Inhibitive Role Of 15 KDa Selenoprotein (Sep15) On Tunicamycin- Induced Disorder Of Human Lens Cell Differentiation

15 kDa selenoprotein (Sep15) is a resident of endoplasmic reticulum (ER). A recent study reported that Sep15 knockout (Sep15 KO) mice developed a prominent nuclear cataract in the 1.5 month after birth. However, the mechanism of Sep15 KO mice causing cataract formation remained unclear. Further analysis showed that Sep15 protein was expressed in the anterior epithelium of mouse lens. The differentiation process of lens epithelial cell transforming to fiber cells, accompanied by synthesis of crystallin proteins and degradation of all cytoplasmic organlles, is necessary for the establishment and maintenance of lens transparency. Moreover, lens epithelial cell apoptosis may be an early and critical event during non-congenital cataract formation. Therefore, it is important to unveil the cause of cataract in Sep15 KO mice that conducts sophisticated investigation on the role of Sep15 in lens epithelial (hLE) cell apoptosis and differentiation. It will be beneficial to understand the biological function of selenium in eyes and display a potential value for cataract prevention.In this thesis, we firstly reviewed the relationship among lens development, ER stress, oxidative stress and biological function of Sep15. Then we used hLE cells as a cell model to study the effect of Sep15 deficiency on Tm-induced apoptosis and differentiation dysfunction in lens epithelial (hLE) cell, and the main results were as follows:(1) Effect of Sep15 gene knockdown on Tm-induced apoptosis in hLE cellsThe effect of Sep15 gene knockdown on Tm-induced apoptosis and redox homeostasis disruption in hLE cells was measured using MTT assay, RT-PCR, western blotting, flow cytometry and fluorescence microscope. Our results suggested that reduction of Sep15 expression increased Tm-induced apoptotic cells even though sole knockdown of Sep15 showed no significant change in hLE cell viability. The result was further supported by decreased mitochondrial membrane potential, release of cytochrome c and activation of caspases in Tm-treated Sep15 siRNA hLE cells. In addition, knockdown of Sep15 aggravated Tm-induced oxidative stress, accompanied by further increase of ROS and MDA content and decrease of total thiols in Tm-treated Sep15 siRNA hLE cells. Interestingly, knockdown of Sep15 expression did not cause ER stress or elevate Tm-induced ER stress in hLE cells even though Sep15 can be regulated by Tm-induced ER stress. In conclusion, the study showed that Sep15 might protect hLE cells against Tm-induced apoptosis via regulating intracellular redox status rather than regulating Tm-induced ER stress.(2) Effect of knockdown of Sep15 and Tm-induced ER stress on differentiation of lens epithelial cellsbFGF, a fibroblast growth factor, was used to induce differentiation of hLE cells, and then the effect of Sep15 gene knockdown and Tm treatment on bFGF-induced differentiation of hLE cells was measured by MTT assay and western blotting. Our results showed that 40 ng/mL bFGF increased the expression of β-crystallin protein synthesized in the early stage of cell differentiation, indicating initiated transformation of epithelial cell to fiber cells. But Tm-induced ER stress decreased bFGF-induced expression of β-crystallin protein in hLE cells. Moreover, hLE cells pretreated with Tm before bFGF stimulation exhibited more significant inhibition on cells differentiation. Similarly, reduction of Sep15 in hLE cells decreased β-crystallin protein and aggravated the inhibition of Tm. Observation on FGF signal in cells differentiation showed that Tm treatment and Sep15 gene knockdown inhibited the bFGF-induced phosphorylation of ERK1/2 and Akt, which were downstream of FGF/MAPK and FGF/PI3K pathway, respectively. In conclusion, hLE cell differentiation was inhibited by Sep1S gene knockdown and Tm-induced ER stress, and the inhibition might be induced via FGF-dependent signaling pathway.(3) Effect of selenium intake on hLE cell apoptosis induced by Sep15 gene knockdown and Tm-induced ER stressSelenium intake was conducted by supplementation of sodium selenite into hLE cell, and the effect was investigated using MTT assay, fluorescence microscope and western blotting. Our results showed that appropriate supplementation of sodium selenite ameliorated viability of hLE cells under modest Tm-induced stress. However, the protection was not sufficient under acute stress when cell was treated with higher concentration of Tm. It was observed that the selenium intake reduced percentage of apoptotic cells, as well as expression of caspase proteins and content of protein carbonyl, but elevated content of total thiols. In addition, the similar reduction effect was also observed on ER stress markers, such as BiP、Ire la and eIF2a protein. Investigation of Sep15,SelS and Se1R in negative siRNA hLE cells and Sep15 siRNA hLE cells showed that selenium intake boosted expression of all the three selenoprotein. In conclusion, sodium selenite supplementation maintained redox and ER homeostasis via increased expression of selenoprotein, that protected hLE cells against oxidative stress and apoptosis induced by Sep15 gene knockdown and Tm treatment, and ameliorated Tm-induced ER stress.