Reverse Transcription Quantitative Polymerase Chain Reaction Data Research Articles

Identification of specific reference gene for normalization of RT-qPCR data in rhythmic gene expression studies of the effect of developmental hormone antagonist in postembryonic development in Bombyx mori.

Bombyx mori is a lepidopteran holometabolous insect with distinct developmental stages: egg, larvae, pupae, and adult. The lepidopteran insect undergoes major modifications in the central nervous system (CNS) so as to adapt to the lifestyle of these distinct stages with specific habitats and functions from voraciously feeding larval stages to flying reproductive adults via dormant pupal stages. Such transitions are linked to transcriptional, epigenetic, and translational complexities. Therefore, studying rhythmic gene expression in CNS of various developmental stages and the effects of antagonists on developmental hormones requires a very stable reference gene (RG). To facilitate rhythmic gene expression studies using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in B. mori and the effect of developmental hormone juvenile hormone (JH) and 20-hydroxy ecdysone hormone (20 HE), antagonists Precocene 1 and testosterone, respectively, were used. Eight candidate RGs, namely, Translational initiation factor 3 subunit 4 (TI3S4), Translational initiation factor 3 subunit 5 (TI3S5), Ribosomal protein subunit 7 (RPs7), TATA-binding protein association factor (TAF13), Translational initiation factor 4 A (TI4A), Ribosomal protein (RPL32), Elongation factor 1 (EF1), and Arginine kinase (AK), were assessed in the CNS of B. mori. The postembryonic developmental (PED) stages used were the fifth late larval instar, early pupa, mid pupa, late pupa, and adult. The assessments were done at four different time points, Zeitgeber time (ZT) 0, 6, 12, and 18, to find stability towards 24-h rhythmic expression. RefFinder, geNorm, and Ct value analysis were performed. RefFinder and geNORM studies suggested stability order as TI3S4 > TI3S5 > RPs7, but Ct value evaluation showed stability order as TI3S5 > TI3S4 > RPs7. We therefore demonstrated that TI3S4, TI3S5, and RPs7 can be used as RG in various PED stages in CNS of B. mori (Strain: CB-hybrid, PM×CSR2) towards studies with effects of JH and 20 HE antagonists.

Integrated bioinformatic analysis reveals NOS2 as a novel ferroptosis-related biomarker for pre-eclampsia

BackgroundPre-eclampsia (PE) is a common condition in pregnancy; however, methods for early diagnosis and effective treatment options are lacking. Ferroptosis is a newly identified iron-dependent cell death pathway. The aim of this study was to investigate the role of ferroptosis-related genes in PE, the underlying mechanism, and their potential diagnostic value using a bioinformatics approach.MethodsWe downloaded the GSE48424 and GSE98224 datasets from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between PE and healthy pregnancy samples were identified in the GSE48424 dataset and subjected to weighted gene co-expression network analysis; the most relevant modules were intersected with known ferroptosis-related genes to distinctly identify the role of ferroptosis in PE. We further searched transcription factors and microRNAs that are predicted to regulate these ferroptosis-related genes, and patients in the GSE48424 dataset were divided into two groups according to high or low expression of the key ferroptosis-related genes associated with PE. To obtain robust key ferroptosis-related genes in PE, we validated their expression levels in the external dataset GSE98224. Finally, the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay was utilized to access the expression of these genes in the PE and normal blood samples.ResultsSix ferroptosis-related genes involved in PE were obtained by overlapping 3661 genes most associated with PE, 565 DEGs between PE and normal samples, and 259 known ferroptosis-related genes. Among these genes, patients with PE displaying lower expression levels of NOS2 and higher expression levels of PTGS2 had a higher ferroptosis potential index. The expression pattern of NOS2 was consistent in the GSE48424 and GSE98224 datasets. RT-qPCR data confirmed that NOS2 expression was more significantly elevated in patients with PE than in those with a normal pregnancy.ConclusionsOur study explored the diagnostic value of ferroptosis-related genes in PE, and identified NOS2 as the key gene linking ferroptosis and PE, suggesting a new candidate biomarker for early PE diagnosis.

Evaluation of candidate reference genes stability for gene expression analysis by reverse transcription qPCR in Clostridium perfringens

Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase + . These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-h period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.

