The Oncogenicity of Human Papillomavirus Type 58 E7 Variants

• Other Authors: Hu, Chenghua (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292325

由人類乳頭瘤病毒58 型（HPV58）引起的子宮頸癌在東亞地區以及部分拉丁美洲地區占有全部子宮頸癌相當的比例，然而在其他地區卻比較少見。這種地域上的差別至今還沒有得到很好的解釋。我們此前的流行病學研究辨認出一種HPV58 的變體（V1），在其致癌基因E7 中產生了二個錯意突變（T20I 與G63S），與增加宮頸上皮內瘤變第三期以及宮頸癌的形成顯著相關。更重要的是，比起其他地方，V1 在東亞地區更加流行。因此，我們假設比起其他HPV58 變體，V1 擁有更強的致癌性，從而能夠解釋HPV58在東亞地區子宮頸癌中的高流行率。 === 本研究通過HPV58 致癌蛋白E7 的亞細胞定位，蛋白穩定性試驗，抑癌蛋白pRb 的降解試驗以及軟瓊脂集落形成試驗描素其致癌能力的特性。除變體1 之外，變體2（G41R/ G63D）與3（T74A/D76E），分別為人類乳頭瘤病毒58 型最常見以及第三常見的變體也被包含在內。並且，人造的單一突變V1A（T20I）和V1B（G63S）也被用來研究是否其中某一個突變使V1 擁有更強的致癌性。 === 結果表明，在我們檢驗的HPV58 E7 原型以及變體中，E7 以點狀形式集中在宿主細胞核內，但也少量分佈在細胞質當中。無論HPV58 E7 帶有何種突變，它們的半衰期都在二十分鐘左右。因此這些突變並沒有通過影響E7 的亞細胞定位以及蛋白穩定性來增加E7 的致癌性。然而，在抑癌蛋白pRb 的降解試驗中，HPV58 原型與變體1 比起其他變體降解了更大一部分的外源性pRb（HPV58 原型，變體1，變體1A，變體1B，變體2 和變體3 分別降解了84.99 ± 3.82%，84.29 ± 3.56%，68.70 ± 1.97%，65.80 ±4.37%，66.36 ± 2.59%以及68.82 ± 1.43%的外源性pRb）（P 值小於0.001）。最後也是最重要的是，在軟瓊脂集落形成試驗中，HPV58 E7 原型以及變體1 比起其他變體使NIH/3T3 細胞在軟瓊脂培養板中形成更多數量的集落（HPV58 原型：28.76 ± 5.97，變體1：31.44 ± 3.15，變體1A：16.33 ± 2.62，變體1B：16.11 ± 1.50，變體2：16.22 ± 1.34，變體3：16.44 ± 2.08:），表示它們有更強的細胞轉型能力。 === 總而言之，與HPV16 E7 相似，HPV58 E7 原型以及變體有降解pRb 以及細胞轉型的能力。在我們檢驗的HPV58 E7 原型以及變體中，HPV58 E7 原型以及變體1 可能是通過pRb 信號途徑使致癌蛋白E7 具有更強的致癌性。這個結論，至少部分解釋了HPV58 在東亞地區的子宮頸癌中的高流行率。為子宮頸癌的臨床診斷以及治療提供重要的參考。 === Human papillomavirus (HPV) type 58 accounts for a remarkable proportion of cervical cancers in East Asia and parts of Latin America but it is uncommon elsewhere. The reason for such geographical predilection is not fully understood. Our previous epidemiological study identified an HPV58 variant, which harbored two amino acid substitutions T20I and G63S in the E7 protein designated as V1, to be significantly associated with higher risks for developing cervical intraepithelial neoplasia III and invasive cervical cancers. Moreover, this variant was more commonly detected in East Asia than other regions of the world. These observations led us hypothesize that V1 possessed stronger oncogenicity than other variants, which might explain the high prevalence of HPV58 in cervical cancer in East Asia. === The current study aims at characterizing the oncogenicity of HPV58 E7 variants by protein localization assay, protein stability assay, retinoblastoma protein (pRb) degradation assay and soft agar colony formation assay. Apart from V1, two other HPV58 E7 variants, namely V2 (G41R/ G63D) and V3 (T74A/D76E), which were respectively the most and the third common HPV58 E7 variants, were also included. In addition, the oncogenicity of artificial single mutants V1A (T20I) and V1B (G63S) were studied to examine if only one of these two mutations contributes to the carcinogenic phenotype of V1. === Results showed that HPV58 E7 prototype and variants were predominantly localized in the nucleus with a punctate pattern, and could also be detected in the cytoplasm. The half-life of HPV58 E7, regardless of the variations they carried, was approximately 20 minutes, implicating that they did not confer their oncogenicity through affecting E7 localization and stability. However, in pRb degradation assay, HPV58 E7 prototype and V1 degraded a larger portion of exogenous pRb (84.99 ± 3.82% and 84.29 ± 3.56%, respectively) than V1A, V1B, V2 and V3 (68.70 ± 1.97%, 65.80 ± 4.37%, 66.36 ± 2.59% and 68.82 ± 1.43%, respectively) (P < 0.001). Last and foremost, in the soft agar colony formation assay, HPV58 E7 prototype and V1 conferred a higher colony number (28.76 ± 5.97 and 31.44 ± 3.15, respectively) in NIH/3T3 cells grown in soft agar than V1A, V1B, V2 and V3 (16.33 ± 2.62, 16.11 ± 1.50, 16.22 ± 1.34 and 16.44 ± 2.08, respectively) (P < 0.001), implicating their stronger transforming ability. === In sum, like HPV16 E7, HPV58 E7 prototype and variants are capable to degrade pRb and possess transforming ability. Moreover, HPV58 E7 prototype and V1 exerted higher oncogenicity than other variants possibly through the pRb pathway. This information can explain, at least in part, the high prevalence of HPV58 in East Asia. It also serves a very important indication in clinical screening, diagnosis and therapeutic management, particularly for East Asian countries. === Hu, Chenghua. === Thesis M.Phil. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.