慢性乙型肝炎microRNA诊断标志物及其与乙型肝炎病毒复制相关性研究CSCD

目的筛选慢性乙型肝炎（慢乙肝）microRNA（miRNA）诊断标志物,并揭示miRNA与乙型肝炎病毒（HBV）复制的相关性。方法采用荧光定量PCR法,对50例慢乙肝患者和50例健康人血浆中10条miRNA进行定量检测,通过分析表达水平差异,获得慢乙肝miRNA诊断标志物,建立回归诊断模型。斯皮尔曼等级相关性分析方法对miRNA表达量及HBV-DNA病毒载量进行相关性分析。结果8条miRNA在慢乙肝患者和健康人间的表达水平差异显著,其中miR-451a在慢乙肝患者中表达水平显著下降,其他7条miRNA表达水平显著升高。选择miR-122-5p、miR-27a-3p和miR-451a建立回归模型,曲线下面积（AUC）达到1,敏感性和特异性均达到100%,具有显著诊断价值。分析miRNA与HBV的相关性,发现miR-122-5p与HBV病毒载量呈正相关。结论miR-122-5p、miR-27a-3p和miR-451a在慢乙肝的发生与发展中起到重要作用,可作为慢乙肝诊断的新型标志物。     

食管鳞癌肿瘤微环境中miRNA与TGF-β1的相关性分析

目的 检测mir-18a-5p、mir-23a-3p、mir-24-3p、mir-25-3p和TGF-β1在食管鳞癌(ESCC)患者肿瘤微环境中的表达水平,分析它们在ESCC发生发展中的作用及相互关系.方法 收集52例ESCC患者癌组织、癌旁组织手术标本;实时荧光定量PCR检测ECA-109、TE-1细胞株、正常食管上皮细胞和52例ESCC患者手术标本的mir-18a-5p、mir-23a-3p、mir-24-3p、mir-25-3p和TGF-β1的mRNA表达水平;Western blot法检测TGF-β1在上述细胞以及组织中的蛋白表达情况.结果 与正常食管上皮细胞相比,mir-18a-5p、mir-24-3p、mir-25-3p、mir-23a-3p在ESCC细胞株ECA-109中的表达明显增高(P＜0.05),其中前3种miRNA在ESCC细胞株TE-1的表达明显增高(P＜0.05);TGF-β1在ESCC细胞株ECA-109、TE-1中明显低表达(P＜0.05).与癌旁组织对照组相比,52例ESCC患者癌组织标本的mir-18a-5p、mir-23a-3p、mir-24-3p、mir-25-3p显著升高[上调比例分别为86.5％ (45/52)、63.5％ (33/52)、78.8％(41/52)、86.5％(45/52),P＜0.05],TGF-β1显著降低(P＜0.05).ESCC患者mir-18a-5p、mir-23a-3p、mir-24-3p、mir-25-3p的高表达和TGF-β1的低表达与患者性别、年龄、肿瘤大小、肿瘤位置等无明显关系,但mir-18a-5p、mir-23a-3p与患者癌组织分化程度相关,mir-25-3p与患者癌组织分化程度、浸润深度和范围相关(P＜0.05).癌组织中TGF-β1的低表达与患者癌组织分化程度、浸润深度和范围相关(P＜0.05).mir-18a-5p、mir-23 a-3p与TGF-β1的表达无相关性,mir-24-3p、mir-25-3p与TGF-β1的表达呈负相关.结论 ESCC肿瘤微环境中mir-18a-5p、mir-23a-3p、mir-24-3p、mir-25-3p和TGF-β 1可能在ESCC的发生发展中有一定作用.     

