Mechanism Of MiR-31 Driving Colitis Via Targeting SATB2

Objective:1.To investigate whether miR-31 driving colon colitis via targeting SATB2.2.Using miR-31 overexpressing transgenic mice to explore the molecular mechanism of miR-31 on driving colitis.Methods:1.SATB2 knock-down experiment:The human colon epithelial cells HCT 116 were divided into 4 groups:control group,miR-31 over-expression group?transfection of miR-31mimic to make miR-31 overexpression?,miR-31 knock-down group?transfection of miR-31 inhibiror to inhibit the expression of miR-31?and si-SATB2 group?transfection of SATB2 siRNA to inhibit the expression of SATB2?.The cells were incubated in CO₂ incubator after transient transfection using liposomes and collected after 72 h.qRT-PCR was used to detect the transfection efficiency and optimal transfection concentration of siRNA.Western blot was used to detect the expression of SATB2,NF-?B,COX-2,TGF-?1 protein.2.SATB2 overexpression experiment:Human colon epithelial cells HCT 116 were divided into control group,pEXP-RB-Mam group?transfected with empty vector?,and pEXP-RB-Mam-SATB2 group?transfected with SATB2 overexpression plasmid?.After the cells were adhered,the cells were transfected with lip2000 and cells were incubated in a CO₂ incubator.After 6 hours,the medium waschanged,and cells were collected after72 hours.Western Blot was used to detect the expressions of SATB2,NF-?B,COX-2,and TGF-?1 in four groups of cells.3.Animal experiment:,14 non-transgenic mice were randomly divided into control group?n=6?,DSS group?n=8?,16 transgenic mice with miR-31 overexpression were randomly divided into miR-31 overexpression control group?n=8?,and miR-31overexpression group?n=8?,the DSS group and the miR-31 overexpressing DSS group drank 1%of the DSS in the first week and the normal aseptic water in the second week was one cycle,repeating the end of two semi-cycle modeling.Mouse colon tissue was retained,western blot was used to detect the expression of SATB2,TLR4,NF-?B,COX-2,TNF-?,i NOS,Bax,Bcl-2 protein in mouse colon tissue.Immunohistochemical?IHC?method was used to detect the expression of SATB2,TLR4,NF-?B,COX-2,TNF-?,Bax,Bcl-2 protein in mouse colon tissue.In situ terminal transferase labeling method?TUNEL?was used to detect apoptosis of mouse colon tissue.Results:1.siRNA transfection efficiency and optimal transfection concentration:qRT-PCR results showed that SATB2 expression was significantly decreased after HCT 116 cells were transfected with SATB2 siRNA?P<0.01?,which proved that SATB2 siRNA transfection was effective.When the siRNA concentration increase from 3nM to 5nM,the expression of SATB2 reduced significantly compared to control.However,when the siRNA concentration is increased to 10nM,the expression of SATB2 was not inhibited more than in 5nM dose,so we used 5nM as the optimal concentration for subsequent transfection of SATB2 siRNA.2.SATB2 protein expression results:Western Blot results showed that after transfection of HCT 116 cells,compared with the control group,the expression levels of SATB2 protein in the mimic group and si-SATB2 group were significantly reduced?P<0.01?,and the expression levels of SATB2 protein in the inhibitor group significantly increased?P<0.05?,further confirming the successful transfection.3.Expression of inflammatory cytokines NF-?B,COX-2,TGF-?1 protein:Western Blot results showed that compared with the control group,the expression of NF-?B and COX-2 protein in the mimic group was significantly increased?P<0.05?,the expression of TGF-?1 protein was significantly reduced?P<0.01?,the expression of NF-?B and COX-2 protein was significantly reduced in inhibitor group?P<0.01?,and the expression of NF-?B and COX-2 protein was significantly increased in si-SATB2 group High?P<0.01?,TGF-?1 protein expression was significantly reduced?P<0.01?.4.Transfection efficiency of SATB2 overexpression plasmid:Western Blot results showed that after transfection