miR-15a-5p和miR-16-5p在骨肉瘤组织中的表达分析

目的:使用microRNAs基因芯片及实时定量PCR法测定骨肉瘤组织中miR-15a-5p和miR-16-5p的相对表达含量,并与瘤旁组织对比,分析骨肉瘤细胞内miR-15a-5p和miR-16-5p的表达变化。方法:选取34例骨肉瘤组织蜡块样本,使用microRNAs基因芯片观察miR-15a-5p和miR-16-5p在骨肉瘤和瘤旁组织内的表达差异;实时定量PCR法测定骨肉瘤组织和瘤旁组织中miR-15a-5p和miR-16-5p的相对表达含量,并将两种结果对比分析。结果:microRNAs基因芯片结果显示,在骨肉瘤组织中,miR-15a-5p在肿瘤中的表达较瘤旁组织低1.79倍,miR-16-5p较瘤旁组织低1.62倍。实时定量PCR实验结果表明,miR-15a-5p和miR-16-5p表达较瘤旁组织降低,差异有统计学意义(P0.05)。经过统计学计算,miR-15a-5p在肿瘤中的表达较瘤旁组织低3.14倍,miR-16-5p较瘤旁组织低5.65倍。结论:在骨肉瘤中,miR-15a-5p和miR-16-5p表达含量降低,提示这两种microRNAs在骨肉瘤中可能做为抑癌因子存在。     

Circulating miR-3613-5p but not miR-125b-5p,miR-199a-3p,and miR-451a are biomarkers of endometriosis

ObjectiveThis study aimed to assess the utility of circulating miR-125b-5p, miR-199a-3p, miR-451a, and miR-3613-5p as biomarkers of endometriosis.Study designPatients with stage III or IV of endometriosis according to the revised American Society of Reproductive Medicine (rASRM) staging classification, as well as control women, were recruited. We created a prospective study conducted on a group of 48 patients (n = 25 controls, n = 24 endometriosis) who had laparoscopic surgery. Blood samples were taken and plasma miRNA levels were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and assessed with AUC and ROC curves.ResultsMiR-451a and miR-3613-5p were significantly decreased in the plasma of endometriosis patients. miR-451a had a receiver-operating characteristic (ROC) area under the curve 0.8283 and miR-3613-5p had a ROC area under the curve 0.7617. The concentration of circulating miR-125b-5p and miR-199-3p did not differ between endometriosis patients and controls. Plasma miRNA levels did not change with BMI, smoking status, fertility problems, or menstrual pain according to the VAS scale (p > 0.05).ConclusionCirculating miR-451a and miR-3613-5p levels significantly differed between endometriosis and controls. However, the levels of miR-451a were discordant with previous studies. Therefore, miR-3613-5p may have better potential as the endometriosis biomarker. Circulating miR-125b-5p and miR-199a-3p cannot be used as reliable markers of endometriosis.     

microRNA-16-5p enhances radiosensitivity through modulating Cyclin D1/E1–pRb–E2F1 pathway in prostate cancer cells

Prostate cancer (CaP) is the second most common cancer in men worldwide in 2012, and radiation therapy is one of the most common definitive treatment options for localized CaP. However, radioresistance is a major challenge for the current radiotherapy, accumulating evidences suggest microRNAs (miRNAs), as an important regulator in cellular ionizing radiation (IR) responses, are closely correlated with radiosensitivity in many cancers. Here, we identified microRNA-16-5p(miR-16-5p) is significantly upregulated in CaP LNCaP cells following IR and can enhance radiosensitivity through modulating Cyclin D1/E1–pRb–E2F1 pathway. To identify the expression profile of miRNAs in CaP cells exposed to IR, we performed human miRNA probe hybridization chip analysis and miR-16-5p was found to be significantly overexpressed in all treatment groups that irradiated with different doses of X-rays and heavy ions (¹²C⁶⁺). Furthermore, overexpression of miR-16-5p suppressed cell proliferation, reduced cell viability, and induced cell cycle arrest at G0/G1 phase, resulting in enhanced radiosensitivity in LNCaP cells. Additionally, miR-16-5p specifically targeted the Cyclin D1/E1–3′-UTR in LNCaP cells and affected the expression of Cyclin D1/E1 in both mRNA and protein levels. Taken together, miR-16-5p enhanced radiosensitivity of CaP cells, the mechanism may be through modulating Cyclin D1/Cyclin E1/pRb/E2F1 pathway to cause cell cycle arrest at G0/G1 phase. These findings provided new insight into the correlation between miR-16-5p, cell cycle arrest, and radiosensitivity in CaP, revealed a previously unrecognized function of miR-16-5p–Cyclin D1/E1–pRb–E2F1 regulation in response to IR and may offer an alternative therapy to improve the efficiency of conventional radiotherapy.     

