hPuf-A與乳癌腫瘤生成之關係探討

Abstract

Human Puf-A (hPuf-A) 是近幾年被發現的RNA結合蛋白Puf家族的新成員。Puf這一類蛋白會透過與目標mRNA的結合進而去調控目標mRNA的穩定性及促進其降解。藉由電腦模擬分析顯示hPuf-A的Puf domain含有6個Puf repeat且其蛋白質構形明顯有別於含有8個Puf repeat的典型Puf蛋白。而本實驗室先前的研究發現, 當細胞遭受到基因毒性物質傷害壓力時, 原本位處於核仁中的hPuf-A會釋出到核質, 進而去抑制PARP-1蛋白被凋亡蛋白酵素 caspases的切割､分解, 從而提高細胞的存活率。目前為止, 我們對於hPuf-A的了解並不多, 其在細胞裡所扮演的角色及生理上的調控功能尚存在很多的未知, 且有待我們去發掘。從Ductal carcinoma in situ (DCIS)､第一期至第四期的乳癌病人腫瘤組織的免疫組織染色結果中, 顯示hPuf-A的表現隨著乳癌期數的增加而增加, 並且hPuf-A的表現程度與乳癌臨床期的進程具有統計上的關聯性 (p=0.005)。將乳癌細胞株MDA-MB-231及T47D的內源性hPuf-A表現抑制後,發現這會降低癌細胞形成細胞群落的能力。相反的, 在MDA-MB-231乳癌細胞中大量表現hPuf-A則會促進癌細胞的群落形成能力且形成較大的細胞群落。在異種移植實驗 (xenograft assay)中, 我們將hPuf-A表現抑制 (hPuf-A-silenced)及hPuf-A大量表現的MDA-MB-231 乳癌細胞經由皮下注射植入裸鼠身上後發現, hPuf-A的大量表現會促進癌細胞的腫瘤生成。值得一提的是, DDX3及RbAp48的蛋白隨著hPuf-A表現被抑制後, 它們的表現也跟著下降了。其原因有可能是hPuf-A藉由結合DDX3及RbAp48蛋白的mRNA進而調控其表現。另外, 透過次世代定序, 發現hPuf-A會與許多核糖蛋白(ribosomal proteins)的mRNA結合。而此結合是否具有調控的功能或生理上的意義尚有值得被探討的空間。 總結, 據我們的研究顯示hPuf-A的大量表現會促進乳癌的腫瘤生成,且其機制有可能是藉由結合DDX3及RbAp48的mRNA進而影響其蛋白表現。

Human Puf-A (hPuf-A) is a novel member of the RNA-binding protein, Puf family which regulates mRNA translation and decay. Computer modeling reveals that hPuf-A contains 6 Puf repeats instead of the 8-repeat Puf repeat domain and is structurally distinct from the canonical Puf proteins. In the previous study, we discovered that hPuf-A translocates from the nucleolus to the nucleoplasm upon genotoxic stress to prevent PARP-1 from caspases cleavage and degradation, thus promotes cell viability. However, the physiological functions of hPuf-A are still remained many to be discovered. In this study, we investigated the role of hPuf-A in promotion of breast cancer tumorigenesis. The immunohistochemical stainings of breast cancer specimens with clinical stage of Ductal carcinoma in situ (DCIS), stage I, II, III, IV showed that hPuf-A was upregulated in tumors with more advanced clinical stage. The expression levels of hPuf-A were significantly correlated with the clinical stage progression (P=0.005). Silencing the endogenous hPuf-A expression reduced the colony forming ability of MDA-MB-231 and T47D breast cancer cells. By contrast, overexpression of hPuf-A in MDA-MB-231 cells led to enhanced colony formation and colony size. Xenograft assays by subcutaneous injection of hPuf-A-silenced and hPuf-A overexpressing MDA-MB-231 cells into nude mice further demonstrated the tumorigenicity of hPuf-A. Interestingly, the silence of hPuf-A resulted in reduced expressions of DDX3 and RbAp48. The reduced expressions of DDX3 and RbAp48 might be regulated by the association of hPuf-A with DDX3 and RbAp48 mRNAs. Besides, the next generation sequencing revealed new potential mRNA targets of hPuf-A, and many of them belong to ribosomal proteins. Overall, our results indicated that the upregulation of hPuf-A promotes breast cancer tumorigenesis. The underlying mechanism might be the regulation of hPuf-A to its associated mRNAs, which are DDX3 and RbAp48.