Expression Of IL-9 In The Development Of Colitis-associated Colorectal Cancer In Mice

Background and ObjectiveMalignances are the dangerous diseases that threat the life of humanbeing.How to arevent and decrease the incidence of malignances has become the main task of clincians and researchers in China.Colorectal cancer(CRC)is one of the most common malignances with a gradual increasing incidence in China.The causes of CRC is cyrrently unclear and probably related to heredity,diet(such as lack of dietary fiber or high fat),living environment,chronic inflammation,and immune factors.Studies have shown that chronic inflammation is a potential risk factor for CRC dvelopment,and ulcerative colitis(UC)is closely related to the occurrence of CRC.Recent researches have revealed that chrnic inflammation is a key promoter for CRC development,the inflammation elicited dysregulation of immune mediators such as cytokines not only play the critical role in the development and maintance of inflammation,but also in the development of inflammation-dysplasia(precancerous lesions)-CRC sequence.Through the administration of periodicative inflammatory and chemical staffs,strong intestinal inflammation was observed in the colonic mucosa,and CAC after 14 weeks in mice,the pathological features are very similar to to the manifestation of human CRC occurred in UC.This mouse model provides the advantages to avoid collecting specimens in a long time period and easy to simulate the inflammation-dysplasia(precancerous lesion)-CAC sequence.Accumulative evidences have shown that interleukin(IL)-9 is an important pro-inflammatory cytokine in the development of chronic inflammation whereas inflammation is closely related to the development of CAC.Therefore,IL-9 might be involved in the development of CAC in patients with UC.We have previously demonstrated that IL-9 antibody infusion can significantly block IL-9 signaling,inhibit intestinal inflammation and experimental UC.Since inflammation is the main driver of CAC and IL-9 has significant pro-inflammatory capabilities,the relationship between IL-9 and inflammation-induced CRC may plays an important role in the development of CAC.At present,most of the research is mainly focused on the research of SCRC.Studies on the relationship between IL-9 and CAC has largely ignored and not been reported.In this study,we have,at the first time,explored the dynamic changes of IL-9 expression in the tumor microenvironment from the precancerous stage to the cancer stage in the mouse CAC model.Materials and methods(1)Establishment of DMH+DSS induced CAC mouse model: 60 ICR male mice(5 weeks,body weight 25-30 g)were randomly divided into CAC group and control group respectively.Among them,30 in the experimental group and 30 in the control group.The mice in the CAC group were intraperitoneally injected with 0.4% DMH once a week,the dosage was 25 mg/kg,then the diet was given for 7 days,and then changed to free drinking 1% DSS for 7 days.This is a cycle.The mice in the control group were injected with saline once a week in the peritoneal cavity at a dose of 25 mg/kg,and the diet was given water regularly.The CAC group and the control group were sacrificed at 14,18,and 22 weeks,and the number of tumors was counted and their pathological properties were identified.(2)Immunohistochemistry(IHC)was used to detect IL-9 protein content: the large intestine tissue was fixed with formalin and embedded in paraffin for 4um thickness.Follow the instructions of the ABC kit manufacturer.Tissue sections were repaired by xylene dewaxing,gradient ethanol hydration and boiling heat,3% hydrogen peroxide-non-specific IgG blocking and blocking,primary antibody overnight at 4 °C;secondary antibody incubation at 37 °C for 30 min;AEC Color,hematoxylin counterstaining,water-soluble gum 50 ul seal,observed under the microscope.Positive cells were observed in the colon tissue with red.(3)Western blotting(WB)was used to detect IL-9 protein content: the above large intestine tissues were taken out,washed twice with pre-cooled PBS,and 30 mg of large intestine liquid nitrogen homogenate was added,RIPA lysate was added,and cleavage was performed on ice for 30 min.After centrifugation at 12000 rpm for 5 min,the supernatant was taken,and the cell protein concentration was measured by BCA method.5 ?l of sample was added per well,?-tublin was used as internal reference,goat anti-mouse IL-9 antibody(1:500)and rabbit anti-mouse ?-The tubin antibody(1:1000)was incubated at 4 ° C overnight,rewarmed at 37 ° C for 30 min,HRP-labeled horse anti-goat antibody(1:200),HRP-labeled goat anti-rabbit antibody(1:10000)was incubated at 37 ° C for 1 h.Exposure,using image analysis software Quantity One to determine the average value of the gray value of the color bands of IL-9 and ?-tublin,the expression level of each group is the ratio of the average value of the target band to the gray value of the internal reference band.ResultsTumors in the colorectal mucosa began to appear at 14 weeks,and the number of tumors increased significantly from 18 weeks to 22 weeks.The average numbers of tumors in the CAC group at 14 weeks,18 weeks,and 22 weeks were 4.20 ±1.92,6.60 ± 1.52,30.20 ± 3.56.There was no significant difference between 14 weeks and 18 weeks(P>0.05),but did have significances between 14 weeks and 22 weeks,18 weeks and 22 weeks(P<0.05).IHC results showed that IL-9 in the control mice was expressed in a small amount in both epithelial and mesenchymal cells,and in the CAC mice it was significantly increased in transformed epithelial cells and stromal cells;WB results confirmed a dradual increase of IL-9 expression from 14 weeks to 22 weeks.Although IL-9 in the control group increased slightly fllowing the age increase,however,it was significantly increased in the CAC group mice at the treatment in different time points(P<0.01).Conclusions(1)IL-9 has an increasing trend in the development of CAC following a inflammation-dysplasia(precancerous lesion)-CAC sequence.(2)IL-9 is involved in the pathogenesis of mouse CAC and play an important role.