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標題: | 探討NRIP、DDB2和AR三者在攝護腺癌中的關係 The correlation of NRIP, DDB2 and AR in prostate cancers |
作者: | Ping Fan 范萍 |
指導教授: | 陳小梨(Show-Li Chen) |
關鍵字: | 核受體交互作用蛋白,雄激素受體,受損DNA結合蛋白2,攝護腺癌, NRIP,AR,DDB2,prostate cancers, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 之前我們實驗室發現新的基因核受體交互作用蛋白(nuclear receptor interaction protein, NRIP),又名IQWD1。另外也屬於 DDB1 和 CUL4 相關因子(DCAF), 所以又名 DCAF6。 NRIP含有7個WD40 domain,是類固醇受體(雄激素受體和糖皮質激素受體)的共活化劑。雄激素受體 (androgen receptors; AR)曾被報導會影響攝護腺的發育,而我們先前的研究也發現NRIP可以穩定AR蛋白; 而最近的研究發現8周大的NRIP基因剃除小鼠的前側攝護腺發育較野生型遲緩,但是腹側和背外側攝護腺的發育和野生型比較則沒有差別。野生型小鼠的前側攝護腺的AR表現量為三者中最低,NRIP基因剃除小鼠前側攝護腺的AR表現量又比野生型小鼠的前側攝護腺更低。這表示NRIP 也許會和AR交互作用,進而調控前側攝護腺的發育而非腹側和背外側攝護腺。另一方面,在前側攝護腺的AR 蛋白比在腹側和背外側攝護腺少,因此在8周NRIP 基因剃除小鼠前側攝護腺的AR蛋白不足以超越小鼠前攝護腺生長所需的閾值。
另外,我們發現受損DNA結合蛋白2 (damge-specific DNA binding protein 2; DDB2)又名p48、XPE,會調控AR的泛素化和降解。像NRIP一樣,DDB2也屬於DDB1 和 CUL4 相關因子(DCAF)並含有WD40 domain。DDB2會和DDB1-CUL4E3 ligase結合並泛素化目標蛋白AR,最後透過蛋白酶體降解AR。目前我們發現NRIP會跟DDB2競爭和AR的結合,防止DDB2-DDB1-CUL4 E3 ligase複合體降解AR蛋白。 因此為了更進一步探討在攝護腺癌病人中NRIP, DDB2和AR的表現,我們使用了免疫組織化學染色實驗。Pearson's 卡方檢定結果顯示,和非癌化的對照組相比,在攝護腺癌化病人中,NRIP的表現量顯著增加,而DDB2的表現量則降低(n=260; 樣本來源:台大醫院和US Biomax Inc.)。在攝護腺癌化病人中,AR的表現量也是顯著增加。NRIP和AR的表現呈現正相關,進一步支持我們在in vitro 的實驗:NRIP會穩定AR蛋白。然而DDB2和AR的表現也呈現正相關,這可以解釋為在AR表現高的病人中,DNA修補機制會被啟動,因此增加DDB2表現。另一方面,NRIP 和 DDB2 的蛋白質表現也是正相關,可能是NRIP表現高的病人,DNA修補機制會被啟動,因此增加DDB2表現。NRIP、DDB2和AR的蛋白質表現也沒有顯著的差異,但是當我們將NRIP表現量高、AR高和DDB2低的檢體分為一組,而其他七種組合為一組,依照癌症型態分為篩狀(cribriform) 和非篩狀(non-cribriform),並用卡方檢定計算p值。結果顯示在相同位點的篩狀病灶顯著觀察到NRIP表現量很高、AR很高而DDB2低 (勝負比: 2.64; 95%信賴區間: 1.02~6.59)。有趣的是,當我們將NRIP表現量高、AR高和DDB2低的檢體分為一組,而其他三種DDB2表現皆低的組合為一組,用卡方檢定計算的p值比前一個更顯著 (勝負比: 4.82; 95%信賴區間: 1.48~16.9) 這表示在DDB2表現低的篩狀攝護腺病灶NRIP和AR表現量高。因此在DDB2表現低時NRIP會保護AR。至於DDB2表現高時NRIP是否會保護AR不被DDB2 透過Cul4-DDB1 E3 ligase複合體降解還未知。 The gene, nuclear receptor interaction protein (NRIP, also named IQWD1) was found in our lab. NRIP is in DDB1 and CUL4-associated factors (DCAF) family, so it's also known as DCAF6. NRIP contains seven WD40 repeats and functions as the coactivator of steroid receptors (androgen receptor and glucocorticoid receptor). Androgen receptor (AR) reportedly participates in the development of prostate. Currently, NRIP can stabilize AR protein. From NRIP knock out mice (NRIP KO), we found that the anterior prostate (AP) development of 8-week NRIP KO mouse was slower than that of wild type (WT), but the development of lateral prostate (LP) and dorsal-lateral prostate (DLP) did not differ for their development as wild type. And the expression of AR of AP was the lowest among the three prostate lobes in WT mice; and AR expression in AP of NRIP KO mouse was also lower than the counterpart of WT. These imply that NRIP may regulate AR and then regulate the development of anterior prostate but not LP and DLP. On the other hand, AR protein in AP was less than that in LP or DLP. Hence, AR protein in AP of 8-week NRIP KO mouse was not enough to cross the threshold of the AP