血清外泌体miRNAs在鉴别诊断子宫内膜重度不典型增生和高分化子宫内膜癌中作用研究

目的 研究血清外泌体miRNAs表达谱对子宫内膜重度不典型增生和高分化子宫内膜癌的鉴别诊断价值。方法 前瞻性研究2020年6月至2021年6月期间来我院就诊的子宫内膜重度不典型增生（n=9）和高分化子宫内膜癌组患者（n=9）,以及同期正常子宫内膜组（n=3）,通过miRNA微阵列芯片筛选差异性表达的血清外泌体miRNAs,荧光实时定量RT-PCR进一步检测微阵列芯片已筛选出显著差异的血清外泌体miRNAs,ROC曲线检测血清外泌体miRNA在子宫内膜重度不典型增生和高分化子宫内膜癌鉴别诊断效能。结果 通过微阵列芯片分析,本研究发现8个在高分化子宫内膜癌与子宫内膜正常中差异表达的血清外泌体miRNA（如miRNA-31、miRNA-92a、miRNA-115、miRNA-125、miRNA-142-3p、miRNA-155、miRNA-21和miRNA-451等表达水平不同）;与子宫内膜重度不典型增生相比,高分化子宫内膜癌患者血清外泌体中表达较低的miRNA（如miRNA-26、miRNA-221、miRNA-518d-5p和miRNA-708）,表达较高的miRNA(miRNA-10a...     

结外NK/T细胞淋巴瘤（鼻型）microRNA的表达研究

目的 通过分析结外NK/T细胞淋巴瘤（鼻型）肿瘤组织中microRNA( miRNA)的表达情况,探索其分子改变特征。方法 对1例结外NK/T细胞淋巴瘤（鼻型）和1例炎性鼻黏膜组织应用TaqMan低密度芯片分析665种miRNA的表达差异,发现95种miRNA存在变化。结合文献复习和数据库检索,从其中筛选差异明显、重要的8种miRNA。应用即时定量聚合酶链反应方法进一步对15例结外NK/T细胞淋巴瘤（鼻型）与3例炎性鼻黏膜组织验证这8种miRNA的表达情况。结果 8种miRNA验证结果与表达谱结果对比表达趋势一致,经统计学处理,其中3种重要miRNA[miR-223和miR-886-3p在结外NK/T细胞淋巴瘤（鼻型）组织中表达上调,miR-34c-5p表达下调]表达差异有统计学意义（P值分别为0.002、0.010和0.017）。结论 造血系分化发育、细胞增殖凋亡有关的miRNA：miR-223、886-3p和34c-5p在结外NK/T细胞淋巴瘤（鼻型）表达有明显异常,其是否参与肿瘤分子遗传学的改变及其意义值得深入探讨。     

以IGF-1、NOS、FGF23和硬骨素为靶基因的骨细胞力学响应微小RNA的筛选

目的 筛选骨细胞分泌因子相关力学响应微小RNA(microRNA, miRNA)。方法 分别向体外培养骨细胞和成骨细胞施加周期性张应变（ε=2.5,f=0.5 Hz）,采用miRNA芯片筛选出仅在骨细胞中差异表达的miRNA。通过生物信息学技术,从这些miRNA中进一步筛选出靶基因为胰岛素样生长因子1（insulin-like growth factor-1）、NO合成酶（nitric oxide synthesase, NOS）、成纤维细胞生长因子23(fibroblast growth factor 23, FGF23)和硬骨素(sclerostin, SOST)等分泌因子的miRNA,与小鼠跑台锻炼后芯片检测出的小鼠股骨组织差异表达miRNA进行比较,然后随机选4个miRNA进行定量PCR验证。结果 仅在体外经力学刺激的骨细胞中差异表达的77个miRNA中,筛选出22个靶基因为4种分泌因子（IGF-1、NOS、FGF23、SOST）的miRNA,并进一步筛选出11个在跑台锻炼后的小鼠骨组织中以同样趋势差异表达的miRNA,其中随机选出的miR-361-3p、miR-3082-5p、miR-6348和miR-706,也在骨细胞和骨组织中以同样趋势同样差异表达。结论 诸如miR-361-3p、miR-3082-5p、miR-6348和miR-706仅在骨细胞中差异表达的力学响应miRNA,很可能通过调节分泌因子影响成骨分化或骨代谢。     

2型糖尿病患者血浆microRNA差异表达及生物信息学分析

目的:构建2型糖尿病(type 2 diabetes mellitus,T2DM)患者血浆microRNA(miRNA)差异表达谱,探讨miRNA作为T2DM诊疗靶标的可能性。方法:采用miRNA芯片技术检测T2DM患者血浆中miRNA差异表达情况,用实时荧光定量PCR(real-time quantitative p...     

