糖皮质激素抵抗的溃疡性结肠炎患者microRNA的筛选和机制研究
发布时间：2018-08-27 07:00
【摘要】：[目的]探寻糖皮质激素抵抗的溃疡性结肠炎患者不同组织中差异表达的microRNA,并通过生物信息学分析寻找其与糖皮质激素抵抗相关靶基因、信号通路之间可能存在的调控网络;再从细胞实验入手初步探索目标microRNA的功能,进而从分子生物学层面探讨炎症性肠病患者糖皮质激素抵抗的预测因子和发病机制。[方法]本课题分为两部分:第一部分:首先,治疗前收集溃疡性结肠炎激素敏感患者血清标本9份,激素抵抗患者血清标本9份,采用microRNA PCR芯片技术检测血清中差异表达的microRNAs,通过生物信息学分析进行靶基因的预测和靶基因GeneOntology(GO)分析,靶基因pathway分析,寻找与溃疡性结肠炎激素抵抗相关的microRNAs及其靶基因和信号通路。再者,治疗前收集溃疡性结肠炎患者血清76份(激素敏感组39份,激素抵抗组37份),肠粘膜标本30份(激素敏感组18份,激素抵抗组12份),采用实时荧光定量PCR (qRT-PCR)验证microRNA PCR芯片筛选出的microRNAs的可靠性。最后,采用ROC曲线分析差异microRNAs预测溃疡性结肠炎患者糖皮质激素抵抗的相关性。第二部分:通过miRNAmimic-lipo2000复合物转染THP-1细胞培养24h,运用荧光定量PCR检测转染后THP-1细胞内miR-150的表达水平,确认miR-150转染成功。然后经佛波酯(PMA) 160nM诱导24h使THP-1细胞分化为巨噬细胞后,使用脂多糖(LPS) 100ng/ml刺激细胞1h,再加入地塞米松(Dex) 10μM处理 24h。实验分为 6 组:Ctrl 组、miR-NC 组、miR-150 组、Ctrl+LPS+Dex 组、miR-NC+LPS+Dex组、miR-150+LPS+Dex组。先通过荧光定量PCR检测各组别细胞中HSP90和HSF1 mRNA的表达水平;其次通过western blot检测各组别细胞中 MAPK 家族成员(p38/p-p38、JNK/p-JNK、ERK1/2/p-ERK1/2、MEK/p-MEK、p-AKT/t-AKT)的蛋白表达水平;再通过ELISA检测各组别细胞培养基上清中IL-10, IL-2, IL-17, TNF-α等炎症因子的表达水平;最后使用流式细胞技术检测各组别细胞表面TLR4的表达水平,从激素抵抗机制的各个环节来寻找miR-150可能的作用环节,从而证明miR-150在溃疡性结肠炎患者激素抵抗中的可能机制。[结果]第一部分:1.激素抵抗组和激素敏感组性别、年龄、Mayo评分和EBV及CMV感染情况均无明显统计学差异(P 0.05)。2.microRNA PCR芯片筛选结果。以Fold change2为显著差异性,激素抵抗组和激素敏感组相比较筛选出128个差异表达的microRNAs,其中50个microRNAs表达上调,78个microRNAs表达下调。进一步分析发现仅有16个表达下调的microRNAs具有统计学显著差异性(P 0.05; Fold change平均4.58倍);而表达上调的50个microRNAs均无统计学意义(P 0.05)。3.Gene Ontology分析(GO分析)结果。上调的microRNAs在生化过程方面主要参与解剖形态结构的变化,在细胞组分方面主要参与细胞内成分的变化,在分子功能方面主要蛋白结合过程;下调的microRNAs在生化过程方面主要参与细胞代谢过程的调节,在细胞组分方面主要细胞内部分的变化,在分子功能方面主要蛋白结合过程。4.Pathway分析结果。上调的microRNAs与43条信号通路有关,其中相关性最高的是"Neurotrophin signaling pathway";下调的 microRNAs 与 116 条信号通路有关,其中相关性最高的是“Pathways in cancer"。值得注意的是在与下调的microRNAs相关的信号通路中“PI3K-Akt signaling pathway” 和 “MAPK signaling pathway"与糖皮质激素抵抗是相关的。5.靶基因预测结果。在综合芯片结果和pathway分析的基础上,筛选出8个下调的 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)作为目标 microRNAs 进行靶基因预测(fold changes 2.0 and P 0.05),结果发现 HSP90B1,MAPK13,MAPK14, MAPK9, PIK3AP1,TLR4等靶基因与糖皮质激素抵抗有关。5.差异microRNAs在血清中检测结果。激素抵抗组和激素敏感组相比较,miR-16-2-3p, miR-30e-3p, miR-32-5p,miR-642a-5p, miR-150-5p,miR-224-5p 的表达水平是显著降低并具有统计学意义的(P0.05)。而miR-425-5p和miR-486-3p的表达差异不具有统计学意义(P 0.05)。6.差异microRNAs在肠粘膜中检测结果。激素抵抗组和激素敏感组相比较,miR-16-2-3p,miR-642a-5p,miR-150-5p,miR-224-5p 的表达水平是显著降低并具有统计学意义的(P 0.05)。而 miR-30e-3p,miR-32-5p,miR-425-5p 和miR-486-3p的表达差异不具有统计学意义(P 0.05)。7.ROC曲线分析结果。选取血清检测结果中的差异表达的microRNAs进行ROC 曲线分析。miR-16-2-3p,miR-30e-3p,miR-32-5p,miR-642a-5p, miR-150-5p,miR-224-5p 的曲线下面积(AUC)分别是 0.94 (95% 可信区间[CI] 0.891-0.986, P0.0001),0.93 (95% 可信区间[CI] 0.878-0.983,P 0.0001),0.85 (95% 可信区间[CI] 0.766-0.932,P 0.0001), 0.87 (95% 可信区间[CI] 0.794-0.950,P0.0001),0.92(95% 可信区间[CI] 0.858-0.974, P0.0001),0.99(95% 可信区间[CI] 0.968-1.00,P 0.0001);特异性分别是 97.30%,89.20%,59.50%,73.00%, 97.30%, 97.30%;敏感性分别是 74.40%, 84.60%, 97.40%, 92.30%,66.70%, 89.70%。第二部分:1.各组THP-1细胞中HSP90和HSF1 mRNA表达水平。分析结果进行组间比较,miR-150 组与 Ctrl 组和 miR-NC 组、miR-150+LPS+Dex 组与 Ctrl+LPS+Dex组和miR-NC+LPS+Dex组间相比较时HSP90的mRNA表达水平均无明显变化(P 0.05 ),表明miR-150没有调节HSP90的mRNA表达的作用。与此同时通过Western blot对处理前后的THP-1细胞中HSP90蛋白的表达水平进行了检测发现各组间比较发现miR-150组HSP90的表达水平较miR-NC组有下降,但不具有统计学意义(P= 0.094, 0.05)。而各组别中HSF1 mRNA表达水平进行组间比较未发现显著性差异(P 0.05)。2.各组别 THP-1 细胞中 p38/p-p38、JNK/p-JNK、ERK1/2/p-ERK1/2、MEK/p-MEK、p-AKT/t-AKT)的蛋白表达水平。