development in mouse. In addition, we found that damage-specific DNA binding protein 2 (DDB2, also named p48 and XPE) interacts with AR and mediates its ubiquitination and degradation. DDB2, like NRIP, is a DCAF containing protein. DDB2 binding to DDB1-CUL4 E3 ligase can ubiquitinate AR for proteasomal degradation. Currently, we found that NRIP competed with DDB2 for AR binding to avoid DDB2-DDB1-CUL4 E3 ligase complex degrading AR protein. Hence, in this study to further investigate NRIP, DDB2, and AR expression in prostate cancer clinical patients, we performed immunohistochemistry (IHC) assay. The results revealed that NRIP protein expression was significantly increased in human prostate cancer patients, while DDB2 expression was decreased in compared to non-neoplastic control by Pearson's chi-square (n=260, from NTUH and US Biomax Inc.). As to expression of AR, it was significantly increased in prostate cancer patients compared with non-neoplastic controls by Pearson's chi-square analysis. When analyzing the correlation between NRIP and AR, there was positive correlation between NRIP and AR. It further supports our in vitro demonstration of NRIP-stabilizing AR protein. However, the correlation between DDB2 and AR also revealed positive correlation; it can explain AR high expression patients; that may induce DNA repair mechanism that drives DDB2 expression for repair. On the other hand, the relation between NRIP and DDB2 also exhibited positive correlation; the explanation is that NRIP high expression patients may stimulate DNA repair mechanism that drives DDB2 expression for repair. Moreover, to analyze NRIP, DDB2 and AR, there was no correlation among these three proteins’ expression in human prostate cancer samples. But when we further categorized high NRIP and high AR expression along with low DDB2 expression as a group, and the other seven combinations as one group; and analyzed p value between these two groups with cribriform and non-cribriform prostate cancer by Chi-square assay. The results showed the high NRIP and AR and low DDB2 expression at the same region was more significantly found in the cribriform tumors than non-cribriform tumors (odds ratio: 2.64; 95% Conf. Interval: 1.02~6.59). Intriguingly, we only analyzed low DDB2 with high NRIP and high AR as a group, and low DDB2 with other three combinations as another group; and the p value of latter by Chi-square assay (odds ratio: 4.82; 95% Conf. Interval: 1.48~16.9) was more significant than the former. This indicates in case of low DDB2 in cribriform prostate cancer, NRIP and AR could be high expression. Hence NRIP protects AR in low DDB2 condition. As to in high DDB2, whether NRIP can protect AR from degradation by DDB2 in Cul4-DDB1 E3 ligase complex system remains unclear. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50230 |
DOI: | 10.6342/NTU201601800 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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