Genetic Expression Screening of Arsenic Trioxide-Induced Cytotoxicity in KG-1a Cells Based on Bioinformatics Technology

Acute myeloid leukemia (AML) is a malignant tumor of the hematopoietic system, and leukemia stem cells are responsible for AML chemoresistance and relapse. KG-1a cell is considered a leukemia stem cell-enriched cell line, which is resistant to chemotherapy. Arsenic trioxide (ATO) is effective against acute promyelocytic leukemia as a first-line treatment agent, even as remission induction of relapsed cases. ATO has a cytotoxic effect on KG-1a cells, but the mechanism remains unclear. Our results demonstrated that ATO can inhibit cell proliferation, induce apoptosis, and arrest KG-1a cells in the G2/M phase. Using transcriptome analysis, we investigated the candidate target genes regulated by ATO in KG-1a cells. The expression profile analysis showed that the ATO had significantly changed gene expression related to proliferation, apoptosis, and cell cycle. Moreover, MYC, PCNA, and MCM7 were identified as crucial hub genes through protein–protein interaction network analysis; meanwhile, the expressions of them in both RNA and protein levels are down-regulated as confirmed by quantitative polymerase chain reaction and Western blot. Thus, our study suggests that ATO not only inhibits the expression of MYC, PCNA, and MCM7 but also leads to cell cycle arrest and apoptosis in KG-1a cells. Overall, this study provided reliable clues for improving the ATO efficacy in AML.

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Mdm2 Is Required for Inhibition of Cdk2 Activity by p21, Thereby Contributing to p53-Dependent Cell Cycle Arrest

Molecular and Cellular Biology ◽

10.1128/mcb.01967-06 ◽

2007 ◽

Vol 27 (11) ◽

pp. 4166-4178 ◽

Cited By ~ 40

Author(s):

Luciana E. Giono ◽

James J. Manfredi

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Target Genes ◽

P53 Protein ◽

Protein Levels ◽

Cycle Arrest ◽

Cdk2 Activity ◽

Dependent Cell ◽

Interfering Rna

ABSTRACT p53 is extensively posttranslationally modified in response to various types of cellular stress. Such modifications have been implicated in the regulation of p53 protein levels as well as its DNA binding and transcriptional activities. Treatment of cells with doxorubicin causes phosphorylation and acetylation of p53, transcriptional upregulation of p21 and other target genes, and growth arrest. In contrast, downregulation of Mdm2 by a small interfering RNA (siRNA) approach led to increased levels of p53 lacking phosphorylation at serine 15 and acetylation at lysine 382. Levels of binding of p53 to the p21 promoter were comparable following treatment with doxorubicin or Mdm2 siRNA. Moreover, p53 was transcriptionally active and capable of inducing or repressing a variety of its target genes. Surprisingly, p53 upregulated by Mdm2 siRNA had no effect on cell cycle progression. Although comparable in level to that achieved by treatment with the p53 activators actinomycin D and nutlin-3, the increases in p53 and p21 after downregulation of Mdm2 were not sufficient to trigger cell cycle arrest. This version of p21 was capable of interacting with cyclin-dependent kinase 2 (Cdk2) but failed to inhibit its activity. Taken together, these results argue that Mdm2 is needed for full inhibition of Cdk2 activity by p21, thereby positively contributing to p53-dependent cell cycle arrest.

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VprBP/DCAF1 Regulates the Degradation and Nonproteolytic Activation of the Cell Cycle Transcription Factor FoxM1

Molecular and Cellular Biology ◽

10.1128/mcb.00609-16 ◽

2017 ◽

Vol 37 (13) ◽

Cited By ~ 13

Author(s):

Xianxi Wang ◽

Anthony Arceci ◽

Kelly Bird ◽

Christine A. Mills ◽

Rajarshi Choudhury ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Cell Cycle Progression ◽

