Zkscan3 Gene Is A Potential Negative Regulator Of Plasma Cell Differentiation

We previously showed that the ZKSCAN3 gene codes for a zinc-finger transcription factor that regulates the expression of important genes and plays crucial roles in the development,metastasis,and pathogenesis of rectal cancer,prostate cancer,myeloma,etc.and in the regulation of autophagy.However,its biological functions under normal physiological conditions remain unclear.Additionally,our previous studies showed that the ZKSCAN3 gene may negatively regulate B cell functions.Therefore,we constructed a zkscan3-knockout mouse model.Methods:1.UBC-Cre tool mice were crossed with zkscan3 flox+/+mice to obtain zkscan3 knockout mice.2.We tested the expression of zkscan3 at the DNA level,MRNA level,and protein level to detect the efficiency of zkscan3 gene knockout.3.We statistically analyzed the number and weight of each genotype mice and observed the phenotypic changes of the mice.4.In wild and knockout mice,the number of cells at each stage of B cell development was detected by flow.5.We constructed a mouse model of MCA38 colorectal cancer,and analyzed the number of each stage of B cell development in a knock-out mouse and wild mice.6.We used LPS to stimulate B cell differentiation in vitro and analyzed the number of germinal center B cells and plasma cells and measured the concentration of lgG?lgA and lgM in the supernatant of cells culture by ELISA.7.We sorted the splenic B cells for RNA-sequence analysis and verified the results of RNA-sequence with Q-PCR.8.We constructed a zkscan3 overexpressing CT26-zkscan3 cell line and used the luciferase reporter system to detect the relationship between xist and zkscan3.Result:1.Zkscan3 is not expressed in mRNA levels?DNA levels and protein levels in knockout mice.2.In the zkscan3 knockout mice,their phenotypes are normal,and the birth rate of the mice is consistent with Mendel's law of inheritance.3.In the normal physiological conditions,the number of plasma cells increased in zksean3 knockout mice compared with wild mice.4.In the MCA38 colorectal cancer mouse model,the number of plasma cells increased in zkscan3 knockout mice compared with wild mice.5.After stimulation with LPS,compared with WT mice,the number of B cells and plasma cells in germinal centers increased,and more plasmablasts secreted more immunoglobulins 1gG?1gA and 1gM,eompared with WT mice.6.RNA-sequence and Q-PCR showed that xist expression was up-regulated in zkscan3 knockout mice.7.Luciferase reporter gene results indicate that zkscan3 regulates the expression of xistCluclusion:We observed that knockout mice had a greater number of plasma cells than wild-type mice.We also found that the number of plasma cells was significantly increased in either colorectal cancer xenografts or under lipopolysaccharide-induced conditions.RNA-seq and quantitative-polymerase chain reaction assay indicated that the X-inactive specific transcript is up-regulated in B cells of zkscan3 knockout mice,which may represent a potential mechanism how zkscan3 modulates plasma cells differentiation.