The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3'-terminal nucleotides via 2'-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2'-O-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3'-terminal 2'-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3'-terminal 2'-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3'-terminal 2'-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.
Animals ; High-Throughput Nucleotide Sequencing ; Humans ; Methylation ; Mice ; MicroRNAs/genetics* ; Oxidative Stress ; RNA, Small Interfering/metabolism* ; Real-Time Polymerase Chain Reaction

Objective To evaluate the development level of public health in Zhejiang Province during China's 12th Five-Year Plan period (from 2011 to 2015)for health policy making in the future. Methods Totally 30 indexes were first summarized into 4 components, including the residents' health level, the allocation of public health resource, the level of public health service and public health security. The evaluation index system was developed by Delphi method. The indicators of 2010 were selected as the baseline for comparison with those corresponding indicators from 2011 to 2015. Each index(the comprehensive development index, the developmental speed, as well as the indicator index)calculated during 12th Five-Year Plan period was evaluated after the targeted indicators were weighed. Results Overall, at a provincial level of Zhejiang, the comprehensive development index was increased from 100 in 2010 to 138.44 in 2015, representing an annual average development speed of 6.72%. The annual average development speed of 13 indexes, ranging from 6.72% to 26.18%, were higher than those in the China's 11th Five-Year Plan period(6.72%); while the annual average development speed of another 17 indexes was lower, with 1 index below zero. However, the number of medical staff per 10000 population(5.23), the treatment success rate among new smear-positive cases (90.76%)and the number of annual outpatient visits in primary hospitals(49.45%)in Zhejiang Province were lower than those in China during the same period(6.39, 92.49%, 56.40%). At a prefectural level, the comprehensive development index fluctuated from 128.65 in Quzhou to 156.35 in Jiaxing and the developmental speed was from 3.90% to 11.26%. These results indicated that the coefficient variant(CV)was 5.76 during China's 12 th Five-year Plan period, which had recorded the historically lowest level since 2010. Conclusion During the China's 12th Five-year Plan period, the development of public health in Zhejiang Province was steadily enhanced. Public health variably developed across 11 prefectures with shrinking gaps. Nevertheless, the number of medical staff per 10 000 population, the treatment success rate among new smear-positive cases and the number of annual outpatient visits in primary hospitals should be improved.

Adaptation, Psychological ; Adult ; Anxiety ; epidemiology ; psychology ; Depression ; epidemiology ; psychology ; Female ; Humans ; Intensive Care Units ; Male ; Middle Aged ; Nurses ; psychology ; statistics & numerical data ; Physicians ; psychology ; statistics & numerical data ; Prevalence ; Singapore ; epidemiology ; Stress Disorders, Post-Traumatic ; epidemiology ; psychology ; Young Adult

The BCL6 (B-Cell Lymphoma 6) gene is a proto-oncogene that is often expressed in diffuse large B-cell lymphomas (DLBCLs). BCL6 loss of function can kill DLBCL cells, demonstrating that BCL6 is necessary for the survival of DLBCL cells and could be a therapeutic target. In this study, we found that BCL6 protein levels were consistently upregulated in DLBCL tissues, whereas its mRNA levels varied randomly in tissues, suggesting that a post-transcriptional mechanism was involved in BCL6 regulation. We used bioinformatics analysis to search for miRNAs, which potentially target BCL6, and identified specific targeting sites for miR-10a in the 3'-untranslated region (3'-UTR) of BCL6. We further identified an inverse correlation between miR-10a levels and BCL6 protein levels, but not mRNA levels, in DLBCL tumor tissue samples. By overexpressing or knocking down miR-10a in DLBCL cells, we experimentally validated that miR-10a directly recognizes the 3'-UTR of the BCL6 transcript and regulated BCL6 expression. Furthermore, we demonstrated that negatively regulating BCL6 by miR-10a suppressed the proliferation and promoted apoptosis of DLBCL cells.
3' Untranslated Regions ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; therapy ; MicroRNAs ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-6 ; biosynthesis ; genetics

OBJECTIVETo study the characteristics of phenylalanine hydroxylase gene (PAH) mutations in patients with PAH deficiency in Fujian population.

METHODSPeripheral blood samples of 36 patients and their parents with classical type phenylketouria (PKU) were collected. Genomic DNA was extracted. Following PCR amplification, DNA sequencing was carried out to identify the origins of mutations.

