臺灣博碩士論文加值系統

巨核球乃血小板的前驅細胞，在人體的凝血功能中扮演必要角色。K562細胞為慢性骨髓性白血病細胞，受到PMA (phorbol 12-myristate 13-acetate) 誘導可走向巨核球系分化，乃研究巨核球系分化的常用模型。Rho家族為small GTPase一員，而RhoGDIα(Rho GDP dissociation inhibitor α) 可抑制Rho GTPases的活 化。過去實驗室研究中發現RhoGDIα能夠正向調控K562走向巨核球系分化。先前文獻指出RhoGDIα在第111、152、180有三個被雙甲基化的精胺酸 (arginine, R) ，但其精胺酸甲基化的作用功能未被探討。而過去實驗室研究發現蛋白精胺酸甲基轉移酶6 (protein arginine methyltransferase 6, PRMT6) 能正調控巨核球系分化，因此本研究著重於探討RhoGDIα的精胺酸甲基化在巨核球系分化中可能扮演的角色，並探討PRMT6是否經由甲基化RhoGDIα的精胺酸而促進巨核球系分化。
本研究中經由將RhoGDIα上的精胺酸突變成離胺酸 (lysine, K) ，阻止這三個位點甲基化來探討甲基化可能的影響。以劉氏染色法發現，在K562細胞中，此三個精胺酸位點無論單點、雙點或三點突變皆造成RhoGDIα促進巨核球系分化的能力降低，其中除了R180K外，其它突變在內生性RhoGDIα存在的情況下，都具有顯性抑制巨核球系分化的效果。在RhoGDIα缺失細胞株中，R111K及R152K兩個單點突變促進分化的能力較野生型低，R111/152K與R111/152/180K則完全失去促進分化的效果，R111/180K及R152/180K的分化率則與R111K及R152K相似，顯示第111及152個精胺酸甲基化在RhoGDIα促進分化的功能扮演較重要的角色。研究並發現在RhoGDIα缺失細胞株中過度表現PRMT6，則PRMT6不再能促進巨核球系分化，而同時將外生性RhoGDIα過度表現則PRMT6可回復促進分化的能力；但若過度表現外生性RhoGDIα R111/152K則無法回復。另外試管中甲基化實驗發現PRMT6能夠甲基化RhoGDIα。綜合以上結果顯示PRMT6可能經甲基化RhoGDIα上的第111及152個精胺酸而影響巨核球分化。

Megakaryocytes are the precursors of platelets and thus play an essential role in blood clotting. K562 is a human leukemia cell line that can be induced by PMA (phorbol 12-myristate 13-acetate) to undergo megakaryocytic (MK) differentiation and has been used as a cell model for studying MK differentiation. The Rho family belongs to the small GTPase superfamily and GDP-dissociation inhibitor α (RhoGDIα) suppresses the activity of Rho. Our previous study has shown that RhoGDIα promoted MK differentiation. RhoGDIα has been reported to contain three di-methylated arginines (R111, R152 and R180) however their function is not reported. In addition, our previous results also showed that protein arginine methyltransferase 6 (PRMT6) played a positive role in MK differentiation. This study thus aimed to investigate the functional role of arginine methylation of RhoGDIα and whether PRMT6 mediates methylation of RhoGDIα.
In this study, the arginine residues (R111, R152 and R180) were mutated to lysines to mimic the non-methylated state. By ectopic expression, my results showed that single, double and triple mutations lost, to different extent, their stimulatory effects on MK differentiation of K562 cells. Besides R180K, all the mutants appeared to have a dominant negative effect. In the RhoGDIα knockdown (KD) cells, R111K and R152K single mutants still promoted MK differentiation however to a lower degree than wild type did. R111/152K and R111/152/180K double mutants completely lost their stimulatory effects. R111/180K and R152/180K had a similar effect to R111K and R152K single mutants. Together, these results suggest methylation on R111 and R152 plays a more significant role than R180 in promoting MK differentiation. Overexpression of PRMT6 in RhoGDIα KD cells could no longer promoted MK differentiation. When co-expressed with RhoGDIα, PRMT6 regains its ability to promote differentiation in RhoGDIa knockdown cell; while co-expression with RhoGDIα R111/152K did not help regaining the ability. Notably, PRMT6 methylated RhoGDIα in in vitro methylation. These results suggest that PRMT6 may be responsible for methylation of R111 and R152 of RhoGDIα which then promote PMA-induced megakaryocytic differentiation of K562 cells. This study provides links suggesting a positive role of RhoGDIα arginine methylation in MK differentiation.

目 錄
目 錄 ………………………………………………………… 1
縮 寫 表 ………………………………………………………… 2
圖次目錄 ………………………………………………………… 4
附圖目錄 ………………………………………………………… 4
中文摘要 ………………………………………………………… 5
英文摘要 ………………………………………………………… 6
緒 論 ………………………………………………………… 8
研究目標 ………………………………………………………… 17
實驗材料 ………………………………………………………… 18
實驗方法 ………………………………………………………… 23
結 果 ………………………………………………………… 35
討 論 ………………………………………………………… 43
參考文獻 ………………………………………………………… 47
圖 表 ………………………………………………………… 54
附 圖 ………………………………………………………… 66

圖次目錄
Figure 1. Ectopic expression of Flag-RhoGDIα increased PMA-induced megakaryocytic differentiation
of K562 cells. ....................................... 54
Figure 2. Ectopic expression of Flag-RhoGDIα single mutants affected PMA-induced megakaryocytic differentiation of K562 cells. ........................................55
Figure 3. Flag-RhoGDIα double and triple mutants suppressed PMA-induced megakaryocytic differentiation of K562 cells. ........................................... 56
Figure 4. Effects of Flag-RhoGDIα single mutants on PMA-induced megakaryocytic differentiation of GDIα-KD1 cells. ................................................ 57
Figure 5. Effects of Flag-RhoGDIα double and triple mutants on PMA-induced megakaryocytic differentiation of GDIα-KD1 cells. ...................................... 58
Figure 6. RhoGDIα was required for the stimulatory effect of PRMT6 on PMA-induced megakaryocytic differentiation of K562 cells. ........................ 59
Figure 7. Ectopic overexpression of RhoGDIα regained inducible effect on megakaryocytic differentiation mediated by PRMT6 overexpression. .............................. 60
Figure 8. Expression and purification of His-RhoGDIα. ......................................... 61
Figure 9. RhoGDIα protein were methylated by PRMT6 and PRMT1 in vitro. ................................... 62
Figure 10. Establishment of RhoGDIα-overexpressing stable clones. ........................................ 63
Figure 11. Stable expression of RhoGDIα did not affect either cell growth or viability. ............... 64
Figure 12. The stable overexpression of RhoGDIα promoted PMA-induced megakaryocytic differentiation. .. 65

附圖目錄
Figure S1 Schematic representation of plasmids of RhoGDIα. ............................................. 66

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