Assessing the Emergence of Resistance in vitro and Invivo: Linezolid Combined with Fosfomycin Against Fosfomycin-Sensitive and Resistant Enterococcus.

PurposeWe aimed to evaluate the synergistic effect of linezolid and fosfomycin on fosfomycin-sensitive and -resistant Enterococcus clinical isolates in vitro and in vivo and whether the emergence of fosfomycin resistance in Enterococcus is associated with changes in strain virulence, from the perspective of fitness cost.MethodsThe synergistic effect of linezolid and fosfomycin was studied via in vitro checkerboard and static time-kill assays, as well as based on the in vivo survival rate and hemolymph load of a Galleria mellonella infection model. Fosfomycin resistance was induced via a stepwise increase in concentration. Changes in the virulence of the strains after drug resistance were investigated using the G. mellonella infection model and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In vitro and in vivo growth curves and competitive experiments were used to study the fitness cost of the strain. Finally, a static time-kill assay was performed to explore the effect of the combined medication.ResultsIn vitro and in vivo data showed that linezolid combined with fosfomycin had a good synergistic effect on Enterococcus treatment. The G. mellonella infection model and RT-qPCR data showed that the virulence of the resistant strains was weakened to varying degrees. A survival curve and competition experimental data showed that this was related to the fitness cost of strains while acquiring resistance and negatively impacted linezolid treatment; however, the combination still showed a good synergistic effect in drug-resistant strains.ConclusionLinezolid combined with fosfomycin had a synergistic effect on both fosfomycin-sensitive and -resistant Enterococcus strains. Strains incur fitness costs as they develop drug resistance, which leads to a decrease in virulence. There is an interaction between fitness cost, virulence, and drug resistance, which indirectly affects drug treatment.

Selection of internal reference gene for normalization of reverse transcription-quantitative polymerase chain reaction analysis in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), which resulting in considerable economic losses in pig farming globally. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a major tool for gene expression studies. However, no internal reference genes for normalization of RT-qPCR data of M. hyopneumoniae have been reported. The aim of this study was to screen the most stable genes for RT-qPCR analysis in M. hyopneumoniae under different conditions. Therefore, a total of 13 candidate internal reference genes (rpoC, Lipo, sgaB, oppB, hypo621, oppF, gyrB, uvrA, P146, prfA, proS, gatB, and hypo499) of M. hyopneumoniae filtered according to the reported quantitative proteomic analysis and the 16S rRNA internal reference gene frequently used in other bacteria were selected for RT-qPCR analysis. The mRNAs from different virulence strains (168, 168 L, J, NJ, and LH) at five different growth phases were extracted. The corresponding cycle threshold (Ct) values of the 25 reverse transcribed cDNAs using the 14 candidate genes were determined. Different internal reference genes or combinations were then screened for expression stability analysis using various statistical tools and algorithms, including geNorm, BestKeeper, and NormFinder software, to ensure the reliability of the analysis. Through further comprehensive evaluation of the RefFinder software, it is concluded that the gatB gene was the most suitable internal reference gene for samples of the different virulence strains in different growth phases for M. hyopneumoniae, followed by prfA, hypo499, and gyrB.

Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly Aquatica leii (Coleoptera: Lampyridae).

Simple SummaryThe purpose of this paper was to determine which genes in Aquatica leii, an endemic Chinese firefly, are suitable for use as reference genes in future RT-qPCR studies. This is the first example of an effective knockdown in fireflies and an important methodological advance for continuing research in A. leii. The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in A. leii for further molecular analysis. Since luciferase is very abundant in the adult light organ of fireflies generally, this is an encouraging result and of interest not just for A. leii researchers but other researchers who plan to perform RNAi or RT-qPCR in fireflies, or other nonmodel insects, as well.Aquatica leii Fu and Ballantyne is a species of rare aquatic firefly and endemic in China. It is considered good material to study the molecular mechanism of sexual flash communication systems. To improve conservation and behavioral research strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been very few reports on appropriate reference genes in any species of firefly. Here, we evaluated eight widely utilized reference genes including 18S, Actin, Reep5, Odc1, Tub, Gapdh, Ef1a and S27Ae for their expression stabilities in A. leii under three different conditions, i.e., life stage, tissue and dsRNA injection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend S27Ae and Reep5 as the most appropriate reference genes for molecular studies in different life stages; Ef1a and Odc1 for different tissues; Tub and Odc1 for RNAi studies. The most appropriate reference genes in all treatments are S27Ae and Tub. The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in A. leii for further molecular analysis.

Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone flaccida Fr. Schmidt at Different Growing Stages.

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

MiRNA-16 как внутренний контроль в исследованиях рака молочной железы: систематический обзор и мета-анализ

Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a method of choice for quantifying micro RNAs (miRNAs). Typically, RT-qPCR data are normalized to reference genes. While miRNAs are used for diagnosing and subtyping breast cancer, various studies show their deregulation in this condition, thus, undermining miRNAs' utility as a reference. This review examines the expression pattern of miR-16 and suggests normalization approaches for breast cancer. We analyzed the data from selected peer-reviewed studies to calculate the standardized mean difference (SMD) with subsequent Chi-square testing and identified the difference in miR-16 expression between breast cancer patients and healthy controls. With a negative SMD value of-0.56 and Chi-square of 62.62 (p-value = 0.05), the deregulation of miR-16 in breast cancer was confirmed. High variance in the stability value (SV) of miR-16 expression levels confirmed its inappropriateness as a control gene in breast cancer. The combination of miR-16 and miR-425 was confirmed as an accurate endogenous control.

Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in Apolygus lucorum (Hemiptera: Miridae).

Apolygus lucorum (Meyer-Dür) is a destructive pest to >280 plants. Major economic significance and pesticide resistance issues have created a need for integrated pest management (e.g., RNAi, entomopathogen-based bioinsecticides) for A. lucorum. To better develop these control strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been no reports on appropriate reference genes in A. lucorum. Here, we evaluated nine widely utilized reference genes including EF1γ, RPL32, RPL27, SDH, TBP, ACT, ACT2, GAPDH, and βTUB for their expression stabilities in A. lucorum under five different conditions i.e., life stage, tissue, sex, dsRNA injection, and entomopathogen infection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend RPL27 and RPL32 as the most appropriate reference genes for molecular studies in different life stages and tissues; GAPDH and EF1γ for different sexes and entomopathogen infection studies; and RPL27 and EF1γ for RNAi studies. The results of this study will help improve the accuracy and reliability for normalizing the RT-qPCR data for further molecular analysis in A. lucorum.

Iridoid glycosides from Radix Scrophulariae attenuates focal cerebral ischemia‑reperfusion injury via inhibiting endoplasmic reticulum stress‑mediated neuronal apoptosis in rats.

Iridoid glycosides of Radix Scrophulariae (IGRS) are a group of the major bioactive components from Radix Scrophulariae with extensive pharmacological activities. The present study investigated the effects of IGRS on cerebral ischemia-reperfusion injury (CIRI) and explored its potential mechanisms of action. A CIRI model in rats was established by occlusion of the right middle cerebral artery for 90 min, followed by 24 h of reperfusion. Prior to surgery, 30, 60 or 120 mg/kg IGRS was administered to the rats once a day for 7 days. Then, the neurological scores, brain edema and volume of the cerebral infarction were measured. The apoptosis index was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The effects of IGRS on the histopathology of the cortex in brain tissues and the endoplasmic reticulum ultrastructure in the hippocampus were analyzed. Finally, the expression of endoplasmic reticulum stress (ERS)-regulating mediators, endoplasmic reticulum chaperone BiP (GRP78), DNA damage-inducible transcript 3 protein (CHOP) and caspase-12, were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The volume of cerebral infarction and brain water content in the IGRS-treated groups treated at doses of 60 and 120 mg/kg were decreased significantly compared with the Model group. The neurological scores were also significantly decreased in the IGRS-treated groups. IGRS treatment effectively decreased neuronal apoptosis resulting from CIRI-induced neuron injury. In addition, the histopathological damage and the endoplasmic reticulum ultrastructure injury were partially improved in CIRI rats following IGRS treatment. RT-qPCR and western blot analysis data indicated that IGRS significantly decreased the expression levels of GRP78, CHOP and caspase-12 at both mRNA and protein levels. The results of the present study demonstrated that IGRS exerted a protective effect against CIRI in brain tissue via the inhibition of apoptosis and ERS.