采用非标记microRNA芯片筛选早产胎盘差异表达microRNA

目的应用非标记microRNA芯片筛选在早产胎盘组织中差异表达的miRNA,并分析其相关的靶基因。方法收集早产儿16例为病例组,同期健康足月产儿18例为对照组,取胎盘组织。通过非标记miRNA芯片技术构建早产胎盘组织miRNA表达谱,采用荧光实时定量PCR进行验证,并分析差异表达miRNAs的靶基因。结果 miRNA芯片结果显示44种miRNAs在早产胎盘组织和对照组胎盘组织中差异表达,其中下调表达的11种,上调表达的33种,荧光实时定量PCR验证出miR-493-3p显著下调,mi R-4632-5p显著上调,与芯片结果一致。经软件分析,miRNA靶基因有IL16、SH2D2A、CCNG2、PHB、WNT1、CCDC92、GIT1和ST7L等相关靶基因。结论早产胎盘组织中存在差异表达的miRNAs,提示这些差异表达的miRNAs在早产的发生中有重要的调控作用。     

心房颤动患者心房组织中差异表达miRNA的初步研究

目的：应用基因芯片技术研究心房颤动（房颤）患者心房组织中miRNA的表达谱,分析差异表达的miRNA,为进一步研究miRNA在房颤发生、发展中的作用奠定基础。方法：采用miRNA基因芯片技术检测房颤患者和非房颤患者心房组织样本中miRNA的表达水平;采用实时定量PcR（quantitativereal,timePCR,qRT．PCR）对部分差异表达的miRNA进行验证。结果：MiRNA基因芯片分析结果显示,与非房颤组织相比,房颤组织中差异表达的miRNA共有26个,其中16AmiRNA表达上调,10个miRNA表达下调。qRT．PCR验证结果与芯片结果相一致。结论：MiRNA在房颤患者心房组织中存在差异性表达。     

血浆mir-142-5p 与冠状动脉粥样硬化程度的关系及其作用机制的研究

目的:探讨血浆mir-142-5p与冠状动脉粥样硬化程度的关系及其作用机制。方法:选择42例冠心病患者和45例健康志愿者,冠心病组均进行Gensini评分,抽取空腹静脉血后应用RT-PCR法检测血浆mir-142-5p水平;应用mir-142-5p minics和inhibitor对ox-LDL处理的血管内皮细胞进行转染,Western blot法检测内皮细胞IGF-1R蛋白的表达情况,RT-PCR检测IGF-1R的相对表达量,硝酸盐还原酶法检测细胞培养上清液中NO的含量。结果:(1)冠心病组血浆mir-142-5p水平高于健康对照组(P0. 05);(2)冠心病组血浆mir-142-5p水平和Gensini评分呈正相关(P0. 01);(3)ox-LDL处理后内皮细胞IGF-1R的表达和NO的合成升高(P0. 01); mir-142-5p inhibitor转染的内皮细胞IGF-1R表达上调(P0. 01)、NO的合成升高(P0. 05),而mir-142-5p minics转染的内皮细胞IGF-1R表达下调(P0. 01)、NO的合成减少(P0. 05)。结论:冠心病患者血浆mir-142-5p水平较健康人群升高,且其升高程度可反映冠脉病变程度; mir-142-5p通过靶定IGF-1R基因损伤血管内皮功能,可能具有促进动脉硬化进展的作用。     