miR-15a 和miR-16-1 模拟物对人骨肉瘤细胞系SOSP-9607 凋亡 和增殖的影响

目的:探讨miR-15a和miR-16-1模拟物对于人骨肉瘤细胞系SOSP-9607凋亡和增殖的影响。方法:将SOSP-9607细胞分为实验组和对照组。实验组分为miR-15a组、miR-16-1组、miR-15a+miR-16-1组。以miR-15a组为例,采用miR-15a模拟物(hsa-miR-15a mimics)上调SOSP-9607细胞内的miR-15a表达量。对照组分为阴性对照组和空白对照组。采用流式细胞仪测定细胞凋亡率,四甲基偶氮唑蓝(MTT)法测定细胞增殖,并计算细胞增殖效率。结果:通过统计学分析,实验组凋亡率与阴性对照组凋亡率相比明显增高(P<0.05);实验组的细胞增殖率明显低于对照组(P<0.05)。结论:上调SOSP-9607细胞内miR-15a和miR-16-1的表达量可促进SOSP-9607细胞的凋亡并抑制其增殖。     

Exosomal small RNA sequencing uncovers the microRNA dose markers for power frequency electromagnetic field exposure

Purpose: The potential health risks caused by power frequency electromagnetic field (PFEMF) have led to increase public health concerns. However, the diagnosis and prognosis remain challenging in determination of exact dose of PFEMF exposure.

Materials and methods: Mice were exposed to different magnetic doses of PFEMF for the following isolation of serum exosomes, microRNAs (miRNAs) extraction and small RNA sequencing. After small RNA sequencing, bioinformatic analysis, quantitative real-time PCR (qRT-PCR) validation and serum exosomal miRNA biomarkers were determined.

Results: Significantly changed serum exosomal miRNA as biomarkers of 0.1, 0.5, 2.5?mT and common PFEMF exposure were confirmed. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) pathway analysis of the downstream target genes of the above-identified exosomal miRNA markers indicated that, exosomal miRNA markers were predicted to be involved in critical pathophysiological processes of neural system and cancer- or other disease-related signalling pathways.

Conclusions: Aberrantly-expressed serum exosomal miRNAs, including miR-128-3p for 0.1?mT, miR-133a-3p for 0.5?mT, miR-142a-5p for 2.5?mT, miR-218-5p and miR-199a-3p for common PFEMF exposure, suggested a series of informative markers for not only identifying the exact dose of PFEMF exposure, also consolidating the base for future clinical intervention.     

Circulating microRNA signatures serve as potential diagnostic biomarkers for Helicobacter pylori infection

Helicobacter pylor (H pylori), a Gram-negative, microaerobic human pathogen, has been found to be involved in many gastroduodenal diseases. Accurate diagnosis of H pylori infection is a vital part of the effective management of gastroduodenal diseases. Circulating microRNAs (miRNAs) have shown the potential to be used as noninvasive biomarkers for the diagnosis of infectious diseases. The aim of this study was to explore plasma miRNAs as noninvasive biomarkers for H pylori infection. We performed a plasma miRNA expression profile using Illumina high-throughput sequencing and validated the levels of differentially expressed miRNAs in the plasma of 63 H pylori-infected patients and 41 healthy volunteers by quantitative real-time polymerase chain reaction (qRT-PCR). The sequencing results showed that 37 miRNAs were upregulated in the H pylori-infected patients compared with that in the healthy volunteers, while six miRNAs were downregulated. qRT-PCR and receiver operator characteristic analysis suggested that the expression of miR-28-3p, miR-143-3p, miR-151a-3p, and miR-148a-3p were closely associated with H pylori infection. Therefore, the four plasma miRNA panels mentioned above could serve as promising noninvasive biomarkers of H pylori infection.     

Expression of miR-15/107 Family MicroRNAs in Human Tissues and Cultured Rat Brain Cells

The miR-15/107 family comprises a group of 10 paralogous microRNAs （miRNAs）,sharing a 5＇ AGCAGC sequence.These miRNAs have overlapping targets.In order to characterize the expression of miR-15/107 family miRNAs,we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members,and other selected miRNAs,in 11 human tissues obtained at autopsy including the cerebral cortex,frontal cortex,primary visual cortex,thalamus,heart,lung,liver,kidney,spleen,stomach and skeletal muscle.miR-103,miR-195 and miR-497 were expressed at similar levels across various tissues,whereas miR-107 is enriched in brain samples.We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations （enriched for cortical neurons,astrocytes and microglia,respectively）.In primary cultures of rat brain cells,several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system （CNS）.In addition to mature miRNAs,we also examined the expression of precursors （pri-miRNAs）.Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors.In summary,we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.     

