Serum miR-146a and miR-223 as potential new biomarkers for sepsis

Objective

Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified.

Methods

Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay.

Results

Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively.

Conclusion

Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.)     

Differentially expressed miR-20, miR-21, miR-100, miR-125a and miR-146a as a potential biomarker for prostate cancer

Prostate cancer is the leading cause of death among men worldwide. Deregulation of microRNAs has been reported in many cancers. Expression of microRNAs miR-20a-5p, miR-21-5p, miR-100-5p, miR-125a-5p and miR-146a-5p in tissue blocks of histologically confirmed prostate cancer patients compared with BPH patients, to identify potential microRNA biomarker for prostate cancer. MicroRNA was isolated and expression was quantified by qRT-PCR using Taqman Advanced microRNA assay kits. The interactions between the microRNA:target mRNA were predicted by using bioinformatics tools such as miRwalk and miRTargetlink. The experimentally validated targets were analysed using gprofiler to identify their molecular function, biological process and related pathways. The expression analysis revealed that miR-21 and miR-100 were significantly down-regulated whereas miR-125a was up-regulated in prostate cancer patients. Comparative analysis of the expression levels with tumor grading reveal that miR-100 was significantly down-regulated (p?<?0.05) in high grade tumor, indicating that miR-100 associated with prostate cancer. ROC analysis revealed that combined analysis of down-regulated miRNAs (miR-21 and miR-100) shown AUC of 0.72 (95% CI 0.65–0.79). The combined analysis of all five miRNAs showed AUC of 0.87 (95% CI 0.81–0.92). The targets prediction analysis revealed several validated targets including BCL2, ROCK1, EGFR, PTEN, MTOR, NAIF1 and VEGFA. Our results provide evidence that combined analysis of all the five miRNAs as a panel can significantly improve the prediction level of the presence of prostate cancer and may be used as a potential diagnostic biomarker.

     

血浆miR-106b、miR-146a表达水平与癫痫患儿脑电图参数、Th17细胞和凋亡分子的相关性分析

摘要 目的：分析血浆miR-106b、miR-146a表达特点及其与脑电图参数、辅助性T细胞17（Th17）和凋亡分子的相关性以及诊断癫痫的价值。方法：选择2018年1月至2020年10月我院收治的癫痫患儿75例作为癫痫组,检测受试者血浆miR-106b、miR-146a表达,外周血Th17细胞占比、血清B细胞淋巴瘤/白血病-1（Bcl-1）、BCL2-Associated X蛋白（Bax）、Survivin、半胱氨酸天冬酰胺酶（Caspase-3）水平和脑电图参数α、β、 δ、θ波功率。分析miR-106b、miR-146a与Th17细胞占比、Bcl-1、Bax、Survivin、Caspase-3以及α、β、 δ、θ波功率的相关性,受试者工作特征（ROC）曲线分析miR-106b、miR-146a诊断癫痫的价值。结果：癫痫组血浆miR-106b、miR-146a表达、Th17细胞占比、Bax、Caspase-3水平高于对照组（P＜0.05）,α波功率、θ波功率、Bcl-1、Survivin水平低于对照组（P＜0.05）。miR-106b、miR-146a表达与Th17细胞占比、Bax、Caspase-3呈正相关（P＜0.05）,与α波功率、θ波功率、Bcl-1、Survivin呈负相关（P＜0.05）。联合miR-106b和miR-146a诊断癫痫的曲线下面积（AUC）为0.975,高于单独miR-106b和miR-146a诊断的0.884、0.835。结论：癫痫患儿血浆miR-146a、miR-106b表达增高,miR-146a、miR-106b高表达与脑电图异常、Th17细胞功能障碍以及神经细胞凋亡有关,miR-146a、miR-106b有望成为癫痫诊断的新生物学标志物。     

Analyses in human urothelial cells identify methylation of miR-152, miR-200b and miR-10a genes as candidate bladder cancer biomarkers

