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Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).
Assuntos
Leucemia Mieloide Aguda , Biologia de Sistemas , Tretinoína , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Tretinoína/farmacologia , Biologia de Sistemas/métodos , Células HL-60 , Perfilação da Expressão Gênica , Células K562 , Descoberta de Drogas/métodos , Transcriptoma , Linhagem Celular Tumoral , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 µM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 µL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.
Assuntos
Dickeya , Polifosfatos , Recombinases , Solanum tuberosum , DNA , Enterobacteriaceae , Nucleotídeos , Desoxiuridina , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e EspecificidadeRESUMO
The long-read RNA sequencing developed by Oxford Nanopore Technologies provides a direct quantification of transcript isoforms, thereby making it possible to present alternative splicing (AS) profiles as arrays of single splice variants with different abundances. Additionally, AS profiles can be presented as arrays of genes characterized by the degree of alternative splicing (the DAS-the number of detected splice variants per gene). Here, we successfully utilized the DAS to reveal biological pathways influenced by the alterations in AS in human liver tissue and the hepatocyte-derived malignant cell lines HepG2 and Huh7, thus employing the mathematical algorithm of gene set enrichment analysis. Furthermore, analysis of the AS profiles as abundances of single splice variants by using the graded tissue specificity index τ provided the selection of the groups of genes expressing particular splice variants specifically in liver tissue, HepG2 cells, and Huh7 cells. The majority of these splice variants were translated into proteins products and appeal to be in focus regarding further insights into the mechanisms underlying cell malignization. The used metrics are intrinsically suitable for transcriptome-wide AS profiling using long-read sequencing.
RESUMO
RNA modifications, particularly N6-methyladenosine (m6A), are pivotal regulators of RNA functionality and cellular processes. We analyzed m6A modifications by employing Oxford Nanopore technology and the m6Anet algorithm, focusing on the HepG2 cell line. We identified 3968 potential m6A modification sites in 2851 transcripts, corresponding to 1396 genes. A gene functional analysis revealed the active involvement of m6A-modified genes in ubiquitination, transcription regulation, and protein folding processes, aligning with the known role of m6A modifications in histone ubiquitination in cancer. To ensure data robustness, we assessed reproducibility across technical replicates. This study underscores the importance of evaluating algorithmic reproducibility, especially in supervised learning. Furthermore, we examined correlations between transcriptomic, translatomic, and proteomic levels. A strong transcriptomic-translatomic correlation was observed. In conclusion, our study deepens our understanding of m6A modifications' multifaceted impacts on cellular processes and underscores the importance of addressing reproducibility concerns in analytical approaches.
Assuntos
Nanoporos , Metilação , Proteômica , Reprodutibilidade dos Testes , RNA/metabolismo , Adenosina/metabolismo , Linhagem CelularRESUMO
The long-read RNA sequencing developed by Oxford Nanopore Technology provides a direct quantification of transcript isoforms. That makes the number of transcript isoforms per gene an intrinsically suitable metric for alternative splicing (AS) profiling in the application to this particular type of RNA sequencing. By using this simple metric and recruiting principal component analysis (PCA) as a tool to visualize the high-dimensional transcriptomic data, we were able to group biospecimens of normal human liver tissue and hepatocyte-derived malignant HepG2 and Huh7 cells into clear clusters in a 2D space. For the transcriptome-wide analysis, the clustering was observed regardless whether all genes were included in analysis or only those expressed in all biospecimens tested. However, in the application to a particular set of genes known as pharmacogenes, which are involved in drug metabolism, the clustering worsened dramatically in the latter case. Based on PCA data, the subsets of genes most contributing to biospecimens' grouping into clusters were selected and subjected to gene ontology analysis that allowed us to determine the top 20 biological processes among which translation and processes related to its regulation dominate. The suggested metrics can be a useful addition to the existing metrics for describing AS profiles, especially in application to transcriptome studies with long-read sequencing.
