Project description:To investigate the inside and exported miRNA in cell culture system. We characterized the miRNA spectra of cell lines when deprived of serum. The absence of miRNAs present in bovine serum is a distinct advantage of using serum depletion to study extracellular miRNA as it removes potential source of interference.
Project description:Purpose: To filter genes that may contribute to introcellualr survival of B. bronchiseptica inside Dictyostelium discoideum, the genes that differently expressed when bacteria inside amoebae or in culture medium are selected as target genes.
Project description:To determine the importance of RNase1 on cellular miRNA function, we used a lentivirus system to stably express RNase1 in dicer wt and dicer ex5 RKO cells in serum free culture. RNase1 was found to govern miRNA that reside outside the pureview of Dicer. Assignment of RNase1 as miRNA-generating enzyme suggests that, like Dicer, RNaseA enzymes mediate essential physiological functions through regulatory RNA.
Project description:Transcriptional profiling of Caco-2 cells comparing Caco-2 monolayers cultured in a custom built co-culture chamber, either inside a 5% CO₂ incubator (conventional cell culture environment) or an anaerobic workstation (apical anaerobic environment) for 12 hours.
Project description:Neural Stem Cells (NSCs) of biological behaviors are frequently regulated by the three-dimensional (3D) niches that they located in. The impact of cell density cue inside niche on cellular outcomes are usually underestimated and understudied. In this study, a facile NSCs of 3D culture system was developed using collagen self-assembled fibril hydrogel. We used RNA sequencing and molecular biology technologies to investigate the NSCs of cell contacts and differentiations in developed 3D hydrogel culture system with low (0.75 million/mL), medium (1.5 million/mL), and high (3 million/mL) cell packing density. Compared with low cell density system, cytoskeletal spreading, gap junction, and tight junction mediated cell contacts were significantly improved in high cell density culture. Moreover, high cell density increased NSCs differentiation into immature (Tuj1) neuron, while there is no discrepancy of NSCs differentiation into astrocytes in different cell densities of 3D culturing system. This study provides new understanding in the regulation of 3D cultured NSCs behaviors through niche of cell density cue.
Project description:MicroRNAs (miRNAs) are an endogenous conserved class of non-coding 20–22 nt small RNAs that regulate gene expression at post-transcriptional level by mostly binding to 3′-UTR of target mRNAs, leading to mRNA degradation or translation inhibition. Recent reports demonstrate a role for miRNA expression in the disease progression and outcome. By now, many researcher focusing on miRNA expression profiles in Barrett's esophagus and esophageal adenocarcinoma have been reported. Nevertheless, there is still a little information available about specific miRNA expression pattern and their roles in ESCC. To develop novel diagnostic and therapeutic targets for esophageal squamous cancer, we first investigated the expression profile of miRNA in three pairs of ESCC clinical samples. Tissues of ESCC and the matched normal counterparts were obtained from surgical specimens immediately after resection from patients undergoing primary surgical treatment of esophageal carcinoma from the Department of Tumor Surgery of Shantou Central Hospital, China. RNA labeling and hybridization were completed by KangChen Bio-tech Inc. (Shanghai, China) according to the manufacturer's instructions. Briefly, total RNA from three pairs of esophageal carcinoma and matched normal tissues were isolated by Trizol (Invitrogen, USA) and purified by RNeasy mini kit (QIAGEN, German). The concentration and quality of total RNA were measured by NanoDrop ND-1000 at 260 and 280 nm (A260/280) and checked by gel electrophoresis. Each RNA sample from three pairs of ESCC was separately labeled either using the miRCURY Hy3/Hy5 labeling kit and hybridized on the six miRCURYTM locked nucleic acid (LNA) array version 11.0 (Exiqon, Denmark), which contains probes for 1700 mature miRNA. Scans were quantified by using GenePix software (Molecular Devices). The data were exported to Microsoft Excel worksheets, log2 transformed, normalized using global Lowess (Locally Weighted Scatter plot Smoothing) regression algorithm (MIDAS, TIGR Microarray Data Analysis System).
Project description:tRNA methylguanosine methyltransferase 1 (METTL1) is the prominent m7G writer protein in yeast and human cells. While several studies documented the transcriptome-wide distributions of METTL1-installed m7G, very little is known about the protein-protein interactions (PPI) involving METTL1. To fill in this knowledge gap, we adopted an engineered ascorbate peroxidase (APEX)-based proximity labeling, followed by LC-MS/MS analysis, to investigate the METTL1 interactome in HEK293T cells. We identified more than 70 unique proteins enriched in the proximity proteome of METTL1, and verified METTL1’s interaction with one of these proteins, exportin 5 (XPO5) through co-immunoprecipitation followed by immunoblot analysis. We also revealed that genetic ablation of METTL1 led to elevated distribution of XPO5 in the cytosol, thereby enhancing pre-miRNA export and miRNA maturation. Mechanistically, METTL1 promotes ERK-mediated phosphorylation of XPO5, which leads to its augmented nuclear retention. Ectopic expression of a constitutively active form of MEK, i.e., MEKDD, restored XPO5’s nuclear localization in METTL1-deficient cells, suggesting that METTL1 modulates miRNA processing through a mechanism independent of its catalytic activity. Together, we uncovered a novel role of METTL1 in modulating XPO5’s subcellular distribution and pre-miRNA export through its interaction with XPO5, and we also revealed a novel mechanism of miRNA maturation and expanded METTL1’s function beyond m7G methylation.
Project description:MicroRNA (miRNA/miR) miR526b and miR655 overexpressed tumor cell-free secretions promote breast cancer phenotypes in the tumor microenvironment (TME). However, the mechanisms of miRNA regulating TME have never been investigated. With mass spectrometry analysis of MCF7-miRNA-overexpressed versus miRNA-low MCF7-Mock tumor cell secretomes, we identified 34 novel secretory proteins coded by eight genes YWHAB, TXNDC12, MYL6B, SFN, FN1, PSMB6, PRDX4, and PEA15 those are differentially regulated. We used bioinformatic tools and systems biology approaches to identify these markers’ role in breast cancer. Gene ontology analysis showed that the top functions are related to apoptosis, oxidative stress, membrane transport, and motility, supporting miRNA-induced phenotypes. These secretory markers expression is high in breast tumors, and a strong positive correlation exists between upregulated markers’ mRNA expressions with miRNA cluster expression in luminal A breast tumors. Gene expression of secretome markers is higher in tumor tissues compared to normal samples, and immunohistochemistry data supported gene expression data. Moreover, both up and downregulated marker expressions are associated with breast cancer patient survival. miRNA regulates these marker protein expressions by targeting transcription factors of these genes. Premature miRNA (pri-miR526b and pri-miR655) are established breast cancer blood biomarkers. Here we report novel secretory markers upregulated by miR526b and miR655 (YWHAB, MYL6B, PSMB6, and PEA15) are significantly upregulated in breast cancer patients’ plasma, and are potential breast cancer biomarkers.