抑癌基因p53调控的微RNA-miR-34基因家族

微RNA（microRNA,miRNA）是一种内源性非编码小RNA,在转录后水平调控基因表达,在肿瘤的发生发展过程中起重要作用。p53是重要的抑癌基因,在DNA损伤和癌基因等刺激下活化,诱导下游基因表达,使细胞周期阻滞、DNA修复并促进细胞衰老或凋亡。本文主要介绍近期发现的直接受p53调控的miR-34基因家族,及其在生长阻滞和细胞凋亡方面的研究进展,揭示了蛋白质与非编码RNA在重要的p53抑癌网络中的相互关系,为肿瘤的研究提供了新的思路。     

p53/miR-30a-5p/ SOX4 feedback loop mediates cellular proliferation,apoptosis, and migration of non-small-cell lung cancer

Many microRNAs (miRNAs) play vital roles in the tumorigenesis and development of cancers. In this study, we aimed to identify the differentially expressed miRNAs and their specific mechanisms in non-small-cell lung cancer (NSCLC). Based on data from the GSE56036 database, miR-30a-5p expression was identified to be downregulated in NSCLC. Further investigations showed that overexpression of miR-30a-5p inhibited cell proliferation, migration, and promoted apoptosis in NSCLC. Increase of miR-30a-5p level could induce the increase of Bax protein level and decrease of Bcl-2 protein level. In addition, chromatin immunoprecipitation assays showed that miR-30a-5p expression was induced by binding of p53 to the promoter of MIR30A. Bioinformatics prediction indicated that miR-30a-5p targets SOX4, and western blot analysis indicated that overexpression of the miRNA decreases the SOX4 protein expression level, which in turn regulated the level of p53. Thus, this study provides evidence for the existence of a p53/miR-30a-5p/SOX4 feedback loop, which likely plays a key role in the regulation of proliferation, apoptosis, and migration in NSCLC, highlighting a new therapeutic target.     

Increased Arf/p53 activity in stem cells,aging and cancer

Arf/p53 pathway protects the cells against DNA damage induced by acute stress. This characteristic is the responsible for its tumor suppressor activity. Moreover, it regulates the chronic type of stress associated with aging. This is the basis of its anti‐aging activity. Indeed, increased gene dosage of Arf/p53 displays elongated longevity and delayed aging. At a cellular level, it has been recently shown that increased dosage of Arf/p53 delays age‐associated stem cell exhaustion and the subsequent decline in tissue homeostasis and regeneration. However, p53 can also promote aging if constitutively activated. In this context, p53 reduces tissue regeneration, which correlates with premature exhaustion of stem cells. We discuss here the current evidence linking the Arf/p53 pathway to the processes of aging and cancer through stem cell regulation.     

Down regulation of miR-30a-5p and miR-182–5p in gastric cancer: Clinical impact and survival analysis

Background and aimGastric Cancer (GC) is a leading cause of morbidity and mortality worldwide, particularly in developing nations, only a few suitable gastric cancer serum biomarkers with acceptable sensitivity and specificity exist. This work aims to highlight and uncover miR-30a-5p and miR-182–5p′s diagnostic roles regarding gastric cancer and their roles in predicting prognosis.Methods148 patients participated in this study. Groups I, II, and III had 47 patients with GC, 54 patients with benign gastric lesions, and 47 apparently healthy subjects of coincided age and gender as controls, respectively. All participants were clinically evaluated and subjected to CBC, serum CEA, and CA19-9 by ELISA, and real-time PCR tests of miR-30a-5p and miR-182–5p.ResultsMiR30a-5p and miR-182–5p were down regulated in gastric cancer patients in Group I more than Groups II and III (P < 0.001). ROC curve analysis revealed that miR30a-5p had better AUC, sensitivity, and specificity (0.961%, 93.62%, and 90.74%respectively). When miR-182–5p was gathered with CEA and CA19-9, specificity raised to 98.15% and PPV to 97.6%. Lower miR-30a-5p levels are linked with the presence of distant metastases, advanced TNM stage, and degree of pathological differentiation of tumors in GC patients (p = 0.034, 0.019, 0.049) respectively. According to the multivariate analysis, miR30a-5p expression level could be an independent predictor of GC.ConclusionOur results exhibited that miRNAs, miR-30a-5p and miR182–5p, gene expression have a diagnostic power and can identify patients with GC. MiR-30a-5p displayed the highest diagnostic specificity and sensitivity. Besides other known tumor markers, they could offer simple noninvasive biomarkers that predict gastric cancer.     

