Differential microRNA expression tracks neoplastic progression in inflammatory bowel disease-associated colorectal cancer

One of the most serious complications faced by patients with inflammatory bowel disease (IBD) is the potential development of colorectal cancer (CRC). There is a compelling need to enhance the accuracy of cancer screening of IBD patients. MicroRNAs (miRNAs) are small nonprotein-coding RNAs that play important roles in CRC oncogenesis. In this study, we report differential miRNA expression in IBD patients with associated CRC from non-neoplastic tissue to dysplasia and eventually cancer. In addition, we identify and examine the role of dysregulated miRNAs in the TP53 pathway. In our CD patients, six miRNAs were upregulated from non-neoplastic tissue to dysplasia, but downregulated from dysplasia to cancer (miR-122, miR-181a, miR-146b-5p, let-7e, miR-17, miR-143) (P < 0.001). Six differentially expressed miRNAs affected the TP53 pathway (miR-122, miR-214, miR-372, miR-15b, let-7e, miR-17) (P < 0.001). Using two human colon cancer cell lines (HT-29 and HCT-116), E2F1, an upstream regulator of TP53, was downregulated in both cell lines transfected with let-7e (P < 0.05) as well as in HCT-116 cells transfected with miR-17 (P < 0.05). Additionally, cyclin G, a cell-cycle regulator miR-122 target was downregulated in both cell lines (P < 0.05). Unique differentially expressed miRNAs were observed in CD-associated CRC progression. Six of these miRNAs had a tumorigenic effect on the TP53 pathway; the effect of three of which was studied using cell lines.     

Expression profile of microRNAs in fetal lung development of Sprague-Dawley rats

As well-known regulators of gene expression, microRNAs (miRNAs) play an important role not only in cell proliferation and differentiation, but also in tumorigenesis and organ development. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Simultaneously, in the clinic, with advances in neonatal care, a larger number of premature infants are being saved, and thus diseases of lung development, including bronchopulmonary dysplasia (BPD) have become more and more common. However, only a few miRNA studies have studied their connection with diseases of lung development. In our study, we used a miRNA microarray including more than 1891 capture probes to profile the expression of miRNAs at three time points of rat lung development [embryonic (E) Day 16 (E16), E19, E21]. miRNAs found to have consistent fold-changes (fold-change>2.0) during all three time points were selected and validated by real-time PCR. As a result, 167 differentially expressed miRNAs were found during rat lung organogenesis, including 81 upregulated and 86 downregulated miRNAs. Seven miRNAs were selected and characterized by having a consistent >2-fold changes between all three groups. Among these 7 miRNAs, except for let-7a, the other 6 miRNAs (miR-1949, miR-125b-5p, miR-296, miR-93, miR-146b, miR-3560) are all first reported for the first time in lung development. Finally, due to the fact that they demonstrated higher fold changes, from these 7 miRNAs we selected miR-125b-5p, miR-296, miR-93, miR-146b and miR-3560 for real-time PCR. We hypothesized that these newly identified miRNAs may play an important role in fetal lung development, and this experimental result could help us to further clarify the mechanism of normal lung development including the development of type?II pneumocytes. This may provide a physiological basis for future research on diseases of lung development.     

减体积肝移植模型大鼠肝脏组织miRNAs表达谱变化

BACKGROUND: Liver regeneration is the key factor influencing the prognosis of living donor liver transplantation. There has not been the research on special miRNA of liver regeneration after living donor liver transplantation. OBJECTIVE: To analyze the variation of miRNAs expression profile after rat reduced-size liver transplantation at certain time point, select and verify target miRNA which can provide targeting intervention strategies in liver regeneration after rat reduced-size liver transplantation and provide theoretical evidence for liver regeneration after living donor liver transplantation. METHODS: The reduced-size liver transplantation models were established. miRNAs microarray was used to detect miRNA expression. In differentially expressed microRNAs, real-time quantitative PCR was utilized to detect target miRNAs. The credibility of miRNAs microarray results was verified. RESULTS AND CONCLUSION: Compared with rat liver tissue in the sham operation group, 11 miRNAs up-regulated in reduced-size liver transplantation, including let-7b-5p, let-7c-5p, miR-101a-3p, miR-103-3p, miR-130a-3p, miR-142-5p, miR-186-5p, miR-199a-3p, miR-21-5p, 221-3p and miR-34a-5p. Four miRNAs were down-regulated, including miR-26b-5p, miR-150-5p, miR-19a-3p and rno-miR-146-5p. PCR test further verified that miR-221-3p and miR-199a-3p expression changes approximated the chip results at 24, 48 hours and 1 week, indicating that results of miRNA microarray were believable. These results verified that it exists variation of miRNAs expression profile after rat reduced-size liver transplantation, which picked out and verified the target miRNAs.        

