| Objectives:In this study,the correlation between the miR-642a-5p and glucocorticoid resistance in UC was determined from the clinical level.The efficacy of miR-642a-5p in DSS colitis model mice was observed at the animal level.The cellular level revealed the mechanism by which miR-642a-5p participates in glucocorticoid resistance through the TLR4 signaling pathway.This study aims to elucidate the mechanism of miR-642a-5p in regulating GC response and provide a theoretical basis for new therapeutic targets of glucocorticoid resistant UC.Methods:This study is divided into three parts.Part Ⅰ20 patients with glucocorticoid sensitivity and 20 patients with glucocorticoid resistance were enrolled,and intestinal mucosal specimens were collected before treatment with glucocorticoid.The correlation between miR-642a-5p and glucocorticoid resistance in UC was determined by the mRNA levels of TLR4,MyD88 and the inflammatory factors TNF-α,IL-1β,IL-6 and IL-12 by real-time fluorescent quantitative PCR(qRT-PCR).Part Ⅱ(1)DSS mouse model construction and Lentivirus intervention with miR-642a-5p precursors and inhibitors140 BALB/C mice weighing 20-25g were randomly divided into control group,DSS group,pre-miR-642a-5p group,miR-642a-5p inhibitor group,Dex group,Dex+pre-miR-642a-5p group and Dex+miR-642a-5p inhibitor group.Normal feeding was used as the control group,while mice in the experimental group were molded with 3%DSS solution.For 7 consecutive days,mice were given intravenous injection of lentivirus or/and intraperitoneal injection of dexamethasone on day 8,and then sacrificed on day 15 to obtain colon specimens.(2)The DAI,colon damage score and histopathology score were recorded.(3)The mRNA levels of TLR4 and the inflammatory factors TNF-α,Il-1,IL-6 and IL-12 in the colonic tissue samples of each group were detected by PCR.(4)Locations of NF-κb subtype was detected by Western Blot.(5)The expressions of NF-icb and GR by immunofluorescence staining.Part Ⅲ(1)Expressions of key proteins and inflammatory factors in each group were detected after transfection with miR-642a-5p mimics.Design and synthesis of mir-642a-5p mimics.THP-1 cells were divided into 6 groups:Control+miR-NC group,LPS+miR-NC group,DEX+LPS+miR-NC group,Control+miR-642a-5p mimics group,LPS+miR-642a-5p mimics group,and DEX+LPS+miR-642a-5p mimic group.The mRNA levels of TLR4 and the inflammatory factors TNF-α,II-1,IL-6 and IL-12 in the samples of each group were detected by PCR.Cell apoptosis was detected by flow cytometry.The cell survival rate was determined by MTT.The levels of TNF-α,Il-1,IL-6 and IL-12 was detected by ELISA.The nucleation of GR and NF-B p65 was detected by immunofluorescence.Western Blot was used to detect the expressions of the related proteins in the TLR4-MyD88-TAK1/p38/JNK signaling pathway.(3)To determine whether TLR4 is a target gene of miR-642a-5p:THP-1 cells were treated with LPS,different TLR activators,DEX and TGF TGF(negative regulators of the TLR signaling pathway)to determine whether miR-642a-5p was related to the TLR4 signaling pathway.The luciferase report detected whether TLR4 had binding sites with miR-642a-5p.After transfection with miR-642a-5p mimics into TLR4-overexpressed cells,it was explored whether it could inhibit the inflammatory response.Results:Clinical trials:Compared with the colonic mucosa samples of GCS group,the expression of miR-642a-5p and TLR4 in GCR group decreased significantly,while the expression of other inflammatory factors IL-1β,IL-6,IL-12 and TNF-α increased correspondingly.Animal experiments:(1)Compared with the control group,the DAI score,colon injury score and histopathology score of the DSS model group were higher than those of the experimental groups;compared with the DSS model group,the DAI score,colon injury score and histopathology score of the DEX group,DEX+pre miR-642a-5p group,DEX+miR-642a-5p inhibitor group were significantly lower,and the DEX+pre miR-642a-5p group was most obvious.(2)The results of qRT-PCR showed that the expression of miR-642a-5p in the intestinal mucosa of pre-miR-642a-5p group was higher than that of DSS group,and the expression of miR-642a-5p in the intestinal mucosa of miR-642a-5p inhibitor group was lower than that of DSS group(P<0.05).At the same time,TNF-α,IL-1β,IL-6,IL-12 and TLR4 mRNA were negatively correlated with miR-642a-5p expression,DEX and pre-mir-642a-5p enhanced the inhibition of TLR4 transcription and anti-inflammatory effect.