Gaofeng Liang

Exosome Uptake through Clathrin-mediated Endocytosis and Macropinocytosis and Mediating miR-21 Delivery

Background: Exosomes can transfer information between cells and facilitate tumor development. Results: PC12 cell-derived exosomes enter into BMSCs through clathrin-mediated endocytosis and macropinocytosis, and decrease the expression of TGFβRII and TPM1 through miR-21. Conclusion: The results dissect the pathway of exosome internalization and demonstrate their regulation ability. Significance: These findings enhanced our knowledge of the internalization and function of tumor exosomes. Exosomes are nanoscale membrane vesicles secreted from many types of cells. Carrying functional molecules, exosomes transfer information between cells and mediate many physiological and pathological processes. In this report, utilizing selective inhibitors, molecular tools, and specific endocytosis markers, the cellular uptake of PC12 cell-derived exosomes was imaged by high-throughput microscopy and statistically analyzed. It was found that the uptake was through clathrin-mediated endocytosis and macropinocytosis. Furthermore, PC12 cell-derived exosomes can enter and deliver microRNAs (miRNAs) into bone marrow-derived mesenchymal stromal cells (BMSCs), and decrease the expression level of transforming growth factor β receptor II (TGFβRII) and tropomyosin-1 (TPM1) through miR-21. These results show the pathway of exosome internalization and demonstrate that tumor cell-derived exosomes regulate target gene expression in normal cells.

Aberrant expression of serum miRNAs in schizophrenia

The circulating miRNAs are sufficiently stable and detectable to serve as clinical biomarkers as recent studies have revealed that the aberrant expression of circulating miRNAs can directly reflect disease status. Based on the analysis of the data (using miRanda software, TargetScan software and SOLID high-throughput sequencing) obtained from the literature, Schizophrenia Gene database, NCBI database, the quantification of the nine miRNAs in the serum samples of 115 patients suffering from schizophrenia and 40 healthy individuals using qRT-PCR and semi-nested qRT-PCR was conducted. The results suggested that the miR-181b, miR-219-2-3p, miR-346, miR-195, miR-1308, miR-92a, miR-17, miR-103 and let-7g are the key players to reflect the schizophrenia illnesses status and may serve as candidate biomarkers for diagnosis of schizophrenia. In addition, we also found that the risperidone improved the serum miR-346 level of schizophrenia significantly, and therefore may not be an effective drug in regulating serum miR-346 level of schizophrenia. Furthermore, the expression level of serum miRNAs levels and schizophrenia patients were regardless of family history subtypes, ages, and gender. Collectively, these findings suggested that the serum miRNAs have strong potential to reflect schizophrenia disease status. To the best of our knowledge, this is the first report demonstrating the analysis of the circulating miRNAs in schizophrenia.

Assessing the survival of exogenous plant microRNA in mice

MicroRNAs (miRNAs), a class of small RNAs, are important molecules that influence several developmental processes and regulate RNA interference (RNAi), and are abundant in animals, plants, and plant tissues that are traditionally consumed in the diet. The survival of plant small RNAs from the diet in animals, however, remains unclear, and the persistence of miRNAs from dietary plants in the animal gastrointestinal (GI) tract is still under debate. In this study, ICR mice were fed plant total RNAs in quantities of 10–50 μg, extracted from Brassica oleracea. Serum, feces, and various tissues were collected from the mice after RNA consumption and analyzed for several miRNAs. Exogenous plant miRNAs were present in the sera, feces, and tissues of animals and these exogenous plant miRNAs were primarily acquired orally. MiR-172, the most highly enriched exogenous plant miRNA in B. oleracea, was found in the stomach, intestine, serum, and feces of mice that were fed plant RNA extracts including miR-172. The amount of miR-172 that survived passage through the GI tract varied among individuals, with a maximum of 4.5% recovered at the stomach of one individual, and had a range of 0.05–4.5% in different organs. Furthermore, miR-172 was detected in the blood, spleen, liver, and kidney of mice.

MicroRNAs as participants in cytotoxicity of CdTe quantum dots in NIH/3T3 cells

Epigenetic aspects of the cytotoxicity of CdTe quantum dots (QDs) recently have attracted more attention for their ability to reprogram gene expression after initial signals have been removed. And the involvement of epigenetic mechanisms in microRNA (miRNA) biogenesis suggests that miRNAs act as participants in the cytotoxicity of CdTe QDs. According to the results of SOLiD sequencing, the expression patterns of miRNAs are widely affected after CdTe QD exposure, resulting in the apoptosis-like cell death. Compared with 86 miRNAs with down-regulated expression, the expression levels of 121 miRNAs are up-regulated by CdTe QD treatment. The Z-test is used to find out miRNAs with significantly regulated expression, and the results indicate that the expression levels of 16 and 35 miRNAs are down- and up-regulated, respectively. And the expression levels of some significantly regulated miRNAs have time- and dose-dependent tendencies, which are similar to cell survival ratios affected by CdTe QDs. The fluctuations of miRNA expression start from the transcription of pri-miRNA, and are strengthened by the processing of pri-miRNA to pre-miRNA. As a regulator in miRNA biogenesis, p53 is involved in the transcription and processing of pri-miRNA. With no significant changes in the mRNA levels of p53, the increase in overall p53 protein levels and its post-translational modification by phosphorylation at Ser-15 are induced by CdTe QD treatment. Therefore, the differential expression of miRNAs are induced by CdTe QDs at the processing of miRNA biogenesis, which is an adaptive process of cells to external stimuli.