Assuntos
Processamento Alternativo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Componente Principal , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Perfilação da Expressão Gênica/métodos , Transcriptoma , Análise de Sequência de RNA/métodos , Fígado , Isoformas de Proteínas/genética , Hepatócitos , Linhagem CelularRESUMO
Bacteria are the constant companions of the human body throughout its life and even after its death. The history of a human disease such as cancer and the history of microorganisms, particularly bacteria, are believed to closely intertwined. This review was conceived to highlight the attempts of scientists from ancient times to the present day to discover the relationship between bacteria and the emergence or development of tumors in the human body. Challenges and achievements of 21st century science in forcing bacteria to serve for cancer treatment are considered. The future possibilities of bacterial cancer therapy, including the creation of bacterial microrobots, or "bacteriobots", are also discussed.
Assuntos
Medicina , Neoplasias , Humanos , Bactérias , Neoplasias/terapiaRESUMO
Although modern biology is now in the post-genomic era with vastly increased access to high-quality data, the set of human genes with a known function remains far from complete. This is especially true for hundreds of mitochondria-associated genes, which are under-characterized and lack clear functional annotation. However, with the advent of multi-omics profiling methods coupled with systems biology algorithms, the cellular role of many such genes can be elucidated. Here, we report genes and pathways associated with TOMM34, Translocase of Outer Mitochondrial Membrane, which plays role in the mitochondrial protein import as a part of cytosolic complex together with Hsp70/Hsp90 and is upregulated in various cancers. We identified genes, proteins, and metabolites altered in TOMM34-/- HepG2 cells. To our knowledge, this is the first attempt to study the functional capacity of TOMM34 using a multi-omics strategy. We demonstrate that TOMM34 affects various processes including oxidative phosphorylation, citric acid cycle, metabolism of purine, and several amino acids. Besides the analysis of already known pathways, we utilized de novo network enrichment algorithm to extract novel perturbed subnetworks, thus obtaining evidence that TOMM34 potentially plays role in several other cellular processes, including NOTCH-, MAPK-, and STAT3-signaling. Collectively, our findings provide new insights into TOMM34's cellular functions.
RESUMO
Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3-12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3-12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated p53 gene.
Assuntos
Núcleo Celular , Granulócitos , Leucemia Promielocítica Aguda , Proteínas Nucleares , Proteoma , Humanos , Caspase 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Proteômica , Tretinoína/farmacologia , Tretinoína/metabolismo , Proteína Supressora de Tumor p53/genética , Células HL-60 , Granulócitos/metabolismo , Granulócitos/patologia , Núcleo Celular/metabolismoRESUMO
CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity.
Assuntos
Antígeno AC133/genética , Células-Tronco Neoplásicas/citologia , Proteína 28 com Motivo Tripartido/metabolismo , Antígeno AC133/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Proteômica , Fatores de Transcrição/metabolismoRESUMO
Induced granulocytic differentiation of human leukemic cells under all-trans-retinoid acid (ATRA) treatment underlies differentiation therapy of acute myeloid leukemia. Knowing the regulation of this process it is possible to identify potential targets for antileukemic drugs and develop novel approaches to differentiation therapy. In this study, we have performed transcriptomic and proteomic profiling to reveal up- and down-regulated transcripts and proteins during time-course experiments. Using data on differentially expressed transcripts and proteins we have applied upstream regulator search and obtained transcriptome- and proteome-based regulatory networks of induced granulocytic differentiation that cover both up-regulated (HIC1, NFKBIA, and CASP9) and down-regulated (PARP1, VDR, and RXRA) elements. To verify the designed network we measured HIC1 and PARP1 protein abundance during granulocytic differentiation by selected reaction monitoring (SRM) using stable isotopically labeled peptide standards. We also revealed that transcription factor CEBPB and LYN kinase were involved in differentiation onset, and evaluated their protein levels by SRM technique. Obtained results indicate that the omics data reflect involvement of the DNA repair system and the MAPK kinase cascade as well as show the balance between the processes of the cell survival and apoptosis in a p53-independent manner. The differentially expressed transcripts and proteins, predicted transcriptional factors, and key molecules such as HIC1, CEBPB, LYN, and PARP1 may be considered as potential targets for differentiation therapy of acute myeloid leukemia.
