Housekeeping Gene Selection Advisory: Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and β-Actin Are Targets of miR-644a

Abstract

Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.

Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation

“…Even in studies involving adipocyte differentiation, GAPDH was still seleced as an endogenous reference gene (30,31). However, substantial evidence has revealed that the GAPDH gene expression level varies considerably in adipose tissue (27,32) and in other tissues or cultured cells under different situations (9,33,34). As demonstrated in differentiating adipocytes (6,7) and in adipose tissue derived from genetically obese rats (35,36), the upregulation of GAPDH parallels the acquisition of the full lipogenic phenotype.…”

Total protein is an effective loading control for cerebrospinal fluid western blots

“…The assumption of this method is that the housekeeping genes (often, beta-actin, beta-tubulin, or GAPDH) are highly expressed at relatively constant levels across cells, tissues, and disease/injury types. Increasingly, however, the validity of this assumption in the analysis of cultured cells [10-12], tissue types [11, 13, 14, 15], and disease/injury states [11, 13, 14, 16, 17] has been criticized. As none of the above housekeeping proteins are considered secreted proteins, their validity as loading controls for WB of biological fluids can likewise be questioned.…”

Crosstalk between the microbiome and cancer cells by quorum sensing peptides

“…The upregulation of HIST1H4 can be associated with increased Notch1 stimulation, again connected to cancer metastasis, together with angiogenesis and NF-B production [30,44]; NF-B in turn contributes to the progression of colorectal cancer by regulating cell proliferation, angiogenesis and tumor metastasis [45]. Micro-RNA's block mRNA translation and thus impede the synthesis of specific proteins by recruiting the micro-RNA-induced silencing complex (miRISC) to target mRNAs [46]; downregulation of micro-RNA 222 and 644a thus increases KIT and -actin protein expression, respectively, thereby promoting endothelial cell proliferation and migration (angiogenesis) [36,47] and tumor cell invasiveness and motility [34]. -actin can be upregulated through the IL-6 receptor pathway as well: the increased IL-6 expression after quorum sensing peptide treatment, as observed with our cytokine array, activates -actin phosphorylation, again promoting tumor cell migration [48].…”

“…Moreover, downregulation of micro-RNA 558 (miR-558) is associated with a bad prognosis of colon cancer outcome, indicating tumor metastases and thus EMT-like behaviour [33]. Micro-RNA 644a (miR-644a) functions by directly binding to its target site in the 3' untranslated region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and -actin, thereby significantly repressing their expression [34]. Downregulation of miR-664a thus upregulates -actin levels, leading to increased cell motility and cell migration [31].…”