| Objective: The present study aim to explore the differentially expressed microRNAs in different tissues of ulcerative uolitis patients with glucocorticoid resistance and try to find the possible regulatory networks and signaling pathways between glucocorticoid resistance associated genes and these microRNAs. Cell test will be used to explore the function of the target microRNA and pathogenesis of glucocorticoid resistance of inflammatory bowel disease are discussed in the view of molecular biology level.Methods: This study is divided into two parts.Part ?First, serum samples were collected from 9ulcerative uolitis patients with glucocorticoid sensitive and 9 ulcerative uolitis patients with glucocorticoid resistant before treatment. The different expressions of microRNAs detected by microRNA panel PCR chip were analysed to predict target genes and target gene pathway by bioinformatics analysis, such as Gene Ontology (GO) analysis and pathway analysis.Second, serum samples were collected before treatment from 76 ulcerative uolitis patients who were divided into two groups: glucocorticoid sensitive group(n=39) and glucocorticoid resistant group (n=37). Intestinal mucosal specimens were collected before treatment from 30 ulcerative uolitis patients who were divided into two groups:glucocorticoid sensitive group(n=18) and glucocorticoid resistant group (n=12). The validity of the different expressions of microRNAs were verified by qRT-PCR testing.Last, ROC curve analysis was used to get access to the reliability of the prediction of glucocorticoid resistance with microRNAs in ulcerative colitis patients.Part ?THP-1 cells were transfected with miRNA mimic-lipo2000 and cultured for 24h,and then the expression level of miR-150 in the transfected THP-1 cells was detected by fluorescence quantitative PCR. The results showed that miR-150 transfection was successful. Then THP-1 cells were induced with Phorbol 12-Myristate 13-Acetate(PMA) 160nM for 24h to differentiate into macrophages. And then the cells were stimulated with lipopolysaccharide (LPS) 100ng/ml for 1h and incubated with Dexamethasone (Dex) 10 ?M for 24h. This study have 6 groups: Ctrl group, miR-NC group, miR-150 group, Ctrl+LPS+Dex group, miR-NC+LPS+Dex group and miR-150+LPS+Dex group. The expression levels of HSP90 and HSF1 mRNA in each group were detected by fluorescence quantitative PCR and the expression levels of MAPK family members (p38/p-p38, JNK/p-JNK, ERK1/2/p-ERK1/2, MEK/p-MEK,p-AKT/t-AKT) were detected using Western blot. ELISA was used in the detection of the expression levels of IL-2, IL-17, IL-10 and TNF-? in cell culture medium supernatant and the expression levels of cell surface TLR4 were detected useing flow cytometry. We tried to find out the possible role of miR-150 from multiple aspects of the mechanism of glucocorticoids resistance and discuss the possible mechanisms of miR-150 in ulcerative colitis patients with glucocorticoids resistance.Results:Part ?1. The sex, age, Mayo score and the infection of epstein-barr virus (EBV) and cytomegalovirus (CMV) were not statistically significant (P > 0.05) between GC-resistant group and GC-sensitive group.2. Profiling of Serum microRNAs by microRNA PCR Panel. 128 miRNAs were identified differentially expressed between two groups with Fold change > 2. Among these 128 miRNAs, 50 miRNAs were upregulated and 78 downregulated. We observed that only 16 miRNAs were significantly downregulated (P < 0.05; 4.58fold on average) in GC-resistant group when compared with GC-sensitive group. All of the expression of the 50 upregulated microRNAs were not statistically significant (P >0.05).3. Gene Ontology (GO) analysis, upregulated transcripts were highly enriched for anatomical structure morphogenesis (ontology: biological process), intracellular(ontology: cellular component), and