Determination of Suitable RT-qPCR Reference Genes for Studies of Gene Functions in Laodelphax striatellus (Fallén).

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Lsα-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Lsβ-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.

Identification of reference genes for RT-qPCR data normalisation in aging studies

Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies.

Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

The precision and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) depend mainly on suitable reference genes; however, reference genes have not yet been identified for Liriodendron chinense (Hemsl.) Sarg. In this study, the expression stability of 15 candidate reference genes, ACT7, ACT97, UBQ1, eIF2, eIF3, HIS, BIG, AGD11, EFG, GAPDH, CYP, RPL25, UBC, RPB1, and TUB, was tested across multiple organs of L. chinense using four algorithms, geNorm, NormFinder, BestKeeper, and RefFinder. To understand the difference between the selected reference genes and the unsuitable candidate reference genes, the expression level of a target gene, LcPAT7, was normalized across various plant samples. ACT97 and eIF3 represented the best combination across all samples tested, while AGD11 and UBQ1 were unsuitable for normalization in this case. In the vegetative organ subset, ACT97, ACT7, and GAPDH showed the highest expression stability. For floral organs, UBC and eIF3 were the most stable reference genes. Unsuitable reference genes underestimated the expression levels of a target gene, LcPAT7. This study identified two reference genes (ACT97 and eIF3) for the precise and reliable normalization of L. chinense RT-qPCR data across various organs. Our work provides an effective framework for quantifying gene expression in L. chinense.

PTEN Expression in Prostate Cancer: Relationship With Clinicopathologic Features and Multiparametric MRI Findings.

OBJECTIVE. The objective of our study was to investigate whether phosphatase and tensin homolog (PTEN) expression is associated with clinicopathologic features and multiparametric MRI findings in prostate cancer. MATERIALS AND METHODS. Forty-three patients with prostate cancer who underwent radical prostatectomy were included. Index tumor was identified on pretreatment MRI and delineated in the area that correlated best with histopathology results. The apparent diffusion coefficient (ADC) from DWI and pharmacokinetic parameters derived from dynamic contrast-enhanced MRI (DCE-MRI) using the extended Tofts model (Ktrans, kep, ve, and vp) within the tumor were estimated. The following clinicopathologic parameters were assessed: pretreatment serum levels of prostate-specific antigen, disseminated tumor cell status, age, Gleason score, tumor size, extraprostatic extension (EPE), tumor location, and lymph node metastases. Gene expression profiles were acquired in biopsies from the tumor using bead arrays, and validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a different part of the biopsy. RESULTS. Based on bead arrays (p = 0.006) and RT-qPCR (p = 0.03) data, a significantly lower ADC was found in tumors with low PTEN expression. Moreover, PTEN expression was negatively associated with lymph node metastases (bead arrays, p = 0.008; RT-qPCR, p < 0.001). A weak but significant association between PTEN expression, EPE (p = 0.048), and Gleason score (p = 0.028) was revealed on bead arrays. ADC was negatively correlated with Gleason score (p = 0.001) and tumor size (p = 0.023). No association among DCE parameters, PTEN expression, and clinicopathologic features was found. CONCLUSION. ADC derived from DWI may be useful in selecting patients with potentially aggressive tumor caused by PTEN deficiency.

Reference MicroRNAs for RT-qPCR Assays in Cervical Cancer Patients and Their Application to Studies of HPV16 and Hypoxia Biomarkers

MicroRNA (miRNA) expressions in tumor biopsies have shown potential as biomarkers in cervical cancer, but suitable reference RNAs for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays in patient cohorts with different clinicopathological characteristics are not available. We aimed to identify the optimal reference miRNAs and apply these to investigate the potential of miR-9-5p as human papilloma virus (HPV) 16 biomarker and miR-210-3p as hypoxia biomarker in cervical cancer. Candidate reference miRNAs were preselected in sequencing data of 90 patients and ranked in a stability analysis by RefFinder. A selection of the most stable miRNAs was evaluated by geNorm and NormFinder analyses of RT-qPCR data of 29 patients. U6 small nuclear RNA (RNU6) was also included in the evaluation. MiR-9-5p and miR-210-3p expression was assessed by RT-qPCR in 45 and 65 patients, respectively. Nine candidates were preselected in the sequencing data after excluding those associated with clinical markers, HPV type, hypoxia status, suboptimal expression levels, and low stability. In RT-qPCR assays, the combination of miR-151-5p, miR-152-3p, and miR-423-3p was identified as the most stable normalization factor across clinical markers, HPV type, and hypoxia status. RNU6 showed poor stability. By applying the optimal reference miRNAs, higher miR-9-5p expression in HPV16- than HPV18-positive tumors and higher miR-210-3p expression in more hypoxic than less hypoxic tumors were found in accordance with the sequencing data. MiR-210-3p was associated with poor outcome by both sequencing and RT-qPCR assays. In conclusion, miR-151-5p, miR-152-3p, and miR-423-3p are suitable reference miRNAs in cervical cancer. MiR-9-5p and miR-210-3p are promising HPV16 and hypoxia biomarkers, respectively.

