| 摘要: | 脂滴被視為新脂質生成的重要指標，不僅作為過量脂質的儲存庫，也能調控脂質的運輸與利用，進而滿足能量產生、抗氧化壓力保護，以及快速細胞增殖期間所需的膜形成。粒線體Lon蛋白在維持粒線體健康中扮演多功能角色，能促進蛋白質降解、協助蛋白質折疊並調控粒線體DNA的完整性。在癌症中，Lon蛋白對於維持細胞增殖至關重要，使細胞能夠抵禦諸如缺氧、蛋白毒性及氧化損傷等壓力。此外，我們的研究發現，在癌細胞中，Lon蛋白過度表現會促進脂滴的形成。本研究旨在探討Lon表現與脂滴生成之間的潛在關聯。根據我們的結果，在HCT15細胞株中，當Lon蛋白酶過度表現時，與對照組及CoCl2處理組相比，通過Plin2表現及Bodipy 493/503螢光染色觀察到脂滴的出現。此外，當細胞接受粒線體呼吸鏈抑制劑魚藤酮（Rotenone）處理時，也觀察到 Plin2 上調與脂滴的生成。然而，在 Lon 過度表現與 Rotenone 處理的細胞中添加抗氧化劑 NAC（N-乙醯半胱氨酸）後，脂滴數量明顯下降，顯示脂滴形成可能受到氧化壓力調控。此外，我們亦通過使用shLon質體抑制Lon表現，觀察脂滴形成的情況，結果顯示，在Lon表現被抑制的樣本中，脂滴沒有形成。為進一步確認Lon上調時癌症細胞中脂滴的形成，我們使用另一口腔鱗狀細胞癌（OSCC）細胞株HSC-3進行實驗。螢光影像結果顯示，與對照組相比，Lon 過表現顯著促進脂滴形成，且脂滴的尺寸亦明顯增大。鑑於新脂質生成與脂滴形成之間的密切關係，我們觀察了兩種關鍵酶的表現，包括脂肪酸合成酶（FASN）及二醯甘油-O-醯基轉移酶2（DGAT2）。結果顯示，在Rotenone及Lon過度表現條件下，與對照組相比，兩種酶的表現均增加。雖然Rotenone樣本經NAC處理後，兩種酶的表現均下調，但在Lon過度表現並經NAC處理的樣本中，FASN的表現與未經NAC處理的Lon過度表現樣本相比呈現相反的上調趨勢。而DGAT2在Lon過度表現並經NAC處理後的表現則下降，與脂滴數量的下調相關。 最後，我們確定了脂滴與粒線體的定位關係，即所謂的「脂滴周邊粒線體形成」，為後續實驗奠定基礎，以進一步證明這兩個細胞器之間的關聯性。;Lipid droplets are considered a hallmark of de novo lipogenesis and serve as reservoirs for excess lipids, regulating lipid trafficking and utilization for processes such as energy production, oxidative stress protection, and membrane formation during rapid cell proliferation. Mitochondrial Lon plays a multifunctional role in maintaining mitochondrial health, facilitating protein degradation, assisting in protein folding, and regulating mitochondrial DNA integrity. In cancer, Lon is crucial for sustaining proliferation, enabling cells to withstand stressors such as hypoxia, proteotoxicity, and oxidative damage. In addition, we found that overexpression of Lon in cancer cells triggers the formation of lipid droplets (LDs). In this work, we aim to explore the link between Lon expression and lipid droplet formation. According to our results, in the HCT15 cell line, once Lon protease was overexpressed, we observed the appearance of lipid droplets via the expression of Plin2 expression and fluorescent staining of Bodipy 493/503 compared to the control sample and CoCl2. Furthermore, lipid droplets also appeared when the cell line was treated by rotenone by observing the increase of Plin2. However, in the NAC treatment samples for both Lon overexpression, and rotenone, the disappearance of lipid droplets was witnessed when compared to their counterpart without the NAC treatment. Besides, we also decided to run the inhibition of Lon expression to check the lipid droplet formation. The results showed that when we terminated the expression of Lon by using a shLon plasmid, there were no lipid droplets in the Lon-inhibited sample. In addition, we tried to experiment with another cancer cell line called HSC-3 (an OSCC cancer) to confirm the lipid droplet formation in cancer when Lon is upregulated. The related images displayed the increase of lipid droplet formation under Lon overexpression condition compared to its control sample. In addition, under Lon overexpression, the size of lipid droplets is enlarged compared to the size of lipid droplets in the control sample. Moreover, due to the closed relationship between de novo lipogenesis and lipid droplet formation, we observed the expression of two key enzymes, including Fatty acid synthase (FASN), and Diacylglycerol O-Acyltransferase 2 (DGAT2). The data indicated that under rotenone and Lon overexpression, the expression of both enzymes was increased when compared to the control. Although the NAC treatment for the rotenone sample showed the downregulation of both enzymes, the Lon overexpression sample with NAC treatment showed the opposite results in the expression of FASN when the enzyme outnumbered the Lon overexpression samples without treatment. With NAC treatment for the Lon overexpression, the expression of DGAT2 showed the decrease correlating with the downregulation of the number of lipid droplets. Finally, we identified the localization of lipid droplets and mitochondria called peridroplet mitochondrial formation, to establish the base for further experiments to prove the connection between the two organelles. |