Urinary miRNAs are discussed as potential biomarkers for bladder cancer. The majority of miRNAs, however, are downregulated, making it difficult to utilize reduced miRNA signals as reliable diagnostic tools. Because the downregulation of miRNAs is frequently associated with hypermethylation of the respective regulative sequences, we studied whether DNA hypermethylation might serve as an improved diagnostic tool compared to measuring downregulated miRNAs. miRNA expression arrays and individual qPCR were used to identify and confirm miRNAs that were downregulated in malignant urothelial cells (RT4, 5637 and J82) when compared to primary, non-malignant urothelial cells (HUEPC). DNA methylation was determined by customized PCR-arrays subsequent to methylation-sensitive DNA-restriction and by mass spectrometry. miRNA expression and DNA methylation were determined in untreated cells and in cultures treated with the demethylating agent 5-Aza-2′-deoxycytidine. miR-200b, miR-152 and miR-10a displayed differential expression and methylation among untreated cancer cell lines. In addition, reduced miRNA expression of miR-200b, miR-152, and miR-10a was associated with increased DNA methylation in malignant cells versus HUEPC. Finally, the demethylation approach revealed a causal relationship between both parameters for miR-152 in 5637 and also suggests a causal connection of both parameters for miR-200b in J82 and miR-10a in 5637. In conclusion, our studies in multiple bladder cancer cell lines and primary non-malignant urothelial cells suggest that hypermethylation of miR-152, miR-10a and miR-200b regulative DNA sequences might serve as epigenetic bladder cancer biomarkers.     

MicroRNAs as biomarkers of cervical cancer development: a literature review on miR-125b and miR-34a

MicroRNAs are non-coding RNAs with important functions in several biological processes, such as, regulation of cell cycle, immune response, inflammation, and apoptosis. In fact, deregulation and abnormal expression of these molecules is associated with human pathologies including cancer and several have already emerged as potential prognostic biomarkers in different neoplasias. miR-34a is directly regulated by p53 and acts as tumor suppressor while miR-125b plays a significant role in immune response and apoptosis. In cervical carcinogenesis, HPV proteins seem to interact with both miR-34a and miR-125b changing its expression and promoting persistent infection and cervical cancer development. In this review we describe the potential role of miR-125b and miR-34a in cervical carcinogenesis, including interaction with HPV and mechanism of deregulation. Additionally, their clinical applications in cervical cancer as prognostic/predictive biomarkers are also briefly discussed.     

The clinical significance of circulating miR-21, miR-142, miR-143, and miR-146a in patients with prostate cancer

BackgroundProstate cancer (PCa) is the most common type of solid tissue cancer among men in western countries. In this study, we determined the levels of circulating miR-21, miR-142, miR-143, miR-146a, and RNU 44 levels as controls for early diagnosis of PCa.MethodsThe circulating miRNA levels in peripheral blood samples from 43 localized PCa patients, 12 metastatic PCa (MET) patients, and a control group of, 42 benign prostate hyperplasia (BPH) patients with a total of 97 volunteers were determined the by PCR method.ResultsNo differences in the DCT values were found among the groups. In PCa and PCaMet groups the expression of miR21 and miR142 were higher compared to the BHP group. No other differences were observed among the other groups. miR21 expression in the PCa group was 6.29 folds upregulated whereas in the PCaMet group 10.84 folds up-regulated. When the total expression of miR142 is evaluated, it showed a positive correlation with mir21 and mir 146 (both p<0.001). Also, the expression of miR146 shows a positive correlation with both miR21 and miR143 (both p<0.001). Expression of miRNAs was found to be an independent diagnostic factor in patients with Gleason score, PSA, and free PSA levels.ConclusionsOur study showed that co-expression of miR21, miR-142, miR-143, and miR-146a and the upregulation of miR-21 resulted in increased prostate carcinoma cell growth. In the PCaMet group, miR21 is the most upregulated of all miRNAs. These markers may provide a novel diagnostic tool to help diagnose PCa with aggressive behavior.     