Assuntos
Diferenciação Celular/fisiologia , Redes Reguladoras de Genes/genética , Leucemia Mieloide/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteômica/métodos , Fatores de Transcrição/metabolismoRESUMO
Information about the characteristics of measuring chips according to their storage conditions is of great importance for clinical diagnosis. In our present work, we have studied the capability of chips to detect nanowire biosensors when they are either freshly prepared or have been stored for either one or two years in a clean room. Potential to detect DNA oligonucleotides (oDNAs)-synthetic analogues of microRNAs (miRNAs) 198 and 429 that are associated with the development of prostate cancer (PCa)-in buffer solution was demonstrated using a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). To provide biospecific detection, nanowire surfaces were sensitized with oligonucleotide probes (oDNA probes) complimentary to the known sequences of miRNA 183 and 484. In this study it is demonstrated that freshly prepared SOI-NW biosensor chips with n-type conductance and immobilized oDNA probes exhibit responses to the addition of complimentary oDNAs in buffer, leading to decreases in chips' conductance at a concentration of 3.3 × 10-16 M. The influence of storage time on the characteristics of SOI-NW biosensor chips is also studied herein. It is shown that a two-year storage of the chips leads to significant changes in their characteristics, resulting in "inverse" sensitivity toward negatively charged oDNA probes (i.e., through an increase in chips' conductance). It is concluded that the surface layer makes the main contribution to conductance of the biosensor chip. Our results indicate that the detection of target nucleic acid molecules can be carried out with high sensitivity using sensor chips after long-term storage, but that changes in their surface properties, which lead to inversed detection signals, must be taken into account. Examples of the applications of such chips for the detection of cancer-associated microRNAs in plasma samples of patients with diagnosed prostate cancer are given. The results obtained herein are useful for the development of highly sensitive nanowire-based diagnostic systems for the revelation of (prostate) cancer-associated microRNAs in human plasma.
Assuntos
Biomarcadores Tumorais/sangue , Técnicas Biossensoriais/métodos , MicroRNAs/sangue , Neoplasias da Próstata/sangue , Humanos , Masculino , Nanofios/química , Ácidos Nucleicos/sangue , Ácidos Nucleicos/isolamento & purificação , Silício/químicaRESUMO
The Chromosome-centric Human Proteome Project aims at characterizing the expression of proteins encoded in each chromosome at the tissue, cell, and subcellular levels. The proteomic profiling of a particular tissue or cell line commonly results in a substantial portion of proteins that are not observed (the "missing" proteome). The concurrent transcriptome profiling of the analyzed tissue/cells samples may help define the set of untranscribed genes in a given type of tissue or cell, thus narrowing the size of the "missing" proteome and allowing us to focus on defining the reasons behind undetected proteins, namely, whether they are technical (insufficient sensitivity of protein detection) or biological (correspond to not-translated transcripts). We believe that the quantitative polymerase chain reaction (qPCR) can provide an efficient approach to studying low-abundant transcripts related to undetected proteins due to its high sensitivity and the possibility of ensuring the specificity of detection via the simple Sanger sequencing of PCR products. Here we illustrated the feasibility of such an approach on a set of low-abundant transcripts. Although inapplicable to the analysis of whole transcriptome, qPCR can successfully be utilized to profile a limited cohort of transcripts encoded on a particular chromosome, as we previously demonstrated for human chromosome 18.
Assuntos
Proteoma/genética , Proteômica/métodos , Cromossomos Humanos , Cromossomos Humanos Par 18 , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
Chronic liver diseases constitute a significant economic, social, and biomedical burden. Among commonly adopted approaches, only organ transplantation can radically help patients with end-stage liver pathologies. Cell therapy with hepatocytes as a treatment for chronic liver disease has demonstrated promising results. However, quality human hepatocytes are in short supply. Stem/progenitor cells capable of differentiating into functionally active hepatocytes provide an attractive alternative approach to cell therapy for liver diseases, as well as to liver-tissue engineering, drug screening, and basic research. The application of methods generally used to isolate mesenchymal stem cells (MSCs) and maintain them in culture to human liver tissue provides cells, designated here as liver MSCs. They have much in common with MSCs from other tissues, but differ in two aspects-expression of a range of hepatocyte-specific genes and, possibly, inherent commitment to hepatogenic differentiation. The aim of this review is to analyze data regarding liver MSCs, probably another type of liver stem/progenitor cells different from hepatic stellate cells or so-called hepatic progenitor cells. The review presents an analysis of the phenotypic characteristics of liver MSCs, their differentiation and therapeutic potential, methods for isolating these cells from human liver, and discusses issues of their origin and heterogeneity. Human liver MSCs are a fascinating object of fundamental research with a potential for important practical applications.
