Expression Levels Of Β-actin Research Articles

β-Actin: Not a Suitable Internal Control of Hepatic Fibrosis Caused by Schistosoma japonicum.

Schistosomiasis japonica is a significant health problem that leads to morbidity and mortality of humans. It is characterized by hepatic granulomatous response and fibrosis caused by eggs deposition in the liver. β-actin, a traditional housekeeping gene, is widely used as an internal control to normalize gene and protein expression. However, β-actin expression can fluctuate upon the treatment with pharmacological agents or under some physiological and pathological conditions. In this study, we found that the expressions of both β-actin mRNA and protein increased significantly with hepatic fibrosis formation after 6 weeks infection with Schistosoma japonicum and kept high level during the progression of hepatic fibrosis, while the levels of β-Tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) remained stable. The dynamic change of β-actin was similar with the profibrogenic factors, including α-SMA, Collagen I, and Collagen III. We employed immunofluorescence staining and further showed that the expression level of β-actin was positively correlated with α-SMA. What is more, there was a positive correlation between the level of β-actin mRNA and the content of hydroxyproline in liver. This study provides evidences that β-actin is variable and unsatisfied for application as an internal control in hepatic fibrosis induced by S. japonicum infection.

Correlation between RNA Degradation Patterns of Rat's Brain and Early PMI at Different Temperatures.

To explore the correlation between early postmortem interval （PMI） and eight RNA markers of rat's brain at different temperatures. Total 222 SD rats were randomly divided into control group （PMI=0 h） and four experimental groups. And the rats in the experimental groups were sacrificed by cervical dislocation and respectively kept at 5 ℃, 15 ℃, 25 ℃ and 35 ℃ in a controlled environment chamber. The RNA was extracted from brain tissues, which was taken at 9 time points from 1 h to 24 h postmortem. The expression levels of eight markers, β-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, were detected using real-time fluorescent quantitative PCR, respectively. Proper internal reference was selected by geNorm software. Regression analysis of normalized RNA markers was performed by SPSS software. Mathematical model for PMI estimation was established using R software. Another 6 SD rats with known PMI were used to verify the mathematical model. 5S rRNA, miR-9 and miR-125b were suitable as internal reference markers for their stable expression. Both β-actin and GAPDH had well time-dependent degradation patterns and degraded continually with prolongation of PMI in 24 h postmortem. The mathematical model of the variation of ΔCt values with PMI and temperature was set up by R software and the model could be used for PMI estimation. The average error rates of model validation using β-actin and GAPDH were 14.1% and 22.2%, respectively. The expression levels of β-actin and GAPDH are well correlated with PMI and environmental temperature. The mathematical model established in present study can provide references for estimating early PMI under various temperature conditions.

Effects of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin-resistant adipocytes

Adipocytokines are closely associated with insulin resistance (IR) in adipose tissues, and they are more and more seriously taken in the study of the development of diabetes. This experiment was mainly to study the effect of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin resistant adipocytes, and investigate the molecular mechanism of berberine in enhancing insulin sensitization and application advantages of droplet digital PCR (ddPCR). ddPCR absolute quantification analysis was taken in this experiment to simply and intuitively determine the appropriate reference genes. ddPCR and quantitative Real-time PCR (qPCR) were used to compare the effect of different doses of berberine (10, 20, 50, 100 μmol•L⁻¹) on mRNA expression levels of PPARγ, adiponectin, resistin and leptin in IR 3T3-L1adipocytes. Antagonist GW9662 was added to study the inherent correlation between PPARγ and adiponectin mRNA expression levels. ddPCR results showed that the expression level of β-actin in adipocytes was stable, and suitable as reference gene for normalization of quantitative PCR data. Both of ddPCR and qPCR results showed that, as compared with IR models, the mRNA expression levels of adiponectin were decreased in the treatment with berberine (10, 20, 50, 100 μmol•L⁻¹) in a dose-dependent manner (P<0.01); the expression of PPARγ was decreased by 20, 50, 100 μmol•L⁻¹ berberine in a dose-dependent manner in qPCR assay (P<0.01) and decreased only by 50 and 100 μmol•L⁻¹ berberine in ddPCR assay (P<0.05). PPARγ specific antagonist GW9662 intervention experiment showed that adiponectin gene expression was directly relevant with PPARγ (P<0.05). ddPCR probe assay showed that various doses of berberine could significantly reduce mRNA expression levels of resistin and leptin (P<0.01) in a dose-dependent manner. In conclusion, berberine enhanced insulin sensitization effect not by up-regulating adiponect in expression of transcriptional level in PPARγ-dependent manner, but may by the elevated multimerization of adiponectin in the posttranslational regulation level. Berberine down-regulated the resistin and leptin expression levels, which could alleviate lipolysis and improve IR in adipocytes. ddPCR provided better sensitivity and linear range than qPCR, with obvious technical advantages for the detection of low abundance expression of target genes.

GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.

BackgroundQuantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR.Materials and methodsAdipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon.ResultsThe expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively.ConclusionThis study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

Downregulation of β-Actin Gene and Human Antigen R in Human Keratoconus

The purpose of this study was to determine the expression levels and regulation of β-actin in the stroma of keratoconus (KC) and normal corneas. A total of 15 different human corneas from both KC and normal individuals were used for this study. Additionally, 3 Fuch's dystrophic corneas were also used. The β-actin gene expression was analyzed at the transcriptional and translational levels in the epithelium and stroma of the KC and normal corneas. The human antigen R (HuR) gene expression was analyzed by real-time PCR in the stroma of five KC and five normal corneas. The keratocytes from three normal and three KC corneas were cultured in the presence of serum, and the expression levels of β-actin and human antigen R (HuR) were analyzed by using confocal imaging in both normal and KC fibroblasts. The expression of the β-actin gene was downregulated in the stroma of the six KC corneas but not in the stroma of six normal and Fuchs' dystrophic corneas. Immunofluorescence detection of β-actin showed that it was absent in the KC fibroblast. The real-time PCR analysis of the HuR gene showed a relative 4.7-fold lower expression in KC corneas relative to the normal corneas, which was further confirmed by the immunofluorescence detection of HuR in fibroblasts of KC corneas. Although ubiquitous β-actins are essential for cell survival during early embryogenesis, the effects on various stages of development are not well understood. Our results show that β-actin is downregulated in the corneal stroma of patients with KC, which may be related to reduced levels of a stabilizing factor (HuR) for β-actin mRNA. We propose that loss of β-actin in the corneal stroma might be a triggering factor in the development of KC.

MYC Protein Inhibits Transcription of the MicroRNA Cluster MC-let-7a-1∼let-7d via Noncanonical E-box

The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

Down-Regulation of the Monocarboxylate Transporter 1 Is Involved in Butyrate Deficiency During Intestinal Inflammation

Butyrate oxidation is impaired in intestinal mucosa of patients with inflammatory bowel diseases (IBD). Butyrate uptake by colonocytes involves the monocarboxylate transporter (MCT) 1. We aimed to investigate the role of MCT1 in butyrate oxidation deficiency during colonic inflammation. Colonic tissues were collected from patients with IBD or healthy controls and from rats with dextran sulfate sodium (DSS)-induced colitis. The intestinal epithelial cell line HT-29 was treated with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). MCT1 expression was analyzed by real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence. Butyrate uptake and oxidation in HT-29 cells was assessed using [(14)C]-butyrate. The mechanism of MCT1 gene regulation was analyzed by nuclear run-on and reporter gene assays. MCT1 messenger RNA (mRNA) and protein levels were markedly decreased in inflamed colonic mucosa of IBD patients and rats. In HT-29 cells, down-regulation of MCT1 mRNA and protein abundance by IFN-gamma and TNF-alpha correlated with a decrease in butyrate uptake and subsequent oxidation. IFN-gamma and TNF-alpha did not affect MCT1 mRNA stability but rather down-regulated gene transcription. We demonstrate that the cytokine response element is located in the proximal -111/+213 core region of the MCT1 promoter. The data suggest that butyrate oxidation deficiency in intestinal inflammation is a consequence of reduction in MCT1-mediated butyrate uptake. This reinforces the proposition that butyrate oxidation deficiency in IBD is not a primary defect.

Role of the CCAAT/Enhancer-binding Protein NFATc2 Transcription Factor Cascade in the Induction of Secretory Phospholipase A2

Inflammatory cytokines such as interleukin-1 and tumor necrosis factor-alpha modulate a transcription factor cascade in the liver to induce and sustain an acute and systemic defense against foreign entities. The transcription factors involved include NF-kappaB, STAT, and CCAAT/enhancer-binding protein (C/EBP). Whether the NFAT group of transcription factors (which was first characterized as playing an important role in cytokine gene expression in the adaptive response in immune cells) participates in the acute-phase response in hepatocytes is not known. Here, we have investigated whether NFAT is part of the transcription factor cascade in hepatocytes during inflammatory stress. We report that interleukin-1 or tumor necrosis factor-alpha increases expression of and activates NFATc2. C/EBP-mediated NFATc2 induction is temporally required for expression of type IIA secretory phospholipase A2. NFATc2 is also required for expression of phospholipase D1 and the calcium-binding protein S100A3. Thus, a C/EBP-NFATc2 transcription factor cascade provides an additional means to modulate the acute-phase response upon stimulation with inflammatory cytokines.

Laser Capture Microdissection Combined with RNA in vitro Linear Amplification Detecting the Relevant Genes of Bladder Cancer

Objective: Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma (BTCC). However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determine a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cells from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods: Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results: That RNA integrity was good after LCM was confirmed by control experiment I; By control experiment II, the correlation between the number of LCMshooting and RNA quantity under arranged conditions was preliminarily confirmed. About 0.5-2.5 kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion: Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.