Construction And Validation Of Stem Cell YAP1 Conditional Knockout Transgenic Prostate Cancer Mouse Model

Prostate cancer is one of the most common malignancies in men and is a major cause of tumor death in men.Although prostate cancer was controlled to varying degrees at the beginning of androgen deprivation therapy?ADT?,after a sensitive period of approximately 18 months,the disease in most patients gradually and irreversibly progresses to castration-resistant prostate cancer.The mechanisms of CRPC formation include androgen receptor?AR?-related mechanisms,stem cell formation mechanisms and neuroendocrine transformation mechanisms.Prostate cancer stem cells are the main cause of failure of ADT treatment.YAP1?Yes-associated protein 1?is a core element in the Hippo signaling pathway,regulates cell proliferation and apoptosis,maintains the normal size of tissues and organs,and the homeostasis of the body's environment.Increased expression of YAP1 is associated with a variety of tumors,including prostate cancer.The expression of YAP1 was significantly increased in CRPC tissues,and it played an important role in regulating the stemness of prostate cancer stem cells.The purpose of this study was to establish a transgenic mouse model of conditionally knockout YAP1 gene on CD133-labeled stem cells in order to observe the effect of knockout YAP1 on stem cells for the progression of prostate cancer.The target transgenic mice were established by gene editing techniques,fertilized egg injection techniques,and crossbreeding techniques.According to the characteristics of spontaneous tumorigenesis of TRAMP mice,YAP1 gene was knocked out at different time points to observe its effect on the progression of prostate cancer.In this study,the CRISPR/Cas9 system was used to integrate YAP1 Exon2 with Loxp sequences on both sides into the mouse genome through homologous recombination to establish homozygous YAP1^fl/fl mice.Next,we introduced TRAMP mice that can spontaneously develop prostate cancer,CD133-Cre mice that can express Cre recombinase on stem cells,and mT/mG mice that can fluoresce,and establishCD133-TRAMP-mTmGmouse.Wethencrossedthe CD133-TRAMP-mT/mG male hybrid mouse with the homozygous YAP1^fl/fll/fl female mouse to finally obtain a CD133-YAP1^fl/fl-TRAMP-mT/mG transgenic mouse capable of inducing knockout of the YAP1 gene on stem cells.Finally,we used the established CD133-YAP1^fl/fl-TRAMP-mT/mG transgenic mice to knockout YAP1 of CD133-labeled cells in stages of prostatic hyperplasia,PIN formation,and cancer progression.Time of sacrifice of mice at 16w,22w,and 34w,validation of YAP1knockout effect and preliminary observation of the effect of knockout on prostate cancer development.The constructed YAP1^fl/fl mice,CD133-TRAMP-mT/mG mice and CD133-YAP1^fl/fl-TRAMP-mT/mG mice were all verified by PCR genotyping.Tamoxifen induced CD133-YAP1^fl/fl-TRAMP-mT/mG mice in different stage resulted in YAP1 knockout on stem cells.After a short-term induction of YAP1 knockout on prostatic proliferative stem cells,although there was no significant change in the morphology of the prostate,knockout mice lost their progenitor cell proliferation and increased apoptosis.During prostate PIN formation,YAP1 was knocked out after a long period of time,prostate intraepithelial neoplasia?PIN?was reduced,prostate tissue cells were reduced in proliferation,and increased in apoptosis.In the advanced stage of prostate cancer,YAP1 was knocked out after a long time,the tumor of the prostate was reduced,the tumor progression was delayed.In this study,a transgenic mouse model that can knock out YAP1 gene from stem cells was established and verified.Preliminary results showed that knocking out YAP1 gene on CD133 stem cells slowed down the progression of prostate cancer,which provides a new direction for the treatment of prostate cancer.