Assuntos
Fígado/citologia , Fígado/fisiologia , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo/citologia , Adulto , Diferenciação Celular/fisiologia , Doença Hepática Terminal/patologia , Doença Hepática Terminal/fisiopatologia , Doença Hepática Terminal/terapia , Hepatócitos/fisiologia , Humanos , Imunomodulação/fisiologia , Transplante de Células-Tronco MesenquimaisRESUMO
Targeted mass spectrometry represents a powerful tool for investigation of biological processes. The convenient approach of selected reaction monitoring using stable isotope-labeled peptide standard (SIS) is widely applied for protein quantification. Along with this method, high-resolution parallel reaction monitoring has been increasingly used for protein targeted analysis. Here we applied two targeted approaches (selected reaction monitoring with SIS and label-free parallel reaction monitoring) to investigate expression of 11 proteins during all-trans retinoic acid-induced differentiation of HL-60 cells. In our experiments, we have determined the proteins expression ratio at 3, 24, 48, and 96 h after all-trans retinoic acid treatment in comparison with 0 h, respectively. Expression profiles of four proteins (VAV1, PRAM1, LYN, and CEBPB) were highly correlated ( r > 0.75) and FGR expression was detected on proteome level starting from 24 h by both techniques. For prominent differences (fold change ≥ 2) label-free parallel reaction monitoring is not inferior to selected reaction monitoring with isotopically labeled peptide standards. Differentially expressed proteins, that have been determined in our study, can be considered as potential drug targets for acute myeloid leukemia (AML) treatment.
Assuntos
Diferenciação Celular/fisiologia , Células HL-60/citologia , Células HL-60/metabolismo , Espectrometria de Massas/métodos , Diferenciação Celular/efeitos dos fármacos , Humanos , Proteoma/análise , Proteoma/metabolismo , Proteômica , Tretinoína/farmacologiaRESUMO
Amyloid-ß peptide (Aß) plays a central role in Alzheimer's disease (AD) pathogenesis. Besides extracellular Aß, intraneuronal Aß (iAß) has been suggested to contribute to AD onset and development. Based on reported in vitro Aß-DNA interactions and nuclear localization of iAß, the interference of iAß with the normal DNA expression has recently been proposed as a plausible pathway by which Aß can exert neurotoxicity. Employing the sedimentation assay, thioflavin T fluorescence, and dynamic light scattering we have studied effects of zinc ions on binding of RNA and single- and double-stranded DNA molecules to Aß42 aggregates. It has been found that zinc ions significantly enhance the binding of RNA and DNA molecules to pre-formed ß-sheet rich Aß42 aggregates. Another type of Aß42 aggregates, the zinc-induced amorphous aggregates, was demonstrated to also bind all types of nucleic acids tested. To evaluate the role of the Aß metal-binding domain's histidine residues in Aß-nucleic acid interactions mediated by zinc, Aß16 mutants with substitutions H6R and H6A-H13A and rat Aß16 lacking histidine residue 13 were used. The zinc-induced interaction of Aß16 with DNA was shown to critically depend on histidine residues 6 and 13. However, the inclusion of H6R mutation in Aß42 peptide did not affect DNA binding to Aß42 aggregates. Since oxidative and/or nitrosative stresses implicated in AD pathogenesis are known to release zinc ions from metallothioneins in cytoplasm and cell nuclei, our findings suggest that intracellular zinc can be an important player in iAß-nucleic acid interactions.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Histidina/fisiologia , Ácidos Nucleicos/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos/fisiologia , Zinco/metabolismo , Células Hep G2 , Humanos , Ligação Proteica/fisiologia , Zinco/farmacologiaRESUMO
