Identification Of EIF3H SNP And Its Regulatory Mechanism In Colorectal Cancer

Colorectal cancer（CRC）is one of the most common malignancies.Statistics show that the morbidity and mortality of CRC continue to rise,posing a serious threat to human gastrointestinal health.The occurrence and development mechanism of CRC is complex and various.Environmental factors are only the initiators of CRC.Genetic characteristics among individuals determine their susceptibility to CRC tumors,resulting in that not all individuals suffering from CRC at the same environmental risk factors.Single Nucleotide Polymorphism（SNP）is the most common genetic variation in human genome,which plays an important role in complex traits,susceptibility to diseases,individualized and precise treatment of tumors,etc.Genome-wide association study（GWAS）is the study of the association between SNP and diseases at the whole genome level,so as to obtain pathogenic genes and susceptible sites related to complex diseases.As the most powerful means to study SNP,GWAS has identified a large number of CRC related risk genes and functional SNP such as EIF3H（rs11986063）,SMAD7（rs12953717）,CDH1（rs9929218）,SLC22A3（rs7758229）,VTI1A（rs12241008）and so on.Eukaryotic translation initiation factors of subunit 3H is a member of the family of eukaryotic translation initiation factors,mainly involved in the initiation process of protein translation.The activity of EIF3H in normal cells is low,and its increase is related to the occurrence and development of tumor.Many researches have found that EIF3H is highly expressed in many tumor cells,which is a tumor susceptible gene.GWAS research shows that EIF3H may be involved in the process of specific gene expression,regulation,cell proliferation and migration through its functional SNP,which is an important driving factor of cancer cell proliferation and survival.However,most GWAS data are still in the stage of bioinformatics analysis and prediction,and lack of reliable biological function experiments to verify the regulatory function and molecular mechanism of SNP.Therefore,it’s of great significance to study the function of SNP at EIF3H locus.In this study,we investigated 9 SNPs in EIF3H locus and their functions in CRC,which were mainly divided into the following two parts:Part I,in the early stage,we obtained two candidate functional SNP rs1347626 and rs1317271 at EIF3H locus by high-throughput Reel-seq screening.EMSA and double luciferase reporter gene experients proved that rs1347626 was a functional SNP,and the same method was used to verify seven SNP related to LD（r~2>0.8）on rs1347626,which were selected from latest databases and then we obtained four functional SNP:rs76316943,rs16892766,rs4422805 and rs4289846.Finally,we selected three SNP with most significance in binding to nucleoprotein for SDCP-MS and finally obtained 18 specific regulatory proteins.It provides an idea for the functional study of SNP.Part II,we selected five proteins from 18 specific binding proteins obtained above,NONO/HNRNPL,MDC1/PSIP1 and RPS9,for the following functional research.The result of AIDP-WB revealed that NONO and HNRNPL can specifically bind to rs1347626,MDC1 and PSIP1 to rs76316943,rs16892766 to RPS9.Subsequently,the study found that the expression of five proteins in DLD1 cells was higher than that in normal colorectal cells by WB and q PCR.In addition,we found that NONO and MDC1 had a certain regulatory effect on EIF3H through siRNA knock-down,WB and q PCR.After knock-down of the former,the expression of EIF3H decreased,while the latter increased.At the same time,through cell proliferation and cell wound healing assay,we explored their roles in CRC and found that NONO can promote the proliferation and migration of CRC cells,while MDC1 has the opposite effect.On the basis,the study reveals that maybe there’s a regulatory relationship between NONO and MDC1 through WB and q PCR.In summary,the study has verified the function SNP and its specific-binding regulatory protein at EIF3H locus through Reel-seq high-throughput screening,EMSA in vitro,double luciferase reporter gene in vivo,SDCP-MS,AIDP-WB and other series of functional experiments as well as transformed the early data obtained by GWAS of CRC into specific biological functional mechanism.It provides a new theoretical basis and reference idea for early screening of CRC,development of tumor drugs and individualized precise treatment.