Circular RNAs (circ RNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome. They share a stable structure, high expression and often exhibit tissue/developmental-stage-specific expression. Emerging evidence indicates that circRNAs might play important roles in human disease, such as cancer, neurological disorders and atherosclerotic vascular disease risk. The huge potentials of circRNAs are recently being discovered from the laboratory to the clinic. CircRNAs might be developed as a potential novel and stable biomarker and potential drugs used in disease diagnosis and treatment. Here, we review the current understanding of the roles of circRNAs in human disease and their potential clinic significance in disease.
Biomarkers ; analysis ; Disease ; genetics ; Gene Expression Regulation ; Humans ; Neoplasms ; diagnosis ; genetics ; therapy ; RNA ; genetics ; RNA Stability ; Signal Transduction ; genetics

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

Mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of several metabolic diseases. Recently, we demonstrated that C1qTNF-related protein-6 (CTRP6) is involved in fatty acid metabolism in muscle cells. In this study, we showed that expression of CTRP6 was up-regulated in mtDNA-depleted C2C12 cells, which displayed a marked decrease in cellular mtDNA and ATP content. Replacement of mtDNA normalized the expression level of CTRP6 similar to that in normal C2C12 cells, indicating that CTRP6 expression was up-regulated by mtDNA depletion. However, CTRP6 promoter activity remained unchanged in mtDNA-depleted cells. We also found that mtDNA depletion inhibited decay of CTRP6 mRNA. Taken together, mtDNA depletion induces an increase in CTRP6 expression by increasing mRNA stability.
Adiponectin/*genetics/metabolism ; Animals ; Cell Line ; DNA, Mitochondrial/*metabolism ; Mice ; Promoter Regions, Genetic ; RNA Stability ; RNA, Messenger/*metabolism ; Up-Regulation

The let-7 miRNA was one of the first miRNAs discovered in the nematode, Caenorhabditis elegans, and its biological functions show a high level of evolutionary conservation from the nematode to the human. Unlike in C. elegans, higher animals have multiple isoforms of let-7 miRNAs; these isoforms share a consensus sequence called the 'seed sequence' and these isoforms are categorized into let-7 miRNA family. The expression of let-7 family is required for developmental timing and tumor suppressor function, but must be suppressed for the self-renewal of stem cells. Therefore, let-7 miRNA biogenesis must be carefully controlled. To generate a let-7 miRNA, a primary transcript is produced by RNA polymerase II and then subsequently processed by Drosha/DGCR8, TUTase, and Dicer. Because dysregulation of let-7 processing is deleterious, biogenesis of let-7 is tightly regulated by cellular factors, such as the RNA binding proteins, LIN28A/B and DIS3L2. In this review, we discuss the biological functions and biogenesis of let-7 miRNAs, focusing on the molecular mechanisms of regulation of let-7 biogenesis in vertebrates, such as the mouse and the human.
Animals ; Base Sequence ; Gene Expression Regulation ; Humans ; MicroRNAs ; biosynthesis ; chemistry ; genetics ; metabolism ; RNA Processing, Post-Transcriptional ; RNA Stability ; Transcription, Genetic

