Identification of valid reference genes for circadian gene-expression studies in human mammary epithelial cells

ABSTRACT

The circadian clock controls most of the physiological processes in the body throughout days and nights’ alternation. Its dysregulation has a negative impact on many aspects of human health, such as obesity, lipid disorders, diabetes, skin regeneration, hematopoiesis and cancer. To date, poor is known on the molecular mechanisms that links mammary gland homeostasis to the circadian clock but recent reports highlight the importance of loss of circadian genes for mammary gland development and during tumour progression in breast cancer. Gene expression studies are then required to clarify how the circadian clock can modulates the human mammary gland development during ontology and its behaviour in physiological and oncogenic context. For this, in addition to genome-wide studies, real-time quantitative RT-PCR (qPCR) is a powerful and pertinent technique to quantify the expression of a reduced set of genes of interest in many different samples. Relative quantification of qPCR data requires the use of reference genes for normalisation. For circadian studies, reference genes expression must not oscillate in mirror of the circadian clock and must not be affected by the synchronisation protocols required in vitro to reset the circadian clock. Inappropriate selection of reference genes can consequently affect the amplitude of gene expression oscillation and bias data interpretation. Currently, no standard reference genes have been validated regarding these criteria for human mammary epithelial cells and the purpose of this study was to fill this gap. For this, we used the RefFinder tool, which combines four different algorithms, on 9 candidate reference genes. We compared reference genes stability using three different synchronisation protocols applied on four different mammary epithelial cell lines. This allowed us to define a set of reference genes in human mammary epithelial cells whose expression remains stable despite synchronisation protocols. We observed that the synchronisation of cells by serum shock was the most suitable procedure for maintaining the amplitude of oscillation of clock genes over time and we identified RPL4, RPLP0, HSPCB and TBP as an optimal combination of reference genes for the normalisation of the oscillatory expression of clock genes in human mammary epithelial cells.     

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Background

Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.

Results

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.

Conclusion

We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.     

Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages

Background  

Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by phorbol 12-myristate 13-acetate (PMA).     

Filament-directed intercellular contacts during differentiation of cultured chick myoblasts

Detergent-extracted, critical point dried chicken myoblasts at early stages of development in tissue culture were observed by electron microscopy. It was found that the organization of filaments within these cells changes significantly during development. A particular specialization of the cellular filament framework is the formation of microprocesses; long extensions of the cellular filament system. These microprocesses appear to be involved in cell-to-cell contact. The filaments of these processes appear to integrate with the filament system of a contacted cell, and possibly transmit tension from one cell to another. The role of these structures in effecting muscle differentiation by forming cytoplasmic connections and the implications for muscle gene expression are discussed.     

Expression of type I and III collagen genes during differentiation of embryonic chicken myoblasts in culture.

Expression of type I and III procollagen genes was studied in embryonic chicken myoblast cell cultures, obtained from thigh muscles of 11-day-old embryos. Differentiation initiated by the addition of ovotransferrin (30 micrograms/ml) was followed visually by phase-contrast microscopy. Myoblast fusion and myotube formation were detected by day 3 and appeared to be complete by day 7. The synthesis of procollagens was monitored by labeling cell cultures for 1 h with [3H]proline and determining the radioactivity in procollagen chains by scanning densitometry of the fluorograms of the sodium dodecyl sulfate-polyacrylamide gels. A 10- to 20-fold increase in the rate of pro alpha-1(I), pro alpha-2(I), and pro alpha-1(III) collagen synthesis was observed, with the greatest increase occurring between days 3 and 9. Collagen mRNA levels in the myoblast cultures were examined by Northern blot and dot blot hybridization assays. The 10- to 20-fold increased rate of protein synthesis was accompanied by a 15-fold increase in the steady-state levels of pro alpha-1(I) and pro alpha-2(I) mRNAs and a 10-fold increase in the steady-state levels of pro alpha-1(III). As a correlate to the studies of collagen expression during myoblast differentiation, the expression of actin mRNAs was examined. Although alpha actin could be detected by day 4, a complete switch from lambda and beta to alpha actin was not observed in the time periods examined. Similar results were obtained in the analysis of RNA extracted from embryonic legs at days 12 and 17 of gestation. Myoblast differentiation is manifested by the accumulation of both muscle-specific mRNAs, such as actin, and type I and III procollagen mRNAs.     

