【摘要】：[目的]探尋糖皮質(zhì)激素抵抗的潰瘍性結(jié)腸炎患者不同組織中差異表達的microRNA,并通過生物信息學(xué)分析尋找其與糖皮質(zhì)激素抵抗相關(guān)靶基因、信號通路之間可能存在的調(diào)控網(wǎng)絡(luò);再從細胞實驗入手初步探索目標(biāo)microRNA的功能,進而從分子生物學(xué)層面探討炎癥性腸病患者糖皮質(zhì)激素抵抗的預(yù)測因子和發(fā)病機制。[方法]本課題分為兩部分:第一部分:首先,治療前收集潰瘍性結(jié)腸炎激素敏感患者血清標(biāo)本9份,激素抵抗患者血清標(biāo)本9份,采用microRNA PCR芯片技術(shù)檢測血清中差異表達的microRNAs,通過生物信息學(xué)分析進行靶基因的預(yù)測和靶基因GeneOntology(GO)分析,靶基因pathway分析,尋找與潰瘍性結(jié)腸炎激素抵抗相關(guān)的microRNAs及其靶基因和信號通路。再者,治療前收集潰瘍性結(jié)腸炎患者血清76份(激素敏感組39份,激素抵抗組37份),腸粘膜標(biāo)本30份(激素敏感組18份,激素抵抗組12份),采用實時熒光定量PCR (qRT-PCR)驗證microRNA PCR芯片篩選出的microRNAs的可靠性。最后,采用ROC曲線分析差異microRNAs預(yù)測潰瘍性結(jié)腸炎患者糖皮質(zhì)激素抵抗的相關(guān)性。第二部分:通過miRNAmimic-lipo2000復(fù)合物轉(zhuǎn)染THP-1細胞培養(yǎng)24h,運用熒光定量PCR檢測轉(zhuǎn)染后THP-1細胞內(nèi)miR-150的表達水平,確認miR-150轉(zhuǎn)染成功。然后經(jīng)佛波酯(PMA) 160nM誘導(dǎo)24h使THP-1細胞分化為巨噬細胞后,使用脂多糖(LPS) 100ng/ml刺激細胞1h,再加入地塞米松(Dex) 10μM處理 24h。實驗分為 6 組:Ctrl 組、miR-NC 組、miR-150 組、Ctrl+LPS+Dex 組、miR-NC+LPS+Dex組、miR-150+LPS+Dex組。先通過熒光定量PCR檢測各組別細胞中HSP90和HSF1 mRNA的表達水平;其次通過western blot檢測各組別細胞中 MAPK 家族成員(p38/p-p38、JNK/p-JNK、ERK1/2/p-ERK1/2、MEK/p-MEK、p-AKT/t-AKT)的蛋白表達水平;再通過ELISA檢測各組別細胞培養(yǎng)基上清中IL-10, IL-2, IL-17, TNF-α等炎癥因子的表達水平;最后使用流式細胞技術(shù)檢測各組別細胞表面TLR4的表達水平,從激素抵抗機制的各個環(huán)節(jié)來尋找miR-150可能的作用環(huán)節(jié),從而證明miR-150在潰瘍性結(jié)腸炎患者激素抵抗中的可能機制。[結(jié)果]第一部分:1.激素抵抗組和激素敏感組性別、年齡、Mayo評分和EBV及CMV感染情況均無明顯統(tǒng)計學(xué)差異(P 0.05)。2.microRNA PCR芯片篩選結(jié)果。以Fold change2為顯著差異性,激素抵抗組和激素敏感組相比較篩選出128個差異表達的microRNAs,其中50個microRNAs表達上調(diào),78個microRNAs表達下調(diào)。進一步分析發(fā)現(xiàn)僅有16個表達下調(diào)的microRNAs具有統(tǒng)計學(xué)顯著差異性(P 0.05; Fold change平均4.58倍);而表達上調(diào)的50個microRNAs均無統(tǒng)計學(xué)意義(P 0.05)。3.Gene Ontology分析(GO分析)結(jié)果。上調(diào)的microRNAs在生化過程方面主要參與解剖形態(tài)結(jié)構(gòu)的變化,在細胞組分方面主要參與細胞內(nèi)成分的變化,在分子功能方面主要蛋白結(jié)合過程;下調(diào)的microRNAs在生化過程方面主要參與細胞代謝過程的調(diào)節(jié),在細胞組分方面主要細胞內(nèi)部分的變化,在分子功能方面主要蛋白結(jié)合過程。4.Pathway分析結(jié)果。上調(diào)的microRNAs與43條信號通路有關(guān),其中相關(guān)性最高的是"Neurotrophin signaling pathway";下調(diào)的 microRNAs 與 116 條信號通路有關(guān),其中相關(guān)性最高的是“Pathways in cancer"。