E-box Mutation In The 5’UTR Of Nle1 Effect Embryonic Death And Intestinal Tumorigenesis

Background:Colorectal cancer（CRC）normally develops from polyps,adenoma andadenocarcinoma in a sequential manner,giving rise to a pressing need for the diagnosis of the disease at its early stages that would substantially benefit treating patients and reducing mortality.In this respect,identifying pivotal biomarkers has become clinically significant especially in early diagnoses and targeted therapy.There have also been deliberate attempts on screening differentially expressed genes in different stages of CRC development,in which a wide series of biomarkers have stood out.Among those,the expression level of Nle1 is reportedly elevated in colorectal adenomas and adenocarcinomas,indicating its involvement in the initiation and progression of CRC.In earlier studies we found that the canonical transcription factor MYC binds to the E-box（5′-CACGTG-3′）region in the 5’UTR（5’untranslated region）adjacent to the promoter.We deduce accordingly that E-box might come as a cis-regulating element of Nle1 bound by MYC,and thus take part in the intricate molecular crosstalks of CRC development.Objectives:for the sake of verifying this hypothesis,we established the mutant mice Nle1^{（-16_-11）delins TATTTA/+}by shuffling the E-box DNA sequence(named as Nle1^mut/+hereafter)in situ,and subsequently introduced a malignant background genetically by crossing Nle1^mut/+with Apc ^Min/+mice,and ultimately analyzed phenotypes typical of CRC between Nle1^mut/+,Apc ^Min/+and Nle1^+/+,Apc ^Min/+mice in the progeny with the aim of exploring the effect（s）of MYC-bound E-box on the tumorigenesis of CRC.Results:We adopted a breeding strategy that involves initially expanding the Nle1^mut/+and Apc ^Min/+population seperately,followed by crossing male Apc ^Min/+and female Nle1^mut/+mice from the progeny of both genotypes.To our total surprise,no Nle1^mut/mutoffspring was identified,indicating the homozygous E-box mutation might cause embryonic lethality somehow.We also performed whole genome sequencing of Nle1^mut/+mice attempting to exclude the possibility of bringing unexpected major structual mutations into the genome in the course of mutant mice engineering,which would eventually disturb or alter the natural phenotypes.Consequently we spotted few critical structural variations other than the E-box mutation.On this account,we presume the embryonic lethality is mainly caused by the E-box mutation on Nle1.Moreover,we closely examined the genotype profiles among E3.5,E4.5,E7.5 and E8.5 embryos.It is proved that embryos of all the three genotypes(Nle1^mut/mut,Nle1^mut/+,WT)survived through 3.5 dpc（days post coitum）and 4.5 dpc but only Nle1^mut/+and WT mice remained by 7.5 dpc and 8.5 dpc.In this regard we infer that Nle1^mut/mutembryos are most likely to be dying during the peri-implantation period.To understand the effect of mutations on Nle1 gene expression,in the second part of the study we will contain the Nle1 c DNA of the target E-box,The expression of Nle1 at the transcriptional level was detected in both low-throughput（a random monoclonal method for ligation of c DNA containing target E-box region of Nle1 to vector）and high-throughput RNA sequencing techniques,and the expression level of the mutant Nle1 was found to be reduced.After that,the expression of Nle1 mutated protein was detected by overexpressing the target E-box two allele containing Nle1 in eukaryotic cell HEK-293.q PCR and Western Blot were used to find that the expression level of Nle1 mutated protein was reduced.Therefore,in vivo experiments of mice and in vitro experiments of cells proved that the expression of Nle1 at both transcription level and protein level was decreased after E-box mutation on the 5’UTR of Nle1 gene.In line with the above studies,we furthered our focus on the E-box mutation-induced occurrence of intestinal tumorigenesis in vivo.We collected Nle1^mut/+,Apc ^Min/+and Nle1^+/+,Apc ^Min/+offsprings out of the male Apc ^Min/+and female Nle1^mut/+crossing progeny,and analyzed the intestinal polyp counts and blood cell profiles of both Nle1^mut/+,Apc ^Min/+and Nle1^+/+,Apc ^Min/+mice.There were though only subtle differences between the mice of two genotypes in terms of polys counts and blood cell profiling.Despite this,due to the limited sample size in previous tests,further studies are necessary to underlie the exact correlation between the E-box mutation and the etiology of CRC.Conclutions:In general,we primarily based our study on the established mouse model with the targeted E-box mutation in the 5’UTR of Nle1,which was used to investigate the roles of the E-box in the development of both CRC and embryos.In spite of the lower expression of Nle1 upon E-box mutation,the Nle1^mut/+mice exhibited minor differences in the formation of polyps on the APC-min backgroud.Additionally,the mutation would also lead to embryonic death during the peri-implantation period,underpinning for the first time the crucial parts the E-box plays in embryonic development.