经western blot检测后利用Image J条带灰度分析进行结果分析,LPS诱导前后相比较(Ctrl+LPS+Dex组与Ctrl组、miR-NC+LPS+Dex 组与 miR-NC 组、miR-150+LPS+Dex 组与 miR-150 组),p38、JNK、ERK1/2、MEK、t-AKT蛋白的表达水平无明显变化(P0.05),而LPS诱导后p38、JNK和ERK1/2的磷酸化水平均有明显升高,这种升高具有统计学意义(P 0.05 )。而在 miR-150+LPS+Dex 组与 Ctrl+LPS+Dex 组和miR-NC+LPS+Dex组相比较时发现,p38和Erk1/2的磷酸化水平却显著降低,这种差异也具有统计学意义(P 0.05)。3.各组别THP-1细胞中培养基上清中IL-10, IL-2, IL-17, TNF-α等炎症因子的表达水平。在LPS诱导炎症反应前,Ctrl组、miR-NC组、miR-150组中各个炎症因子的分泌水平均较低,各组间的水平均无显著性差异(P均0.05)。而在LPS诱导后IL-2和TNF- α的分泌水平较诱导前显著升高,差异具有统计学意义(P 0.05 ),其中miR-150+LPS+Dex组IL-2和TNF- α的分泌水平较Ctrl+LPS+Dex组和miR-NC+LPS+Dex组均显著降低,差异同样具有统计学意义(P0.05)。另外,IL-10在miR-150+LPS+Dex组的分泌水平较其他各组是明显增高的(P 0.05)。而IL-17在各组中分泌水平均较低,各组间的水平均无显著性差异(P均0.05)。4.各组别THP-1细胞表面TLR4的表达水平。分析比较后发现经LPS诱导后THP-1细胞表面TLR4的表达明显上调,对应各组间(Ctrl组与Ctrl+LPS+Dex组、miR-NC 组与 miR-NC+LPS+Dex 组、miR-150 组与 miR-150+LPS+Dex 组)相比较均显著升高且具有统计学意义(P 0.05);而miR-150+LPS+Dex组THP-1细胞表面TLR4的表达较Ctrl+LPS+Dex组和miR-NC+LPS+Dex组显著降低,这种差异具有统计学意义(p 0.05)。[结论]1.糖皮质激素抵抗的溃疡性结肠炎患者血清中存在16个表达下调的microRNAs具有统计学显著差异性。经过生物信息学分析有可能参与糖皮质激素抵抗的有 8 个 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)。差异表达的 microRNAs 可能参与调节的糖皮质激素抵抗的靶基因有HSP90B1,MAPK13, MAPK14,MAPK9,PIK3AP1,TLR4;信号通路有“PI3K-Akt signaling pathway” 和 “MAPK signaling pathway"。2.经扩大样本检测,糖皮质激素抵抗的溃疡性结肠炎患者血清中表达下调的microRNAs 共有 6 个(miR-16-2-3p,miR-30e-3p, miR-32-5p,miR-642a-5p,miR-150-5p,miR-224-5p),对预测溃疡性结肠炎患者的糖皮质激素抵抗有一定的价值。3.肠粘膜中表达下调的 microRNAs 共有 4 个(miR-16-2-3p,miR-642a-5p,miR-150-5p,miR-224-5p)。为进一步研究溃疡性结肠炎患者糖皮质激素抵抗的分子生物学机制提供了理论基础。4.细胞实验证实miR-150升高可提高糖皮质激素治疗的敏感性,可能是通过调节p38 MAPK和ERK1/2的磷酸化水平,或细胞表面TLR4表达水平等关键环节来实现的。为溃疡性结肠炎患者糖皮质激素抵抗的分子生物学机制研究提供了新的思路。
[Abstract]:[Objective] To explore the differentially expressed microRNAs in different tissues of patients with glucocorticoid-resistant ulcerative colitis, and to identify the target genes related to glucocorticoid resistance and the possible regulatory networks between the signal pathways by bioinformatics analysis, and then to explore the function of target microRNAs by cell experiments. [Methods] This study was divided into two parts: First, nine serum samples from patients with glucocorticoid-sensitive ulcerative colitis and nine serum samples from patients with glucocorticoid resistance were collected before treatment, and the serum samples from patients with glucocorticoid resistance were detected by microRNA PCR chip. The differentially expressed microRNAs were predicted by bioinformatics analysis, the target gene Gene Ontology (GO) analysis and the target gene pathway analysis. The microRNAs and their target genes and signaling pathways related to hormone resistance in ulcerative colitis were searched. Furthermore, 76 serum samples of ulcerative colitis patients (39 hormone sensitive group) were collected before treatment. Thirty (18 in the hormone sensitive group and 12 in the hormone resistant group) and thirty (37 in the hormone resistant group) intestinal mucosa samples were used to verify the reliability of microRNAs screened by microRNA PCR chip. Finally, ROC curves were used to analyze the correlation between differential microRNAs and glucocorticoid resistance in ulcerative colitis patients. Part: THP-1 cells were transfected with microRNAmimic-lipo2000 complex and cultured for 24 hours. The expression of microRNA150 in THP-1 cells was detected by fluorescence quantitative PCR to confirm the success