Establishment And Application Of A High-throughput-Screening Model For Detecting The Stability Of Pluripotency Transcription Factors

Key transcriptional factors of ESCs are essential for the maintenance of pluripotency,and play an important role in the somatic cell reprogramming.Cancer stem cell is the key factor for drug resistance,latency and metastasis.Meanwhile,in cancer stem cells,the expression of key transcriptional factors can have an impact on cell differentiation,thereby affecting the development of tumor.Small molecule compounds,as non peptide organic compounds that can recognize and bind specific high structure of protein,are used in the pluripotent regulation of stem cells and targeted therapy in the treatment of cancer.So far,some small molecule compounds have been used in promoting somatic cell reprogramming and inducing differentiation of stem cells.The protein expression of key transcriptional factors is one of important indexes of pluripotency's change.This study attempts to establish a simple,reliable and high-througbput screening system for small molecule compounds that affect the expression level of core transcription factor proteins,and we take Nanog,one of the key transcriptional factors,as an example to apply.We construct a stable cell line that expresses Luciferase-Ub(k0)-Nanoluc-Nanog fusion protein,which can detect the stability of Nanog,thereby achieving high throughput screens.Nanoluc has advantages including small size,strong signal,broad,linearity,and high stability with high sensitive quantitation,etc.By fusing Nanoluc with the target protein,it can reflect the change of proteh stability to a certain extent,while Luciferace acts as a intrinsic parameter,False positives in the screening process can be reduced by using dual-luciferase reporter assay system.More than ten thousands of compounds from The Chinese National Compound Library are evaluated with the screening model preliminary,and some compounds have been found that they are able to significantly change the amount of Nanog protein expression.This study provides a system to screen the small molecular compounds based on the protein stability of key transcriptional factors and lays a foundation for the stem cell research and the treatment of tumor.