Target Genes ◽

Chromosome Instability ◽

Vital Role ◽

Activation Mechanism ◽

Cycle Progression ◽

Protein Levels ◽

Ubiquitin System

ABSTRACT The oncogenic transcription factor FoxM1 plays a vital role in cell cycle progression, is activated in numerous human malignancies, and is linked to chromosome instability. We characterize here a cullin 4-based E3 ubiquitin ligase and its substrate receptor, VprBP/DCAF1 (CRL4VprBP), which we show regulate FoxM1 ubiquitylation and degradation. Paradoxically, we also found that the substrate receptor VprBP is a potent FoxM1 activator. VprBP depletion reduces expression of FoxM1 target genes and impairs mitotic entry, whereas ectopic VprBP expression strongly activates a FoxM1 transcriptional reporter. VprBP binding to CRL4 is reduced during mitosis, and our data suggest that VprBP activation of FoxM1 is ligase independent. This implies a nonproteolytic activation mechanism that is reminiscent of, yet distinct from, the ubiquitin-dependent transactivation of the oncoprotein Myc by other E3s. Significantly, VprBP protein levels were upregulated in high-grade serous ovarian patient tumors, where the FoxM1 signature is amplified. These data suggest that FoxM1 abundance and activity are controlled by VprBP and highlight the functional repurposing of E3 ligase substrate receptors independent of the ubiquitin system.

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Identification of circRNA–miRNA–mRNA networks contributes to explore underlying pathogenesis and therapy strategy of gastric cancer

Journal of Translational Medicine ◽

10.1186/s12967-021-02903-5 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Zhijie Dong ◽

Zhaoyu Liu ◽

Min Liang ◽

Jinhui Pan ◽

Mingzhen Lin ◽

...

Keyword(s):

Gastric Cancer ◽

Target Genes ◽

Trichostatin A ◽

Quantitative Polymerase Chain Reaction ◽

Human Tumor ◽

Interaction Network ◽

Enrichment Analysis ◽

The Cancer Genome Atlas ◽

Circular Rnas ◽

Therapy Strategy

Abstract Background Circular RNAs (circRNAs) are a new class of noncoding RNAs that have gained increased attention in human tumor research. However, the identification and function of circRNAs are largely unknown in the context of gastric cancer (GC). This study aims to identify novel circRNAs and determine their action networks in GC. Methods A comprehensive strategy of data mining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and computational biology were conducted to discover novel circRNAs and to explore their potential mechanisms in GC. Promising therapeutic drugs for GC were determined by connectivity map (CMap) analysis. Results Six overlapped differentially expressed circRNAs (DECs) were screened from selected microarray and RNA-Seq datasets of GC, and the six DECs were then validated by sanger sequencing and RNase R treatment. Subsequent RT-qPCR analysis of GC samples confirmed decreased expressions of the six DECs (hsa_circ_0000390, hsa_circ_0000615, hsa_circ_0001438, hsa_circ_0002190, hsa_circ_0002449 and hsa_circ_0003120), all of which accumulated preferentially in the cytoplasm. MiRNA binding sites and AGO2 occupation of the six circRNAs were predicted using online databases, and circRNA–miRNA interactions including the six circRNAs and 33 miRNAs were determined. Then, 5320 target genes of the above 33 miRNAs and 1492 differently expressed genes (DEGs) from The Cancer Genome Atlas (TCGA) database were identified. After intersecting the miRNA target genes and the 889 downregulated DEGs, 320 overlapped target genes were acquired. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that these target genes were related to two critical tumor-associated signaling pathways. A protein–protein interaction network with the 320 target genes was constructed using STRING, and fifteen hubgenes (ATF3, BTG2, DUSP1, EGR1, FGF2, FOSB, GNAO1, GNAI1, GNAZ, GNG7, ITPR1, ITPKB, JUND, NR4A3, PRKCB) in the network were identified. Finally, bioactive chemicals (including vorinostat, trichostatin A and astemizole) based on the fifteen hubgenes were identifed as therapeutic agents for GC through the CMap analysis. Conclusions This study provides a novel insight for further exploration of the pathogenesis and therapy of GC from the circRNA-miRNA-mRNA network perspective.

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BCL6 Regulates Diffuse Large B-Cell Lymphoma Cell Cycle and Apoptosis Checkpoints through Direct Repression of the p300 Histone Acetyl-Transferase.

Blood ◽

10.1182/blood.v108.11.1413.1413 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1413-1413

Author(s):

Leandro Cerchietti ◽

Maria E. Figueroa ◽

Rita Shaknovich ◽

Ari Melnick

Keyword(s):