微小RNA活性分析法筛选前列腺癌去势抵抗转化相关微小RNA的实验研究

目的 探讨微小RNA(miRNA)活性分析法筛选前列腺癌去势抵抗转化相关miRNA的效率。方法 应用miRNA活性分析法筛选出在前列腺癌去势抵抗过程中潜在的发挥活性作用的miRNAs。培养人激素敏感型前列腺癌LNCAP细胞(对照组)及人去势抵抗型前列腺癌C4-2细胞(C4-2组)、PC-3细胞(PC-3组)、DU-145细胞(DU-145组),提取各组细胞总RNA,采用实时荧光定量PCR(qPCR)检测miRNAs,比较各组细胞miRNAs的表达情况。结果 应用miRNA活性分析法,依据筛选流程,共选出9个差异表达的miRNAs,分别为miR-1、miR-122、miR-218、miR-145、miR-155、miR-210、miR-197、 miR-346、let-7b。采用qPCR检测这9个miRNAs,结果显示,7个miRNAs在两种不同状态下的前列腺癌细胞中存在差异表达;在不同去势抵抗型前列腺癌细胞中,miR-210、miR-197、miR-346、miR-218均明显高表达,而miR-122、miR-145、let-7b均明显低表达。结论 采用miRNA活性分析法筛选前列腺癌去势抵抗转化相关miRNA,有着较高的准确性与可信度;其具体转化过程还需进一步证实。     

Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome

Psoriasis is a chronic and complex inflammatory skin disease with lesions displaying dramatically altered mRNA expression profiles. However, much less is known about the expression of small RNAs. Here, we describe a comprehensive analysis of the normal and psoriatic skin miRNAome with next-generation sequencing in a large patient cohort. We generated 6.7 × 10(8) small RNA reads representing 717 known and 284 putative novel microRNAs (miRNAs). We also observed widespread expression of isomiRs and miRNA*s derived from known and novel miRNA loci, and a low frequency of miRNA editing in normal and psoriatic skin. The expression and processing of selected novel miRNAs were confirmed with qRT-PCR in skin and other human tissues or cell lines. Eighty known and 18 novel miRNAs were 2-42-fold differentially expressed in psoriatic skin. Of particular significance was the 2.7-fold upregulation of a validated novel miRNA derived from the antisense strand of the miR-203 locus, which plays a role in epithelial differentiation. Other differentially expressed miRNAs included hematopoietic-specific miRNAs such as miR-142-3p and miR-223/223*, and angiogenic miRNAs such as miR-21, miR-378, miR-100 and miR-31, which was the most highly upregulated miRNA in psoriatic skin. The functions of these miRNAs are consistent with the inflammatory and hyperproliferative phenotype of psoriatic lesions. In situ hybridization of differentially expressed miRNAs revealed stratified epidermal expression of an uncharacterized keratinocyte-derived miRNA, miR-135b, as well as the epidermal infiltration of the hematopoietic-specific miRNA, miR-142-3p, in psoriatic lesions. This study lays a critical framework for functional characterization of miRNAs in healthy and diseased skin.     

MicroRNA Expression Abnormalities in Limited Cutaneous Scleroderma and Diffuse Cutaneous Scleroderma

Scleroderma (systemic sclerosis, SSc) is a complex autoimmune disease caused by progressive fibrotic replacement of normal tissue architecture, a progressive and ultimately fatal process that currently has no cure. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in SSc is unclear. In comparison with the normal skin tissues, miRNAs were aberrantly expressed in limited cutaneous scleroderma and diffuse cutaneous scleroderma skin tissues. We also identified miRNAs whose expressions were correlated with SSc fibrosis: miR-21, miR-31, miR-146, miR-503, miR-145, and miR-29b were predicted to be involved. This study further confirmed that miR-21 was increased whereas miR-145 and miR-29b were decreased both in the skin tissues and fibroblasts. As predicted target genes, SMAD7, SAMD3, and COL1A1 were regulated by these miRNAs. After stimulation with transforming growth factor β, the expression of miR-21 was increased and that of SMAD7 mRNA was decreased. MiR-145 was upregulated whereas the mRNA level of SMAD3 was downregulated. The downregulation of miR-29b was correlated with the upregulation of COL1A1 mRNA. MiRNAs might play an important role in the pathogenesis of SSc and suggest a potential therapy.     