of microRNA150 transfection. The experiment was divided into six groups: Ctrl group, microRNAs-NC group, microRNAs-150 group, Ctrl+LPS+Dex group, microRNAs-NC+LPS+Dex group, microRNAs-150+LPS+Dex group. The expression of HSP90 and HSF1 mRNA in cells of each group was detected by fluorescence quantitative PCR, and the MAPK family members (p38/p-p38, JN-p38, JN-p38) were detected by Western blot. The protein expression levels of K/p-JNK, ERK1/2/p-ERK1/2, MEK/p-MEK, p-AKT/t-AKT were detected by ELISA, and the expression levels of inflammatory factors such as IL-10, IL-2, IL-17 and TNF-alpha were detected by flow cytometry. Finally, the expression level of TLR4 on the cell surface of each group was detected by flow cytometry to find out the various links of hormone resistance mechanism. [Results] Part 1: 1. There was no significant difference in sex, age, Mayo score, EBV and CMV infection between steroid resistant group and steroid sensitive group (P 0.05). 2. MicroRNA PCR chip screening results. Fold change 2. For significant differences, 128 differentially expressed microRNAs were screened out between the hormone-resistant group and the hormone-sensitive group, of which 50 were up-regulated and 78 were down-regulated. Gene Ontology analysis (GO analysis) showed that the up-regulated microRNAs were mainly involved in the changes of anatomical morphology and structure in the biochemical process, mainly in the changes of cellular components, mainly in the protein binding process in the molecular function; the down-regulated microRNAs were biochemical. Pathway analysis showed that the up-regulated microRNAs were related to 43 signaling pathways, of which the highest correlation was "Neurotrophin signaling pathway"; the down-regulated microRNAs were 1 and 1. Of the 16 signaling pathways involved, Pathways in cancer was the most relevant. Notably, PI3K-Akt signaling pathway and MAPK signaling pathway were associated with glucocorticoid resistance in the down-regulated microRNAs-related signaling pathways. 5. Target gene predictions. On the basis of analysis, eight down-regulated microRNAs (microRNAs, including microRNAs (microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs and microRNAs) were screened for target gene prediction (fold changes 2.0 and P 0.05). Differential microRNAs were found in serum. Compared with hormone-sensitive group, the expression levels of microRNAs in the hormone-resistant group and hormone-sensitive group were significantly lower and statistically significant (P 0.05). However, the expression levels of microRNAs in the hormone-resistant group and hormone-sensitive group were not significantly different (P 0.05). Significance (P Significance (P 0.05). 7. ROC curve analysis. The ROC curve analysis was performed on differentially expressed microRNAs from serum test results. The area under the curve (AUC) of microRNAs was 0.94 (95% confidence interval [CI] 0.891-0.986, P 0.0001), 0.93 (95% confidence interval [CI] 0.878-5 p, Mi-32-5 p, Mi-64 2a-5 p, Mi-150-5 p, and Mi-224-5 p, respectively. 