子宫内膜异位症组蛋白修饰异常及血清microRNA表达异常的研究
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摘要
第一部分子宫内膜异位症组蛋白修饰异常的研究
目的研究子宫内膜异位症(endometriosis, EMs)患者异位内膜及在位内膜组蛋白乙酰化和甲基化状态,组蛋白乙酰化酶(HATs),组蛋白去乙酰化酶(HDACs)及组蛋白甲基转移酶(HMTs)在异位内膜及在位内膜中的表达及其与基因组乙酰化和甲基化水平之间的相互关系。
方法利用EpiQuikTMC H3K4/H3K9甲基化及H3/H4乙酰化)试剂盒对15例EMs患者异位内膜、在位内膜及11例正常对照组内膜进行组蛋白抽提和H3/H4乙酰化,H3K4/H3K9甲基化检测,同时通过Real-time PCR检测HATs(P300,CREBBP和PCAF), HDACs (HDAC1, HDAC2, HDAC4, HDAC5, HDAC7和SIRT1)及HMTs (SUV39H1, SUV39H2和G9a)的mRNA的表达水平。
结果EMs患者在位内膜及异位内膜组蛋白H4整体乙酰化水平明显低于正常对照组(p=0.005),而组蛋白H3整体乙酰化水平三组之间无明显统计学差异(p>0.05)。在位内膜及异位内膜组蛋白H3K4甲基化总水平明显低于正常对照组(p<0.001),异位内膜H3K9甲基化水平明显低于在位内膜组及正常对照组(p<0.001),另两组间无明显统计学差异(p>0.05)。在HATs中,三组之间p300表达水平均无明显统计学差异(p>0.05),CREBBP在位内膜组表达明显高于异位内膜组(p=0.001),PCAF异位内膜组高于正常对照组及在位内膜组(p=0.047,p=0.005),而另两组间无明显统计学差异(p>0.05)。在HDACs中,HDAC4、HDAC5、HDAC7表达水平三个组之间无明显统计学差异(p>0.05),HDAC1异位内膜组表达水平明显低于正常对照组(p=0.006),在位内膜组与其余两组间均无明显统计学差异(p>0.05),HDAC2在位内膜组表达均明显高于正常对照组及异位内膜组(p<0.001,p<0.001),另两组间无统计学意义,SIRT1在位内膜组表达低于正常对照组及异位内膜组(p=0.037,p=0.039),另两组之间无明显统计学差异(p>0.05)。在HMTs中,SUV39H1异位内膜组表达明显低于正常对照组(p=0.006),其余各组间无明显统计学意义,SUV39H2异位内膜组表达水平明显低于正常对照组及在位内膜组(p=0.002,p=0.012),且另两组间表达水平无统计学差异(p>0.05),G9a异位内膜组表达均低于正常对照组(p<0.001),余各组间无明显统计学差异(p>0.05)。
结论EMs患者在位和异位内膜组织组蛋白呈低乙酰化和低甲基化状态,组蛋白乙酰化酶,去乙酰化酶和甲基化酶表达异常,提示其可能参与EMs的发病。
第二部分子宫内膜异位症患者血清microRNA表达的变化
目的研究子宫内膜异位症患者血清中microRNA (miRNA)表达的变化。
方法分离9例子宫内膜异位症患者和9例正常对照者血清,用Trizol试剂提取总RNA及1miRNA,应用高通量miRNA测序方法检测血清中异常表达的miRNA,并应用Real-time PCR法对30例子宫内膜异位症患者和20例正常对照者血清扩大样本进行初步验证。
结果和正常对照组相比较,EMs组中98个miRNAs表达显著下调,1O个miRNAs表达显著上调。挑选EMs组中表达下调的3个miRNAs分子hsa-miR-99b-5p、hsa-miR-127-3p、hsa-miR-30c-5p和表达上调的2个miRNAs分子hsa-miR-424-3p和hsa-miR-185-5p,通过Real-time PCR法进行验证。结果显示：与正常对照组相比较,hsa-miR-99b-5p、hsa-miR-127-3p、hsa-miR-30c-5p表达水平均显著下调,而hsa-miR-424-3p和hsa-miR-185-5p表达均显著上调(p<0.05)。
结论子宫内膜异位症患者血清中miRNA表达异常。
Part Ⅰ Aberrant histone acetylation and methylation levels in endometriosis
Objective To investigate the alterations in histone modifications in woman with endometriosis.