protein binding (ontology: molecular function).The downregulated transcripts were highly enriched for regulation of cellular metabolic processes (ontology: biological process), intracellular part (ontology:cellular component), and protein binding (ontology: molecular function).4. In pathway analysis, 43 pathways corresponded to upregulated transcripts, but the "Neurotrophin signaling pathway" was the most represented pathway. With respect to downregulated transcripts, there were 116 pathways represented and the most highly enriched was the "Pathways in cancer". In particular,"PI3K-Akt signaling pathway" and "MAPK signaling pathway" which were associated with downregulated transcripts, have been shown to be involved in the GCs mechanisms of action.5. microRNAs target prediction. Based on the results of the microRNAs Microarrays and the pathway analysis, we carefully selected 8 downregulated miRNAs (miR-16-2-3p, miR-30e-3p, miR-32-5p, miR-425-5p, miR-642a-5p,miR-150-5p, miR-224-5p, miR-486-3p) as the objective miRNAs whose fold changes were > 2.0 and P < 0.05. The predicted target genes of objective miRNAs were shown in Figure 2. The analysis found that some important genes related to the GCs mechanisms of action,such as HSP90B1,MAPK13, MAPK14, MAPK9, PIK3AP1,TLR4.6.The sex, age,Mayo score and the infection of epstein-barr virus (EBV) and cytomegalovirus (CMV) were not statistically significant (P > 0.05) between GC-resistant group and GC-sensitive group.7. Validation of selected serum microRNAs. The expression levels of miR-16-2-3p, miR-30e-3p, miR-32-5p, miR-642a-5p, miR-150-5p, miR-224-5p in GC-resistant patients were significantly lower than in GC-sensitive patients (P < 0.05).While, the expression levels of miR-425-5p and miR-486-3p were no statistical difference (P > 0.05).8. Validation of selected intestinal mucosa microRNAs. the expression levels of miR-16-2-3p, miR-642a-5p, miR-150-5p, miR-224-5p in GC-resistant patients were significantly lower than in GC-sensitive patients (P < 0.05). While, the expression levels of miR-30e-3p,miR-32-5p,miR-425-5p and miR-486-3p were no statistical difference (P > 0.05).9.Receiver Operating Characteristic (ROC) Analysis. The differential expression of microRNAs in the serum test results was analyzed by ROC curve. ROC analysis showed that the area under curve (AUC) of miR-16-2-3p, miR-30e-3p, miR-32-5p,miR-642a-5p, miR-150-5p and miR-224-5p was 0.94 (95% confidence interval [CI]0.891-0.986, P < 0.0001), 0.93 (95% confidence interval [CI] 0.878-0.983, P <0.0001), 0.85 (95% confidence interval [CI] 0.766-0.932, P < 0.0001), 0.87 (95%confidence interval [CI] 0.794-0.950, P < 0.0001), 0.92 (95% confidence interval [CI]0.858-0.974, P < 0.0001) and 0.99 (95% confidence interval [CI] 0.968-1.00, P <0.0001), with specificity of 97.30%, 89.20%, 59.50%, 73.00%, 97.30% and 97.30%,and sensitivity of 74.40%,84.60%,97.40%,92.30%,66.70% and 89.70%,respectively.Part ?1.The expression levels of HSP90 mRNA and HSF1 mRNA in different groups. The result indicated that miR-150 did not regulate the expression of HSP90 mRNA. At the same time, the expression of Hsp90 in THP-1 cells were detected by Western blot in each groups. We found that the expression level of Hsp90 was decreased in miR-150 group than in mir-NC group , but not statistically significant(p= > 0.094, 0.05). However, there was no significant difference in the expression level of HSF1 mRNA in the groups (P > 0.05).2.The expression levels of p38/p-p38, JNK/p-JNK, ERK1 /2/p-ERK 1 /2,MEK/p-MEK, p-AKT/t-AKT) in different groups. After Western blot detection, the results were analyzed using image J gray level analysis. Before and after the induction of LPS (Ctrl+LPS+Dex group and Ctrl group,miR-NC+LPS+Dex group and mir-NC group, miR-150+LPS+Dex group and miR-150 group), The expression level of