Different Sex-based Responses Of Gut Microbiota During The Development Of Hepatocellular Carcinoma In Liver-specific Tsc1-Knockout Mice

Research BackgroundStudies have shown that many human diseases such as type 2 diabetes,obesity,Crohn's disease are closely related to the intestinal flora.The liver and the intestine are interconnected through the portal vein.The toxins and microbial metabolites absorbed by the intestine depend on the metabolism of the liver.When the intestinal micro-ecological disorders,various liver diseases may occur,such as alcoholic fatty liver,non-Alcoholic fatty liver,cirrhosis,HCC,etc.These data have led to a growing interest in gut microbiota and their metabolites as a new therapeutic target for the prevention,diagnosis,and treatment of HCC.However,despite considerable progress,most studies focused on comparing differences in gut microbiota between Male patients or animal models with HCC and healthy controls.Although differences in phylogenetic and functional compositions have been found,the following question remains unclear:Is the incidence of different genders of HCC related to intestinal flora?Are there differences in the intestinal flora between different stages of HCC?ObjectiveIn the present study,we evaluated the changes occurring in the gut microbiota of LTsc1KO mice and wide-type Tsclfl/fl mice with different ages and genders by using MiSeq high-throughput sequencing of the 16S rRNA gene.To provide a new perspective for understanding the pathogenesis of HCC from the perspective of intestinal microecology and explore new strategies for prevention and treatment.Results1.HCC development of LTsc1KO miceLiver histology showed no significant pathological change in the livers of the 6-month-old LTsc1KO mice.Likewise,the serum concentrations of the liver enzymes ALT and AST show no significant change at the 6-month-old.At 10 and 14 months of age,an increase in the serum concentrations of the liver enzymes ALT and AST was observed in the LTsc1KO mice.However,HCC was detected at 10 months and 14 months of age.No pathological change was detected in the livers of the Tsc1fl/fl mice at any age.2.Repeatability of the results using sequencing technology to analyze fecal microbiotaDespite using only 5,000 sequences of each specimen,MiSeq sequencing still exhibited a forceful repeatability for each sample.3.Changes in the gut microbiota between LTsclKO and wide-type Tsc1fl/fl mice at different agesThe relative abundance of aerobic bacteria in the LTsclKO mice in group 5 was obviously higher than that in the Tsc1fl/fl mice among the females.In contrast,the1 relative abundance of anaerobic bacteria was obviously decreased in the female LTsc1KO mice in group 3 compared with that in the corresponding female Tsc1fl/fl cohorts.Such obviously decreases were also observed in male LTsc1KO mice in groups 1 and 5.we did not find any significant changes in the relative abundance of facultative anaerobic bacteria between the LTsc1KO and Tsc1fl/fl mice in any of the groups,except in group 3,where it was obviously increased in female LTsc1KO mice compared with that in corresponding female Tsc1fl/fl mice.Moreover,no significant difference was detected between the relative abundance of gram-negative or gram-positive bacteria from the LTsc1KO and Tsclfl/fl mice in any of the groups.For LPS metabolism,no significant difference was detected in the relative abundance of the genes that participate in the KEGG pathways of LPS biosynthesis,LPS transport system,or LPS export system between the LTsc1KO and Tsc1fl/fl mice in any of the groups.4.Changes in gut microbiota between LTsc1KO and wide-type Tsc1fl/fl mice with the development of HCCFor all the genera that showed significant differences in their relative abundance,from female LTsc1KO mice were significant higher than those from others in group 4,including Allobaculum,Erysipelotrichaceae,Neisseriaceae,Sutterella,Burkholderiales,Prevotella.However,no other differences was evident,with the exception of gram-positive Allobaculum that showed significantly lower abundance in female Tsc1fl/fl mice than in male mice.The relative abundance of Paraprevotellaceae?Parapervotella?Prevotella of gram-negative bacteria were significantly higher in the gut microbiota from male LTsc1KO mice than in the other mice.Conclusionwe detected different changing patterns between female and male LTsc1KO mice of different ages in our study with the development of HCC.Our results also suggest that the decrease in the relative abundance of anaerobic bacteria and the increase in the relative abundance of facultative anaerobic bacteria have potential for use as risk indexes of female HCC.The increased abundances of Allobaculum,Erysipelotrichaceae,Neisseriaceae,Sutterella,Burkholderiales,and Prevotella species might also be applied as risk indicators of female HCC,and for male HCC,increased abundances of Paraprevotellaceae?Parapervotella?Prevotella might be potential risk indicators.