Methods Global histone H3/H4acetylation and H3K4/H3K9methylation in eutopic and ectopic endometrium from15endometriosis patients were assayed using the EpiQuikTM global histone H3/H4acetylation and H3K4/H3K9methylation assay kits. Quantitative real-time RT-PCR (reverse transcriptase-polymerase chain reaction) was applied to measure mRNA levels of12members of histone related chromatin modifier genes.
Results Histone H4hypoacetylation was detected both in eutopic and ectopic endometrium. There were no difference between patients with endometriosis and controls on global levels of H3acetylation. Furthermore, global histone H3K4hypomethylation and H3K9hypomethylation were detected both in ectopic and eutopic endometrium (p=0.000), and in ectopic endometrium (p=0.000), respectively. SIRT1mRNA level was significantly decreased in eutopic endometrium, while mRNA levels of HDAC1, SUV39H1, SUV39H2and G9a were significantly downregulated in ectopic endometrium. HDAC2mRNA level was significantly increased in eutopic endometrium. PCAF mRNA level was significantly increased in ectopic endometrium.
Conclusions Global histone acetylation and methylation were decreased and the expression levels of histone acetyltransferases, histone deacetylases and histone methyltransferases were aberrant in endometriosis, suggesting their possible involvement in the pathogenesis of endometriosis.
Part Ⅱ Abnormal microRNA expression in serum from patients with endometriosis
Objective To investigate the alterations of microRNA (miRNA) in serum from patient with endometriosis.
Methods Serum from9patients with endometriosis and9normal controls were extracted. Total RNA including miRNAs was extracted using Trizol reagent. MiRNAs with differential expression were detected by high-throughput miRNA sequencing method and the results were confirmed in30patients with endometriosis and20healthy controls by Real-time PCR.
Results The results showed that98miRNAs expression was downregulated and10miRNAs expression was significantly upregulated in serum from patients with endometriosis compared to normal controls. Downregulated molecules of miRNAs (hsa-miR-99b-5p, hsa-miR-127-3p, hsa-miR-30c-5p) and upregulated molecules of miRNAs (hsa-miR-424-3p and hsa-miR-185-5p) were seleted to confirmed in30patients and20healthy controls by Real-time PCR. Hsa-miR-99b-5p, hsa-miR-127-3p and hsa-miR-30c-5p expression level was significantly reduced, whereas hsa-miR-424-3p and hsa-miR-185-5p expression level were significantly upregulated in serum from endometriosis patients (p<0.05)
Conclusions Aberrant miRNA expression may play a role in the pathogenesis of endometriosis.
目的研究子宫内膜异位症(endometriosis, EMs)患者异位内膜及在位内膜组蛋白乙酰化和甲基化状态,组蛋白乙酰化酶(HATs),组蛋白去乙酰化酶(HDACs)及组蛋白甲基转移酶(HMTs)在异位内膜及在位内膜中的表达及其与基因组乙酰化和甲基化水平之间的相互关系。
方法利用EpiQuikTMC H3K4/H3K9甲基化及H3/H4乙酰化)试剂盒对15例EMs患者异位内膜、在位内膜及11例正常对照组内膜进行组蛋白抽提和H3/H4乙酰化,H3K4/H3K9甲基化检测,同时通过Real-time PCR检测HATs(P300,CREBBP和PCAF), HDACs (HDAC1, HDAC2, HDAC4, HDAC5, HDAC7和SIRT1)及HMTs (SUV39H1, SUV39H2和G9a)的mRNA的表达水平。
结果EMs患者在位内膜及异位内膜组蛋白H4整体乙酰化水平明显低于正常对照组(p=0.005),而组蛋白H3整体乙酰化水平三组之间无明显统计学差异(p>0.05)。在位内膜及异位内膜组蛋白H3K4甲基化总水平明显低于正常对照组(p<0.001),异位内膜H3K9甲基化水平明显低于在位内膜组及正常对照组(p<0.001),另两组间无明显统计学差异(p>0.05)。在HATs中,三组之间p300表达水平均无明显统计学差异(p>0.05),CREBBP在位内膜组表达明显高于异位内膜组(p=0.001),PCAF异位内膜组高于正常对照组及在位内膜组(p=0.047,p=0.005),而另两组间无明显统计学差异(p>0.05)。在HDACs中,HDAC4、HDAC5、HDAC7表达水平三个组之间无明显统计学差异(p>0.05),HDAC1异位内膜组表达水平明显低于正常对照组(p=0.006),在位内膜组与其余两组间均无明显统计学差异(p>0.05),HDAC2在位内膜组表达均明显高于正常对照组及异位内膜组(p<0.001,p<0.001),另两组间无统计学意义,SIRT1在位内膜组表达低于正常对照组及异位内膜组(p=0.037,p=0.039),另两组之间无明显统计学差异(p>0.05)。在HMTs中,SUV39H1异位内膜组表达明显低于正常对照组(p=0.006),其余各组间无明显统计学意义,SUV39H2异位内膜组表达水平明显低于正常对照组及在位内膜组(p=0.002,p=0.012),且另两组间表达水平无统计学差异(p>0.05),G9a异位内膜组表达均低于正常对照组(p<0.001),余各组间无明显统计学差异(p>0.05)。