慢性神经病理性疼痛对大鼠脊髓背角miRNA表达的影响

目的 筛选慢性神经病理性疼痛大鼠脊髓背角差异表达的miRNA,并预测其调控的靶基因.方法 建立大鼠坐骨神经慢性压迫损伤CCI模型,在术后疼痛高峰期取腰膨大脊髓背角,用miRNA芯片筛选CCI大鼠差异表达的miRNAs,再用荧光实时定量RT-PCR验证差异表达的miRNAs,并利用MIRANDA、TARGETSCAN、PICTAR 3个数据库找出这些miRNA可能调控的靶基因.结果 CCI大鼠表达上调的有miR-99b,表达下调的有miR-674-3p、miR-879与miR-325-5p.RT-qPCR验证结果与芯片基本相符.预测这些miRNA可能的靶基因约26个,这些基因功能广泛.结论 慢性神经病理性疼痛可导致miRNA的表达发生变化,这些miRNA及其调控的靶基因为进一步研究奠定了基础.     

PLGF参与Ang II诱导的心脏成纤维细胞激活

 目的：探讨胎盘生长因子（PLGF）在血管紧张素II（Ang II）激活心脏成纤维细胞(CFs)中的表达及其作用。方法：原代分离培养并鉴定新生SD大鼠CFs。蛋白免疫印迹检测α-平滑肌肌动蛋白（α-SMA）、PLGF和磷酸化ERK1/2(p-ERK1/2),免疫荧光观察α-SMA的表达,WST-1法检测细胞增殖,RT-PCR检测PLGF、I型和III型胶原蛋白的mRNA表达水平。结果：(1)Ang II组PLGF mRNA 表达明显高于对照组（P<0．01),联用替米沙坦后,PLGF mRNA表达水平下降;Ang  II组PLGF蛋白表达水平高于对照组（P<0.05); (2)PLGF诱导CFs增殖及α-SMA蛋白表达增加（P<0.05）;PLGF干预CFs 60 min后,p-ERK1/2蛋白水平表达明显高于对照组（P<0.01);(3)Ang II+anti-PLGF组与Ang II组比较,细胞增殖和α-SMA蛋白表达水平下降（P<0.05）,I型和III型胶原蛋白mRNA表达水平亦下调（P<0.05）。结论：PLGF可能参与Ang II诱导的CFs增殖和纤维化过程。     

miRNA 表达与免疫细胞因子相关性在原发性肝癌早期诊断和预后中的作用

目的:检测miR-146a、miR-224、miR-34c、miR-200a、miR-148b、miR-375 六种miRNAs 在原发性肝癌中的表达变化及其与HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc 和IL-12、IL-4、IL-6、IL-10、IFN-γ、TNF-α等炎症因子表达的相关性,以验证循环miRNA 是否可作为理想的血源性新型生物标志物用于原发性肝癌的早期检测。方法:收集肝炎、肝硬化患者及健康对照组静脉血,并收集原发性肝癌患者癌组织和癌旁组织。提取总RNA 后通过实时定量PCR 检测并比较各组miRNA 的相对表达水平,同时检测miRNA 表达水平变化与血清肿瘤标志物AFP、CEA、CA19-9、CA125 表达的关系;并检测miRNAs 表达水平变化与HBsAg、抗-HBs、HBeAg、抗-HBe、抗-HBc 和炎症因子IL-12、IL-4、IL-6、IL-10、IFN-γ、TNF-α表达相关性。结果:相对于健康组,miR-34c、miR-224、miR-146a 在PHC 组血清和组织中表达显著上调;miR-200a、miR-148b、miR-375 在PHC 组血清和组织中表达显著下调,差异具有统计学意义。HBsAg 与血清miR-375 和miR-146a 存在回归关系,miR-375 随HBsAg 表达水平升高而降低,而miR-146a 随HBsAg 表达升高而升高。IFN-γ与miR-146a 存在回归关系, miR-146a 随IFN-γ表达水平降低而升高,miR-375 和miR-146a 诊断能力大于CA19-9 和AFP。结论:miR-146a、miR-224、miR-34c、miR-200a、miR-148b、miR-375 在原发性肝癌血清和组织中存在表达差异,其中miR-375 和miR-146a 诊断能力优于AFP 和CA19-9,血清miR-375 和miR-146a 可能成为新的肝癌早期诊断标志。     

