Mechanism Study Of Patient-derived IPSCs On DNA Repair Pathway Of XPC Gene In Xeroderma Pigmentosa

[Background and Significance]Xeroderma pigmentosum is an autosomal recessive disease associated with DNA repair.Its symptoms are mainly manifested in the skin,and there are even eye,neurological and other symptoms.XP patients cannot be repaired by ultraviolet radiation,and are prone to cancerous changes.Therefore,it is very important for the prevention and treatment of this disease.One of the causes of XP is the inability of XPC protein to act on DNA repair pathways,so studying the mechanism of XPC gene at the level of DNA repair is of great significance for the provision of therapeutic strategiesiPSCs are induced by the transfer of exogenous transcription factors into somatic cells,and are currently a hot topic in the field of regenerative medicine.Like embryonic stem cells,induced pluripotent stem cells(iPSCs cells)can not only multiply,divide and self-renew continuously in vitro,but also have the ability to differentiate into various types of cells.[Objective]In this study,the disease model of iPSCs was established by in vitro induction,and the mechanism related to DNA repair pathway was studied at this level,so as to simulate the expression of the disease at the embryonic level.On this basis,the mechanism of XPC gene in DNA repair pathway is studied,which lays the foundation for gene therapy and drug screening of this disease.[Method]1.Sample collection: The mutation site was obtained after the whole genome sequencing of the blood sample from the proband,then peripheral blood of other members of the proband’s family was collected and sequenced at 200 bp upstream and downstream of the mutation site.2.Construction of iPSCs cell lines: The plasmid was used to reprogram the proband source PBMC,establish the disease model of XPC gene mutant iPSCs,and test their pluripotency.3.Research on xpc protein in DNA repair pathway: Select proteins related to XPC protein expression in DNA repair pathway,conduct q PCR and wb detection,and study the mechanism of xpc protein in DNA repair pathway at the level of gene expression and protein.4.Research on signal pathways and molecular mechanisms related to XPC gene:High-throughput sequencing(RNA-seq)and transcriptional expression profile analysis were used to screen out genes related to XPC gene defects,and the expression of related genes was compared at the iPSCs level,so as to analyze the mechanism and explore therapeutic methods.[Results]1.Family analysis: Two heterozygous variants of the proband XPC gene c879+1G> C and c2107＿2109del GAG(p.E703del).The mutation c879+1G>C was inherited from the father,and mutation c2107＿2109del GAG(p.E703del)was inherited from the mother,and the family map was obtained.2.The cell line was established: normal karyotype was detected;Alkaline phosphatase staining was strongly positive.The results of RT-q PCR showed that the pluripotent genes OCT4,SOX2,NANOG and LIN28 were highly expressed.Immunofluorescence staining could express the pluripotent markers OCT4,SOX2,SSEA4 and TRA181.Teratoma can differentiate into three germ layers.3.Study on the DNA repair pathway of XPC protein: The genes related to different repair pathways ATR,XPA,MSH and DDB2 were obtained after screening at the protein and gene levels,and the above genes were significantly expressed in patients with XPC gene defects compared with normal people.4.Studies on the signaling pathway and molecular mechanism related to XPC gene:m RNA and ribosome transcriptional activity levels in mutant group were significantly improved compared with control group,but there were defects in the material transfer relationship of intercellular interactions.[Discussion]1.The mutation site and family genetic map of patients with XPC gene mutation were obtained to provide clinical basis for the incidence of xeroderma pigmentation in this family.2.The iPSCs disease model with XPC gene mutation was established and its pluripotency was verified,which provided the research basis for the subsequent research on the related mechanisms of genotype and phenotype of xeroderma pigmentum.3.In the NER repair pathway,XPC protein has an obvious up-regulation effect,suggesting the interaction of XPC gene with other proteins in the DNA repair pathway after mutation.[Conclusion]In conclusion,the mutant group is more active in energy consumption and protein synthesis,which is more likely to cause cancer and autoimmune diseases.However,in terms of intercellular signal transduction and substance delivery,the mutant group had relatively poor function,and the secretion of cell matrix was blocked,which led to the slowing down of immune response and thus led to diseases.