RESULTSTwenty types mutations were identified in 63 of the 72 alleles. The most common mutations were R241C, R408Q and Ex6-96A>G, which respectively accounted for 15.9%, 12.7% and 11.1% of all mutant alleles. The c.189_190dupTGAC mutation was first reported. R241C was associated with 28% of mild hyperphenylalaninemia and R408Q is associated with 25% of classical PKU.

CONCLUSIONThere is a specific spectrum of PAH gene mutation in Fujian region. R241C, R408Q and Ex6-96A>G are the most common mutations.

Adolescent ; Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; China ; Female ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; enzymology ; genetics

Amniotic Fluid ; drug effects ; metabolism ; Female ; Fetus ; drug effects ; metabolism ; Gene Expression Regulation, Developmental ; drug effects ; genetics ; Humans ; MicroRNAs ; genetics ; pharmacology ; Placenta ; metabolism ; Pregnancy ; RNA, Plant ; genetics ; pharmacology ; Umbilical Cord ; drug effects ; metabolism

Avian Myeloblastosis Virus ; enzymology ; Escherichia coli ; genetics ; metabolism ; Genome, Bacterial ; MicroRNAs ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA

MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression. The deregulated expression of miRNAs is associated with a variety of diseases, including breast cancer. In the present study, we found that miR-495 was markedly up-regulated in clinical breast cancer samples by quantitative real time-PCR (qRT-PCR). Junctional adhesion molecule A (JAM-A) was predicted to be a potential target of miR-495 by bioinformatics analysis and was subsequently verified by luciferase assay and Western blotting. JAM-A was found to be negatively correlated with the migration of breast cancer cells through loss-of-function and gain-of-function assays, and the inhibition of JAM-A by miR-495 promoted the migration of MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of JAM-A could restore miR-495-induced breast cancer cell migration. Taken together, our findings suggest that miR-495 could facilitate breast cancer progression through the repression of JAM-A, making this miRNA a potential therapeutic target.
3' Untranslated Regions ; genetics ; Adult ; Aged ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Adhesion Molecules ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; MCF-7 Cells ; MicroRNAs ; genetics ; Middle Aged ; RNA Interference ; Receptors, Cell Surface ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells (MDSCs) could prevent the concanavalin A (ConA)-induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConA-mediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell (Treg)-depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.
Adoptive Transfer ; Animals ; Blotting, Western ; Bone Marrow Cells ; immunology ; CD11b Antigen ; immunology ; metabolism ; Cell Movement ; immunology ; Cell Proliferation ; Chemical and Drug Induced Liver Injury ; etiology ; immunology ; prevention & control ; Concanavalin A ; toxicity ; Dexamethasone ; pharmacology ; Flow Cytometry ; Glucocorticoids ; pharmacology ; Liver ; immunology ; pathology ; Male ; Mice, Inbred C57BL ; Mitogens ; administration & dosage ; toxicity ; Myeloid Cells ; immunology ; metabolism ; transplantation ; Receptors, Chemokine ; immunology ; metabolism ; Spleen ; immunology ; pathology ; T-Lymphocytes ; immunology ; T-Lymphocytes, Regulatory ; immunology

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that are involved in post-transcriptional gene regulation. It has long been assumed that miRNAs exert their roles only in the cytoplasm, where they recognize their target protein-coding messenger RNAs (mRNAs), and result in translational repression or target mRNA degradation. Recent studies, however, have revealed that mature miRNAs can also be transported from the cytoplasm to the nucleus and that these nuclear miRNAs can function in an unconventional manner to regulate the biogenesis and functions of ncRNAs (including miRNAs and long ncRNAs), adding a new layer of complexity to our understanding of gene regulation. In this review, we summarize recent literature on the working model of these unconventional miRNAs and speculate on their biological significance. We have every reason to believe that these novel models of miRNA function will become a major research topic in gene regulation in eukaryotes.
Cell Nucleus ; genetics ; Cytoplasm ; genetics ; Eukaryota ; genetics ; Gene Expression Regulation ; Humans ; MicroRNAs ; genetics ; RNA Stability ; genetics ; RNA, Long Noncoding ; genetics ; RNA, Messenger ; genetics ; metabolism