急性心肌梗死大鼠缺血心肌中差异microRNA的表达谱分析

 目的：筛选急性心肌梗死（AMI）大鼠缺血心肌中差异表达的microRNAs（miRNAs）,预测其靶基因并分析其可能的生物学功能。方法：结扎冠状动脉左前降支建立雄性Wistar大鼠AMI模型,同时检测其心电图和颈总动脉血压变化,并用TTC法测定心肌梗死面积;假手术（sham）组除不结扎冠状动脉左前降支外,其它实验程序与AMI组相同。心肌缺血4 h后取梗死区心肌组织,提取总RNA进行microRNA芯片杂交检测,并用real-time PCR进行验证;生物信息学方法预测差异miRNAs的靶点并分析其生物学功能。结果：心电图、血压检测及病理切片证实AMI模型制备成功。Microarray检测结果表明,与sham组相比,获得11个与急性心肌梗死相关的miRNAs,其中6个miRNAs上调表达,5个miRNAs下调表达;已知3个miRNAs（rno-miR-181c、rno-miR-146b和rno-miR-208）参与了心血管功能的调节,8个miRNAs（rno-miR-672*、rno-miR-743b、rno-miR-128、rno-miR-138-1*、rno-miR-336、rno-miR-138-2*、rno-miR-325-3p和rno-miR-3572）是否与心血管功能有关尚不清楚,可能是心肌梗死相关的新的生物标志物。预测的miRNA靶基因中的一部分亦与心血管功能相关。结论：本研究获得的与AMI相关的差异miRNAs,可能是急性心肌梗死新的生物标志物和潜在的治疗靶点。     

Reprint of: MicroRNA profiling of human intermediate monocytes

Among the three human monocyte subsets, intermediate CD14++CD16+ monocytes have been characterized as particularly proinflammatory cells in experimental studies and as potential biomarkers of cardiovascular risk in clinical cohorts. To further substantiate the distinct role of intermediate monocytes within human monocyte heterogeneity, we assessed subset-specific expression of miRNAs as central epigenetic regulators of gene expression. We hypothesized that intermediate monocytes have a distinct miRNA profile compared to classical and non-classical monocytes.By using small RNA-seq we analyzed 662 miRNAs in the three monocyte subsets. We identified 38 miRNAs that are differentially expressed in intermediate monocytes compared to both classical and non-classical monocytes with a p value of <10⁻¹⁰, of which two miRNAs – miR-6087 (upregulated) and miR-150-5p (downregulated) – differed in their expression more than ten-fold. Pathway analysis of the 38 differentially expressed miRNAs linked intermediate monocytes to distinct biological processes such as gene regulation, cell differentiation, toll-like receptor signaling as well as antigen processing and presentation. Moreover, differentially expressed miRNAs were connected to those genes that we previously identified as markers of intermediate monocytes.In aggregation, we provide first genome-wide miRNA data in the context of monocyte heterogeneity, which substantiate the concept of monocyte trichotomy in human immunity. The identification of miRNAs that are specific for intermediate monocytes may allow to develop strategies, which particularly target this cell population while sparing the other two subsets.     

Altered microRNA expression signature in Chikungunya-infected mammalian fibroblast cells

Chikungunya virus (CHIKV) infection can cause severe arthralgia and chronic arthritis in humans. MicroRNAs (miRNA) have demonstrated their potential use as biomarker in variety of human pathologies and infections. This study was conducted to understand the miRNA signature in early CHIKV infection stages. In the current study, we used TaqMan-based quantitative PCR method to identify the miRNA signature of host response upon CHIKV infection in human and mouse fibroblast cells. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are to be involved in RIG-I pathway, TGF-beta-signaling pathway, JAK–STAT-signaling pathway, MAPK-signaling pathway, cytokine–cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The results obtained in the current study and earlier studies indicate the potential use of miR15, miR-16, miR-17, let-7e, miR-125, miR-99, and miR-23a as a biomarker in CHIKV infection. miRNAs such as miR-15a, miR-16, miR-140, miR-146a, miR-155, miR203, miR223, miR-499, and miR-363 which are implicated in rheumatoid arthritis showed differential regulation in CHIKV infection. The data obtained in this study provide valuable information on CHIKV-induced miRNA expression in mammalian fibroblast cells, and suggest that CHIKV may establish infection by regulating miRNA expression profile.     

