195 TRANSCRIPTOME PROFILING OF BOVINE ENDOMETRIUM BASED ON THE PREGNANCY OUTCOME AFTER TRANSFER OF IN VIVO-DERIVED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 196
Author(s):  
D. Salilew-Wondim ◽  
N. Ghanem ◽  
C. Grosse-Brinkhaus ◽  
A. Becker ◽  
F. Rings ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Pregnancy Outcome ◽  
Embryo Transfer ◽  
Transcriptome Profiling ◽  
R Package ◽  
Differentially Expressed ◽  
Regulation Of Transcription ◽  
Protein Amino Acid ◽  
Expression Of Genes

Transcriptome profiling of pre-transfer cycle endometrium in relation to its ability to sustain the upcoming pregnancy may pave the way to develop or identify molecular markers that can be utilized to detect and select receptive endometrium before embryo transfer. Here, we aim to show differential expression of genes between endometrium biopsies derived from recipients during pre-transfer cycle based on the pregnancy successes after embryo transfer. For this, endometrium biopsies were taken from 56 Simmental cyclic heifers of the same age at day 7 and 14 of the estrous cycle. On the next cycle, in vivo-produced day 7 blastocysts were transferred to all animals at day 7 of the estrous cycle. Pregnancy diagnosis was done at 28, 42, and 56 days of gestation. Thirty-two cows were returned to heat after 21 days, 7 were pregnant until day 42 but no pregnancy after that, and 15 resulted in calf delivery. Subsequently, the endometrium biopsies sampled during the pre-transfer period were categorized based on the pregnancy outcome. Those endometrial biopsies taken at days 7 and 14 during the pre-transferred period from those that resulted in successful calf delivery were named as d7CD and d14CD, respectively, and those endometrial samples collected at days 7 and 14 during pre-transferred period from those groups that resulted in no pregnancy were named as d7NP and d14NP, respectively. Total RNA was extracted from 3 pools of each experimental group in 3 replicates using RNeasy mini kit (Qiagen, Hilden, Germany). A total of 12 biotin-labeled cRNA samples were hybridized on 12 bovine Affymetrix arrays (Affymetrix, Santa Clara, CA, USA) consisting of 24128 probe sets. The microarray data normalization and background correction was performed using guanine cytosine robust multi-array analysis, and the data analysis was performed using LIMMA written on R package, which maintained the bioconductor. The results showed that 1130 transcripts were differentially expressed between d7CD and d7NP, of which 626 and 504 were up- and down-regulated, respectively, in d7CD. Genes that involve in regulation of transcription (PPARA, NR2F1, MYB, MYB, and CHF2) and the collagen families (COL1A1 and COL1A2) were enriched in d7CD. A total of 234 transcripts were differentially expressed between d14CD and d14NP, of which 94 and 140 were up- and down-regulated, respectively, in d14CD compared to d14NP. Transcripts involved in protein amino acid phosphorylation (MA2K6, GATM, AK3L1, and MAPK10) were found to be enriched in d14CD compared to d14NP. In conclusion, pre-transfer endometrium biopsies showed significant differences in transcriptome profile depending on the pregnancy outcome after transfer of in vivo-derived blastocysts and enable to identify transcripts related to pregnancy establishment.

248 DIESTRUS TRANSCRIPTOME DYNAMICS OF BOVINE ENDOMETRIUM IN RELATION TO PREGNANCY SUCCESS AFTER EMBRYO TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 281
Author(s):  
D. Salilew-Wondim ◽  
N. Ghanem ◽  
M. Hoelker ◽  
F. Rings ◽  
C. Phatsara ◽  
...  
Keyword(s):  
Estrous Cycle ◽  
Embryo Transfer ◽  
System Development ◽  
Mapk Signaling ◽  
Nervous System Development ◽  
Differentially Expressed ◽  
Regulation Of Transcription ◽  
Transferase Activity ◽  
And Function

