Sustained viral activity of epstein-Barr virus contributes to cellular immortalization of lymphoblastoid cell lines

EBV-transformed lymphoblastoid cell lines (LCLs) are used as a resource for human genetic, immunological, and pharmacogenomic studies. We investigated the biological activity of 20 LCL strains during continuous long-term subculture up to a passage number of 160. Out of 20 LCL strains, 17 proliferated up to a passage number of 160, at which point LCLs are generally considered as “immortalized”. The other three LCL strains lost the ability to proliferate at an average passage number of 41, during which these LCLs may have undergone cellular crisis. These non-immortal LCL strains exhibited no telomerase activity, decreased EBV gene expression, and a lower copy number of the EBV genome and mitochondrial DNA when compared with immortal LCLs. Thus, this study suggests that sustained EBV viral activity as well as telomerase activity may be required for complete LCL immortalization. These authors contributed equally to this work.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

Three common functional polymorphisms in microRNA encoding genes in the susceptibility to hepatocellular carcinoma: A systematic review and meta-analysis

Emerging evidences have shown that common genetic polymorphisms in microRNAs may be associated with the development of hepatocellular carcinoma (HCC); but individually published studies and previous meta-analyses revealed inconclusive results. The aims of this review and meta-analysis are to assess whether common single-nucleotide polymorphisms (SNPs) in the genes encoding the microRNAs are associated with susceptibility to HCC development and clinicopathologic characteristics of hepatitis B virus (HBV) related HCC. A computerized search was performed in PubMed, Embase, Web of Science and China BioMedicine (CBM) databases to identify relevant articles published before January 1st 2013. Ten case–control studies were assessed with a total of 3437 cases and 3437 healthy controls. Three common functional SNPs in miRNA-encoding genes were found, including miR-146a G > C (rs2910164), miR-196a-2 C > T (rs11614913) and miR-499 T > C (rs3746444). This meta-analysis revealed that the miR-146a*C variant was associated with a decrease in HCC risk, especially among Asian and male populations; while the miR-196a-2*T variant was associated with susceptibility to HCC among Caucasian populations. However, we failed to find any significant correlations between the miR-499*C polymorphism and HCC risks. When further stratification on HBV status was conducted, a similar trend of association between the three SNPs and the HBV-related HCC risks was observed, but these results were not statistically significant due to small sample sizes. The current meta-analysis demonstrates that SNPs contained in the genes encoding miR-146a and miR-196a-2 may play a major role in genetic susceptibility to HCC.     

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits

Objective: MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein–Barr virus (EBV)‐transformed lymphoblastoid cell line (LCL) and associated clinical traits. Material and Methods: Hence, we performed miRNA microarray approach with early‐ (p6) and late‐passage (p161) LCLs. Results and Conclusion: Microarray data showed that nine miRNAs (miR‐20b*, miR‐28‐5p, miR‐99a, miR‐125b, miR‐151‐3p, miR‐151:9.1, miR‐216a, miR‐223* and miR‐1296) were differentially expressed in most LCLs during long‐term culture. In particular, miR‐125b was up‐regulated in all the tested late‐passage LCLs. miR‐99a, miR‐125b, miR‐216a and miR‐1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long‐term culture in a previous study. Linear regression analysis showed that miR‐200a and miR‐296‐3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor’s health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV‐mediated tumorigenesis.     

The peripheral blood mononuclear cell microRNA signature of coronary artery disease

Background

MicroRNAs are being used in the oncology field to characterize tumors and predict the survival of cancer patients. Here, we explored the potential of microRNAs as biomarkers for coronary artery disease (CAD) and acute coronary syndromes.

Methods and results

Using real-time PCR-based profiling, we determined the microRNA signature of peripheral blood mononuclear cells (PBMCs) from stable and unstable CAD patients and unaffected controls. 129 of 157 microRNAs measured were expressed by PBMCs and low variability between separate PBMC pools was observed. The presence of CAD in general coincided with a marked 5-fold increase (P < 0.001) in the relative expression level of miR-135a, while the expression of miR-147 was 4-fold decreased (P < 0.05) in PBMCs from CAD patients as compared to controls, resulting in a 19-fold higher miR-135a/miR-147 ratio (P < 0.001) in CAD. MicroRNA/target gene/biological function linkage analysis suggested that the change in PBMC microRNA signature in CAD patients is probably associated with a change in intracellular cadherin/Wnt signaling. Interestingly, unstable angina pectoris patients could be discriminated from stable patients based upon their relatively high expression level of a cluster of three microRNAs including miR-134, miR-198, and miR-370, suggesting that the microRNA signatures can be used to identify patients at risk for acute coronary syndromes.