Cell Cycle ◽

B Cell ◽

Target Genes ◽

B Cell Lymphoma ◽

Dominant Negative ◽

Mrna Levels ◽

Histone Acetyl Transferase ◽

Protein Levels ◽

P53 Target Genes ◽

Large B Cell

Abstract The BCL6 oncogenic transcriptional repressor protein is frequently constitutively expressed in Diffuse Large B-cell Lymphomas (DLBCLs). A BCL6 peptidomimetic inhibitor (BPI) that specifically inhibits the repressor activity of BCL6 can induce cell death in DLBCL cell lines and primary tumor tissue, both in vitro and in vivo. Many genes involved in DNA damage, cell cycle and others are targets of BCL6. Among these is the p53 tumor suppressor gene. However, we find that p53 mRNA levels are actually higher in the subset of DLBCL patients with higher BCL6 expression (n=176 cases). Overall, we could readily detect p53 protein expression by immunohistochemistry in 50% of BCL6 positive DLBCL samples (n=350 cases). By studying expression levels of p53 target genes, we show that even in DLBCLs expressing wild-type p53, the protein is not fully active, and a p53 activating peptide was required to trigger p53 activity and execute cellular checkpoints. Accordingly, even though p53 was already present, BCL6 blockade by BPI could still induce a p53 response in DLBCL cells (with only small changes in p53 levels). Based on these results we speculated that BCL6 might inhibit p53 activity through an alternative mechanism such as regulating its activity through post-translational modifications. In accordance with this prediction, we found that BPI can strongly induce expression of the p300 histone acetyl-transferase, which can activate p53 by acetylation. The p300 promoter has two BCL6 binding sites and by chromatin immunoprecipitation (ChIP) assays we show that BCL6 directly binds to these sites. We found that 80% of DLBCL (n=70) express low protein levels of p300 (compared with other B-lymphomas) and the same is apparent from mRNA studies. By performing kinetic studies in DLBCL cells with multiple time points, we show that after BPI treatment, p300 mRNA and then protein levels are induced, after which p53 becomes acetylated and after which p53 target genes (p21, PUMA, NOXA, GADD45 and PIG3) are upregulated. These changes are partially or totally overcome by expression of either a p53-dominant negative or p300-dominant negative construct. In DLBCL cells with p53 mutations, this program is preferentially executed trough p73 and/or p63, which in turn become acetylated by p300. Interestingly, after BPI treatment p300 acetylates BCL6 itself, which further reduces BCL6 activity. This leads to higher BCL6 inhibition and triggering of a signal amplification loop. These findings have significant therapeutic implications, since co-treatment of DLBCL cells with BPI plus histone deacetylase inhibitors such as Trichostatin A or SAHA (i.e. that hyperacetylate p53 and BCL6) resulted in a synergistic effect in killing DLBCL cells. Our studies demonstrated that p300 is a direct target gene of BCL6 with a critical role in determining DLBCL response to treatments that require activation of p53 and/or p53-family members. This can be capitalized on to develop powerful biological therapeutic regiments for DLBCL.

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Target genes of the largest human SWI/SNF complex subunit control cell growth

Biochemical Journal ◽

10.1042/bj20101358 ◽

2011 ◽

Vol 434 (1) ◽

pp. 83-92 ◽

Cited By ~ 18

Author(s):

Hiroko Inoue ◽

Stavros Giannakopoulos ◽

Christopher N. Parkhurst ◽

Tatsushi Matsumura ◽

Evelyn A. Kono ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Target Genes ◽

Control Cell ◽

Cycle Phase ◽

Chromatin Remodelling Complex ◽

Baf Complex ◽

Protein Levels ◽

Hela Cell Lines ◽

Reporter Assays

The largest subunit of the mammalian SWI/SNF-A or BAF (BRG1-associated factor) chromatin-remodelling complex is encoded by two related cDNAs hOsa1/BAF250a and hOsa2/BAF250b that are unique to the BAF complex and absent in the related PBAF (Polybromo BAF). hOsa/BAF250 has been shown to interact with transcriptional activators and bind to DNA suggesting that it acts to target the remodelling complex to chromatin. To better understand the functions of hOsa2, we established inducible stable HeLa cell lines over-expressing FLAG–hOsa2 or a derivative lacking the ARID (AT-rich interactive domain) DNA-binding domain. Immunopurification of complexes containing hOsa2 that was followed by mass spectrometry and immunoblotting demonstrated the presence of BRG1 and known BAFs, but not hOsa1 or hBRM. Deletion of the ARID did not compromise the integrity of the complex. Induction of hOsa2 expression caused impaired cell growth and accumulation of cells in the G0/G1 cell cycle phase. Elevated levels of the p53 and p21 proteins were detected in these cells while c-Myc mRNA and protein levels were found to decrease. Chromatin immunoprecipitation and reporter assays suggested that hOsa2 had a direct effect on c-myc and p21 promoter activity. Thus hOsa2 plays an important role in controlling genes regulating the cell cycle.

