMT3A, the de novo methyltransferase, as a direct target of miR-199a-3p using a 3’-UTR reporter assay. In NT2 (NTera 2) and HT (Hs 1.Tes) cells, miR-199a-3p regulated the expression of endogenous DNMT3A (both DNMT3A1 and DNMT3A2 isoforms), especially DNMT3A2 isoform. In clinical samples, the expression of DNMT3A2 and miR-199a-3p were reciprocally regulated. However, DNMT3A did not regulate miR-199a expression. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation partly through targeting DNMT3A. MiR-199a-3p could restore the expression of APC and MGMT via de-methylation in their promoters. Our studies demonstrated the dysregulation of miR-199a-3p in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potential therapeutic use of synthetic miR-199a-3p oligonucleotides as effective demethylation agent in the treatment of TGCT. However, since DNMT3A expression did not regulate miR-199a expression, the mechanism of promoter DNA hypermethylation of miR-199a in TGCT needs further investigation. / MiR-199a is encoded by two loci in the human genome, namely, miR-199a-1 on chromosome 19 and miR-199a-2 on chromosome 1. Another microRNA, miR-214, also locates on chromosome 1. Previous study revealed that it is co-transcribed with miR-199a-2, which is directed by miR-199a-2 promoter. However, the biological significance of the co-expression of miR-199a and miR-214 remains largely unknown. In this project, it was determined that miR-199a and miR-214 were concordantly expressed in TGCT. Silencing of DNMT1 increased the expression of miR-199a and miR-214, accompanied by de-methylation in the promoters of miR-199a-1/2. Overexpression of TP53 down-regulated the expression of DNMT1 and increased the expression of mature miR-199-3p/5p and miR-214. In addition, silencing of PSMD10 up-regulated the expression of TP53, while miR-214 over-expression resulted in PSMD10 down-regulation and TP53 up-regulation. Collectively, our findings highlighted a miR-199a/miR-214/PSMD10/TP53/DNMT1 self-regulatory network, which caused the down-regulation of miR-199a, miR-214 and TP53, as well as the up-regulation of DNMT1 and PSMD10 in TGCT. These observations partly explain the mechanism of promoter DNA hypermethylation in miR-199a in TGCT. They also suggest a potential therapeutic approach by targeting the miR-199a/miR-214/PSMD10/TP53/DNMT1 regulatory network in the treatment of TGCT. / 先前的研究證實miR-199a在睾丸生殖細胞腫瘤 (簡稱睾丸癌) 中是低表達的，部分歸因於其啟動子區域過度甲基化。對其功能研究發現miR-199a能抑制睾丸癌細胞的生長，侵襲和轉移，且miR-199a的抑癌屬性應歸功於它的兩個衍生物之一miR-199a-5p。然而，miR-199a的另一個衍生物miR-199a-3p在睾丸癌中的生物學功能仍然在很大程度上是未知的。此研究中，DNMT3A被鑒定為miR-199a-3p的直接靶定目標。在NT2和HT細胞中，miR-199a-3p能調控內源性DNMT3A(DNMT3A1和DNMT3A2)的表達水準，尤其是DNMT3A2。在臨床樣本中，DNMT3A2的表達水準與miR-199a-3p的表達水準呈負相關。但DNMT3A並不能調控miR-199a的表達水準。進一步研究顯示過表達miR-199a-3p能減少APC和MGMT啟動子區域甲基化而恢復其表達水準。研究證實異常表達的miR-199-3p可能在睾丸癌的癌變過程中發揮作用，並提出一個潛在的治療方案，即使用miR-199a -3p作為有效的去甲基化藥劑治療睾丸癌。然而睾丸癌中導致miR-199a啟動子區域過度甲基化的機制有待進一步研究。 / 在人類基因組中，miR-199a-1(位於19號染色體)和miR-199a-2(位於1號染色體)都編碼miR-199a。同時miR -214也位於1號染色體，研究表明miR-214與miR-199a-2由miR-199a-2啟動子介導共同轉錄，但miR-199a和miR- 214共同表達的生物學意義仍未知。此研究中，miR-199a和miR-214在睾丸癌中的表達呈現一致性。沉默DNMT1後miR-199a和miR-214的表達水準顯著提高，並伴隨著miR-199a-1/2啟動子區域的DNA去甲基化。在NT2細胞中。過表達TP53能下調DNMT1的表達水準，同時上調miR-199-3p/5p和miR- 214的表達水準。此外，過表達miR -214能導致PSMD10表達水準的下調以及TP53表達水準的上調。綜上所述，我們提出一個miR-199a/miR-214/PSMD10/TP53/DNMT1自我調控網路，此調控通路能引起睾丸癌中miR-199a，miR-214和TP53表達水準的下調，以及DNMT1和PSMD10表達水準的上調，且部分解釋睾丸癌中miR-199a啟動子區域過度甲基化的機制，同時該調控網路可作為治療睾丸癌的一個潛在靶點。 / Chen, Bifeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 103-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 20, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1290659 |
| Date | January 2014 |
| Contributors | Chen, Bifeng (author.), Chan, Wai-Yee (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (xvi, 127 leaves) : illustrations, computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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