Crosstalk between circulating microRNAs and chronotypical features in subjects with metabolic syndrome

ABSTRACT

Circulating microRNAs (miRNAs) are valuable biomarkers that may provide important insight into the pathogenesis of metabolic syndrome (MetS). Moreover, there is an association between chronotypical characteristics and MetS predisposition. Considering that expression of some miRNAs is circadian-rhythm-dependent, the aim of this study was to investigate the circulating miRNA profile in subjects with and without MetS in association with chronotype. The expression of 86 metabolic syndrome-related miRNAs was investigated in the plasma of 21 subjects with MetS and in 82 subjects without MetS using miRCURY LNA miRNA PCR System technology. Chronotype was assessed using the Horne and Östberg Morningness-Eveningness Questionnaire. Bioinformatic analyses were performed to explore the target genes and biological pathways regulated by the selected miRNAs. Subjects with MetS were more often evening chronotype compared to non-MetS controls. Additionally, four miRNAs (miR-140-3p, miR-150-5p, miR-375, and miR-29 c-3p) demonstrated interaction with MetS and chronotype. Interestingly, the target genes of these four miRNAs participate in pathways related to the circadian clock. In conclusion, we identified four circulating miRNAs whose circulating levels could interact with MetS and chronotype.     

miR-181a/b-5p regulates human umbilical vein endothelial cell angiogenesis by targeting PDGFRA

Type 2 diabetes mellitus (T2DM) is a growing burden in low-and middle-income countries. Changing lifestyles and lack of physical activity are some of the reasons contributing to this epidemic increase. Co-morbidities associated with T2DM are largely due to the complications which arise as a consequence of endothelial dysfunction. Platelet derived growth factor-alpha (PDGFRA) is a protein responsible for cell proliferation, angiogenesis, migration and invasion. Increased levels of PDGFRA have been reported in T2DM. This study assessed the epigenetic regulation of PDGFRA through microRNAs (miR-181a/b-5p).Using a bioinformatics-based approach, we assessed the binding of miR-181a/b-5p to PDGFRA. Experimentally, this binding was confirmed using a dual luciferase reporter assay. Further, we overexpressed miR-181a/b-5p in Human umbilical vein endothelial cells (HUVECs) and the influence of over-expression on cell proliferation, migration and angiogenesis was assessed using in-vitro approaches. The influence of miR-181a/b-5p over expression on cellular apoptosis was ascertained using a TUNEL assay with concomitant changes being observed in the levels of Bcl-2 and cleaved Caspase-3.In HUVECs, PDGFRA is a direct target for miR-181a/b-5p. Over expression of miR-181a/b-5p decreased cellular proliferation, migration, invasion, and tube formation—a surrogate marker for angiogenesis. miR-181a/b-5p may be used as a therapeutic intervention to restrict uncontrolled levels of PDGFRA and thereby rescue the phenotypes of increased cell proliferation, migration, invasion and tube formation. miR-181a/b negatively regulates PDGFRA levels. Significance of the study : T2DM and its associated complications emerge from endothelial dysfunction. The associated phenotypes are regulated by a number of proteins, one such member being, PDGFRA. PDGFRA is in turn regulated by miR-181a/b-5p. Complementation with miR-181a/b-5p resulted in reversion of phenotypes. Thus, miR-181a/b-5p-mediated suppression of PDGFRA may be used as a therapeutic intervention in the management of type 2 diabetes.     

Suppressive effect of exogenous miR-16 and miR-34a on tumorigenesis of breast cancer cells

Recent investigations have shown tumor-suppressive roles for miR-16 and miR-34a. They also share some features in regard to targeting cancer cell signaling pathways which they control. Therefore, in this study, we aimed to further scrutinize whether exogenous induction of mature miR-34a and miR-16 can collaborate in breast tumor suppression. MDA-MB-231 and SK-BR-3 human breast cancer cell lines were cultured and transfected twice with hsa-miR-16-5p and hsa-miR-34a-5p mimics individually or in combination. The cells were analyzed for apoptosis rate and cell cycle indices by flow cytometry. Also, the expression of several invasion and the epithelial-mesenchymal transition markers was evaluated at gene and protein levels by quantitative real-time polymerase chain reaction and western blot analysis, respectively. Assessment of invasiveness and migratory potential of the transfected cells was performed using three-dimensional spheroid formation and wound-healing assay, respectively. In both cell lines, miR-16 and miR-34a induced apoptosis and cell-cycle arrest and also suppressed invasion and migration. Some of these effects, like cell-cycle arrest and induction of apoptosis, were significantly higher when using both microRNAs than when using them individually for transfection of the cells. Our results are indicating that miR-16 and miR-34a can collaborate in breast tumor suppression.