LUAD transcriptomic profile analysis of d-limonene and potential lncRNA chemopreventive target.

d-Limonene, a type of natural extract obtained from citrus oils, was reported to have anti-cancer effects and be well-tolerated by cancer patients. Despite arousing interest as a cancer chemopreventive substance, the transcriptomic profile of d-limonene in humans is poorly understood. Based on the results of the transcriptomic profiling, a lncRNA named protein disulfide isomerase family A member three pseudogene (PDIA3P1) was found to be regulated by d-limonene. PDIA3P1 is an oncogene verified by three lung adenocarcinoma (LUAD) datasets. The knockdown of PDIA3P1 with siRNA decreased the viability, invasion, migration, and proliferation of LUAD cells. Based on The Cancer Genome Atlas (TCGA) LUAD datasets, PDIA3P1 regulates functions and pathways mainly including lipid metabolism, immunity, and the change of the chromosome structure. This study comprehensively performs the transcriptomic analysis of the d-limonene regulation on LUAD, and reveals that PDIA3P1 may be the mediator in helping d-limonene to prevent and suppress LUAD via lipid metabolism, immunity pathway, and the change in the chromosome structure.

Identification of differentially regulated deceitful proteins in SH-SY5Y cells engineered with Tet-regulated protein expression system.

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.

Discrimination of pseudogene and parental gene DNA methylation using allelic bisulfite sequencing.

Determining the methylation status of genes with pseudogenes can be technically challenging due to sequence homology. High sequence homology can result in the amplification of both pseudogene and parental gene alleles, potentially leading to data misinterpretation. Allelic bisulfite sequencing allows for detection of the methylation status of individual alleles at nucleotide resolution and represents the most reliable method for discriminating pseudogene and parental gene sequences. Here, we discuss important points that should be considered when investigating pseudogene and parental gene methylation status and we describe the method of allelic bisulfite sequencing, including assay design.