0.983, P 0.0001, 0.983, P 0.0001, 0.85 (95% confidence interval [CI] 0.766-0.932, P 0.0001), 0.87 (95% confidence interv [CI] 0.794-0.950, P 0.0001), 0.87 (95% confidence interv [CI] 0.794-0.950.950, P 0.0001), 0.92 (95% confidence interv [CI] 0.858-0.978-0.974, P 0.0001), 0.99 (95% confidence interv [CI] 0.968-1.968-1.00, P 0.000 1;97.30%, 89.30%, 89.30.30%, 89.20%, 89.20.20%, 59.20.20.74.40% respectively. The expression levels of HSP90 and HSF1 mRNA in THP-1 cells of each group were 84.60%, 97.40%, 92.30%, 66.70%, 89.70%. The results showed that there was no significant change in the expression level of HSP90 mRNA between the groups of Mi-150 and CTrl, Mi-NC, Mi-150 + LPS + Dex, CTrl + LPS + Dex and Mi-NC + LPS + Dex (P 0.05). The expression level of HSP90 protein in THP-1 cells before and after treatment was detected by Western blot. It was found that the expression level of HSP90 in microwave-150 group was lower than that in microwave-NC group, but not statistically significant (P = 0.094, 0.05). There was no significant difference in mRNA expression between groups (P 0.05). 2. The expression levels of p38/p-p38, JNK/p-JNK, ERK1/2/p-ERK1/2, MEK/p-MEK, p-AKT/t-AKT in THP-1 cells of each group were compared before and after LPS induction (Ctrl+LPS+Dex group and CTrl group). The expression levels of p38, JNK, ERK1/2, MEK, t-AKT protein were not significantly changed in the group of microRNAs-NC+LPS+Dex and the group of microRNAs-NC, the group of microRNAs-150+LPS+Dex and the group of microRNAs-150 (P 0.05), but the phosphorylation levels of p38, JNK and ERK1/2 were significantly increased in the group of microRNAs-150+LPS+Dex and the group of CTrl+LPS+Dex after LPS induction (P 0.05). Compared with the iR-NC+LPS+Dex group, the phosphorylation levels of p38 and Erk1/2 were significantly lower, and the difference was statistically significant (P 0.05). 3. The expression levels of inflammatory factors such as IL-10, IL-2, IL-17, TNF-alpha in the supernatant of THP-1 cells were significantly lower in each group (P 0.05). The secretion levels of IL-2 and TNF-alpha were significantly higher after LPS induction than before induction (P 0.05). The secretion levels of IL-2 and TNF-alpha in Mi-150+LPS+Dex group were significantly lower than those in Ctrl+LPS+Dex group and Mi-NC+LPS+Dex group. In addition, the secretion level of IL-10 in the group of miR-150+LPS+Dex was significantly higher than that in the other groups (P After induction, the expression of TLR4 on THP-1 cells was significantly up-regulated, and the expression of TLR4 on THP-1 cells in corresponding groups (Ctrl group and CTrl+LPS+Dex group, microRNANC group and microRNANC+LPS+Dex group, microRNA150 group and microRNA150+LPS+Dex group) was significantly higher than that in Ctrl+LPS+Dex group and microRNANC+Dex group (P 0.05). LPS + Dex group significantly decreased, the difference was statistically significant (p 0.05). [Conclusion] 1. There were 16 down-regulated microRNAs in the serum of patients with glucocorticoid-resistant ulcerative colitis. There were 8 microRNAs (Mi-16-2-3p, MI) that might be involved in glucocorticoid resistance by bioinformatics analysis. R-30e-3p, microRNAs-32-5p, microRNAs-425-5p, microRNAs-642a-5p, microRNAs-150-5p, microRNAs-224-5p, microRNAs-486-3p). Differentially expressed microRNAs may be involved in the regulation of glucocorticoid resistance target genes HSP90B1, MAPK13, MAPK14, MAPK9, PIK3AP1, TLR4; signal pathways are "PI3K-Akt signaling pathway" and "MAPK