基于microRNA差异表达探讨恶性纤维组织细胞瘤致病机制

目的 应用芯片表达分析技术探讨miRNA在恶性纤维组织细胞瘤发病机制中的作用.方法 利用miRCURYTM LNA miRNA表达谱芯片技术高通量筛选NMFH1与MSC、HSF、MRC-5和HT-1080细胞系差异表达miRNA,筛选靶基因,分析调控作用机制.结果 筛查NMFH1与4种对照组细胞系差异表达miRNA,选取与所有正常样本对比均下调miR-222、miR-186及miR-933,均上调miR-886-3p、miR-886-5p、miR-183、miR-340及miR-BART7进行qRT-PCR实验,验证芯片数据可靠性.基于生物信息学方法挖掘miRNA靶基因,构建基因调控网络,基于功能组注释信息分析NMFH1风险疾病基因及通路,揭示MFH恶性病变机制.结论 miRNA在肿瘤样本与正常细胞系间显著差异表达,是参与MFH发生发展过程的重要调控因子,发挥负调控基因表达作用,使肿瘤细胞逃避正常生长调控机制,无限增殖和转移,出现恶性表型.     

Medpor regulates osteoblast's microRNAs

Porous polyethylene (PP or Medpor) is an alloplastic material worldwide used for craniofacial reconstruction. Although several clinical studies are available, there is a lack as regard the genetic effects. Because PP is always fixed on bone and the mechanism by which PP acts on osteoblasts is unknown, we therefore attempted to address this question by using microRNA microarray techniques to investigate the translation regulation in osteoblasts exposed to PP. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide microRNA profiling in human samples.By using miRNA microarrays containing 329 probe designed from Human miRNA sequence, we identified in osteoblast-like cells line (MG-63) cultured with Medpor (Porex Corporation, Fairburn, Georgia, USA) several miRNA which expression is significantly modified.We identified 16 up-regulated miRNA (i.e. mir-337, mir-515-3p, mir-377, mir-153, mir-367, mir-152, let-7b, mir-92, mir-155, mir-424, mir-148b, mir-368, mir-18b, mir-520d, mir-20b, mir-128a) and 2 down-regulated miRNA (i.e. mir-143, mir-32).The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to PP. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.     

Skewed X-chromosome inactivation is associated with primary but not secondary ovarian failure

Premature ovarian failure (POF) is the occurrence of menopause before the age of 40, and may present with either primary or secondary amenorrhea. Numerous cases of POF in women with X-chromosome deletions or translocations have been reported; thus, it is possible that smaller rearrangements undetectable by conventional cytogenetics may contribute to POF in some patients. In females with an abnormal X chromosome, cells with inactivation of the normal X may be selected against, causing skewed X-chromosome inactivation (XCI). We therefore assessed XCI by methylation sensitive restriction digestion and PCR amplification at the androgen receptor (AR) locus, in 4 primary and 55 secondary POF patients and 109 control women. In samples heterozygous at AR and therefore informative for the skewing assay, the frequency of skewed XCI among the women with secondary amenorrhea was identical to that in control women, with 4 out of 48 (8.3%) secondary ovarian failure patients and 8 out of 97 (8.2%) control women having > or =90% skewing. Notably, all three primary amenorrhea patients that were informative at AR had skewed XCI > or =90% (P = 0.001 vs. control women; Fisher's exact test). To investigate whether X-chromosome copy number alterations were responsible, DNA from selected patients with skewed XCI was examined by high resolution DNA microarray, however no potential regions of DNA addition or deletion were confirmed by FISH or PCR. X-chromosome abnormalities undetectable by array, or reduced follicular pool due to an early trisomic rescue event, may explain the skewed XCI observed in POF patients presenting with primary amenorrhea.     