值得注意的是在與下調(diào)的microRNAs相關(guān)的信號通路中“PI3K-Akt signaling pathway” 和 “MAPK signaling pathway"與糖皮質(zhì)激素抵抗是相關(guān)的。5.靶基因預(yù)測結(jié)果。在綜合芯片結(jié)果和pathway分析的基礎(chǔ)上,篩選出8個下調(diào)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)作為目標(biāo) microRNAs 進行靶基因預(yù)測(fold changes 2.0 and P 0.05),結(jié)果發(fā)現(xiàn) HSP90B1,MAPK13,MAPK14, MAPK9, PIK3AP1,TLR4等靶基因與糖皮質(zhì)激素抵抗有關(guān)。5.差異microRNAs在血清中檢測結(jié)果。激素抵抗組和激素敏感組相比較,miR-16-2-3p, miR-30e-3p, miR-32-5p,miR-642a-5p, miR-150-5p,miR-224-5p 的表達水平是顯著降低并具有統(tǒng)計學(xué)意義的(P0.05)。而miR-425-5p和miR-486-3p的表達差異不具有統(tǒng)計學(xué)意義(P 0.05)。6.差異microRNAs在腸粘膜中檢測結(jié)果。激素抵抗組和激素敏感組相比較,miR-16-2-3p,miR-642a-5p,miR-150-5p,miR-224-5p 的表達水平是顯著降低并具有統(tǒng)計學(xué)意義的(P 0.05)。而 miR-30e-3p,miR-32-5p,miR-425-5p 和miR-486-3p的表達差異不具有統(tǒng)計學(xué)意義(P 0.05)。7.ROC曲線分析結(jié)果。選取血清檢測結(jié)果中的差異表達的microRNAs進行ROC 曲線分析。miR-16-2-3p,miR-30e-3p,miR-32-5p,miR-642a-5p, miR-150-5p,miR-224-5p 的曲線下面積(AUC)分別是 0.94 (95% 可信區(qū)間[CI] 0.891-0.986, P0.0001),0.93 (95% 可信區(qū)間[CI] 0.878-0.983,P 0.0001),0.85 (95% 可信區(qū)間[CI] 0.766-0.932,P 0.0001), 0.87 (95% 可信區(qū)間[CI] 0.794-0.950,P0.0001),0.92(95% 可信區(qū)間[CI] 0.858-0.974, P0.0001),0.99(95% 可信區(qū)間[CI] 0.968-1.00,P 0.0001);特異性分別是 97.30%,89.20%,59.50%,73.00%, 97.30%, 97.30%;敏感性分別是 74.40%, 84.60%, 97.40%, 92.30%,66.70%, 89.70%。第二部分:1.各組THP-1細胞中HSP90和HSF1 mRNA表達水平。分析結(jié)果進行組間比較,miR-150 組與 Ctrl 組和 miR-NC 組、miR-150+LPS+Dex 組與 Ctrl+LPS+Dex組和miR-NC+LPS+Dex組間相比較時HSP90的mRNA表達水平均無明顯變化(P 0.05 ),表明miR-150沒有調(diào)節(jié)HSP90的mRNA表達的作用。與此同時通過Western blot對處理前后的THP-1細胞中HSP90蛋白的表達水平進行了檢測發(fā)現(xiàn)各組間比較發(fā)現(xiàn)miR-150組HSP90的表達水平較miR-NC組有下降,但不具有統(tǒng)計學(xué)意義(P= 0.094, 0.05)。而各組別中HSF1 mRNA表達水平進行組間比較未發(fā)現(xiàn)顯著性差異(P 0.05)。2.各組別 THP-1 細胞中 p38/p-p38、JNK/p-JNK、ERK1/2/p-ERK1/2、MEK/p-MEK、p-AKT/t-AKT)的蛋白表達水平。