The Study On The Mechaninsm Of Initiation And Progression Of Colorectal Carcinoma Involved In Interaction Between Circadian Disruption And Intestinal Dysbacteriosis

Background:CRC,one of the most common malignancies in the world,frequently causes death and is emerging worldwide.It is expected that CRC burden will substantially increase in the next two decades,which can be affected by factors such as host genetic susceptibility,dietary habits,living environment,age,traditions and changing lifestyles.CRC has long been investigated and it is classified into two typical types:colitisassociated colorectal cancer and sporadic colorectal cancer,according to genomic mutation diversity.CRC is usually used to refer to the common colorectal cancer that considered without family heredity.CRC is a malignant disease which involves multiple factors during its multi-stage development.Animal models and epidemiological data suggest that the colonic microbiota have a role in tumorigenesis.The mechanisms underlying intestinal dysbiosis often remain unclear.Any disorder in the gut microbial composition may initiate aberrant intestinal mucosal cell signaling pathways,epigenetic modification and even gene mutations.In addition,microbiota dysbiosis and subquent bacterial metabolite and their surface toxins may induce serious inflammation which can leads to malignancy.Therefore,we first systematically profiled and analyzed the gut microbiomes and mucosal transcriptomes of CRC patients to identify their associations that characterize CRC involved in specific microbiota,providing new markers of diagnosis and therapeutic applications of CRC.Epidemiologic studies have identified several lifestyle factors that affect the risk for developing CRC,which hypothesize the correlation between alteration of the microbiota and circadian rhythms disruption as one of the important causes of colorectal cancer.The disruption of the circadian timing system(CTS),which rhythmically controls cellular metabolism and proliferation,accelerated cancer progression.It has been shown that in the colon cellular proliferation follows a circadian rhythm,showing clock genes modulate the expression of oncogene.Likewise,the studies of circadian oscillations and innate and adaptive gut immunity joint to the host-microbial interactions in CRC should be explored.In this study,to address the confounding variables associated with CRC microbiota studies,we conducted a prospective study of the colonic microbiota using paired mucosal samples from patients undergoing surgery for CRC,and the microbiota composition was correlated with the mutation and expression of specific host response genes.Futhermore,the expression of circadian gene,PER1 was examined in colorectal carcinoma and adjacent normal mucosal tissues.This work provides novel evidence regarding the complex interplay between the circadian clock and gut dybiosis involved in CRC carcinogenesis and progression.Methods:1.Tissues samples were collected from Zibo Municipal Hospital between 2019 and 2020,Mucosal biopsies from CRC patients(including tumor tissues,normal adjacent tissues and peritumoral tissues(n=26)were subjected to microbiome analysis.The variable V3-V4 regions of 16S rRNA gene was amplified using PCR.Sequencing was performed based on the Illumina Hiseq2500 platform.The operational taxonomic units(OTU)clustering and species annotation were processed using FLASH,USEARCH and other software.2.Mucosal biopsies from CRC patients(including tumor tissues and normal adjacent tissues(n=24))were subjected to WES analysis.All clean reads were aligned to the human reference genome using Burrows-Wheeler Aligner.All genomic variations,including SNPs and InDels were detected by the state-of-the-art software to get high-confident variant calls.Identified variants were analyzed using COSMIC and dbSNPv151hg19 databases,to search for rare and novel germline variants in known and novel candidate CRC predisposing genes.3.Mucosal biopsies from CRC patients(including tumor tissues and normal adjacent tissues(n=21))were subjected to transcriptome analysis.The expression of genes was measured using RPKM methods and the differentially expressed genes were compared between the two sets of samples.And the key genes and signal were analyzed by GO and KEGG.Finally,analyze the correlation between gut microbiota and host circadian rhythm genes by R language.4.The expression levels of core genes were confirmed by qRT-PCR and IHC.In vitro experiments showed that both genes overespression and knockout could affect the proliferation of CRC cell lines.Results:1.Amplification sequencing was used to reveal the structure,diversity and differences of colorectal mucosal microbiota among tumor tissues,nomal adjacant tissues and peritumoral tissues.Bacteroides Fragilis were significantly increased in tumor tissues,Roseburia faecis was relatively decreased.The batsroides Fragilis was negatively correlated with Bacteroides Ovatus.2.Ten kinds of colorectal cancer pathogenic gene mutations were detected using whole exon sequencing,These genes were MSH2 c.505A>G,c.1168C>T,TGFBR2 c.1019C>T,POLE c.2974G>A,MLH3 c.2221G>T,AXIN2 c.1250C>T,POLD1 c.56G>A,PARP1c.2819A>G1,MSH6 c.3488A>T,PRDM1c.1901 A>G.Four novel candidate CRC predisposing genes were GPRIN2 c.1333C>T、ZNF488 c.778G>A、NCOA4 c.1886A>C、MA GEA6 c.632A>G.3.Transcriptome sequencing results showed that PER1,APOA4,SLC10A2,APOC3,APOA1,G6PC might play an important role in the initiation and progression of colorectal cancer.The combined analysis of transcriptome and amplicon reveals that the gut microbiota is related to the host circadian rhythm genes.PER1 and BMAL1 are positively correlated with Sutterella.NR1D1 was negatively correlated with Roseburia faecis.And speculated that Roseburia faecis may directly or indirectly affect the initiation and progression of CRC by regulating circadian rhythm genes.4.Transcriptome sequencing results showed that PER1 is downregulated in CRC cell lines and CRC tissues,and clustering analysis revealed that PER1 inhibits CRC cell proliferation.Cell clone formation rate and cell proliferation assay showed that PER1 overexpression can inhibit the proliferation of colorectal cancer cells.Futhermore,the targets of PER1 was determined as miR-423-5p by three databases(TargetScan,miRDB,Starbase),that.The result showed that miR-423-5p promoted the proliferation of colorectal cancer cells by regulating PER1.Conclusion:We identified genetic variants,together with the differences in the expression levels of cell factors,suggesting they might play important roles in colorectal tumorigenesis and contribute to the risk prediction and prognosis of colorectal cancer.Importantly,we presented a large-scale,comprehensive analysis of the microbiome and microbiome-associated host transcriptome in progression of CRC using multi omics analysis.Gene set enrichment analysis found that PER1 was enriched and associated with circadian rhythms.PER1 could inhibit the proliferation and colony formation of colorectal cancer cells,and showed an anti-tumor effect.In addition,our findings identified previously unknown links in which miR-423-5p affected the initiation and progression of colorectal cancer through targeted PER1,providing theoretical basis for the molecular mechanism of CRC.