| Objective:MicroRNAs and IL-6/STAT3 inflammatory signaling pathways play an important role in the development of Colitis-Assosiated Colorectal Cancer(CAC).However,the pathological mechanism of CAC is still unclear.Especially the relationship between microRNAs,their target genes and IL-6/STAT3 signaling pathway has not been thoroughly studied in the development of CAC.Firstly,we screened and validated miR-29a,the key microRNAs associated with CAC.Secondly,we explored the target genes of miR-29a and their correlation with IL-6/STAT3,and revealed their molecular mechanisms in the pathogenesis of CAC.Finally,we explored the intervention mechanism of Pedunculoside.Methods:The CAC model of C57BL/6 mice was established by AOM/DSS method.The differential expression of microRNAs in control group and CAC model mice was detected by miRCURY LNATM miRNAs chip.The screened microRNAs were validated by RT-PCR in colon tissue of clinical patients and model animal samples;The expression of TET1 mRNA,TET2 mRNA and TET3 mRNA were detected by RT-PCR.The expression of 5-hmc and TET1 in colon tissue of model animals and HCT-116,IEC-6 were determined by immunofluorescence assay,the expression of TET3,STAT3 and P-STAT3 protein were detected by Western-Blot,and the proliferation of tumor cells was detected by MTT.Results:1.C57BL/6 mice were injected with AOM once intraperitoneally and stimulated by 3%DSS solution.After 8 weeks(8w),moderate and severe dysplasia of colon mucosa appeared in the model mice,accompanied by a large number of inflammatory cells infiltration.The expression of miR-29a in colon tissue of CAC model was significantly increased by sequencing results.RT-PCR results showed that the expression levels of miR-29a in colon tissue of CAC mice,human colon cancer(operation sample)and colon cancer cell line HCT-116 were significantly higher than those in normal mice,human adjacent colon tissue(operation sample)and IEC-6 cells.2.At the same time,the expression levels of STAT3 and P-STAT3 were significantly increased in CAC mice and human colon cancer tissues,while the expression levels of TET3 and TET3 protein were decreased.Immunofluorescence assay results showed that the expression of 5-hmc and TET1 in colon tissue of CAC mice was lower than normal.3.In vitro,studies showed that the expression of STAT3 protein increased after HCT-116,IEC-6 and RAW264.7 transfected with miR-29a precursor while the expression of TET3mRNA decreased(IEC-6 and HCT-116)and the expression of TET3 protein decreased(HCT-116);The expression of 5-hmc and TET1 decreased(IEC-6 and HCT-116).The expression of STAT3 protein in colon cells(HCT-116 and IEC-6)decreased,while the expression of TET3mRNA increased(IEC-6 and HCT-116)after miR-29a inhibitors were transfected.Stimulation of IL-6(50ng/ml)24h in cells for 24 hours increased STAT3 protein expression(HCT-116,IEC-6,RAW264.7).Moreover,it also increased TET1,TET2,TET3mRNA(HCT-116 and IEC-6).Stimulation of IL-6(50ng/ml)24h in cells for 24 hours increased the level of miR-29a in HCT-116 cells,and the use of STAT3 inhibitors reversed the increase of miR-29a.4.Pedunculoside can reduce the level of miR-29a and STAT3 protein in CAC mice model,while increase TET3 protein.It also inhibit the proliferation of HCT-116 in vitro,inhibit the down-regulation of TET3 protein induced by miR-29a precursor in HCT-116 cellsConclusion:The results showed that miR-29a/STAT3 may form a positive feedback in the process of Colitis-Assosiated Colorectal Cancer.miR-29a activates IL-6/STAT3 signaling pathway and increases the expression of STAT3 protein and P-STAT3 protein.At the same time,IL-6/STAT3 further induces the expression of miR-29a in macrophages and colonic epithelial cells,and intensifies the inhibition of TET3 and TET3 protein expression.The interaction of miR-29a/STAT3 enlarges the inflammatory effect.It is involved in the process of colitis-cancer.The intervention of Pedunculoside may be related to its inhibition of key molecules in miR-29a/STAT3. |
PDF Full Text Request