| Background:Colorectal cancer(CRC)is one of the most common malignant tumors of digestive tract.It is the cancer type with the third incidence rate and the second mortality rate in the world,posing a serious threat to human health.MiRNA is a small non coding RNA,which has been proved to regulate the expression of target genes at the post transcriptional level.More evidence has shown that the abnormal expression and disfunction of miRNAs are involved in the pathophysiological process of many cancers,including CRC.Recently,it is reported that intestinal flora disorder is closely related with the occurrence of CRC.Among them,Fusobacterium nucleatum(Fn)can induce the proinflammatory states in the colon,thus leading to malignant progression of CRC.More importantly,Fn could affect the occurrence and development of colorectal cancer by regulating a variety of miRNAs,but little is known about tumor suppressor miRNAs.Therefore,deeply exploring Fn and the regulatory role of miRNAs helps to reveal the pathogenesis of CRC.Objectives:To explore the regulatory effect of Fn on the expression of miRNAs in CRC cells,and to analyze the mechanism of Fn promoting CRC metastasis.It provides new ideas for CRC treatment.Methods:1.Fn respectively co-cultured with SW480 and HCT116 cells and established Fninfected CRC cell models.The exosomes derived from Fn infected CRC cells were extracted by ultracentrifugation.High throughput sequencing was applied in two panels,including serum exosomes of CRC patients with different Fn abundance and exosomes derived from Fn infected and uninfected CRC cell derived culture supernatant.The intersection of the two sequencing results was analysed to screen out the differentially expressed miRNAs.2.We further verified the above miR-122-5p expression by RT-qPCR.3.The expression of miR-122-5p in CRC clinical samples was detected by RT-qPCR.4.In vitro,transwell assays and wound healing assays were used to evaluate the effects of Fn and/or miR-122-5p mimics or inhibitors on the invasion and migration of CRC cells.In vivo,the liver metastasis model of CRC was established by spleen injection to evaluate the effects of the above different treatment groups on CRC metastasis.5.Combined the increased mRNAs in Fn infected CRC tissues with miR-122-5p target genes predicted by bioinformatics,we selected the downstream target of miR-122-5p in Fn infected CRC cells.6.The regulation of miR-122-5p on FUT8 was verified by double luciferase assays.7.RT-qPCR and Western blot were used to detect the FUT8 expression in CRC tissues and normal adjacent tissues,and CRC cells with or without Fn infection.8.EMT related markers were detected by Western blot and RT-qPCR in overexpression or inhibition of miR-122-5p or FUT8 groups.Results:1.High throughput sequencing analysis showed that miR-122-5p was identified as the most prominently upregulated miRNA in both panels.2.In Fn-positive CRC patients,the expression of miR-122-5p has a significant negative correlation between serum and tissue.And high expression of miR-122-5p in serum exosomes was associated with lymph node metastasis and distant metastasis in CRC patients.3.In vitro experiments showed that overexpression of miR-122-5p significantly reduced the migration and invasion potential of CRC cells.In vivo experiments showed that the number of liver metastatic nodules in mice significantly decreased after miR-122-5p overexpression,and inhibition of miR-122-5p expression would lead to the opposite results.4.RT-qPCR results showed that Fn infection enhanced the exosomal miR-122-5p expression but downregulated its intracellular levels.These results indicated that Fn infection promoted the excretion of exosome-encapsulated tumor suppressive miR-122-5p to maintain the stability of tumor cells.5.RNA binding proteins(RBP)hnRNPA2B1 mediates the extracellular transport of miR-122-5p via exosomes.6.The double luciferase experiments confirmed that FUT8 was the target gene of miR122-5p.RT-qPCR and Western blot The negative correlation between the expression of FUT8 and miR-122-5p was verified by RT-qPCR and Western blot.FUT8 could partially reverse the inhibitory effect of miR-122-5p on colorectal cells.7.Mechanistically,the downregulation of miR-122-5p led to the increase of FUT8 levels and enhanced Smad2/3 protein levels and EMT-related markers.Conclusion:1.Fn infection promotes the efflux of exosome-wrapped tumor suppressive miR-122-5p,leading to the decrease of intracellular miR-122-5p levels.2.The decreased miR-122-5p in CRC cells caused by Fn infection removes inhibition of downstream target gene FUT8,activates TGF-β1/Smads signaling pathway and induces EMT by regulating miR-122-5p/FUT8 axis,thus promoting CRC metastasis. |
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