It has become a hotspot and keystone in gene engineering and bioengineering to produce recombinant proteins through heterologous expression systems. Unfortunately, not all the genes could be successfully and effectively expressed in heterologous hosts. The role of gene itself in regulating translation process through its intrinsic sequence characteristics such as codon bias, codon pair bias, GC content, mRNA secondary structure and mRNA stability, has been gradually elucidated. Here we review these factors that influence the translation processes and their corresponding optimization methods in the process of gene design. We emphatically discussed codon bias and codon pair bias and their optimization methods. In particular, the latest theories of codon harmonization and codon pair harmonization were discussed and compared with the traditional codon and codon pair optimization strategies in gene design.
Codon ; genetics ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; Genes, Synthetic ; genetics ; Protein Biosynthesis ; genetics ; Protein Engineering ; methods ; trends ; RNA Stability ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVE: To explore the feasibility of real-time RT-PCR of housekeeping mRNA degradation in dead rats in order to seek new suitable techniques for post-mortem interval (PMI). METHODS: The levels of GAPDH mRNA and beta-actin mRNA in the brain and spleen of the rats were measured at different times after death by the SYBR Green I real-time RT-PCR. The relative mRNA level was indicated with the Ct value, then the relationship between the Ct value and PMI were analyzed and the corresponding regression equation was obtained. RESULTS: The Ct values of GAPDH mRNA and beta-actin mRNA by SYBR Green I real time RT-PCR correlated well with the PMI. CONCLUSION The SYBR Green I real-time RT-PCR is a reliable technique for studying mRNA degradation. Adoption of the housekeeping genes eliminates systematical errors of other genes which have individual difference. As an objective and dynamic indication, Ct value has a good correlation with PMI, and could be applied for estimating PMI, especially in late period.
Actins/genetics* ; Animals ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Postmortem Changes ; RNA Stability/genetics* ; RNA, Messenger/metabolism* ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction/methods* ; Time Factors

OBJECTIVE: To explore the correlation between postmortem interval (PMI) and five RNA markers of rat's skin--β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA(18S rRNA), 5S ribosomal RNA (5S rRNA), and microRNA-203 (miR-203), at different temperatures. METHODS: Eighteen SD rats were randomly divided into three environmental temperature groups: 4 °C, 15 °C and 35 °C, respectively. Skin samples were taken at 11 time points from 0 h to 120 h post-mortem. The total RNA was extracted from the skin samples and the five RNA levels were detected by real-time fluorescent quantitative PCR. Proper internal reference was selected by geNorm software. Regression analysis of the RNA markers was conducted by GraphPad software. RESULTS: 5S rRNA and miR-203 were most suitable internal references. A good linear relationship between PMI and RNA levels (β-actin and GAPDH) was observed in two groups (4 °C and 15 °C), whereas the S type curve relationship between the expression levels of the two markers (β-actin and GAPDH) and PMI was observed in the 35 °C group. The partial linear relationship between 18S rRNA and PMI was observed in the groups (15 °C and 35 °C). CONCLUSION Skin could be a suitable material for extracting RNA. The RNA expression levels of β-actin and GAPDH correlate well with PMI, and these RNA markers of skin tissue could be additional indice for the estimation of PMI.
Actins ; Animals ; Autopsy ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Postmortem Changes ; RNA ; RNA Stability ; RNA, Ribosomal, 18S ; Rats ; Real-Time Polymerase Chain Reaction ; Regression Analysis ; Skin ; Temperature

With the development of molecular biology, the evidences of genetics has been used widely in forensic sciences. DNA technology has played an important role in individual identification and paternity testing, RNA technology is showing more and more wide application in prospect. This article reviews the application and progress of RNA in forensic science including estimation of postmortem interval, bloodstain age, wound age, as well as determination of cause of death and the source of body fluids.
Actins/metabolism* ; Animals ; Blood Stains ; Body Fluids/metabolism* ; Cause of Death ; Forensic Medicine/methods* ; Gene Expression ; Genetic Markers/genetics* ; Humans ; MicroRNAs/genetics* ; Polymerase Chain Reaction/methods* ; Postmortem Changes ; RNA/genetics* ; RNA Stability ; RNA, Messenger/metabolism* ; RNA, Ribosomal, 18S/genetics*

OBJECTIVETo investigate the effect of deletion of the La protein binding site of HBV RNA, caused by its mutation, on the HBV S-mRNA stability of S gene, to study the role of the site in hepatitis B virus life cycle, and to try to find a new anti-HBV target in the future.

