MicroRNAs (miRNAs) are short 20-25 nucleotides RNA molecules that have been shown to regulate gene expressions in a variety of eukaryotic systems. miRNAs are widespread in eukaryotes and several hundred of miRNAs have been identified, but still a lot of miRNAs have not been detected in various eukaryotic organisms. However, it is not an easy work to clone miRNAs by traditional methods. Here, we describe the identification of 27 miRNAs from a human fetal liver cDNA library by a novel cloning method. Low molecular weight RNA fraction (6200 nt) from fetal liver tissue was extracted, and polyadenylated by poly(A) polymerase. A 5 0 RNA adaptor was ligated to poly(A)-tailed RNA using T4 RNA ligase. After reverse transcription, the cDNA was amplified by PCR with two adaptor primers. The PCR product with a size about 109 bp was recovered and cloned into T vector. After sequencing, database searching, and expression profiling, 5 novel miRNAs were discovered among other 22 known miRNAs in human fetal liver. These finding indicate that a large diverse population of miRNAs may function to regulate gene expression in hepatocyte.

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with important roles in the regulation of gene expression. Although a large number of miRNAs have been identified in a variety of eukaryotic systems, the function of the vast majority of these molecules remains unknown. To study the functions of miRNAs, it is crucial to determine their spatial and temporal expression patterns. Although there are some existing methods that can analyze the expression of miRNAs, it is not an easy task for routine gene-expression studies. In this study, we have established a simple method to detect the expression of mature miRNAs. Total RNA was polyadenylated by poly(A) polymerase, and then cDNA was synthesized by a specific reverse transcriptase (RT) primer and reverse transcriptase using the poly(A)-tailed total RNA as templates. The expression of several mature miRNAs was assayed by this method. The expression profile of two miRNAs, determined by the polymerase chain reaction (PCR) assay, was identical to that determined by Northern blotting. All these data show that the poly(A)-tailed RT-PCR is a convenient method to detect the expression of miRNAs.

Mutations in POLG disrupt mtDNA replication and cause devastating diseases often with neurological phenotypes. Defining disease mechanisms has been hampered by limited access to human tissues, particularly neurons. Using patient cells carrying POLG mutations, we generated iPSCs and then neural stem cells. These neural precursors manifested a phenotype that faithfully replicated the molecular and biochemical changes found in patient post-mortem brain tissue. We confirmed the same loss of mtDNA and complex I in dopaminergic neurons generated from the same stem cells. POLG-driven mitochondrial dysfunction led to neuronal ROS overproduction and increased cellular senescence. Loss of complex I was associated with disturbed NAD + metabolism with increased UCP2 expression and reduced phosphorylated SirT1. In cells with compound heterozygous POLG mutations, we also found activated mitophagy via the BNIP3 pathway. Our studies are the first that show it is possible to recapitulate the neuronal molecular and biochemical defects associated with POLG mutation in a human stem cell model. Further, our data provide insight into how mitochondrial dysfunction and mtDNA alterations influence cellular fate determining processes.

The aim of the present study was to compare the method of ultracentrifugation and density gradient separation for isolating Tca8113 human tongue squamous cell carcinoma cell line-derived exosomes. The exosomes were obtained from the culture supernatant of cultured Tca8113 cells, respectively, followed by identification with transmission electron microscopy observation and western blot analysis. The two different methods were then compared by the morphology, the distribution range of the particle size and the concentration of proteins of the extracted exosomes. In vitro, Tca8113 cells can secrete a large amount of vesicle-like structures, which are identified as exosomes by the presence of the surface markers, Hsp-70 and Alix. The protein profile of the two products are almost the same, however the particle size distribution of the exosomes extracted with density gradient centrifugation are more limited, between 40–120 nm, and these have a higher protein concentration. The results indicate that Tca8113 cells can secrete exosomes in vitro, and the density gradient separation methods for purifying exosomes is improved, which is helpful for future research and application of exosomes.

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