结论EMs患者在位和异位内膜组织组蛋白呈低乙酰化和低甲基化状态,组蛋白乙酰化酶,去乙酰化酶和甲基化酶表达异常,提示其可能参与EMs的发病。
第二部分子宫内膜异位症患者血清microRNA表达的变化
目的研究子宫内膜异位症患者血清中microRNA (miRNA)表达的变化。
方法分离9例子宫内膜异位症患者和9例正常对照者血清,用Trizol试剂提取总RNA及1miRNA,应用高通量miRNA测序方法检测血清中异常表达的miRNA,并应用Real-time PCR法对30例子宫内膜异位症患者和20例正常对照者血清扩大样本进行初步验证。
结果和正常对照组相比较,EMs组中98个miRNAs表达显著下调,1O个miRNAs表达显著上调。挑选EMs组中表达下调的3个miRNAs分子hsa-miR-99b-5p、hsa-miR-127-3p、hsa-miR-30c-5p和表达上调的2个miRNAs分子hsa-miR-424-3p和hsa-miR-185-5p,通过Real-time PCR法进行验证。结果显示：与正常对照组相比较,hsa-miR-99b-5p、hsa-miR-127-3p、hsa-miR-30c-5p表达水平均显著下调,而hsa-miR-424-3p和hsa-miR-185-5p表达均显著上调(p<0.05)。
结论子宫内膜异位症患者血清中miRNA表达异常。
Part Ⅰ Aberrant histone acetylation and methylation levels in endometriosis
Objective To investigate the alterations in histone modifications in woman with endometriosis.
Methods Global histone H3/H4acetylation and H3K4/H3K9methylation in eutopic and ectopic endometrium from15endometriosis patients were assayed using the EpiQuikTM global histone H3/H4acetylation and H3K4/H3K9methylation assay kits. Quantitative real-time RT-PCR (reverse transcriptase-polymerase chain reaction) was applied to measure mRNA levels of12members of histone related chromatin modifier genes.
Results Histone H4hypoacetylation was detected both in eutopic and ectopic endometrium. There were no difference between patients with endometriosis and controls on global levels of H3acetylation. Furthermore, global histone H3K4hypomethylation and H3K9hypomethylation were detected both in ectopic and eutopic endometrium (p=0.000), and in ectopic endometrium (p=0.000), respectively. SIRT1mRNA level was significantly decreased in eutopic endometrium, while mRNA levels of HDAC1, SUV39H1, SUV39H2and G9a were significantly downregulated in ectopic endometrium. HDAC2mRNA level was significantly increased in eutopic endometrium. PCAF mRNA level was significantly increased in ectopic endometrium.
Conclusions Global histone acetylation and methylation were decreased and the expression levels of histone acetyltransferases, histone deacetylases and histone methyltransferases were aberrant in endometriosis, suggesting their possible involvement in the pathogenesis of endometriosis.
Part Ⅱ Abnormal microRNA expression in serum from patients with endometriosis
Objective To investigate the alterations of microRNA (miRNA) in serum from patient with endometriosis.
Methods Serum from9patients with endometriosis and9normal controls were extracted. Total RNA including miRNAs was extracted using Trizol reagent. MiRNAs with differential expression were detected by high-throughput miRNA sequencing method and the results were confirmed in30patients with endometriosis and20healthy controls by Real-time PCR.
Results The results showed that98miRNAs expression was downregulated and10miRNAs expression was significantly upregulated in serum from patients with endometriosis compared to normal controls. Downregulated molecules of miRNAs (hsa-miR-99b-5p, hsa-miR-127-3p, hsa-miR-30c-5p) and upregulated molecules of miRNAs (hsa-miR-424-3p and hsa-miR-185-5p) were seleted to confirmed in30patients and20healthy controls by Real-time PCR. Hsa-miR-99b-5p, hsa-miR-127-3p and hsa-miR-30c-5p expression level was significantly reduced, whereas hsa-miR-424-3p and hsa-miR-185-5p expression level were significantly upregulated in serum from endometriosis patients (p<0.05)
Conclusions Aberrant miRNA expression may play a role in the pathogenesis of endometriosis.
引文
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