Effects of Epidermal Growth Factor and Basic Fibroblast Growth Factor on the Proliferation and Osteogenic and Neural Differentiation of Adipose-Derived Stem Cells

Stem cells used for clinical tissue regeneration therapy should have the capacity of self-renewal, high proliferation, and differentiation and be able to be transplanted in large numbers. Although high concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) may induce the differentiation of stem cells, these factors have been widely used to enhance the propagation of stem cells, including adipose-derived mesenchymal stem cells (ASCs). However, the effects of low concentrations of EGF and bFGF on stem cells need to be evaluated carefully. This study illustrates that low concentrations of EGF (5 ng/mL) and bFGF (10 ng/mL) increase the proliferative ability of ASCs and induce the typical spindle-shaped cell morphology. EGF and bFGF added to medium promoted neural lineage differentiation and impaired the mesodermal differentiation ability of ASCs. This study demonstrates that even low concentrations of EGF and bFGF may limit the differentiation ability of stem cells during stem cell expansion in vitro. EGF and bFGF supplementation should be carefully considered in stem cells for clinical applications.

Enhanced Specificity of Multiplex Polymerase Chain Reaction via CdTe Quantum Dots

Nanoparticles were recently reported to be able to improve both efficiency and specificity in polymerase chain reaction (PCR). Here, CdTe QDs were introduced into multi-PCR systems. It was found that an appropriate concentration of CdTe QDs could enhance the performance of multi-PCR by reducing the formation of nonspecific products in the complex system, but an excessive amount of CdTe QDs could suppress the PCR. The effects of QDs on PCR can be reversed by increasing the polymerase concentration or by adding bovine serum albumin (BSA). The mechanisms underlying these effects were also discussed. The results indicated that CdTe QDs could be used to optimize the amplification products of the PCR, especially in the multi-PCR system with different primers annealing temperatures, which is of great significance for molecular diagnosis.

A novel type of self-assembled nanoparticles as targeted gene carriers: an application for plasmid DNA and antimicroRNA oligonucleotide delivery

In this study, a new type of amphiphilic cetylated polyethyleneimine (PEI) was synthesized, and then polylactic-co-glycolic acid (PLGA)/cetylated PEI/hyaluronic acid nanoparticles (PCPH NPs) were developed by self-assembly as a novel type of gene-delivering vehicle. The PCPH NPs showed good DNA-condensation ability by forming polyplexes with small particle size and positive zeta potential. The transfection efficiency and cytotoxicity of PCPH NPs were evaluated as plasmid DNA vectors to transfect HepG2 in vitro. PCPH NPs exhibited much lower cytotoxicity and higher gene-transfection efficiency than PEI (25,000) and commercial transfection reagents. Furthermore, PCPH NPs were used as an anti-miR-221 vector for transfecting HepG2 cells, and anti-miR-221 was effectively transfected into cells and produced a greater inhibitory effect on cancer-cell growth by PCPH NPs. These results demonstrate that PCPH NPs can be a promising nonviral vector for gene-delivery systems.

Deep sequencing reveals complex mechanisms of microRNA deregulation in colorectal cancer

Deregulated microRNAs (miRNAs) have been reported to be of functional relevance for tumor biology. In this study, we analyzed the small RNA transcriptomes of 10 paired colorectal cancer samples using SOLiD next-generation sequencing, and generated a total of >15141000 reads. miRNA expression profiles obtained by SOLiD sequencing correlated well with quantitative PCR results. The results showed that the expression of 36 miRNAs was significantly different between the two groups of samples. Additionally, we explored the global miRNA-mRNA interactions using a statistical model. In-depth analysis reveals a diagram of extensive post-transcriptional miRNA regulations. Signaling pathways associated with the miRNA altered expression signature were identified using gene enrichment analysis. The results suggest that these microRNAs, in the aggregate, regulate signaling pathways, such as MAPK, Wnt and p53 pathways in cancer, which are known to be involved in the transformation of colorectal cancer. This evidence demonstrates that miRNAs can cooperatively regulate a given pathway and play a subtle role by regulating their target genes.

Enrichment of cancer stem cells by cotton fiber

Isolation and expansion of cancer stem cells (CSCs) plays a main role in the better understanding of cancer biology as cancer is maintained by these few cancer cells with stem cell properties. Conventional methods to improve the isolation of CSCs at present are unreliable or lower producing. The hampered development of an effective method to enrich CSCs may create some substantial gaps in the advancement of an effectual therapeutic strategy in the field of cancer biology. In this study, we first obtained PC12 cell spheres using a conventional method and carefully characterized them as CSCs. Then we further developed a new, simple method for selecting and expanding CSCs. In our system, PC12 cells were cultured on a supporting material of cotton fibers treated by NH3 plasma. This allowed the CSCs to grow readily into individual round colonies. Our findings from various experiments such as expression of surface receptor markers, drug resistance ability, gene and protein expression, spheroid colony formation and in vivo tumorigenicity support that the cotton fiber dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. Thus, our study strongly indicates that cotton fiber scaffolds will facilitate the study of CSC biology and provide a new suitable in vitro model for the discovery of more efficacious anti-cancer therapies.