Attenuation of endoplasmic reticulum stress-related myocardial apoptosis by SERCA2a gene delivery in ischemic heart disease

Previous studies suggested that endoplasmic reticulum (ER) stress-associated apoptosis plays an important role in the pathogenesis of ischemic heart disease. Gene transfer of sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) attenuates myocardial apoptosis in a variety of heart failure models. This study is to investigate the effects of SERCA2a gene delivery on the myocardial apoptosis and ER stress pathway in a porcine ischemic heart disease model. Eighteen pigs were either subjected to ameroid implantation in the coronary artery or sham operation. Eight wks after gene delivery, the protein level and activity of SERCA2a were measured. Myocardial apoptosis was determined using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling assay. Regional myocardial perfusion and function were evaluated by (99m)Tc-sestamibi ((99m)Tc-MIBI) single photon emission computed tomography and echocardiography. The ER stress signaling was assessed by Western blot. SERCA2a protein level and activity were significantly decreased in the ischemic myocardium and restored to normal after SERCA2a gene transfer. Restoration of SERCA2a expression significantly improved the cardiac function, although no improvement of regional myocardial perfusion was detected. Restoration of SERCA2a significantly attenuated myocardial apoptosis and reversed the activation of unfolded protein response (UPR) pathway and the ER stress-associated apoptosis pathways. These findings demonstrate a robust role of SERCA2a in attenuation of ischemic myocardial apoptosis, correlating with reverse activation of the ER stress-associated apoptosis pathways, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of ER stress-associated myocardial apoptosis.     

Characterization of an endoplasmic reticulum stress-related signature to evaluate immune features and predict prognosis in glioma

Endoplasmic reticulum (ER) stress has considerable impact on cell growth, proliferation, metastasis, invasion, angiogenesis and chemoradiotherapy resistance in various cancers. However, the effect of ER stress on the outcomes of glioma patients remains unclear. In this study, we established an ER stress risk model based on The Cancer Genome Atlas (TCGA) glioma data set to reflect immune characteristics and predict the prognosis of glioma patients. Survival analysis indicated that there were significant differences in the overall survival (OS) of glioma patients with different ER stress-related risk scores. Moreover, the ER stress-related risk signature, which was markedly associated with the clinicopathological properties of glioma patients, could serve as an independent prognostic indicator. Functional enrichment analysis revealed that the risk model correlated with immune and inflammation responses, as well as biosynthesis and degradation. In addition, the ER stress-related risk model indicated an immunosuppressive microenvironment. In conclusion, we present an ER stress risk model that is an independent prognostic factor and indicates the general immune characteristics in the glioma microenvironment.     

Circulating miR-17, miR-20a,miR-29c,and miR-223 Combined as Non-Invasive Biomarkers in Nasopharyngeal Carcinoma

Background

MicroRNAs have been considered as a kind of potential novel biomarker for cancer detection due to their remarkable stability in the blood and the characteristics of their expression profile in many diseases.

Methods

We performed microarray-based serum miRNA profiling on the serum of twenty nasopharyngeal carcinoma patients at diagnosis along with 20 non-cancerous individuals as controls. This was followed by a real-time quantitative Polymerase Chain Reaction (RT-qPCR) in a separate cohort of thirty patients with nasopharyngeal carcinoma and thirty age- matched non-cancerous volunteers. A model for diagnosis was established by a conversion of mathematical calculation formula which has been validated by analyzing 74 cases of patients with nasopharyngeal carcinoma and 57 cases of non-cancerous volunteers.

Results

The profiles showed that 39 and 17 miRNAs are exclusively expressed in the serum of non-cancerous volunteers and of patients with nasopharyngeal carcinoma respectively. 4 miRNAs including miR-17, miR-20a, miR-29c, and miR-223 were found to be expressed differentially in the serum of NPC compared with that of non-cancerous control. Based on this, a diagnosis equation with Ct difference method has been established to distinguish NPC cases and non-cancerous controls and validated with high sensitivity and specificity.

Conclusions

We demonstrate that the serum miRNA-based biomarker model become a novel tool for NPC detection. The circulating 4-miRNA-based method may provide a novel strategy for NPC diagnosis.     

Identification of a novel chloride channel expressed in the endoplasmic reticulum, golgi apparatus, and nucleus

MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. Using MID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.     

Identification of a G protein in rough endoplasmic reticulum of canine pancreas

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.     

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.     

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.     

MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.     

Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells

The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.