| 摘要: | 在真核細胞中，鋅指蛋白 (zinc finger protein) 是含量最多的蛋白質，依照結構組成的不同，功能也不盡相同。其功能包含調控特定基因的表現，細胞週期的進行、細胞凋亡相關訊號以及DNA修復等。ZFP36蛋白質家族 (ZFP36 protein family) 是屬於CCCH-type 中的一員，其包含一段重複兩次的鋅指蛋白功能區域。ZFP36蛋白質家族包含：ZFP36、ZFP36L1、ZFP36L2和ZFP36L3四個成員。其中ZFP36過去曾經被發現可以透過p53-dependent的機制，抑制細胞的增生。
在本篇研究中，我們以結腸直腸癌細胞為研究目標，來探討ZFP36L1和ZFP36L2抑制細胞生長的影響。首先在人類胚胎腎臟細胞 (human embryonic kidney, HEK 293 cells) 中建立doxycycline-inducible系統，來誘導ZFP36L1或ZFP36L2的大量表現。發現過度表現ZFP36L1或ZFP36L2會降低人類胚胎腎臟細胞的增生能力。由流式細胞儀分析，發現過度表現ZFP36L1或ZFP36L2會造成細胞週期停滯於G1期。以Real-time PCR偵測，當過度表現ZFP36L1或ZFP36L2時，p53的mRNA表現量會增加，其調控細胞週期的相關分子，如cyclin D1、c-Myc和p21的mRNA表現量會下降。進一步，在結腸直腸癌HCT116 p53+/+、HCT116 p53-/-和SW620 (p53突變) 細胞株中，利用慢病毒感染誘導ZFP36L1和ZFP36L2的大量表現，也發現可以抑制這三株細胞的增生能力；以西方墨點法偵測，發現過度表現ZFP36L1和ZFP36L2時，cyclin D1在此三株細胞中蛋白質表現量皆出現下降。本研究中c-Myc的蛋白質表現量下降，只出現在SW620細胞、p21的蛋白質表現量增加，只出現在HCT116 p53+/+細胞。接著在ZFP36L1和ZFP36L2的鋅指區域 (TZF1和TZF2) 做點突變，發現mutation ZFP36L1 (C135/173R) 和ZFP36L2 (C174/212R) 在此三株結腸直腸細胞中，喪失了抑制細胞增生的能力，接著我們在HCT116 p53+/+、HCT116 p53-/-和SW620 中knockdown ZFP36L1和ZFP36L2後，研究中發現會增加細胞增生能力及促進細胞週期的進行。經由這些實驗結果，我們推測ZFP36L1和ZFP36L2蛋白在人類結腸直腸癌細胞中藉由降低cyclin D1的表現，並透過p53-independent機制扮演抑制細胞生長的角色。
The zinc finger proteins (ZFPs) are the most abundant protein in eukaryotic cells. They have been found with different functions including regulation of gene expression, cell cycle, apoptosis, and DNA repair according to their organization of domains. ZFP36 protein family belongs to CCCH-type zinc finger proteins and contains double-repeated zinc finger domains. There are four members in ZFP36 family including ZFP36, ZFP36L1, ZFP36L2, and ZFP36L3 proteins, in which ZFP36 has been found to inhibit cell proliferation through p53-dependent manner.
In this study, we investigated the effects of ZFP36L1 and ZFP36L2 on the inhibition of cell growth in human colorectal cancer cells. First, we used HEK 293 stable cell lines with doxycycline-inducible ZFP36L1 or ZFP36L2. We found that overexpression of ZFP36L1 or ZFP36L2 decreased cell proliferation in HEK293 cells. The flow cytometry analysis indicated overexpression of ZFP36L1 or ZFP36L2 induced cell cycle arrest in G1 phase. Overexpression of ZFP36L1 or ZFP36L2 decreased the mRNA expression of cyclin D1, c-Myc, and p21 but increased p53 mRNA expression by qRT-PCR analysis. Next, we transduced HCT116 p53+/+, HCT116 p53-/- and SW620 (mutated p53) cells with ZFP36L1 or ZFP36L2 gene by lentivirus and found overexpression of ZFP36L1 and ZFP36L2 also inhibited these cell proliferation. Overexpression of ZFP36L1 or ZFP36L2 decreased the cyclin D1 protein expression in these three kinds of cells and the c-Myc expression only in SW620 cells, and increased the p21 protein expression only in HCT116 p53+/+. Mutation in zinc finger domain of ZFP36L1 (C135/173R) and ZFP36L2 (C174/212R) lost the inhibitory ability for cell proliferation in these three kinds of colorectal cancer cells. On the other hand, knockdown of ZFP36L1 and ZFP36L2 increased cell proliferation and cell cycle progression in HCT116 p53+/+, HCT116 p53-/- and SW620 cells. Taken together, these results suggest that ZFP36L1 and ZFP36L2 may play a negative role in cell proliferation of human colorectal cancer cells, and the underlying mechanisms might be mediated through downregulation of cyclin D1 and p53-independent pathway. |