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Transcriptional regulation in the rat prefrontal cortex and hippocampus after a single administration of psilocybin

Journal of Psychopharmacology ◽

10.1177/0269881120959614 ◽

2020 ◽

pp. 026988112095961 ◽

Cited By ~ 1

Author(s):

Oskar Hougaard Jefsen ◽

Betina Elfving ◽

Gregers Wegener ◽

Heidi Kaastrup Müller

Keyword(s):

Prefrontal Cortex ◽

Target Genes ◽

Therapeutic Potential ◽

Quantitative Polymerase Chain Reaction ◽

Dependent Manner ◽

Single Administration ◽

Obsessive Compulsive ◽

Compulsive Disorder ◽

Protein Levels ◽

Subcortical Regions

Background: Psilocybin is a serotonergic psychedelic found in “magic mushrooms” with a putative therapeutic potential for treatment-resistant depression, anxiety, obsessive-compulsive disorder, and addiction. In rodents, psilocybin acutely induces plasticity-related immediate early genes in cortical tissue; however, studies into the effects on subcortical regions, of different doses, and the subsequent translation of corresponding proteins are lacking. Methods: We examined the acute effects of a single administration of psilocybin (0.5–20 mg/kg) on the expression of selected genes in the prefrontal cortex and hippocampus. In total, 46 target genes and eight reference genes were assessed using real-time quantitative polymerase chain reaction. Corresponding protein levels of the three most commonly regulated genes were assessed using Western blotting. Results: In the prefrontal cortex, psilocybin increased the expression of Cebpb, c-Fos, Dups1, Fosb, Junb, Iκβ-α, Nr4a1, P11, Psd95, and Sgk1, and decreased the expression of Clk1. In the hippocampus, psilocybin strongly increased the expression of Arrdc2, Dusp1, Iκβ-α, and Sgk1 in a dose-dependent manner, and decreased the expression of Arc, Clk1, Egr2, and Ptgs2. Protein levels of Sgk1, Dusp1, and Iκβ-α showed only partial agreement with transcriptional patterns, stressing the importance of assessing downstream translation when investigating rapid gene responses. Conclusion: The present study demonstrates that psilocybin rapidly induces gene expression related to neuroplasticity, biased towards the prefrontal cortex, compared to the hippocampus. Our findings provide further evidence for the rapid plasticity-promoting effects of psilocybin.

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Inhibition of mTOR Reverses the Ability of c-MYC To Block the Differentiation of Murine Granulocytes.

Blood ◽

10.1182/blood.v106.11.310.310 ◽

2005 ◽

Vol 106 (11) ◽

pp. 310-310

Author(s):

Meaghan Wall ◽

Gretchen Poortinga ◽

Richard B. Pearson ◽

Ross D. Hannan ◽

Grant A. McArthur

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Target Genes ◽

Terminal Differentiation ◽

S Phase ◽

Brdu Incorporation ◽

Granulocytic Differentiation ◽

Differentiation Potential ◽

Protein Levels ◽

Vehicle Treated Control

Abstract The proto-oncogene c-MYC encodes a bHLH leucine-zipper transcription factor that acts as a potent differentiation inhibitor and as a promoter of cell cycle progression. Additionally, c-MYC and mTOR are widely acknowledged as major regulators of cell growth, the process by which cells accumulate biomass. Both c-MYC overexpression and constitutive activation of the PI 3-kinase/mTOR pathway are frequent events in acute myeloid leukaemia; a haemopoietic malignancy characterised by failure to undergo terminal differentiation. In this study we used the MPRO system, an in vitro murine myeloid terminal differentiation model, to study the relationship between cell growth effectors and differentiation in more detail. Enforced expression of c-MYC by the addition of 4-hydoxytamoxifen (4-OHT) to MPRO cells carrying the inducible mycER fusion protein prevents downregulation of growth and impairs retinoid-induced terminal myeloid differentiation. Because downregulation of mTOR limits growth we hypothesised that inhibition of mTOR may reverse the phenotypic effects of c-MYC. Strikingly, pharmacological inhibition of mTOR by rapamycin, but not growth limitation by other means such as amino acid deprivation or cycloheximide treatment, restores differentiation in granulocytes with enforced c-MYC expression (74.0±2.0% mature granulocytes in the rapamycin+4-OHT-treated mycER MPROs vs 4.5±1.0% in the cells treated with 4-OHT alone and 92.9±1.1% in vehicle-treated controls). Rapamycin restores cell growth parameters to levels equivalent to those of vehicle-treated control cells undergoing physiological granulocytic differentiation (2–3 fold reduction in 35S methionine incorporation in rapamycin+4-OHT-treated and vehicle-treated controls compared to levels in 4-OHT-treated cells) and facilitates cell cycle exit (26.0±7.14% of rapamycin+4-OHT-treated cells and 10.7±2.48% of vehicle-treated control cells in S phase vs 51.9±1.72% of tamoxifen-treated cells in S phase as determined by BrdU incorporation). Interestingly, rapamycin attenuates c-MYC’s ability to activate transcription of its target genes without impairing activation of RARα target genes. c-mycER protein levels are reduced while levels of retrovirus-driven c-mycER mRNA do not fall in rapamycin-treated MPRO mycER cells. Furthermore, c-mycER protein levels decrease in rapamycin-treated MPRO cells expressing the ER fusion of the mycT58A point mutation known to increase protein stability. Overall, this data suggests that rapamycin restores the differentiation potential of granulocytes in the MPRO mycER system by regulating c-MYC at the level of protein translation, rather than at the level of gene transcription or protein degradation. These findings suggest that mTOR could be targeted to influence terminal differentiation regulated by MYC in haemopoietic malignancies.