BACKGROUND: Human hepatoma HepG2 cells are used as an in vitro model of the human liver. High-throughput transcriptomic sequencing is an advanced approach for assessing the functional state of a tissue or cell type. However, the influence of experimental factors, such as the sample preparation method and inter-laboratory variation, on the transcriptomic profile has not been evaluated. RESULTS: The whole-transcriptome sequencing of HepG2 cells was performed using the SOLiD platform and validated using droplet digital PCR. The gene expression profile was compared to the results obtained with the same sequencing method in another laboratory and using another sample preparation method. We also compared the transcriptomic profile HepG2 cells with that of liver tissue. Comparison of the gene expression profiles between the HepG2 cell line and liver tissue revealed the highest variation, followed by HepG2 cells submitted to two different sample preparation protocols. The lowest variation was observed between HepG2 cells prepared by two different laboratories using the same protocol. The enrichment analysis of the genes that were differentially expressed between HepG2 cells and liver tissue mainly revealed the cancer-associated gene signature of HepG2 cells and the activation of the response to chemical stimuli in the liver tissue. The HepG2 transcriptome obtained with the SOLiD platform was highly correlated with the published transcriptome obtained with the Illumina and Helicos platforms, with moderate correspondence to microarrays. CONCLUSIONS: In the present study, we assessed the influence of experimental factors on the HepG2 transcriptome and identified differences in gene expression between the HepG2 cell line and liver cells. These findings will facilitate robust experimental design in the fields of pharmacology and toxicology. Our results were supported by a comparative analysis with previous HepG2 gene expression studies.
Assuntos
Perfilação da Expressão Gênica , Fígado/metabolismo , Análise por Conglomerados , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , TranscriptomaRESUMO
We report the results obtained in 2012-2013 by the Russian Consortium for the Chromosome-centric Human Proteome Project (C-HPP). The main scope of this work was the transcriptome profiling of genes on human chromosome 18 (Chr 18), as well as their encoded proteome, from three types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma. The transcriptome profiling for liver tissue was independently performed using two RNaseq platforms (SOLiD and Illumina) and also by droplet digital PCR (ddPCR) and quantitative RT-PCR. The proteome profiling of Chr 18 was accomplished by quantitatively measuring protein copy numbers in the three types of biomaterial (the lowest protein concentration measured was 10(-13) M) using selected reaction monitoring (SRM). In total, protein copy numbers were estimated for 228 master proteins, including quantitative data on 164 proteins in plasma, 171 in the HepG2 cell line, and 186 in liver tissue. Most proteins were present in plasma at 10(8) copies/µL, while the median abundance was 10(4) and 10(5) protein copies per cell in HepG2 cells and liver tissue, respectively. In summary, for liver tissue and HepG2 cells a "transcriptoproteome" was produced that reflects the relationship between transcript and protein copy numbers of the genes on Chr 18. The quantitative data acquired by RNaseq, PCR, and SRM were uploaded into the "Update_2013" data set of our knowledgebase (www.kb18.ru) and investigated for linear correlations.
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Cromossomos Humanos Par 18 , Fígado/metabolismo , Plasma , Proteoma , Transcriptoma , Células Hep G2 , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
The final goal of the Russian part of the Chromosome-centric Human Proteome Project (C-HPP) was established as the analysis of the chromosome 18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells with the sensitivity of 10(-18) M. Using SRM, we have recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2 cells. On the basis of the results of the survey, the SRM assays were drafted for 250 proteins: 41 proteins were found only in the liver tissue, 82 proteins were specifically detected in depleted plasma, and 127 proteins were mapped in both samples. The targeted analysis of HepG2 cells was carried out for 49 proteins; 41 of them were successfully registered using ordinary SRM and 5 additional proteins were registered using a combination of irreversible binding of proteins on CN-Br Sepharose 4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq and RT-PCR has shown a significant correlation (r = 0.78) for 42 gene transcripts. A pilot affinity-based interactome analysis was performed for cytochrome b5 using analytical and preparative optical biosensor fishing followed by MS analysis of the fished proteins. All of the data on the proteome complement of the Chr 18 have been integrated into our gene-centric knowledgebase ( www.kb18.ru ).