經(jīng)western blot檢測后利用Image J條帶灰度分析進行結(jié)果分析,LPS誘導(dǎo)前后相比較(Ctrl+LPS+Dex組與Ctrl組、miR-NC+LPS+Dex 組與 miR-NC 組、miR-150+LPS+Dex 組與 miR-150 組),p38、JNK、ERK1/2、MEK、t-AKT蛋白的表達水平無明顯變化(P0.05),而LPS誘導(dǎo)后p38、JNK和ERK1/2的磷酸化水平均有明顯升高,這種升高具有統(tǒng)計學(xué)意義(P 0.05 )。而在 miR-150+LPS+Dex 組與 Ctrl+LPS+Dex 組和miR-NC+LPS+Dex組相比較時發(fā)現(xiàn),p38和Erk1/2的磷酸化水平卻顯著降低,這種差異也具有統(tǒng)計學(xué)意義(P 0.05)。3.各組別THP-1細胞中培養(yǎng)基上清中IL-10, IL-2, IL-17, TNF-α等炎癥因子的表達水平。在LPS誘導(dǎo)炎癥反應(yīng)前,Ctrl組、miR-NC組、miR-150組中各個炎癥因子的分泌水平均較低,各組間的水平均無顯著性差異(P均0.05)。而在LPS誘導(dǎo)后IL-2和TNF- α的分泌水平較誘導(dǎo)前顯著升高,差異具有統(tǒng)計學(xué)意義(P 0.05 ),其中miR-150+LPS+Dex組IL-2和TNF- α的分泌水平較Ctrl+LPS+Dex組和miR-NC+LPS+Dex組均顯著降低,差異同樣具有統(tǒng)計學(xué)意義(P0.05)。另外,IL-10在miR-150+LPS+Dex組的分泌水平較其他各組是明顯增高的(P 0.05)。而IL-17在各組中分泌水平均較低,各組間的水平均無顯著性差異(P均0.05)。4.各組別THP-1細胞表面TLR4的表達水平。分析比較后發(fā)現(xiàn)經(jīng)LPS誘導(dǎo)后THP-1細胞表面TLR4的表達明顯上調(diào),對應(yīng)各組間(Ctrl組與Ctrl+LPS+Dex組、miR-NC 組與 miR-NC+LPS+Dex 組、miR-150 組與 miR-150+LPS+Dex 組)相比較均顯著升高且具有統(tǒng)計學(xué)意義(P 0.05);而miR-150+LPS+Dex組THP-1細胞表面TLR4的表達較Ctrl+LPS+Dex組和miR-NC+LPS+Dex組顯著降低,這種差異具有統(tǒng)計學(xué)意義(p 0.05)。[結(jié)論]1.糖皮質(zhì)激素抵抗的潰瘍性結(jié)腸炎患者血清中存在16個表達下調(diào)的microRNAs具有統(tǒng)計學(xué)顯著差異性。經(jīng)過生物信息學(xué)分析有可能參與糖皮質(zhì)激素抵抗的有 8 個 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)。差異表達的 microRNAs 可能參與調(diào)節(jié)的糖皮質(zhì)激素抵抗的靶基因有HSP90B1,MAPK13, MAPK14,MAPK9,PIK3AP1,TLR4;信號通路有“PI3K-Akt signaling pathway” 和 “MAPK signaling pathway"。2.經(jīng)擴大樣本檢測,糖皮質(zhì)激素抵抗的潰瘍性結(jié)腸炎患者血清中表達下調(diào)的microRNAs 共有 6 個(miR-16-2-3p,miR-30e-3p, miR-32-5p,miR-642a-5p,miR-150-5p,miR-224-5p),對預(yù)測潰瘍性結(jié)腸炎患者的糖皮質(zhì)激素抵抗有一定的價值。3.腸粘膜中表達下調(diào)的 microRNAs 共有 4 個(miR-16-2-3p,miR-642a-5p,miR-150-5p,miR-224-5p)。為進一步研究潰瘍性結(jié)腸炎患者糖皮質(zhì)激素抵抗的分子生物學(xué)機制提供了理論基礎(chǔ)。4.細胞實驗證實miR-150升高可提高糖皮質(zhì)激素治療的敏感性,可能是通過調(diào)節(jié)p38 MAPK和ERK1/2的磷酸化水平,或細胞表面TLR4表達水平等關(guān)鍵環(huán)節(jié)來實現(xiàn)的。為潰瘍性結(jié)腸炎患者糖皮質(zhì)激素抵抗的分子生物學(xué)機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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R574.62

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