Developmental regulation of a cadherin during the differentiation of skeletal myoblasts

Cadherins are a family of integral membrane glycoproteins which mediate calcium-dependent intercellular adhesion in vertebrate species. Here we present evidence that fusion-competent rat L6 myoblasts express a cadherin (Mr 127 kDa). The levels of this cadherin were found to be developmentally regulated. Maximal levels were expressed prior to fusion. The increase in cadherin levels observed during differentiation was prevented by the differentiation inhibitor, 5-bromo-2'-deoxyuridine. L6 myoblasts grown in the presence of anti-cadherin antibodies exhibited an altered morphology in comparison to control cultures, coupled with decreased myoblast fusion. These data indicate that the developmental regulation of cadherin is part of the program of terminal differentiation of skeletal myoblasts, and that cadherins are involved in the process of myoblast fusion.     

Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts

Alterations in receptor-independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3, 6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low-and high-affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low- and high-affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p-dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 microM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera- and pertussis toxin-dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.     

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.     

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Although the accuracy of quantitative real-time polymerase chain reaction （qRT-PCR） is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing （RNA-Seq） to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression proffies of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes （B2M, GAPDH, GUSB, HMBS, HPRT1, IP08, PGK1, POLR2A, PPIA, RPLPO, TBP, TFRC, UBC, and YWHAZ）, the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes （ACTB） and the 5 additional candidate reference genes （EEFIA1, FAU, RPS9, RPSll, and RPSI4） were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEFIAI, FA U, RPS9, RPSl l, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.     

Expression of chimeric human beta- and delta-globin genes during erythroid differentiation

To determine whether sequences contained within the small intervening sequence (IVS 1) or large intervening sequence (IVS 2) are involved in the regulated expression of the human beta-globin gene, chimeric genes containing portions of the human beta- and delta-globin genes were stably transfected into mouse erythroleukemia (MEL) cells. Since MEL cells can be induced to differentiate in culture, the expression of the chimeric genes was compared to the expression of beta and delta both before and after the induction of erythroid differentiation. The expression of beta delta 1, a beta-globin gene containing delta IVS 1 in place of beta IVS 1, was comparable to the expression of a beta-globin gene both before and after erythroid differentiation. However, the base-line expression of human beta-globin genes containing delta IVS 2 in place of beta IVS 2 was dramatically decreased. Furthermore, the substitution of delta IVS 2 for beta IVS 2 prevented the regulated increase in expression of the beta-globin gene upon induction. The results also indicate that sequences present in beta IVS 2 are not sufficient for this induced increase in expression since the substitution of beta IVS 2 for delta IVS 2 in a delta gene does not increase the regulated expression of delta during differentiation. These experiments suggest that either the presence of delta IVS 2 in a beta gene interrupts sequences required for the induced expression of beta-globin or that sequences in beta IVS 2 act in concert with other beta globin sequences not present in the delta-globin gene to permit optimal expression.     

Regulation of hexose transport in rat myoblasts during growth and differentiation

We report here the effects of growth conditions and myogenic differentiation on rat myoblast hexose transport activities. We have previously shown that in undifferentiated myoblasts the preferred substrates for the high (HAHT)- and low (LAHT)-affinity hexose transport systems are 2-deoxyglucose (2-DG) and 3-O-methyl-D-glucose (3-OMG), respectively. The present study shows that at cell density higher than 4.4 x 10(4) cells/cm2, the activities of both transport processes decrease with increasing cell densities of the undifferentiated myoblasts. Since the transport affinities are not altered, the observed decrease is compatible with the notion that the number of functional hexose transporters may be decreased in the plasma membrane. Myogenic differentiation is found to alter the 2-DG, but not the 3-OMG, transport affinity. The Km values of 2-DG uptake are elevated upon the onset of fusion and are directly proportional to the extent of fusion. This relationship between myogenesis and hexose transport is further explored by using cultures impaired in myogenesis. Treatment of cells with 5-bromo-2'-deoxyuridine abolishes not only myogenesis but also the myogenesis-induced change in 2-DG transport affinity. Similarly, alteration in 2-DG transport affinity cannot be observed in a myogenesis-defective mutant, D1. However, under myogenesis-permissive condition, the myogenesis of this mutant is also accompanied by changes in its 2-DG transport affinity. The myotube 2-DG transport system also differs from its myoblast counterpart in its response to sulfhydryl reagents and in its turnover rate. It may be surmised from the above observations that myogenesis results in the alteration of the turnover rate or in the modification of the 2-DG transport system. Although glucose starvation has no effect on myogenesis, it is found to alter the substrate specificity and transport capacity of HAHT. In conclusion, the present study shows that hexose transport in rat myoblasts is very sensitive to the growth conditions and the stages of differentiation of the cultures. This may explain why different hexose transport properties have been observed with myoblasts grown under different conditions.