p38?JNK?ERK1/2?MEK?t-AKT protein had no significant change between the Ctrl+LPS+Dex group and the Ctrl group? miR-NC+LPS+Dex group and miR-NC group?miR-150+LPS+Dex group and miR-150 group (P > 0.05). After inducted by LPS, the phosphorylation levels of p38?JNK?ERK1/2 were significantly increased,the increase was statistically significant (P < 0.05). Compared with the Ctrl+LPS+Dex group and miR-NC+LPS+Dex group, we found that the phosphorylation level of p38 and ERK 1/2 was significantly reduced in miR-150+LPS+Dex group, the difference was also statistically significant (P < 0.05).3. The expression levels of IL-10 , IL-2 , IL-17,TNF-a in the culture supernatant of different groups. Before induced by LPS, the expression levels of IL-10,IL-2,IL-17,TNF-a in Ctrl group,mir-NC group,miR-150 group were low,there was no significant difference in the three groups (P > 0.05). But after induced by LPS, the expression levels of IL-2 and TNF-a in the three groups were increased.The difference was statistically significant (P < 0.05). However, compared with Ctrl+LPS+Dex group and miR-NC+LPS+Dex group, the expression levels of IL-2 and TNF-a in the miR-150+LPS+Dex group were significantly decreased. The difference was statistical significance (P < 0.05). In addition, compared with other groups, the secretion level of IL-10 in miR-150+LPS+Dex group was significantly higher (P < 0.05). While the expression levels of IL-17 was low in all groups, and there was no significant difference between the groups each other (P > 0.05,respectively).4. The expression level of TLR4 on the surface of THP-1 cells in different groups. We found that the expression level of TLR4 on TPH-1 cells surface was upregulated after induced by LPS. Compared between ctrl group and Ctrl+LPS+Dex group, miR-NC group and miR-NC+LPS+Dex group,miR-150 group and miR-150+LPS+Dex group, the elevation of TLR4 was statistically significant (P <0.05, respectively). Compared with Ctrl+LPS+Dex group and miR-NC+LPS+Dex group,the expression of TLR4 on TPH-1 cells surface was significantly lower. The difference was statistically significant (P < 0.05).Conclusions:1. There were 16 significantdownregulated expression microRNAs in the serum of the glucocorticoid resistance patients with ulcerative colitis. Through bioinformatics analysis, 8 microRNAs (miR-16-2-3p, miR-30e-3p, miR-32-5p,miR-425-5p, miR-642a-5p, miR-150-5p, miR-224-5p, miR-486-3p) were possible to participate the glucocorticoid resistance in ulcerative colitis. And these microRNAs may be involved in the regulation of the target genes (HSP90B1, MAPK13, MAPK14,MAPK9, PIK3AP1, TLR4) and the signaling pathways ("PI3K-Akt signaling pathway" and "MAPK signaling pathway")associated with glucocorticoid resistance.2. Extended sample detection, there were 6 significant downregulated expression microRNAs in the serum of the glucocorticoid resistance patients with ulcerative colitis (miR-16-2-3p, miR-30e-3p, miR-32-5p, miR-642a-5p, miR-150-5p,miR-224-5p). These microRNAs had a certain value in predicting glucocorticoid resistance with ulcerative colitis.3. There were 4 microRNAs (mir-16-2-3p, mir-642a-5p, mir-150-5p, mir-224-5p)downregulated in intestinal mucosa of the glucocorticoid resistance patients, which provided a theoretical basis for further study of the molecular mechanisms of hormone resistance in patients with ulcerative colitis.4. Cell experiment showed that elevated miR-150 can improve the sensitivity of glucocorticoid. This is probably because of miR-150 can regulate the phosphorylationp level of p38 MAPK and ERK1/2 and the expression level of TLR4 on the cell surface. This study provides a new idea for the molecular mechanism of glucocorticoid resistance in patients with ulcerative colitis and provides a new therapeutic target. |
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