signaling pathway". Six microRNAs were down-regulated in the serum of patients with glucocorticoid-resistant ulcerative colitis (Mi-16-2-3p, Mi-30e-3p, Mi-32-5p, Mi-642a-5p, Mi-150-5p, and Mi-224-5p). The down-regulated microRNAs in the intestinal mucosa were 4.3. Mi-16-2-3p, Mi-642a-5p, Mi-150-5p, and Mi-224-5p. These results provide a theoretical basis for further study of the molecular mechanism of glucocorticoid resistance in patients with ulcerative colitis. The expression level of TLR4 on the cell surface and other key links were achieved, which provided a new idea for the molecular biological mechanism of glucocorticoid resistance in patients with ulcerative colitis.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62
本文编号：2206480
[Abstract]:[Objective] To explore the differentially expressed microRNAs in different tissues of patients with glucocorticoid-resistant ulcerative colitis, and to identify the target genes related to glucocorticoid resistance and the possible regulatory networks between the signal pathways by bioinformatics analysis, and then to explore the function of target microRNAs by cell experiments. [Methods] This study was divided into two parts: First, nine serum samples from patients with glucocorticoid-sensitive ulcerative colitis and nine serum samples from patients with glucocorticoid resistance were collected before treatment, and the serum samples from patients with glucocorticoid resistance were detected by microRNA PCR chip. The differentially expressed microRNAs were predicted by bioinformatics analysis, the target gene Gene Ontology (GO) analysis and the target gene pathway analysis. The microRNAs and their target genes and signaling pathways related to hormone resistance in ulcerative colitis were searched. Furthermore, 76 serum samples of ulcerative colitis patients (39 hormone sensitive group) were collected before treatment. Thirty (18 in the hormone sensitive group and 12 in the hormone resistant group) and thirty (37 in the hormone resistant group) intestinal mucosa samples were used to verify the reliability of microRNAs screened by microRNA PCR chip. Finally, ROC curves were used to analyze the correlation between differential microRNAs and glucocorticoid resistance in ulcerative colitis patients. Part: THP-1 cells were transfected with microRNAmimic-lipo2000 complex and cultured for 24 hours. The expression of microRNA150 in THP-1 cells was detected by fluorescence quantitative PCR to confirm the success of microRNA150 transfection. The experiment was divided into six groups: Ctrl group, microRNAs-NC group, microRNAs-150 group, Ctrl+LPS+Dex group, microRNAs-NC+LPS+Dex group, microRNAs-150+LPS+Dex group. The expression of HSP90 and HSF1 mRNA in cells of each group was detected by fluorescence quantitative PCR, and the MAPK family members (p38/p-p38, JN-p38, JN-p38) were detected by Western blot. The protein expression levels of K/p-JNK, ERK1/2/p-ERK1/2, MEK/p-MEK, p-AKT/t-AKT were detected by ELISA, and the expression levels of inflammatory factors such as IL-10, IL-2, IL-17 and TNF-alpha were detected by flow cytometry. Finally, the expression level of TLR4 on the cell surface of each group was detected by flow cytometry to find out the various links of hormone resistance mechanism. [Results] Part 1: 1. There was no significant difference in sex, age, Mayo score, EBV and CMV infection between steroid resistant group and steroid sensitive group (P 0.05). 