Conclusions

The present study is the first to show that microRNA signatures can possibly be utilized to identify patients exhibiting atherosclerotic CAD in general and those at risk for acute coronary syndromes. Our findings highlight the importance of microRNAs signatures as novel tool to predict clinical disease outcomes.     

Dehydration mediated microRNA response in the African clawed frog Xenopus laevis

Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure > 30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37 ± 6, 64 ± 8, and 80 ± 4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05 ± 0.45, 2.11 ± 0.08, and 1.36 ± 0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40 ± 0.09, 1.31 ± 0.05, and 2.17 ± 0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35 ± 0.05 and 1.74 ± 0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression.     

Decreased circulating miR-375: A potential biomarker for patients with non-small-cell lung cancer

MicroRNAs (miRNAs) are directly involved in cancer initiation, progression and metastasis. Alterations of miRNAs expression in cancer tissue may be reflected in circulation. We attempted to investigate the expression and clinical significance of plasma miR-20a, miR-31 and miR-375 in patients with non-small cell lung cancer (NSCLC). The plasma levels of miR-20a, miR-31 and miR-375 in 164 NSCLC patients and 164 healthy controls (discovery cohort) were evaluated and compared among various clinicopathological characteristics. The relationship between miRNA expression and clinical outcome of NSCLC patients was examined in an independent cohort (53 cases and 53 controls). The expression level of miR-375 in tissue was also examined. Plasma miR-375 levels in NSCLC patients were significantly decreased in both patient cohorts (P < 0.05). In addition, patients with metastatic NSCLC had lower plasma miR-375 expression than those with non-metastatic NSCLC (P < 0.05). Survival analysis showed that patients with low miR-375 expression had worse overall survival rates than those with high miR-375 expression (hazard ratios (HR) = 1.537 (1.046–2.258), P = 0.029). This association was independently validated in a separate cohort of 53 NSCLC patients (HR = 2.406, 95% CI 1.170–4.945, P = 0.017). The expression level of miR-375 was also found to be significantly down-regulated in NSCLC tissues compared with paracancerous tissues (P < 0.001). These findings indicate that miR-375 has an important role in NSCLC initiation and progression, and may be an independent poor prognostic factor in NSCLC patients.     

Exploring differentially expressed genes in the ovaries of uniparous and multiparous goats using the RNA-Seq (Quantification) method

Fecundity improvement is one of the most important objectives for goat breeders as it greatly increases production efficiency. To investigate the genes associated with litter sizes in the Anhui White goat (AWG), gene expression differences in the ovaries of uniparous and multiparous AWG were assessed using the RNA-Seq (Quantification) method. This analysis generated 6,027,714 and 5,884,062 clean reads in uniparous and multiparous libraries, respectively. A total of 2201 differentially expressed genes (DEGs) were thereby identified (FDR ≤ 0.001, |log₂Ratio| ≥ 1). There were 1583 up-regulated and 618 down-regulated genes in the multiparous samples compared with the uniparous samples. A large number of these DEGs were related to the terms cellular process, cell & cell part and binding. Twelve genes which may be associated with the high prolificacy of AWG were identified using a bioinformatic screen. In addition, pathway analysis revealed that these DEGs were significantly enriched in 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including ECM–receptor interactions, focal adhesion, and regulation of the actin cytoskeleton among others. This suggested a role for these pathways in the prolificacy of AWG. These results provide a list of new candidate genes for goat prolificacy.     