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The SIN3/RPD3 Deacetylase Complex Is Essential for G2 Phase Cell Cycle Progression and Regulation of SMRTER Corepressor Levels

Molecular and Cellular Biology ◽

10.1128/mcb.22.14.4965-4976.2002 ◽

2002 ◽

Vol 22 (14) ◽

pp. 4965-4976 ◽

Cited By ~ 43

Author(s):

Lori A. Pile ◽

Erin M. Schlag ◽

David A. Wassarman

Keyword(s):

Cell Cycle ◽

Transcriptional Repression ◽

Cell Cycle Progression ◽

Target Genes ◽

Phase Delay ◽

Phase Cell ◽

Global Changes ◽

Cycle Progression ◽

Protein Levels ◽

Repression Complex

ABSTRACT The SIN3 corepressor and RPD3 histone deacetylase are components of the evolutionarily conserved SIN3/RPD3 transcriptional repression complex. Here we show that the SIN3/RPD3 complex and the corepressor SMRTER are required for Drosophila G2 phase cell cycle progression. Loss of the SIN3, but not the p55, SAP18, or SAP30, component of the SIN3/RPD3 complex by RNA interference (RNAi) causes a cell cycle delay prior to initiation of mitosis. Loss of RPD3 reduces the growth rate of cells but does not cause a distinct cell cycle defect, suggesting that cells are delayed in multiple phases of the cell cycle, including G2. Thus, the role of the SIN3/RPD3 complex in G2 phase progression appears to be independent of p55, SAP18, and SAP30. SMRTER protein levels are reduced in SIN3 and RPD3 RNAi cells, and loss of SMRTER by RNAi is sufficient to cause a G2 phase delay, demonstrating that regulation of SMRTER protein levels by the SIN3/RPD3 complex is a vital component of the transcriptional repression mechanism. Loss of SIN3 does not affect global acetylation of histones H3 and H4, suggesting that the G2 phase delay is due not to global changes in genome integrity but rather to derepression of SIN3 target genes.

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microRNA-133b Regulates Cell Proliferation and Cell Cycle Progression via Targeting HuR in Colorectal Cancer

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2910 ◽

2022 ◽

Vol 12 (2) ◽

pp. 335-345

Author(s):

Xiaoyan Zhang ◽

Wei Zhu ◽

Junjie Lu

Keyword(s):