2. MicroRNA PCR chip screening results. Fold change 2. For significant differences, 128 differentially expressed microRNAs were screened out between the hormone-resistant group and the hormone-sensitive group, of which 50 were up-regulated and 78 were down-regulated. Gene Ontology analysis (GO analysis) showed that the up-regulated microRNAs were mainly involved in the changes of anatomical morphology and structure in the biochemical process, mainly in the changes of cellular components, mainly in the protein binding process in the molecular function; the down-regulated microRNAs were biochemical. Pathway analysis showed that the up-regulated microRNAs were related to 43 signaling pathways, of which the highest correlation was "Neurotrophin signaling pathway"; the down-regulated microRNAs were 1 and 1. Of the 16 signaling pathways involved, Pathways in cancer was the most relevant. Notably, PI3K-Akt signaling pathway and MAPK signaling pathway were associated with glucocorticoid resistance in the down-regulated microRNAs-related signaling pathways. 5. Target gene predictions. On the basis of analysis, eight down-regulated microRNAs (microRNAs, including microRNAs (microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs, microRNAs and microRNAs) were screened for target gene prediction (fold changes 2.0 and P 0.05). Differential microRNAs were found in serum. Compared with hormone-sensitive group, the expression levels of microRNAs in the hormone-resistant group and hormone-sensitive group were significantly lower and statistically significant (P 0.05). However, the expression levels of microRNAs in the hormone-resistant group and hormone-sensitive group were not significantly different (P 0.05). Significance (P Significance (P 0.05). 7. ROC curve analysis. The ROC curve analysis was performed on differentially expressed microRNAs from serum test results. The area under the curve (AUC) of microRNAs was 0.94 (95% confidence interval [CI] 0.891-0.986, P 0.0001), 0.93 (95% confidence interval [CI] 0.878-5 p, Mi-32-5 p, Mi-64 2a-5 p, Mi-150-5 p, and Mi-224-5 p, respectively. 0.983, P 0.0001, 0.983, P 0.0001, 0.85 (95% confidence interval [CI] 0.766-0.932, P 0.0001), 0.87 (95% confidence interv [CI] 0.794-0.950, P 0.0001), 0.87 (95% confidence interv [CI] 0.794-0.950.950, P 0.0001), 0.92 (95% confidence interv [CI] 0.858-0.978-0.974, P 0.0001), 0.99 (95% confidence interv [CI] 0.968-1.968-1.00, P 0.000 1;97.30%, 89.30%, 89.30.30%, 89.20%, 89.20.20%, 59.20.20.74.40% respectively. The expression levels of HSP90 and HSF1 mRNA in THP-1 cells of each group were 84.60%, 97.40%, 92.30%, 66.70%, 89.70%. The results showed that there was no significant change in the expression level of HSP90 mRNA between the groups of Mi-150 and CTrl, Mi-NC, Mi-150 + LPS + Dex, CTrl + LPS + Dex and Mi-NC + LPS + Dex (P 0.05). The expression level of HSP90 protein in THP-1 cells before and after treatment was detected by Western blot. It was found that the expression level of HSP90 in microwave-150 group was lower than that in microwave-NC group, but not statistically significant (P = 0.094, 0.05). There was no significant difference in mRNA expression between groups (P 0.05). 