探討ZFP36L1與ZFP36L2在抗細胞增生中扮演的角色

The roles of ZFP36L1 and ZFP36L2 in the anti-proliferative function

饒家鳳

鋅手指蛋白質 ； 細胞增生 ； ZFP36L1 ； ZFP36L2 ； zinc-finger protein ； proliferation

臺北醫學大學醫學檢驗暨生物技術學系所學位論文

2015年

碩士

梁有志

繁體中文

ZFP36L1 (又稱做BRF1、ERF1或TIS11B)和ZFP36L2 (又稱做BRF2、ERF2或TIS11D)，屬於CCCH-鋅手指蛋白質家族(CCCH-type zinc finger protein family)之成員，具有2個串聯的鋅手指區域(tandem zinc finger domain；TZF)被歸類在ZFP36 (又稱做TTP) 家族成員。ZFP36家族蛋白質屬於RNA-binding protein，透過結合mRNA位在 3’UTR上的AREs (AU-rich elements)進而導致mRNA的降解或者抑制轉譯，這樣的功能已經被證實在發炎分子、細胞激素和調控細胞週期等的相關分子上面。本篇研究利用Tetracycline inducible的T-REx-293細胞，透過doxycycline (Dox)誘導細胞內ZFP36L1或ZFP36L2蛋白質的過度表現，發現細胞的生長明顯被抑制，但卻沒有顯著造成細胞死亡情形的增加。同樣的情形在HCT116人類結腸直腸癌細胞也被觀察到。以西方墨點法偵測，發現過度表現ZFP36L1或ZFP36L2蛋白質後，p53蛋白質表現會增加。相反的，調控細胞週期相關的cyclin B1、cyclin D1、cyclin A2及p21蛋白質的表現則受到抑制。再者，以RT-PCR方法檢測受到ZFP36L1或ZFP36L2影響的與調控細胞週期相關之分子，卻只有cyclin D1和p21在mRNA的表現有受到明顯抑制。將ZFP36L1和ZFP36L2的鋅指區域(TZF1與TZF2)上做點突變(point mutation)，發現TZF突變後的ZFP36L1 (C135/173R)喪失了抑制T-REx-293細胞增生的能力。觀察ZFP36L1 knockdown或ZFP36L2 knockdown後的HCT116人類結腸直腸癌細胞，增加了癌細胞的增生能力。本實驗指出ZFP36L1與ZFP36L2可以藉由對細胞週期相關分子的調控，進而抑制細胞的生長。

ZFP36L1 (also called BRF1, ERF1 or TIS11B) and ZFP36L2 (also called BRF2, ERF2 and TIS11D), classified in ZFP36 family proteins, belong to the family members of CCCH-type zinc finger protein with 2 tandem zinc finger region (TZF). ZFP36 family members include ZFP36 (also named TTP and TIS11), ZFP36L1 and ZFP36L2 proteins. These ZFP36 family proteins can function as RNA-binding protein through binding to AU-rich elements (AREs) in the 3’ untranslated region (3’ UTR) of mRNA, which promote the mRNA degradation or translation repression. These regulatory functions have been demonstrated on many inflammatory-related molecules, including cytokines and cell cycle-related molecules. In this study, we used tetracycline-inducible (Tet-On) system to induce the overexpression of ZFP36L1 or ZFP36L2 proteins in T-REx-293 cells by doxycycline (Dox) treatment. While ZFP36L1 or ZFP36L2 was overexpressed, we found that cell proliferation was dramatically inhibited, but didn’t cause cell death significantly. The effect was also observed in HCT116 human colorectal cancer cells. Using western blot analysis, it was revealed that expression level of p53 protein was increased after the overexpression of ZFP36L1 or ZFP36L2 proteins. Whereas, the protein levels of cell cycle-related proteins including cyclin B1, cyclin D1, cyclin A2 and p21 were decrease. Using RT-PCR, the mRNA levels of cyclin D1 and p21, but not cyclin B1 were decrease. The importance of TZF was confirmed by using TZF mutants, and found that cell proliferation was deprived in HEK-293 cells with overexpression of ZFP36L1 mutant (C135/173R). Conversely, knockdown of ZFP36L1 or ZFP36L2 in HCT116 cells increased the cell proliferation. Taken together, our data indicated that ZFP36L1 and ZFP36L2 may inhibit the cell proliferation through the regulation of cell cycle-related molecule.