Serum anti-Müllerian hormone expression in women with premature ovarian failure

BACKGROUND: Premature ovarian failure (POF) is generally irreversible. However, developing follicles up to the antral stage are reported in POF and anti-Müllerian hormone (AMH) might be a good indicator of follicular presence. This study analysed serum AMH, ovarian histology and AMH immunoexpression in POF patients. METHODS: A cross-sectional study of 48 POF patients in an Endocrinology Department setting. Patients had an ovarian biopsy simultaneously with serum AMH sampling and/or ovarian AMH immunostaining. RESULTS: Mean serum AMH was 1.04 +/- 1.66 ng/ml. Serum AMH was significantly higher in women with 15 or more follicles at ovarian histology (P = 0.001). Comparison of ovarian AMH immunostaining from POF patients and 10 normal controls revealed a normal AMH expression in POF pre-antral follicles, but a decreased expression at the early antral stages. Serum AMH was undetectable in 77% of the patients with 0-5 AMH immunopositive follicles and detectable in 100% of the patients with more than 15 AMH immunopositive follicles. CONCLUSIONS: AMH levels in POF patients could identify women with persistent follicles. The decrease of AMH immunoexpression in POF antral follicles could suggest a defect of antral development.     

Cryptic Xp duplication including the SHOX gene in a woman with 46,X, del(X)(q21.31) and premature ovarian failure

BACKGROUND: Premature ovarian failure (POF) is defined as amenorrhoea for >6 months, occurring before the age of 40, with an FSH serum level in the menopausal range. Although Xq deletions have been known for a long time to be associated with POF, the mechanisms involved in X deletions in order to explain ovarian failure remain unknown. In order to look for potentially cryptic chromosomal imbalance, we used high-resolution genomic analysis to characterize X chromosome deletions associated with POF. METHODS: Three patients with POF presenting terminal Xq deletions detected by conventional cytogenetics were included in the study. Genome wide microarray comparative genomic hybridization (CGH) at a resolution of 1 Mb and fluorescence in situ hybridization (FISH) was performed. RESULTS: Microarray CGH and FISH studies characterized the three deletions as del(X)(q21.2), del(X)(q21.31) and del(X)(q22.33). Microarray CGH showed that the del(X)(q21.31) was also associated with a Xpter duplication including the SHOX gene. In these patients with POF, deletions or duplications of autosomes have been excluded. CONCLUSION: This study is the first one using microarray in patients with POF. It demonstrates that putative X chromosome deletions can be associated with other chromosomal imbalances such as duplications, and therefore illustrates the use of microarray CGH to screen chromosomal abnormalities in patients with POF.     

G769A variation of inhibin alpha-gene in korean women with premature ovarian failure

Premature ovarian failure (POF) is menopause before the age of 40 years. The frequency of POF is about 1% of all women. Recently inhibin alpha gene (INHalpha) has been indicated as candidate in POF pathogenesis. Inhibin, a glycoprotein, is a gonadal hormone, which can inhibit the synthesis and secretion of pituitary follicle-stimulating hormone (FSH), which has an important role in the recruitment and development of ovarian follicles during the folliculogenesis. G769A variation of INHalpha, alanine, is highly conserved across species, and has an important role of its receptor binding. We screened a G769A transition in the INHalpha from the total population of the patients of 84 women with POF and 100 normal fertile women. We found no variation between the normal subjects and the POF patients. G769A variation of INHalpha is rare in Korea women with POF.     

Inhibin: a candidate gene for premature ovarian failure

Premature ovarian failure (POF) occurs in 1% of all women, and in 0.1% of women under the age of 30 years. The mechanisms that give rise to POF are largely unknown. Inhibin has a role in regulating the pituitary secretion of FSH, and is therefore a potential candidate gene for ovarian failure. Using single-stranded conformation polymorphism (SSCP) and DNA sequencing, DNA samples were screened from 43 women with POF for mutations in the three inhibin genes. Two variants were found: a 1032C-->T transition in the INHssA gene in one patient, and a 769G-->A transition in the INHalpha gene in three patients. The INHssA variant appears to be a polymorphism, as there was no change in the amino acid sequence of the gene product. The INHalpha variant resulted in a non-conservative amino acid change, with a substitution from alanine to threonine. This alanine is highly conserved across species, and has the potential to affect receptor binding. The INHalpha variant is significantly associated with POF (3/43 patients; 7%) compared with control samples (1/150 normal controls; 0.7%) (Fisher's exact test, P < 0.035). Further analysis of the inhibin gene in POF patients and matched controls will determine its role in the aetiology of POF.     