2. The expression levels of p38/p-p38, JNK/p-JNK, ERK1/2/p-ERK1/2, MEK/p-MEK, p-AKT/t-AKT in THP-1 cells of each group were compared before and after LPS induction (Ctrl+LPS+Dex group and CTrl group). The expression levels of p38, JNK, ERK1/2, MEK, t-AKT protein were not significantly changed in the group of microRNAs-NC+LPS+Dex and the group of microRNAs-NC, the group of microRNAs-150+LPS+Dex and the group of microRNAs-150 (P 0.05), but the phosphorylation levels of p38, JNK and ERK1/2 were significantly increased in the group of microRNAs-150+LPS+Dex and the group of CTrl+LPS+Dex after LPS induction (P 0.05). Compared with the iR-NC+LPS+Dex group, the phosphorylation levels of p38 and Erk1/2 were significantly lower, and the difference was statistically significant (P 0.05). 3. The expression levels of inflammatory factors such as IL-10, IL-2, IL-17, TNF-alpha in the supernatant of THP-1 cells were significantly lower in each group (P 0.05). The secretion levels of IL-2 and TNF-alpha were significantly higher after LPS induction than before induction (P 0.05). The secretion levels of IL-2 and TNF-alpha in Mi-150+LPS+Dex group were significantly lower than those in Ctrl+LPS+Dex group and Mi-NC+LPS+Dex group. In addition, the secretion level of IL-10 in the group of miR-150+LPS+Dex was significantly higher than that in the other groups (P After induction, the expression of TLR4 on THP-1 cells was significantly up-regulated, and the expression of TLR4 on THP-1 cells in corresponding groups (Ctrl group and CTrl+LPS+Dex group, microRNANC group and microRNANC+LPS+Dex group, microRNA150 group and microRNA150+LPS+Dex group) was significantly higher than that in Ctrl+LPS+Dex group and microRNANC+Dex group (P 0.05). LPS + Dex group significantly decreased, the difference was statistically significant (p 0.05). [Conclusion] 1. There were 16 down-regulated microRNAs in the serum of patients with glucocorticoid-resistant ulcerative colitis. There were 8 microRNAs (Mi-16-2-3p, MI) that might be involved in glucocorticoid resistance by bioinformatics analysis. R-30e-3p, microRNAs-32-5p, microRNAs-425-5p, microRNAs-642a-5p, microRNAs-150-5p, microRNAs-224-5p, microRNAs-486-3p). Differentially expressed microRNAs may be involved in the regulation of glucocorticoid resistance target genes HSP90B1, MAPK13, MAPK14, MAPK9, PIK3AP1, TLR4; signal pathways are "PI3K-Akt signaling pathway" and "MAPK signaling pathway". Six microRNAs were down-regulated in the serum of patients with glucocorticoid-resistant ulcerative colitis (Mi-16-2-3p, Mi-30e-3p, Mi-32-5p, Mi-642a-5p, Mi-150-5p, and Mi-224-5p). The down-regulated microRNAs in the intestinal mucosa were 4.3. Mi-16-2-3p, Mi-642a-5p, Mi-150-5p, and Mi-224-5p. These results provide a theoretical basis for further study of the molecular mechanism of glucocorticoid resistance in patients with ulcerative colitis. The expression level of TLR4 on the cell surface and other key links were achieved, which provided a new idea for the molecular biological mechanism of glucocorticoid resistance in patients with ulcerative colitis.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62
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