Validation of suitable housekeeping genes for 3T3-L1 derived adipocytes cultured in obesity mimicking conditions and RAW 264.7 macrophage cells lines in hypoxic and normoxic conditions

Abstract Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) is the most commonly used method for gene expression analysis, and reliable results depend on proper normalization by stable reference genes. Adipose tissue hypoxia is the new concept in understanding the major disorders associated with obesity and may facilitate cellular mechanisms associated with obesity like chronic inflammation, macrophage infiltration, adiponectin reduction, leptinelevation, adipocyte death, ER stress and mitochondrial dysfunction. The hypoxic conditions in the obese adipose tissue lead to differential expression of genes in the resident cells of adipose tissues including macrophages andhence reliable normalization of RT-qPCR data becomes crucial. In this study, the expression stability of 4 most commonly used candidate reference genes, ACTB, B2M, HMBS and 18SrRNA were evaluated in murine adipocytesand macrophage cell lines, in presence of high free fatty acids, under hypoxia and normoxia conditions. The results were analyzed by manually calculating fold difference using 2−ΔΔCt and validated by using two algorithms, NormFinder, and Bestkeeper. Our results showed HMBS and B2M as the most stably expressed genes in both cell lines under obesity mimicking conditions (in presence of high amount of FFAs and hypoxia). By contrast, ACTB and 18S rRNA were the least stable genes under the tested conditions. Furthermore, the expression levels of FABP4 andPPAR-γ (for adipocytes) and MCP-1 and IL-6 (for macrophages) were profiled in these cells to validate the selected reference genes. Our data provides a basis for gene expression analysis in future studies related to inflammation in hypoxia related obesity pathogenesis.

PML/RARa blocks the differentiation and promotes the proliferation of acute promyelocytic leukemia through activating MYB expression by transcriptional and epigenetic regulation mechanisms.

The promyelocytic leukemia (PML)/retinoic acid receptor-alpha (RARα) onco-fusion protein that is generated from t(15;17) chromosome translocation is crucial for the leukemogenesis of acute promyelocytic leukemia (APL) and is well documented as a transcriptional repressor. To understand the relationship between PML/RARα and the oncogene in the development of APL, we investigate the regulation mechanism of PML/RARα to MYB proto-oncogene and the role of this regulation on the proliferation and differentiation of APL cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays show that MYB expression was significantly higher in PML/RARα positive cell lines. Microarray data verify that the MYB expression was significantly higher in APL patient samples than in normal promyelocyte samples. Further expression analysis from RT-qPCR and microarray data verifies that the expression of MYB is upregulated by PML/RARα. Transcriptional factor binding analysis shows that MYB is directly bound by PML/RARα and its cofactors. Luciferase assays show that PML/RARα transactivated MYB promoter activity through the RARα binding site and the coexistence of CCAAT enhancer binding protein ε. We also find that PML/RARα increases the acetylation level of the promoter region of MYB. Further evidence demonstrates that PML/RARα regulates MYB expression through long-range interaction. Functionally, PML/RARα increases the cell proliferation and blocks the differentiation through activating MYB expression. Collectively, this study uncovers a novel mechanism of PML/RARα-mediated transcriptional activation and enriches our knowledge of the onco-fusion protein-mediated transcription activation.

Expression of galectin-7 in vulvar lichen sclerosus and its effect on dermal fibroblasts.

