Mst3在飢餓引起的自噬作用中所扮演的角色

Abstract

Mst3 在壓力誘導凋亡作用中扮演著重要角色。自噬作用乃一種自我吞噬現象，是透過溶酶體的消化機制，來降解不需要的以及功能不彰的胞器。在養分不足或粒線體有缺陷的情況下，細胞會引發形成Autophagosome，而autophagosome接著會與溶脢體融合，進而去除細胞內的糾結的蛋白，病源體以及損壞之細胞胞器來維持細胞內恆定跟細胞內的品質管理。若細胞自噬作用的調節出問題，則會引起人類的許多疾病，如、神經退化性病變，癌症及老化。在先前的研究中，我們發現當細胞內的的Mst3表現量降低時，細胞株的粒線體的功能會喪失。據此，我們希望了解HeLa和HeLa(siMst3)細胞株在飢餓下產生自噬作用之反應。在與正常細胞株相較下，HeLa(siMst3)細胞株內LC3蛋白的基本表現量是較低的。然而，當HeLa和HeLa(siMst3)細胞株培養在EBSS無胺基酸培養液中時，兩細胞的LC3-II蛋白質的量會在挨餓兩小時後均迅速增加。有趣的是，兩者的LC3II/LC3I比例隨挨餓過程產不同變化和結果；正常HeLa細胞中，在前兩小時有提升跡象，隨後變化變得平緩。而HeLa(siMst3)細胞株在饑餓兩小時後LC3II/LC31比例明顯持續增加著。我們也採用了MDC染色進行驗證，證實細胞挨餓確實誘發了自噬作用的發生。這些結果顯示 HeLa(siMst3)細胞株可能具有更佳的耐餓性與生存能力。隨後的研究還發現HeLa(siMst3)細胞株在饑餓兩小時後ROS會明顯的增加。相對的，HeLa細胞株在挨餓8小時後依然沒有ROS增加的跡象。就這項觀察的結果，我們認為因飢餓所引起的自噬作用或許跟ROS的產生有關。

Mst3 plays important role in stress-induce apoptotic event. Autophagy is a self-eating process to degrade unnecessary or dysfunctional cellular components through the lysosomal machinery. Upon nutrient deprivation or mitochondrial defect cell initiates the formation of autophagosome, which then fuses with lysosome to eliminate intracellular aggregated proteins, pathogens and impaired organelles, serving as the quality control and homeostasis of cells. Dysregulation of autophagy associated many human diseases, including neuron degenerative diseases, cancer and aging. Previous studies have shown that HeLa(siMst3), a stable clone with selective knock down of Mst3, exhibited signs of mitochondrial defect. Accordingly, the autophagic responses of HeLa and HeLa(siMst3) cells to starvation were studied. Compared to normal control, the basal level of LC3 was reduced in HeLa(siMst3) cells. However, LC3-II level increased rapidly in both HeLa and HeLa(siMst3) cells at 2 h after starvation with EBSS. Interestingly, the HeLa and HeLa(siMst3) cells exhibited a differential result in the starvation-mediated LC3-II/LC3-I ratio change. In HeLa cells a moderate increase in LC3-II/LC3-I ratio started 2 h after starvation and remained constant afterward. While, in HeLa(siMst3) cells the LC3-II/LC3-I ratio increased markedly 2 h after starvation. MDC staining on both HeLa and HeLa(siMst3) cells confirmed the induction of autophagic process by starvation. These results suggest that HeLa(siMst3) cells may have better chance to resist starvation and survive. Further study showed that relatively high level of ROS accumulated in HeLa(siMst3) cells started at 2 h of starvation; whereas, ROS was undetectable even after 8 h of starvation. It suggests that starvation-induced autophagic event in HeLa(siMst3) cells may correlate to the elevation of ROS.