Real-time expression profiling of microRNA precursors in human cancer cell lines

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines

Novel clone selection technique reveals heterogeneity among HEK293T cells engineered to produce therapeutic extracellular vesicles

HEK293T cells have been engineered to produce extracellular vesicles (EVs) that deliver miR-199a-3p to CD44+ hepatocellular carcinoma cells. Restoration of this miRNA has been shown to slow cancer progression in-vitro. Isolation and analysis of EVs from cell culture media containing selection agent revealed that the number of miRNA-199a-3p copies was less than the number of cells in culture suggesting that not all cells produce therapeutic EVs. Therefore, therapeutic EV production can be significantly increased by selecting the HEK293T clones that produce the most therapeutic EVs. While clone selection is traditionally accomplished by cell analysis techniques such as fluorescence activated cell sorting (FACS), detection of therapeutic EVs poses a unique challenge in that cellular expression of miRNA-199a-3p does not necessarily correlate to the amount of exosomal miRNA-199a-3p. In response to this challenge, a fibrous microwell array was developed to screen thousands of clones for therapeutic EV productivity (figure 1). The fibrous microwell system is able to evaluate cell growth rate under fluid shear stress, EV productivity and EV characterization using fluorescently labeled antibodies or cationic lipoplex nanoparticles (detect presence of miRNA-199a-3p inside captured EVs produced by single clones). The most productive clones can be released from the microwells and grown in large scale cell culture to significantly increase therapeutic EV production. Please click Additional Files below to see the full abstract

A high-throughput method to monitor the expression of microRNA precursors

microRNAs (miRNAs) are small, functional, non-coding RNAs. miRNAs are transcribed as long primary transcripts (primary precursors) that are processed to the ∼75 nt precursors (pre-miRNAs) by the nuclear enzyme Drosha. The ∼22 nt mature miRNA is processed from the pre-miRNA by the RNase III Dicer. The vast majority of published studies to date have used northern blotting to detect the expression of miRNAs. We describe here a sensitive, high throughput, real-time PCR assay to monitor the expression of miRNA precursors. Gene-specific primers and reverse transcriptase were used to convert the primary precursors and pre-miRNAs to cDNA. The expression of 23 miRNA precursors in six human cancer cell lines was assayed using the PCR assay. The miRNA precursors accumulated to different levels when compared with each other or when a single precursor is compared in the various cell lines. The precursor expression profile of three miRNAs determined by the PCR assay was identical to the mature miRNA expression profile determined by northern blotting. We propose that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila

Dynamics Correlation Network for Allosteric Switching of PreQ1 Riboswitch.

Riboswitches are a class of metabolism control elements mostly found in bacteria. Due to their fundamental importance in bacteria gene regulation, riboswitches have been proposed as antibacterial drug targets. Prequeuosine (preQ1) is the last free precursor in the biosynthetic pathway of queuosine that is crucial for translation efficiency and fidelity. However, the regulation mechanism for the preQ1 riboswitch remains unclear. Here we constructed fluctuation correlation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in the bound riboswitch is distinctly different from that in the apo riboswitch. The community network indicates that the information freely transfers from the binding site of preQ1 to the expression platform of the P3 helix in the bound riboswitch and the P3 helix is a bottleneck in the apo riboswitch. Thus, a hypothesis of "preQ1-binding induced allosteric switching" is proposed to link riboswitch and translation regulation. The community networks of mutants support this hypothesis. Finally, a possible allosteric pathway of A50-A51-A52-U10-A11-G12-G56 was also identified based on the shortest path algorithm and confirmed by mutations and network perturbation. The novel fluctuation network analysis method can be used as a general strategy in studies of riboswitch structure-function relationship