Lichen sclerosus is a chronic and inflammatory disease. Extensive studies have focused on the epidermis, with the dermis or epidermis-dermis receiving less attention. To investigate the role of galectin-7, a keratinocyte protein, in vulvar lichen sclerosus (VLS) and its potential effects on dermal fibroblasts, immunohistochemical staining was performed with VLS tissue samples and normal control samples. The expression of galectin-7 was determined by evaluating the galectin-7 integrated density analysis, and further assessed by western blot analysis. Dermal fibroblasts were isolated from the normal tissue of the female anogenital region following sexual plastic surgery. A cell viability assay was performed on isolated dermal fibroblast cells in the presence or absence of galectin-7. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the transcriptional level of collagen I and collagen III in the response to different doses of galectin-7. In the immunohistochemical analysis, galectin-7 demonstrated a significantly elevated level in VLS, compared to control tissues, which was confirmed by western blot analysis. In the analysis of primary dermal fibroblast cells, galectin-7 significantly inhibited the viability rate of fibroblasts in a dose-dependent manner. RT-qPCR data revealed that the transcription level of collagen I and collagen III were positively associated with the galectin-7 treatment concentration. The overexpression of galectin-7 is associated with the progression of VLS in the epidermis, a high concentration of galectin-7 inhibits the viability of the primary vulvar dermal fibroblasts, and stimulates the accumulation of collagen I and collagen III in dermal fibroblast cultures, thus galectin-7 may serve as a drug target during VLS progression.

Expression of BANCR promotes papillary thyroid cancer by targeting thyroid stimulating hormone receptor.

Papillary thyroid carcinoma (PTC) is the most common form of non-medullary thyroid cancer, accounting for ~80% of all cases of thyroid cancer. The aim of the present study was to explore the role of BRAF-activated long noncoding RNA (BANCR) in the development of PTC. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BANCR, thyroid-stimulating hormone receptor (TSHR) and cyclin D1 between PTC and benign control thyroid nodule tissue samples from 60 patients were determined. Using RT-qPCR and western blot analysis, the expression levels of TSHR and cyclin D1 mRNA and protein were determined in cells transfected with BANCR-small interfering (si)RNA. An MTT assay and flow cytometry were used to analyze the effect of BANCR knockdown on the proliferation and cell cycle distribution of IHH-4 PTC cells. The expression of BANCR, TSHR and cyclin D1 was increased in the PTC group compared with the control group based on the RT-qPCR data. The transfection of IHH-4 cells with BANCR-siRNA induced the inhibition of TSHR and cyclin D1 expression compared with a transfection control. In addition, the proliferation of the IHH-4 cells transfected with BANCR-siRNA was suppressed, relative to the transfection control, and cells arrested in the G0/G1 phase, potentially due to the inhibition of the expression of cyclin D1. The data suggested that the expression of BANCR may promote the development of malignant thyroid nodules via the modulation of TSHR expression and its downstream effector, cyclin D1.

MiR-34c induces apoptosis and inhibits the viability of M4e cells by targeting BCL2.

The present study aimed to investigate microRNA (miR/miRNA)-34c expression and the association of miR-34c with B cell lymphoma 2 (BCL2) in M4e laryngeal carcinoma cell line. M4e laryngeal carcinoma cells were cultured and transfected with lenti-miR-34c or scramble miRNA for 72 h. Cell viability and the percentage of cells undergoing apoptosis of transfected cells were detected using MTT and Annexin V/allophycocyanin and propidium iodide assays, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were performed to determine BCL2 mRNA and protein expression in transfected M4e cells. In addition, luciferase reporter assay was performed to identify whether BCL2 is a direct target of miR-34c. Transfection of lenti-miR-34c was able to significantly inhibit cell viability (P<0.01), increase the percentage of cells undergoing apoptosis (P<0.001) and downregulate BCL2 protein expression (P<0.01) in M4e cells. RT-qPCR data revealed that lenti-miR-34c transfection did not affect BCL2 mRNA expression. However, data from the luciferase reporter assay revealed that transfection with miR-34c negative control decreased luciferase activity in M4e cells co-transfected with pGL3-BCL2-MUT plasmid, compared with miR-34c inhibitor (P<0.01). Collectively, the results from the present study provided evidence that miR-34c may be involved in the pathogenesis of laryngeal cancer, and BCL2 may be negatively regulated by miR-34c in M4e cells.