METHODSA HBV vector with mutation related to the La protein binding site was constructed using molecular cloning and PCR based site directed mutagenesis, and the vector was named pHBV-mLa. The HBV RNA secondary structure of the site was calculated using a computer. Normal HBV vectors and mutant vectors were respectively transfected into HepG2 cells by Lipofectamine 2000. HBV S-mRNA levels in the two groups were analyzed using semi-quantitative RT-PCR, and HBsAg secretion into the culture media was tested using ELISA.

RESULTSA HBV vector with mutation related to the La protein binding site was successfully constructed, and it was identified and confirmed using restriction analysis and sequencing. The HBV RNA secondary structure of the mutant vector was completely different to the stem-loop structure of the normal HBV vector. Semi-quantitative RT-PCR and ELISA analyses showed that the level of HBV S-mRNA in the mutant vector group was significantly lower than that in the normal HBV vector group (t'=12.703, P<0.05), and the expression efficacy of HBsAg was reduced in the mutant vector group (t= 44.648, P<0.01).

CONCLUSIONSThe change of La protein binding site in the HBV RNA caused by the mutation in HBV DNA disorganizes the stem-loop structure in the HBV RNA site. With the structural change, the La protein cannot bind the site and stabilize the HBV RNA (HBV S-mRNA), as the cleavage site in the upstream of the stem-loop structure is exposed to endoribonuclease. This results in HBV S-mRNA decay and affects the expression of the S gene. This study shows that only the sequence of this site in the HBV DNA is reserved, then the stem-loop structure in the La protein binding site will remain intact, and the disorganization of the stem-loop structure affects the stability of the transcripted HBV RNA. The La protein binding site in HBV RNA and the special secondary structure of the site are crucial to the life cycle of the hepatitis B virus.

Binding Sites ; Cell Line, Tumor ; Gene Deletion ; Genetic Vectors ; Hepatitis B virus ; genetics ; Humans ; Mutation ; Nucleic Acid Conformation ; RNA Stability ; RNA, Messenger ; genetics ; RNA, Viral ; genetics ; Viral Envelope Proteins ; genetics

OBJECTIVETo study the effects of licorice on the proliferation of intestinal crypt stem cell line IEC-6 and the expression of p53.

METHODSInduced by difluoro-methylornithine (DFMO), polyamine-depleted IEC-6 cells under growth inhibition were used as the pathological cell model in this study. Cells were divided into four groups, i. e., the control group, the DFMO-treated group, the high dose licorice group, and the low dose licorice group. The control group consisted of IEC-6 cells cultured in normal condition. The other three groups were all treated with 5 mmol/L DFMO. The high dose and low dose licorice groups were supplemented with 40 and 80 microg/mL licorice granule respectively. All the groups were cultured for 6 successive days. The cell number and viability were determined using flow cytometry. The level of p53 protein was detected by Western blot. The p53 mRNA levels and stability were detected using fluorescent quantitative Real-time PCR.

RESULTSCompared with the control group, the cell growth of the DFMO group was obviously inhibited on the 4th day (P < 0.05). The cell number increased more obviously in the low dose licorice and the high dose licorice groups in a dose-dependent way on the 6th day when compared with the DFMO group (P < 0.05). When compared with the control group, significantly elevated expression levels of p53 protein and mRNA in cells of the DFMO group were detected after 6-day treatment (P < 0.05). When compared with the DFMO group, the expression levels of p53 protein and mRNA were significantly down-regulated in the low dose licorice and the high dose licorice groups (P < 0.05). The degradation of p53 mRNA was the fastest in the control group, while the degradation speed of cells in the DFMO group was the slowest.

CONCLUSIONOne of mechanisms for protective and healing effects of licorice on the intestinal mucosa was possibly through down-regulating the stability of p53 mRNA, lowering the expression of p53, thus promoting the proliferation of the intestinal crypt stem cells.

Animals ; Cell Line ; Cell Proliferation ; Glycyrrhiza ; Intestines ; cytology ; metabolism ; RNA Stability ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; metabolism