of SATB2 overexpression plasmid on HCT 116 cells,there was no significant difference in the expression level of SATB2 protein between pEXP-RB-Mam group and control group,pEXP-The expression level of SATB2 protein in RB-Mam-SATB2 group was significantly higher than that in control group?P<0.01?,proved that SATB2 overexpression plasmid transfection was effective.5.Expression of NF-?B,COX-2,TGF-?1 protein after SATB2 overexpression:Western Blot results showed that the expression levels of NF-?B,COX-2,TGF-?1protein in pEXP-RB-Mam group had no significant difference compared with control group,while compared with the control group,the pEXP-RB-Mam-SATB2 group significantly reduced the expression levels of NF-?B?P<0.01?and COX-2?P<0.05?,and significantly increased the expression levels of TGF-?1?P<0.05?.6.SATB2 protein expression in mouse colon tissue:Western Blot results showed that compared with the control group,the expression level of SATB2 protein in the DSS group?P<0.01?and miR-31 overexpression group?P<0.05?decreased significantly,while Compared with DSS group,the expression level of SATB2 protein in miR-31+DSS group also decreased significantly.7.Western Blot detected the expression of TLR4,NF-?B,iNOS,TNF-?protein in mouse colon tissue:The results showed that compared with the control group,the expression of TLR4,NF-?B,iNOS,TNF-?protein in the DSS group was significant increased?P<0.01?,the expression of TLR4?P<0.05?,NF-?B?P<0.05?,and iNOS?P<0.01?in miR-31 overexpression group was also significantly increased,compared with the DSS group The expression levels of TLR4,NF-?B,iNOS,and TNF-?protein in miR-31+DSS group were also significantly increased?P<0.01?.8.IHC detected the expression of TLR4,NF-?B,COX-2,and TNF-?proteins in mouse colon tissue:The results showed that compared with the control group,the expression of TLR4,NF-?B,COX-2,and TNF-?proteins in the DSS group was significantly increased?P<0.01?,the expression of TLR4?P<0.01?,NF-?B?P<0.01?,COX-2?P<0.05?,TNF-??P<0.01?in the miR-31 overexpression group was also significantly increased.Compared with the DSS group,the expression of TLR4,NF-?B,COX-2,and TNF-?proteins in the miR-31+DSS group were also significantly increased?P<0.01?.9.TUNEL method to detect apoptosis of mouse colon tissue cells:The results showed that compared with the control group,apoptotic cells in the DSS group and the miR-31 overexpression group were significantly increased?P<0.01?,compared with the DSS group,the apoptotic cells of miR-31+DSS group also increased significantly?P<0.01?.10.Western Blot detected the expression of Bax and Bcl-2 proteins in mouse colon tissue:The results showed that compared with the control group,Bax protein expression levels in the DSS group increased?P<0.05?,and Bcl-2 protein protein expression levels decreased?P<0.01?,Bcl-2/Bax ratio decreased?P<0.01?,Bax protein expression level of miR-31 overexpression group increased?P<0.05?,Bcl-2/Bax ratio decreased?P<0.01?,and compared with DSS group,the expression level of Bax protein in miR-31+DSS group increased?P<0.05?,the expression level of Bcl-2 protein protein decreased?P<0.01?,and the ratio of Bcl-2/Bax decreased?P<0.01?.11.IHC detected the expression of Bax and Bcl-2 proteins in mouse colon tissue:The results showed that compared with the control group,Bax protein expression levels in the DSS group increased?P<0.01?,and Bcl-2 protein protein expression levels decreased?P<0.01?,the expression level of Bax protein in miR-31 overexpression group increased?P<0.05?,and the expression level of Bcl-2 protein decreased?P<0.01?.At the same time,compared with DSS group,the expression level of Bax protein in miR-31+DSS group increased?P<0.01?,and the expression level of Bcl-2 protein decreased?P<0.01?.Conclusion:1.miR-31 promotes colitis via targeting SATB2.2.miR-31 drives the development of colitis by promoting the TLR4/NF-?B signaling pathway and mediating intestinal epithelial cell apoptosis.