Effects Of Deletion Of Hif-1?/1? Gene In Myeloid Cells On Intestinal Tumor Formation And Intestinal Microbiota In Mice

Background and aimColorectal cancer is one of the most common malignancies in the world,survival and treatment options for patients with colorectal cancer remain poorly.At present,the studies believe that its pathogenesis is closely related to gene mutations and intestinal microbiota.Studies found that HIF-lpathway is actively expressed in colon cancer tissues,and related inhibitors are also under development.However,due to the lack of cell specificity,inhibitors would also affect other cells while blocking target proteins of tumor cell.The current study found that both HIF-1? and HJIF-1?play important roles in myeloid cells,and myeloid cells are closely related to tumor development and intest:inal microbiota.Therefore,we hope to explore whether myeloid cells deletion of Hif-1?or Hif-1? gene would affect intestinal tumors or intestinal microbiota.MethodsWe identified the genotype of the mice by PCR and detected the residual expression of the knockout genes by qPCR.The effect of deletion of Hif-1? or Hif-1?gene in myeloid cells on intestinal tumor formation in mice was observed by using AOM/DSS model.Cytokine levels in mouse serum were detected by liquid protein chip.The proportion of immune cells was detected by flow cytometry.The intestinal barrier function of mice was evaluated by measuring serum DAO and endotoxin levels.Composition of mouse intestinal microbiota was detected by 16s rRNA gene sequencing.RTCA technique was used to detect the effect of mouse fecal metabolites on HT-29 cells,and cytokine secretion by BMDM cells was detected by ELISA.The effects of intestinal microbiota on intestinal tumorigenesis inmice were verified by cohousing CKO and WT mice.ResultsCompared with wt mice,the knockout genes were reduced by about 80%-90%and 60%-70%in BMDM cells ofour bred Hi/Hif-1?f/f,lysM-cre+ and Hif-1?f/f,lysM-cre+ mice,respectively.There were no significant differences in serum DAO and endotoxin levels between the two kinds of CKO mice and their WT littermates.We analyzed the fecal microbiota of two kinds of CKO mice and their WT littermates by 16s rRNA gene sequencing and we did not find significant bacterial species differences between CKO and WT mice of the same litter source.There was no difference in the effects of HT-29 cell growth andthere was no statistical difference in the levels of TNF-a and IL-1?secreted by BMDM cells both after stimulation with fecal metabolites from CKO and their WT littermates.After AOM/DSS modeling,Hif-1?f/f,lysM-cre+ mice and their WT littermates had no significant difference in body weight,DAI score,colon length,the number of colon tumors and the sum of all tumor diameter.However,the number of tumors and the sum of tumor diameter of Hif-1?f/f,lysM-cre+ mice was significantly greater than that of their WT littermates.The correlation between serum IL-9,IL-13 and IL-18 in Hif-1?f/f,lysM-cre+ mice and the severity of tumor formation disappeared after AOM/DSS modeling compared with WT mice.The proportion of Gr1+ CD11b+ cells and CD8+T cells in the blood of Hif-1?f/f,lysM-cre+?mice was lower than that of WT mice after AOM/DSS modeling.The 16s rRNA gene sequencing analysis showed that the intestinal microbiota before and after AOM/DSS modeling changed significantly.The Hif-1?f/f,lysM-cre+ mice and their WT littermate also showed significant different fecal microbiota.The fecal metabolites of Hif-1?f/f,lysM-cre+ mice showed stronger cytotoxicity to HT-29 cells than that of WT mice,but the secretion of IL-1?by BMDM cells after being stimulated by fecal metabolites of Hif-1?f/f,lysM-cre+ mice was significantlyattenuated than that of WT mice.We cohoused Hif-1?f/f,lysM-cre+ mice with WT mice during AOM/DSS modeling.It was found that there was no significant difference in the number of tumors and the sum of the tumors diameter between the two groups after modeling,and there was also no significant difference in the fecal microbiota and effects of metabolites on the cells.Conclusions1?There was no significant difference in the intestinalmicrobiota at the genus level between the Hif-1?f/f,lysM-cre+?Hif-1?f/f,lysM-cre+ and their wild-type littermates detected by 16s rRNA gene sequencing.Their fecal metabolites have the same effect on cells.2?After AOM/DSS modeling,the loss of Hif-1?gene in myeloid cells did not significantly affect mouse colitis,but it would aggravate the occurrence of intestinal tumor in mice;the deletion of Hif-1? gene in myeloid cells did not significantly affect the colitis and intestinal tumor in mice.3?The deletion of Hif-1? in the myeloid cells would affect the production of various cytokines?the proportion of Grl+CD11b cells and CD8+ T cells in the blood and the composition of mouse intestinal microbiota as well as metabolites of intestinal microbiota after colon tumor formation in mice.4?Co-housing could reduce the difference in intestinal tumor formation between Hif-1?f/f,lysM-cre+ mice and WT mice.