Downregulation of microRNA-214 and overexpression of FGFR-1 contribute to hepatocellular carcinoma metastasis

miR-214 is one of the most significantly downregulated microRNAs (miRNAs) in hepatocellular carcinoma (HCC). Fibroblast growth factor receptor 1 (FGFR-1) is a miR-214 target gene implicated in the progression of HCC. However, the roles of miR-214 and FGFR-1 in HCC are not fully understood. Here, we analyzed the expression of miR-214 and FGFR-1 in 65 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR and Western blot (WB), respectively. Our data indicated that miR-214 was downregulated and FGFR-1 was overexpressed in HCC compared to the paired non-neoplastic tissues. The low miR-214 expression was correlated with portal vein invasion (p = 0.016) and early recurrence (p = 0.045) in HCC patients. Moreover, the low miR-214 expression was correlated with high positive rate of FGFR-1 in HCC cases (p = 0.020). Our data further demonstrated that miR-214 overexpression in SK-HEP1 and HepG2 cells downregulated FGFR-1 expression and inhibited liver cancer cell invasion. The Luciferase assay results further demonstrated the targeted regulation of FGFR-1 by miR-214. In conclusion, our data indicate that the downregulation of miR-214 in HCC and the upregulation of its target gene FGFR-1 is associated with HCC progression. Therefore, miR-214 and FGFR-1 are potential prognostic markers and therapeutic targets in HCC.     

Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature

Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs), have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC) system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCL cultures from six unrelated donors to iPSCs on the ensuing gene expression patterns within and between individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures, increasing the number of genes with a detectable donor effect by an order of magnitude. The proportion of variation in gene expression statistically attributed to donor increases from 6.9% in LCLs to 24.5% in iPSCs (P < 10^-15). Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in iPSCs than in LCLs. Our findings indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation.     

Integrated microRNA–mRNA analysis of coronary artery disease

Although patients with coronary artery disease (CAD) have a high mortality rate, the pathogenesis of CAD is still poorly understood. The purpose of this study was to explore the underlying molecular mechanisms and potential target molecules for CAD. The platelet miRNA (GSE28858) and blood mRNA (GSE42148) expression profiles of patients with CAD and healthy controls were downloaded from Gene Expression Omnibus. Differentially expressed miRNAs and genes (DEGs) were identified by significant analysis of microarray algorithm after data preprocessing. Furthermore, the miRNA-target gene regulatory network was constructed based on miRecords database. The spearman correlation coefficients (ρ) between miRNAs and their target genes were calculated. Six up- (miR-340, miR-545, miR-451, miR454-5p, miR-624 and miR-585) and four down-regulated (miR-199a, miR-17-3p, miR-154 and miR-339) miRNAs were screened. Total 295 target genes of miR-545, miR-451, miR-585 and miR-154 were predicted. Among these 295 target genes, 7 genes were DEGs. Further analysis showed miR-545-TFEC and miR-585-SPOCK1 were highly positively correlated (ρ = 0.808091264; ρ = 0.874680776) in CAD samples. Therefore, differentially expressed miRNAs might participate in the pathogenesis of CAD by regulating their target genes.     

Identification and expression of microRNA in the brain of hibernating bats, Myotis lucifugus

Recent research has highlighted roles for non-coding RNA i7n the regulation of stress tolerance in bats. In this study, we propose that microRNA could also play an important role in neuronal maintenance during hibernation. To explore this possibility, RT-PCR was employed to investigate the expression of eleven microRNAs from the brain tissue of euthermic control and torpid bats. Results show that eight microRNAs (miR-21, -29b, -103, -107, -124a, -132, -183 and -501) increased (1.2–1.9 fold) in torpid bats, while the protein expression of Dicer, a microRNA processing enzyme, did not significantly change during torpor. Bioinformatic analysis of the differentially expressed microRNA suggests that these microRNAs are mainly involved in two processes: (1) focal adhesion and (2) axon guidance. To determine the extent of microRNA sequence conservation in the bat, we successfully identified bat microRNA from sequence alignments against known mouse (Mus musculus) microRNA. We successfully identified 206 conserved pre-microRNA sequences, leading to the identification of 344 conserved mature microRNA sequences. Sequence homology of the identified sequences was found to be 94.76 ± 3.95% and 98.87 ± 2.24% for both pre- and mature microRNAs, respectively. Results suggest that brain function related to the differentiation of neurons and adaptive neuroprotection may be under microRNA control during bat hibernation.     

Transcriptional changes in response to X chromosome dosage in the mouse: implications for X inactivation and the molecular basis of Turner Syndrome

Background

In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.

Results

Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.

Conclusions

The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.