ACTB
Actin, cytoplasmic 1 (Beta-actin) [Contains: Actin, cytoplasmic 1, N-terminally processed]
Publications[править]
Endothelial Colony Forming Cells (ECFCs) represent a subset of endothelial progenitors with well-documented vasoreparative capacity. However, cellular senescence, which occurs due to aging, diabetes, smoking, or tissue inflammation, renders these cells dysfunctional. Therefore, there is growing interest in studying expression of senescence markers in ECFCs. RT-qPCR is the most commonly used technique to quantify gene expression and the proper choice of reference genes used for data normalization is critical for accurate quantification. It has been reported that the expression of commonly used housekeeping genes is often unstable in senescence. To identify the most suitable reference genes for ECFC senescence studies, we analyzed a microarray dataset, which compared the gene expression between proliferating and senescent ECFCs. In addition to replicative senescence, the data included X-ray-induced and Etoposide-induced senescence. We used the geNorm algorithm to identify the most stable genes across all studied conditions. Gene Ontology analysis found that the most stable genes belonged to the KEGG category of Genetic Information Processing. The optimal combination of housekeeping genes for ECFC senescence was found to include four ribosomal protein genes; RPL13, RPL31, RPL37, and RPL30. The RT-qPCR validation confirmed that normalization with our novel panel was more sensitive in identifying senescence markers compared to commonly used genes such as ACTB, UBC, and GAPDH.
Keywords
- ECFCs
- RT-qPCR
- computational biology
- housekeeping gene
- senescence
The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo µCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.
MeSH Terms
- Aged
- Aging
- Animals
- Autografts
- Bone Regeneration
- Bone Transplantation
- Humans
- Mice
- Mice, Transgenic
- Middle Aged
- Osteonecrosis
- Wnt Signaling Pathway
- Wnt3A Protein
Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.
MeSH Terms
- Adult
- Aged
- Aging
- Algorithms
- Gene Expression
- Genes
- Humans
- Hypoxanthine Phosphoribosyltransferase
- Leukocytes, Mononuclear
- Middle Aged
- RNA, Messenger
- Reference Standards
- Reproducibility of Results
- Reverse Transcriptase Polymerase Chain Reaction
The aim of this study was to evaluate reference genes for expression studies of human adipose tissue. Using 52 human adipose tissue expression profiles (HU95), 10 putative reference genes with the lowest variation in expression levels were selected for further studies. Expression stability of these 10 novel and 5 previously established reference genes was evaluated by real-time reverse transcriptase-polymerase chain reaction analysis. For this purpose, 44 adipose tissue biopsies from 27 subjects were chosen to include a wide range of parameters such as sex, age, BMI, depot origin, biopsy procedure, and effects of nutrition. LRP10 was identified as the gene with the least variation in expression levels. The frequently used reference genes RPLP0, 18S rRNA, PPIA, ACTB, and GAPD were ranked as 4, 6, 7, 8, and 10, respectively. Our results suggest that LRP10 is a better choice as reference for expression studies of human adipose tissue compared with the most frequently used reference genes.
MeSH Terms
- Adipose Tissue
- Adult
- Aging
- Biopsy
- Body Mass Index
- Female
- Gene Expression
- Gene Expression Profiling
- Humans
- LDL-Receptor Related Proteins
- Male
- Membrane Transport Proteins
- Middle Aged
- Oligonucleotide Array Sequence Analysis
- Receptors, LDL
- Reverse Transcriptase Polymerase Chain Reaction
- Sex Characteristics