| Fusobacterium nucleatum(F.nucleatum)is an obligate anaerobic,Gram-negative bacterium,which usually resides in human mouth.It plays a bridge role in oral biofilm.Different pathogenic microorganisms in the oral cavity are integrated by various adhesins secreted by F.nucleatum.Research shows that in addition to oral diseases,F.nucleatum is also closely related to infectious diseases such as abscess,premature delivery,neonatal sepsis,intrauterine infection associated with pregnancy complications,inflammatory bowel disease and colorectal cancer.Colorectal cancer(CRC)is a serious threat to human health.Among all the malignant tumors,colorectal cancer ranks third incidence and second for mortality.According to WHO statistics,the number of new CRC cases in 2018 was about 1.846 million,with more than 883,000 CRC deaths.Studies have shown that F.nucleatum has positive correlation with lymph node metastasis in colorectal cancer tissues,and also with tumor immune escape and chemotherapy resistance.At present,the molecular mechanism of CRC caused by F.nucleatum infection has not been fully elucidated,and specific tumor molecules related to F.nucleatum-induced colorectal cancer progression have not been identified.Therefore,strengthening the research on F.nucleatum will help us better understand the process of colorectal cancer.The screening and identification of specific tumor molecules involved in the progression of colorectal cancer induced by F.nucleatum is of great significance for the prevention and treatment of colorectal cancer.MicroRNAs(mi RNAs)are noncoding single stranded RNA molecules encoded by endogenous genes,with a length of about 19 to 25 nucleotides.Mi RNAs negatively regulate target gene expression at the post-transcriptional level.MicroRNAs play an important regulatory role in various biological processes and participate in several biological functions of tumor cells(such as migration,proliferation,invasion and differentiation).Previous research has indicated that many miRNAs(such as mi R-21,miR-25 and mi R-92)are involved in the occurrence and progression of human colorectal cancer as oncogenes or tumor suppressor genes.However,few studies have explored the role of aberrant expression of miRNAs induced by F.nucleatum infection in human colorectal cancer.In order to find out whether there are mi RNAs involved in the process of colorectal cancer infected by F.nucleatum,we used the techniques of Affymetrix mi RNA microarray and GeneChip human transcription array 2.0 to study several miRNAs and and mRNAs involved in the development of CRC induced by F.nucleatum.A series of analyses were performed,including differential screening,cluster analysis,GO-Analysis,Pathway-Analysis and microRNA-GO-Network analysis.It is shown that miR-4474,miR-4717,and CREBBP may have important roles in colorectal cancer caused by F.nucleatum infection.Further,the corresponding mi R-4474,mi R-4717,and CREBBP expression in clinical samples was verified by RT-qPCR.Finally,we used bioinformatics and dual luciferase experiments to verify the correlation between miR-4474/4717 and CREBBP.【Objectives】1.To screen the aberrant mi RNAs and mRNAs in F.nucleatum-induced CRC;2.To explore the correlation between miRNAs and mRNAs aberrantly expressed in F.nucleatum-induced CRC.【Methods】1.Screening of mi RNAs and mRNAs aberrantly expressed in F.nucleatum-induced CRC.(1)Affymetrix miRNA microarray technology and Gene Chip Human Transcriptome Array 2.0 were used to screen 25 CRC clinical specimens.After differential screening,clustering analysis,Pathway-Analysis and GO-Analysis,we screened differentially expressed miRNAs and mRNAs.(2)The expression of miR-4474,mi R-4717,and CREBBP in CRC clinical samples was detected by real-time quantitative PCR(RT-qPCR).(3)RT-qPCR was used to detect the expression of miR-4474,mi R-4717,and CREBBP in the F.nucleatum infected group and the corresponding uninfected control group in cell models,respectively.2.Correlation between miR-4474/4717 and CREBBP aberrantly expressed in F.nucleatum-induced CRC.(1)Bioinformatics predicted the downstream target gene of miR-4474 and mi R-4717.(2)The regulation of mi R-4474 / 4717 on CREBBP was verified by luciferase experiments.(3)miR-4474 and mi R-4717 were overexpressed or inhibited,respectively.The correlation between miRNAs and CREBBP was observed by Western Blot and RT-qPCR.【Results】1.The expression of mi R-4474/4717 is up-regulated and CREBBP mRNA is down-regulated in F.nucleatum infected CRC tissues and cells.(1)Gene chip screening of 25 clinical specimens.The results showed that mi R-4474 and miR-4717 were up-regulated in F.nucleatum-infected colorectal cancer tissues compared to the control group(para-cancerous tissues),while other genes related to cancer signaling pathways,including CREB-binding protein(CREBBP),STAT1,PRKACB,CAMK2 B,JUN,TP53 and EWSR1,appeared expression dysregulation.Bioinformatics analysis showed that CREBBP is the main aberrantly expressed gene in CRC induced by F.nucleatum.(2)The results of RT-qPCR on clinical samples demonstrated that mi R-4474/4717 expression was up-regulated and that of CREBBP m RNA was down-regulated in F.nucleatum-positive CRC tissues.And with the progression of CRC,the expression of miR-4474/4717 gradually increased,whereas the expression of CREBBP mRNA gradually decreased.(3)RT-qPCR was used to detect the expression of mi R-4474,miR-4717 and CREBBP mRNA in the F.nucleatum infected cell model,and the results were consistent with the clinical specimen results.2.CREBBP is a new target for miR-4474/4717.(1)mi R-4474 / 4717 was negatively correlated with CREBBP expression in F.nucleatum infected colorectal cancer specimens.(2)Bioinformatics predicts that the downstream target gene of mi R-4474 /4717 is CREBBP.(3)The dual luciferase report experiment confirmed that CREBBP is a downstream target gene of miR-4474 / 4717.(4)MiR-4474 and miR-4717 were over-expressed or inhibited,respectively.Western Blot and RT-q PCR confirmed that mi R-4474 / 4717 can effectively inhibit the expression of CREBBP.【Conclusions】1.In this study,several miRNAs and mRNAs that are highly related to F.nucleatum infection were screened from 25 cases of colorectal cancer and adjacent tissues by gene chip.2.CREBBP is a key gene in colorectal cancer tissues associated with F.nucleatum infection.3.F.nucleatum infection can induce high expression of mi R-4474 / 4717 and low expression of CREBBP.4.CREBBP is a new target of mi R-4474 and miR-4717.【Significance】In this study,microarray screening of clinical specimens was used as the entry point.Bioinformatics analysis was used to screen the aberrantly expressed miRNAs and mRNAs in the F.nucleatum-induced CRC.The correlation between the differentially expressed mi R-4474,miR-4717 and their target gene CREBBP was also explored.The study provides new insights into the pathogenesis of colorectal cancer caused by F.nucleatum infection,and also provides new ideas of using F.nucleatum as a potential target in the clinical diagnosis,efficacy monitoring,and prognostic evaluation of CRC. |
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