Prevention of gonadal toxicity and preservation of gonadal function and fertility in young women with systemic lupus erythematosus treated by cyclophosphamide: the PREGO-Study

BACKGROUND: With dramatically improved survival rates of SLE patients, comorbidity and long-term damage such as premature ovarian failure (POF) gain increasing importance. In the Erlangen cohort, 14% of cyclophosphamide treated patients younger than 41 years have POF, which is a common consequence of cyclophosphamide treatment. PATIENTS AND METHODS: We tested the concentrations of FSH and LH, before, during and after cyclophosphamide treatment in 63 premenopausal women with SLE without ovarian protection and initiated the PREGO-Study (Prospective randomized study on protection against gonadal toxicity) in patients with SLE. RESULTS: In lupus patients treated with cyclophosphamide, 60% suffered from POF and hypergonadotropic amenorrhea. Whereas the POF rate was <50% in women below 30 years, it was 60% between 30 and 40 years. The cumulative dosage of cyclophosphamide also strongly influenced POF rate. CONCLUSIONS: Our present results, with a high POF rate in Cyclophosphamide treated SLE patients demonstrate the urgent need for ovarian protection in this patient group. Besides POF these women are at high risk for premature atherosclerosis which is the major cause of death in lupus. Following preliminary encouraging experience in women with lymphoma, in whom the temporary induction of a prepubertal hormonal milieu during chemotherapy, has significantly decreased the risk of POF, we have initiated the PREGO-Study, comparing randomised monthly injection versus no injection of gonadotropin-releasing hormone analogue (GnRH-a) to young SLE patients during cyclophosphamide therapy.     

Immune disorders in women with premature ovarian failure in initial period

PROBLEM: Premature ovarian failure (POF) may be considered as an autoimmune endocrine disease. Autoantibodies and lymphocyte subset changes are associated with premature ovarian failure. Immune cell parameters were studied in relation with anticardiolipin antibodies (ACAB) classes M and G in the initial period of POF. METHODS: Two-color flow cytometry was used to determine lymphocyte subsets and enzyme-linked immunosorbent assay (ELISA) was used to detect ACAB and hormones in the peripheral blood of 68 POF patients, 32 women with normal menopause (NM) and 13 healthy women as a normal control (NC). RESULTS: Patients in the initial period of POF had decreased levels of CD3+, CD19+, CD3+8+, and CD8+57+ lymphocytes and a high percentage of CD5 positive in CD19+ cell population compared to the control; frequencies of IgM ACAB in POF patients were significantly higher than both IgG ACAB and IgM ACAB in NC; correlation between lymphocyte subsets and hormone levels was absent. Women with early NM showed a low number of CD3+, CD3+4+, and CD3+8+ lymphocytes, a high number of CD3 + DR, and elevation of the percentage of CD5 positive in CD19+ lymphocytes compared with the control. The frequencies of both IgM and IgG ACAB were high; the levels of lymphocyte subsets had correlations with progesterone and estradiol concentrations. CONCLUSIONS: An increase of autoantibody producing B cells (CD5+19+) and a low number of effector suppressor/cytotoxic lymphocytes (CD8+57+) with active production of anticardiolipin autoantibodies class M were found. This suggested a primary autoimmune process in the initial period of POF. Autoimmune defeat of the ovary could be the primary cause of POF, whereas in NM autoimmunity is a result of hormone dysfunction.