9-順式視網酸對粒線體生合成影響之研究
The effects of 9-cis retinoic acid on mitochondrial biogenesis
中文摘要
已知粒線體影響生長發育並參予細胞凋亡過程，而對於粒線體生合成控制路徑 的瞭解將有助於治療因為粒線體缺陷及匱乏所造成的退化性疾病。本實驗之目的 主要建立一個粒線體生合成的細胞模式，探討及篩選影響粒線體生合成的營養 素及其調控機轉。本實驗以濃度為0.25 ~ 25 μM 的 9-順式視網酸（9-cis retinoic acid, 9-cis RA）培養人類骨癌細胞株 143B TK－細胞（human osteosacroma 143B TK－cells）24、48 及 72 小時。之後，以 MTS 試劑偵測視網酸對細胞存活率之影
響，並使用光學顯微鏡觀察細胞形態之變化。利用RT-PCR 及西方墨點法分別分
析細胞中粒線體mRNA 及蛋白質的表現量以評估粒線體生合成之狀況。結果發
現，細胞存活率受9-cis RA 處理濃度與時數之不同而改變。例如：經 25 μM 的 9-cis RA 處理 24 小時，細胞存活率顯著高於控制組。9-cis RA 不改變 143B TK－
細胞的細胞型態。 9-cis RA 顯著地增加粒線體 DNA 轉錄的氧化磷酸化蛋白質 cytochrome c oxidase subunit I （COX I）、 cytochrome c oxidase subunit II （COX II）、 cytochrome c oxidase subunit III （COX III）、NADH dehydrogenase subunit 1（ND I）及 ATP synthase subunit 6（ATPase 6）基因的 mRNA 表現量。例如：
經9-cis RA 處理 24 小時後，COX II 的 mRNA 表現量在溶劑組、0.25 μM 組、0.5 μM 組、1 μM 組、2 μM 組、5 μM 組、10 μM 組及 25 μM 組分別為控制組的 0.97 ± 0.09、1.78 ± 0.07、1.67 ± 0.09、1.68 ± 0.24、2.63 ± 0.30、2.05 ± 0.34、2.53 ± 0.39 及 2.76 ± 0.24 倍。同時，9-cis RA 增加細胞核 DNA 轉譯的氧化磷酸化蛋白質 NADH-ubiquinone oxidoreductase subunit 9（NDUFA 9）及 succinate-ubiquinone oxidoreductase （SDHA）基因的 mRNA 表現量。另外，retinoid X receptor α（RXRα）及 mitochondrial transcription factor A （mtTFA）基因的 mRNA 表現
量亦隨著9-cis RA 濃度的增加而顯著地增加。至於在蛋白質表現量部分，9-cis
RA 增加粒線體 DNA 轉譯的氧化磷酸化蛋白質 COX I、COX II、COX III 及細胞 核DNA 轉譯的氧化磷酸化蛋白質 NDUFA9 及 SDHA 的表現量。例如：經 9-cis RA 處理 72 小時後，COX III 的蛋白質表現量在溶劑組、0.25 μM 組、0.5μM 組、1 μM 組、2 μM 組、5 μM 組、10 μM 組及 25 μM 組分別為控制組的 0.81 ± 0.20、1.73
± 0.50、1.71 ± 0.40、1.88 ± 0.64、1.95 ± 0.51、1.96 ± 0.56、1.71 ± 0.53 及 1.65 ± 0.42 倍。然而，9-cis RA 在高濃度下卻降低了 RXRα 蛋白質的表現量。實驗結果顯示 9-cis RA 可能藉由活化細胞核接受器 RXRα 以及 mtTFA 的大量表現，直接或間 接地調控粒線體之生合成。
英文摘要
In this study, the mRNA and protein levels of mitochondrial DNA-encoded and nuclear DNA-encoded oxidative phosphorylation system subunits were determined by
RT-PCR and Western blotting analysis to evaluate the effects of 9-cis retinoic acid (9- cis RA) on mitochondrial biogenesis.
Various amounts of 9-cis RA (0.25~25 μM) were given to culture human
osteosarcoma 143B TK－ cells for 24, 48, and 72 hours. The MTS assay data showed that the concentration and the treatment time of 9-cis RA would affect the cell
survival rate. For examples, after giving 9-cis RA treatments for 24 hours, the cell survival rate in the 25 μM group was significantly higher than in the control group (p
< 0.05). However, the cell morphology did not change after 72 hours of treatment.
From RT-PCR analysis, the levels of mRNA expression of mitochondrial DNA- encoded cytochrome c oxidase subunit I (COX I), cytochrome c oxidase subunit II (COX II), cytochrome c oxidase subunit III (COX III), NADH dehydro-
genase subunit 1(ND1), and ATP synthase subunit 6 (ATPase 6) were significantly increased by 9-cis RA. For instances, after giving 9-cis RA treatments for 24 hours, the mRNA levels of COX II in the vehicle, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 25 μM groups were 0.97 ± 0.09, 1.78 ± 0.07, 1.67 ± 0.09, 1.68 ± 0.24, 2.63 ± 0.30, 2.05 ± 0.34, 2.53 ± 0.39 and 2.76 ± 0.24 folds of the control group. Also, the levels of mRNA expression of nuclear DNA-encoded NADH-ubiquinone
oxidoreductase subunit 9 (NDUFA 9) and succinate-ubiquinone oxidoreductase (SDHA) were increased by 9-cis RA. As the concentration of 9-cis RA increased, the levels of mRNA expression of the nuclear DNA-encoded retinoid X receptor α (RXRα) and mitochondrial transcription factor A (mtTFA) were significantly increased.
Western blot data revealed that the levels of protein expression of mitochondrial DNA-encoded COX I, COX II and COX III , and nuclear DNA-encoded NDUFA 9 and SDHA were increased by 9-cis RA. For instances, after giving 9-cis RA
treatments for 72 hours, the levels of protein expression of COX IIIin the vehicle, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 25 μM groups were 0.81 ± 0.20, 1.73 ± 0.50, 1.71 ± 0.40, 1.88 ± 0.64, 1.95 ± 0.51, 1.96 ± 0.56, 1.71 ± 0.53 and 1.65 ± 0.42 folds of the control group. However, the levels of protein expression of the nuclear receptor, RXRα, were decreased as the concentration of 9-cis RA increased.
These results provided evidence of elevation effects of retinoic acid on mitochondrial biogenesis. It is suggested that 9-cis RA may induce mitochondrial biogenesis through activating RXRα and increasing the transcription of mtTFA .
