The Study Of The Role And Mechanism Of CUL4B In The Repair Of Intestinal Injury

The intestine is the largest organ in mammals responsible for nutrient absorption and digestion and plays an important role in life.The small intestine epithelium is composed of crypts-villi structure and functions for food digestion,nutrients absorption and anti-bacteria.Adult intestinal adult stem cells maintain the rapid renewal of the intestine,renew every 3-5 days,produce new offspring cells to replace apoptotic epithelial cells at the top of the villi.Intestinal stem cells are very important in the repair of intestinal damage,intestinal stem cells(ISCs)are located at the bottom of the crypts,consisting of the proliferating,irradiation-sensitive active stem cell population(Lgr5+)and a slow-proliferating,irradiation-insensitive resting population(Bmil+).After irradiation,most of Lgr5+stem cells are ablated,and Bmil?resting stem cells and some progenitor cells are mainly responsible for intestinal injury repair.The low-dose X-ray systemic irradiation model of mice can cause cellular DNA damage and rapid repair,which is an ideal model for studying damage repair.This thesis is to use X-ray systemic radiation mice and intestinal cells to build a damage repair model.The CRL complex is one of the largest known E3 ubiquitin ligase that plays a vital role in the ubiquitination of proteins and ubiquitin-mediated protein degradation.CUL4B associates with DDB1 and ROC1 to form the CULLIN-RING ubiquitin ligase complex(CRL4B).Our previous research revealed that CUL4B is involved in regulating various physiological processes including cell cycle,cell signal transduction,and DNA damage repair.Early research in this laboratory found that CUL4B is involved in regulating various physiological processes including cell cycle,signal transduction,and DNA damage repair.Unpublished data from the previous laboratory indicates that CUL4B can promote the self-renewal of intestinal stem cells through the WNT signaling pathway.However,the role of CUL4B in intestinal regeneration is not clear.Therefore,we aim to explore the role of CUL4B in intestinal damage and repair.The following research mainly include the following three aspects:1.The effect of knocking down Cul4b on radiation-induced damage and repair in IEC-6 cells:First,we conducted in vitro experiments in IEC-6 cells to study the role of CUL4B.IEC-6 cells with Cul4b knocked down and its control cells were irradiated with 3Gy X-rays,and cells were harvested at 12,24,36,and 48 hours after irradiation.We found:1)radiation increased CUL4B expression in control cells,indicating CUL4B may play a role in repair after radiation;2)Cell proliferation analysised at different time points showed that cell proliferation is slowed by Cul4b knockdown;3)Cells with reduced Cul4b exhibited less apoptosis after irradiation;4)?-H2AX staining showed cells with Cul4b knocked down had more ?-H2AX than control cells when not irradiated,indicating Cul4b knockdown causes DNA damage.However,y-H2AX level was significantly lower in Cul4b knockdown cells than that in control cells after irradiation,suggesting knocking down Cul4b leads to abnormal DNA repair response.These data demonstrated that CUL4Bparticipates in DNA repair response.2.The effect of specific knockout of Cul4b gene in intestinal epithelial cells on radiation-induced damage and repair:To test the results we got from in vitro experiments,we crossed Cul4bfn/fn mice to pVillin-Cre+/-mice and got the pVillin-Cre+/-;Cul4bfn/Y(KO)mice with Cul4b specifically knocked out in intestinal epithelial cells.The KO and WT mice were irradiated with 12Gy X-rays.The intestines were collected on days 1,3,5,7 after irradiation.We found:1)irradiation significantly increased CUL4B expression in intestine;2)Histological analysis showed that the crypt-villi structure of Cul4b knockout mice was smaller than that of the control group;3)Proliferation analysis showed that loss of Cul4b led to slower proliferation during intestinal regeneration;4)Apoptosis analysis showed that the apoptosis caused by CUL4B irradiation was less;5)?-H2AX protein expression analysis showed that without radiation,KO mice had higher ?-H2AX level than WT mice.However,after radiation ?-H2AX level was lower than that in WT mice,which is consistent with the in vitro data,indicating that loss of Cul4b led to DNA repair deficiency in intestine.3.The preliminary study on the mechanism of CUL4B affecting the repair of radiation-induced intestinal damage:In order to further explore the possible mechanism of CUL4B regulating intestinal damage repair,we performed mRNA sequencing of intestines from Cul4b knockout and control mice on days 0,3,and 7 after irradiation.And bioinformatics analysis found that change in p53 pathway was identified in analysis of the differentially expressed genes at different time points.Western Blot was performed to validate p53 expression.We found that without irradiation p53 expression was significantly increased in Cul4b knockout mice.But on days 1 and 3 after radiation,p53 expression was elevated in WT mice but not in KO mice,suggesting that CUL4B activates the p53 signaling pathway after irradiation and knocks out mice with an abnormal injury response.We further found Puma and Bax,two genes downstream of p53,exhibited radiation-induced change in expression similar to p53,it was significantly down-regulated after Cul4b knockout,suggesting that CUL4B may inhibit apoptosis during injury regulated by p53 signaling pathway.At the same time,we also tested the damage repair protein BRCA1 and found that interfering with CUL4B promoted its phosphorylation during radiation-induced damage and repair,which further indicates that knockdown of Cul4b leads to abnormal damage response.In this study,we used the classic injury model of radiation-irradiated intestine.We studied the intestinal epithelial specific Cul4b knockout mouse and Cul4b knockdown IEC-6 cells.The conclusion of this paper provides the basis for radiation enteritis and the theory for clinical treatment of intestinal injury repair.We will further construct Cul4b overexpression model to confirm the mechnism.Subsequent studies will be further carrird out in vivo and vitro to improve the role of CUL4B in the repair of intestinal injury.