Studies On The Functions And Interacing Proteins Of Stem Cell Markers CD133/prominin-like And CD117/PVR In Drosophila Melanogaster

Cancer stem cells (CSCs) are cancer cells that possess characteristics associated with normal stem cells, specifically the ability to self-renew and differentiation into multiple cell types. CSCs are responsible for tumor initiation, proliferation, metastasis, chemo-and radio-resistance. Thus, it is important to be able to identify these cells in order to better understand the mechanisms of tumorigenesis, and to develop novel therapeutic strategies. This depends largely on discovering specific markers that can be used to identify CSCs. CD 133 and CD117 are not only important CSC markers, but also function in cell growth, development and tumor biology. However there is still much to be known about the interaction proteins and biological function of CD133 and CD117.In this study, we down-regulated the expression of endogenous prominin-like (homologous protein of CD 133) in Drosophila Schneider (S2) cells using RNAi-mediated method. The protein levels of VDAC (mitochondrial outer membrane protein)、NDUFS3(subunit of oxidative phosphorylation complex Ⅰ)、COX IV(subunit of oxidative phosphorylation complex IV), but not of ATP5a (subunit of oxidative phosphorylation complex V), were significantly reduced at day 1 and 2 after prominin-like RNAi treatment, then recovered to normal level at day 3. Correspondingly, the cellular ATP levels were found to drop markedly at day 1 and 2 after RNAi treatment, then to return to normal level at day 3. In contrast, the ROS levels were largely increased at day 1 and 2 after RNAi treatment, then back to normal level at day 3. These results showed that prominin-like depletion results in acute injury of mitochondria, which may be responsible for the dysfunction of mitochondria after prominin-like treatment. We then generated prominin-like mutants drosophila by FLP-FRT mediated mitotic recombination and CRISPR/Cas9 konckout method. The prominin-like mutant adults exhibited weaker loco motion. Transmission electron microscope analysis suggested mitochondria with disorganized and fragmented cristae in mutant adults. Using yeast two-hybird system we identified that ND20 was the new interacting protein of prominin-like, where the interaction domain of prominin-like located at area 55-164aa. GST pull-down and Co-Immunoprecipitation (Co-IP) experiments further confirmed the interaction between prominin-like and ND20. We next investigated the biological function of ND20 in S2 cells. ND20 RNAi treated cells exhibited cell cycle G1 arrest, increased ROS levels and unchanged ATP production. Accordingly the protein levels of VDAC NDUFS3 and COX IV, not ATP5a, gradually dereased in ND20-deficient cells. These results suggested that ND20 deficiency lead to dysfunction of cells and electron transfer of mitochondria. Besides, the damage on mitochondria caused by prominin-like RNAi could be achieved by ND20. In a word, prominin-like is likely involved in adaptive changes in cellular bioenergetic metabolism through ND20.Meanwhile, we identified that Shc directly interact with PVR (homologous protein of CD117) by yeast two-hybird screening, and the interaction domain of PVR located at area 55-164aa. Co-Immunoprecipitation (Co-IP) were employed to further confirm the interaction of PVR and Shc.The results suggested that PVR physically interacts with Shc in S2 cells, which may bring us new ideas for the further research on biological function of PVR.