(3)Western Blot showed that the expression of NF-κb p65 and p50 was higher in pre-miR-642a-5p group and lower in miR-642a-5p inhibitor group than in DSS group.On the contrary,the expression of p65 and p50 in cell nucleus was down regulated in pre-miR-642a-5p group,and the expression of p65 and p50 in miR-642a-5p inhibitor group was higher,the difference was statistically significant(P<0.05).At the same time,compared with miR-642a-5p inhibitor group,the expression of NF-κb p65 and p50 in the cytoplasm of DEX+pre-miR-642a-5p group was higher,and the expression in the nucleus was lower,while the difference was statistically significant(4)Compared with miR-642a-5p inhibitor group,NF-Kb p65 and p50 expression in pre-miP-642a-5p inhibitor group decreased significantly(P<0.05).In addition,compared with DEX group and miR-642a-5p inhibitor group,the expression of GR in Dex+pre-miR-642a-5p group was higher,the difference was statistically significant(P<0.05),suggesting that miR-642a-5p could achieve anti-inflammatory effect by cooperating with DEX to improve the expression of GR.Cell experiments:(1)THP-1 cells were transfected with miR-642a-5p mimics,and their viability was detected by MTT assay and flow cytometry.The results showed that there was no significant difference in cell viability and mortality between the miR-642a-5p and the blank control group or the miR-NC control group at 24h and 48h,indicating that miR-642a-5p had no effect on THP-1 Cell Viability and mortality.(2)After miR-642a-5p mimics was transfected into THP-1 cells alone,it had no significant effect on LPS induced inflammation;however,after miR-642a-5p mimics was transfected into the cells,it could cooperate with DEX to reduce the levels of IL-12,IL-1β,IL-6 and TNF-α induced by LPS.(3)Western Blot and immunofluorescence confirmed the expression level of GR and NF-κB subtypes.The results showed that after miR-642a-5p mimics,with LPS and DEX treatment,the expression of GR in the cytoplasm had no significant change,but LPS could increase the expression of NF-κB p65 and p50 in the nucleus,while DEX could maintain the expression of NF-κB p65 and p50 in the cytoplasm.After treated with miR-642a-5p mimics and DEX,the expression levels of GR and NF-κB p65 and p50 were increased.(4)The results showed that there was no change in the expression of miR-642a-5p by TLR2 agonist pam3csk4,TLR3 agonist poly(I:C),TLR7 agonist imiquimod,TLR9 agonist CpG oligodeoxynucleotide(CpG DNA oligo).LPS could down regulate the expression of miR-642a-5p,DEX and TGF β could up regulate the expression of miR-642a-5p,the results were statistically significant(P<0.05).This suggests that miR-642a-5p may play a role through TLR4.(5)Bioinformatics analysis and luciferase report showed that TLR4 was the target gene of miR-642a-5p.In addition,transfection of miR-642a-5p mimics into cells can reduce the expression level of TLR4 mRNA.miR-642a-5p can enhance the LPS induced inhibition of DEX on TLR4 and inhibit the expression of TLR4 on the cell surface.Compared with wild-type cells,the level of inflammatory factors in TLR4 overexpression cells was higher,while DEX did not reduce the level of inflammatory factors;however,after miR-642a-5p mimics transfection,the level of inflammatory factors decreased(P<0.05).(6)LPS can induce the up-regulation of TLR4,MyD88,TAK1 and other factors,and increase the phosphorylation level of ERK,p38 and JNK.DEX can inhibit the phosphorylation of ERK,p38 and JNK,while miR-642a-5p mimics can further inhibit the phosphorylation level.Conclusions:1.Compared with the GCS group,miR-642a-5p was down regulated in the intestinal mucosa of glucocorticoid resistant in UC,while the expression of IL-1β,IL-6,IL-12,TNF-α and TLR4 were increased,which was negatively correlated with the expression level of miR-642a-5p.2.In vivo,it was confirmed that miR-642a-5p was negatively correlated with the expression of IL-1β,IL-6,IL-12,TNF-α and TLR4 mRNA.3.Vivo and vitro experiments showed that overexpression of miR-642a-5p could cooperate with the anti-inflammatory effect of glucocorticoids,reduce the secretion level of inflammatory factors TNF-α,IL-1β,IL-6,IL-12,inhibit the transfer of NF-κb p65 and p50 into the nucleus,promote the expression of GR,and improve the sensitivity of glucocorticoids.4.Vitro experiments showed that miR-642a-5p could directly target to inhibit TLR4,thus it could affect the expression of TLR4/MyD88/TAK1 signaling pathway induced by LPS,down regulate the phosphorylation level of ERK,p38 and JNK,reduce the expression and nucleus-entry of NF-κB,promote the entry of glucocorticoid receptor into the nucleus and play a synergistic and anti-inflammatory role in UC. |
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