Colorectal Cancer ◽

Cell Cycle ◽

Cell Viability ◽

Cell Lines ◽

Cell Apoptosis ◽

Quantitative Polymerase Chain Reaction ◽

Luciferase Reporter ◽

Protein Levels ◽

Ht 29

MicroRNAs (miRNAs/miRs) have been identified to serve a key role in the development of tumors. However, the role of miR-133b in colorectal cancer (CRC) remains largely unclear. This study will investigate the role and mechanism of miR-133b in CRC. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the level of miR-133b in CRC cell lines. Bioinformatics software TargetScan predicted the potential target genes of miR-133b, and a dual luciferase reporter assay was used to confirm this. To investigate the role of miR-133b in CRC cells, miR-133b was upregulated or downregulated in CRC cell lines (SW620 and HT-29) by transfecting with a miR-133b mimic or inhibitor, respectively. Subsequently, cell viability was analyzed using MTT assay, whereas cell apoptosis and the cell cycle distribution were analyzed by flow cytometry. In addition, the associated protein levels were detected using western blot analysis. The results demonstrated that miR-133b was significantly downregulated in CRC cell lines when compared with the normal colonic epithelial NCM-460 cell line. Human antigen R (HuR; also termed ELAVL1) was demonstrated to be a direct target of miR-133b and was negatively regulated by miR-133b. HuR was also notably upregulated in the CRC cell lines when compared with the normal control. Transfection of SW620 and HT-29 cells with the miR-133b mimic significantly inhibited cell viability, and induced cell apoptosis and G1 phase arrest, while upregulation of HuR demonstrated the opposite effects. Furthermore, the present data demonstrated that the miR-133b mimic significantly enhanced the protein levels of p21 and p27, and downregulated cyclin D1 and cyclin A levels in SW620 and HT-29 cells; the opposite effects were observed following treatment with the miR-133b inhibitor. In conclusion, the data indicate that miR-133b suppressed CRC cell growth by targeting HuR.

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Integrated miRNA-mRNA Expression Profile Analysis Reveals a Role of miR-146a-3p/TRAF6 in Plasma from Gestational Diabetes Mellitus Patients

10.21203/rs.2.19652/v1 ◽

2020 ◽

Author(s):

Min Chen ◽

Jianying Yan

Keyword(s):

Pregnant Women ◽

Target Genes ◽

Profile Analysis ◽

Interaction Network ◽

Enrichment Analysis ◽

Mrna Expression Profile ◽

Hub Genes ◽

Blood Samples ◽

Expression Profile Analysis ◽

Signaling Events

Abstract BACKGROUND: By utilizing an integrative strategy, we screened noninvasive molecular markers for the early detection of GDM and constructed miRNA-mRNA regulatory networks associated with GDM.METHODS: A total of 3 microarray datasets (GSE98043, GSE19649 and GSE92772) of plasma samples comparing GDM pregnant women and healthy control pregnant women were downloaded from the GEO database. The GEO2R online platform was used to identify the differentially expressed genes (DEmRNAs) and the differentially expressed miRNAs (DEmiRNAs). The target genes of DEmiRNAs were identified using two independent and complementary types of information: computational target predictions and expression relationships. KEGG pathway annotation was performed for target genes and DEmiRNAs. An interaction network was constructed to identify hub genes of GDM. Another dataset (GSE92772) was used to externally verify the predictive ability of the hub genes. Gene set enrichment analysis (GSEA) was performed to explore the biological function of hub genes.RESULTS: A total of 264 DEmiRNAs and 1217 DEmRNAs were identified by comparing the microarray data of serum samples of GDM patients and healthy pregnant women. Hsa-miR-146a-3p ranked first because of its lowest P value and was selected for further analysis. A total of 47 target genes, including TRAF6, were shared between the computational target predictions and DEmRNAs and were identified as target genes of hsa-miR-146a-3p. Enrichment analysis indicated that GDM-related miRNAs were mainly enriched in the glypican pathway, proteoglycan syndecan-mediated signaling events, and syndecan-1-mediated signaling events. In addition, the glypican pathway was also one of the pathways regulated by hsa-miR-146a-3p. The interaction network analysis of DEmRNAs identified TRAF6, CASP8, PTPN6, and CHD3 as hub genes involved in the pathophysiological process of GDM. Next, these 4 hub genes were selected for independent external validation using the GSE19649 dataset. The expression of TRAF6, CASP8 and CHD3 in 8 pairs of GDM blood samples was confirmed to be higher than that in healthy pregnant women blood samples. However, the expression of PTPN6 in the blood samples of GDM patients was similar to that of healthy pregnant women blood samples. The AUC for predicting GDM was 0.813, 0.828, 0.813, and 0.703 for CHD3, PTPN6, and CASP8, respectively. GSEA showed that 9 hallmark gene sets of metabolism processes were enriched in TRAF6 function.CONCLUSIONS: Three hub genes, TRAF6, CASP8, and CHD3, were identified and independently externally validated as potential GDM noninvasive serum markers. Enrichment analysis indicated that GDM-related miRNAs were mainly enriched in the glypican pathway, proteoglycan syndecan-mediated signaling events, and syndecan-1-mediated signaling events. In addition, integrated miRNA-mRNA expression profile analysis showed that miR-146a-3p/TRAF6 might play a central role in the pathogenesis of gestational diabetes mellitus by involving the above pathways.

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