Differentially expressed microRNAs in Huh-7 cells expressing HCV core genotypes 3a or 1b: potential functions and downstream pathways

microRNA (miRNA) dysfunction is believed to play important roles in human diseases, including viral infectious diseases. Hepatitis C virus (HCV) infection promotes the development of steatosis, cirrhosis and hepatocellular carcinoma, which is genotype-specific. In order to characterize the miRNA expression profile of Huh-7 cells expressing the HCV core 3a vs. 1b, microarrays and real-time PCR were performed. Consequently, 16 miRNAs (5 miRNAs upregulated and 11 miRNAs downregulated) were found to be dysregulated. In addition, we generated the predicted and validated targets of the differentially expressed miRNAs and explored potential downstream function categories and pathways of target genes using databases of Gene Ontology (GO) and PANTHER and the database for annotation, visualization and integrated discovery (David). The computational results indicated that the dysregulated miRNAs might perform the functions of cellular metabolism and cellular growth. Finally, these biological effects were preliminarily validated. This study identifies a specific miRNA expression profile in cells expressing HCV core proteins of different genotypes (genotype 3a and 1b), which may account for the variable pathophysiological manifestation associated with HCV infection.     

Downregulation of miR-27a* and miR-532-5p and Upregulation of miR-146a and miR-155 in LPS-induced RAW264.7 Macrophage Cells

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the epigenetic regulation of cellular processes. To identify more miRNAs which are involved in the macrophage inflammatory response to lipopolysaccharide (LPS) stimulation and dissect the mechanisms more clearly, microRNA profiling of LPS-treated RAW264.7 macrophage cells was performed by initial high-throughput array-based screen and further real-time RT-PCR validation; bioinformatics approaches were used to analyze the target genes of the differentially expressed miRNAs. Compared to the untreated control, two microRNAs (miR-146a and miR-155) with more than twofold higher expression and two microRNAs (miR-27a* and miR-532-5p) with twofold lower expression were detected by array-based screen, which can be validated by qRT-PCR, and more than 1,000 candidate target genes were detected by at least of one of four different algorithms (TargetScan, PicTar, miRDB, and microRNA.org); with gene ontology classification, we were able to correlate the upregulation and downregulation of miRNA to the differential expression of inflammation-related candidate target gene during LPS-induced inflammation. Our findings may provide the basic information for the precise roles of miRNAs in the macrophage inflammatory response to LPS stimulation in the future.     

MicroRNAs Are Aberrantly Expressed in Hypertrophic Heart: Do They Play a Role in Cardiac Hypertrophy?

MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate gene expression. Although miRNAs are highly expressed in the heart, their roles in heart diseases are currently unclear. Using microarray analysis designed to detect the majority of mammalian miRNAs identified thus far, we demonstrated that miRNAs are aberrantly expressed in hypertrophic mouse hearts. The time course of the aberrant miRNA expression was further identified in mouse hearts at 7, 14, and 21 days after aortic banding. Nineteen of the most significantly dysregulated miRNAs were further confirmed by Northern blot and/or real-time polymerase chain reaction, in which miR-21 was striking because of its more than fourfold increase when compared with the sham surgical group. Similar aberrant expression of the most up-regulated miRNA, miR-21, was also found in cultured neonatal hypertrophic cardiomyocytes stimulated by angiotensin II or phenylephrine. Modulating miR-21 expression via antisense-mediated depletion (knockdown) had a significant negative effect on cardiomyocyte hypertrophy. The results suggest that miRNAs are involved in cardiac hypertrophy formation. miRNAs might be a new therapeutic target for cardiovascular diseases involving cardiac hypertrophy such as hypertension, ischemic heart disease, valvular diseases, and endocrine disorders.     

MicroRNA profiling of human intermediate monocytes

Among the three human monocyte subsets, intermediate CD14++CD16+ monocytes have been characterized as particularly proinflammatory cells in experimental studies and as potential biomarkers of cardiovascular risk in clinical cohorts. To further substantiate the distinct role of intermediate monocytes within human monocyte heterogeneity, we assessed subset-specific expression of miRNAs as central epigenetic regulators of gene expression. We hypothesized that intermediate monocytes have a distinct miRNA profile compared to classical and non-classical monocytes.By using small RNA-seq we analyzed 662 miRNAs in the three monocyte subsets. We identified 38 miRNAs that are differentially expressed in intermediate monocytes compared to both classical and non-classical monocytes with a p value of <10⁻¹⁰, of which two miRNAs – miR-6087 (upregulated) and miR‐150-5p (downregulated) – differed in their expression more than ten-fold. Pathway analysis of the 38 differentially expressed miRNAs linked intermediate monocytes to distinct biological processes such as gene regulation, cell differentiation, toll-like receptor signaling as well as antigen processing and presentation. Moreover, differentially expressed miRNAs were connected to those genes that we previously identified as markers of intermediate monocytes.In aggregation, we provide first genome-wide miRNA data in the context of monocyte heterogeneity, which substantiate the concept of monocyte trichotomy in human immunity. The identification of miRNAs that are specific for intermediate monocytes may allow to develop strategies, which particularly target this cell population while sparing the other two subsets.