This experiment aimed to investigate the diestrus transcriptome dynamics of endometrium that resulted in calf delivery or no pregnancy after embryo transfer. Endometrium biopsies were collected from Simmental cyclic heifers at Days 7 and 14 of estrus cycle. On the next cycle, in vivo-produced Day 7 blastocysts were transferred to all animals at Day 7 of estrous cycle. Following pregnancy diagnosis, the endometrial biopsies collected at Day 7 and 14 were categorized based on the pregnancy success. Those endometrial biopsies collected from heifers that subsequently delivered a calf were assigned to the calf-delivery group, and those collected from heifers that did not conceive were assigned to the no-pregnancy group. The endometrial temporal transcriptome profile was compared between Days 7 and 14 in both heifer groups. Total RNA was isolated from each sample in triplicate. Two rounds of RNA amplification were performed using MEGAscript® T7 Kit (Ambion, Inc., Austin, TX, USA) and GeneChip® IVT Labeling Kit (Affymetrix, Inc., Santa Clara, CA, USA), respectively. Following fragmentation, biotin-labeled cRNA samples were hybridized to Affymetrix bovine gene chip array. The microarray data normalization and background correction were performed using GCRMA, and the differentially expressed genes (DEG) (fold change >2,P < 0.05, FDR < 0.3) were identified using LIMMA written on R package integrated with Bioconductor. The result showed that in the calf-delivery group, there were 1867 DEG, among which 1015 and 852 were up- and down-regulated, respectively, in Day 7 compared with Day 14 of the estrous cycle. Some of those genes are believed to be involved in reproductive system development and function (F3, PTGER2, PTGER4, MFGE8, PTGS2, and TDGF1), embryonic development (ALDH1A1,ALDH1A3, FGF2, TGFBR2, PDGFB, and TGFBR2), and nervous system development and function (CYP3A4, CYP3A4, HSD17B4, FOXA2, MET, TDGF, WNT11). The bioinformatic analysis using KEGG revealed that those DEG were classified into several pathways including the MAPK signaling pathway. On the other hand, in the no-pregnancy group, 254 genes were found to be differentially expressed, of which 160 and 94 were up- and down-regulated, respectively, in Day 7 compared with Day 14 of the estrous cycle. Some of these genes were found to be involved in signal transducer activity (AXIN2, AGTR1, MAPK10, NTRK2, TLR2, DMBT1, IL1RN, CDK5, CHRNE), transferase activity (DGKI, TXNDC6, RPS6KA5, RIOK3, MYLK, CDK5, MET, NTRK2), receptor activity (MET,AGTR1, NTRK2, TLR2, DMBT1, CHRNE), regulation of transcription (FOS, ELF1, BHLHB2,ATF3, HOXA11), signal transduction (TLR2, AGTR1, FCNB, DGK, NOTCH2, ADAM9, PLEK), and transcription regulator activity (BHLHB2, FOS, ELF1,ATF3, HOXA11). Those DEG were found to be involved in different pathways including the focal adhesion pathway. In conclusion, the result of the current study revealed a remarkable transcriptome dynamics between Days 7 and 14 of the estrous cycle in cows resulted in calf delivery compared with those that did not support pregnancy.

scholarly journals Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 651-660 ◽  
Author(s):  
D Corcoran ◽  
T Fair ◽  
S Park ◽  
D Rizos ◽  
O V Patel ◽  
...  
Keyword(s):  
Expressed Sequence Tags ◽  
Differentially Expressed ◽  
Bovine Embryos ◽  
Quantitative Real Time Pcr ◽  
Expression Of Genes ◽  
Mrna Transcripts ◽  
Induced Changes ◽  
Expressed Sequence

In vivo-derived bovine embryos are of higher quality than those derivedin vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived fromin vitroversusin vivoculture. Microarray (BOTL5) comparison betweenin vivo- andin vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated inin vitrocultured blastocysts, showing a much reduced overall level of mRNA expression inin vitro- compared within vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according toPvalue) were verified in new pools ofin vivo- andin vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason whyin vitro-derived embryos are of inferior quality compared within vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.

Gene Expression Profiling in AML and MDS Identifies Genes Located on 5q Which Are Consistently Lower Expressed in Cases with 5q Deletion as Compared to Cases with Normal Karyotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 549-549 ◽  
Author(s):  
Claudia Schoch ◽  
Alexander Kohlmann ◽  
Wolfgang Kern ◽  
Sylvia Merk ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Signal Transduction ◽  
Normal Karyotype ◽  
Differentially Expressed ◽  
Regulation Of Transcription ◽  
Chromosome 5 ◽  
Expression Of Genes ◽  
Lower Expression ◽  
5Q Deletion

Abstract Deletions of the long arm of chromosome 5 occur either as the sole karyotype abnormality in MDS and AML or as part of a complex aberrant karyotype. It was the aim of this study to analyze the impact of the 5q deletion on the expression levels of genes located on chromosome 5q in AML and MDS. Therefore, gene expression analysis was performed in 344 AML and MDS cases using Affymetrix U133A+B oligonucleotide microarrays. The following subgroups were analyzed: AML with sole 5q deletion (n=7), AML with complex aberrant karyotype (n=83), MDS with sole 5q deletion (n=9), and MDS with complex aberrant karyotype (n=9). These were compared to 200 AML and 36 MDS with normal karyotype. In total, 1313 probe sets representing 603 genes cover sequences located on the long arm of chromosome 5. Overall a significant lower mean expression of all genes located on the long arm of chromosome 5 was observed in subgroups with 5q deletion in comparison to their respective control groups (for all comparisons, p&lt;0.05). 36 genes showed a significantly lower expression in all comparisons. These genes are involved in a variety of different biological processes such as signal transduction (CSNK1A1, DAMS), cell cycle regulation (HDAC3, PFDN1) and regulation of transcription (CNOT8). In addition we performed class prediction using support vector machines (SVM). In one approach all 6 different subgroups were analyzed as one class each. While AML and MDS with normal karyotype as well as AML with complex aberrant karyotype were correctly predicted with high accuracies (97%, 81%, and 92%, respectively) AML and MDS with 5q- sole and MDS with complex aberrant karyotype were frequently misclassified as AML with complex aberrant karyotype. In a second approach only two classes were defined: all cases with 5q deletion combined vs. all cases without 5q deletion. 102 out of 108 cases (94%) with 5q deletion were identified correctly supporting the fact that a distinct gene expression pattern is associated with 5q deletion in general. Performing SVM only with genes located on the long arm of chromosome 5 also resulted in a correct prediction of 92 of 108 (85%) stressing the importance of the expression of genes located on chromosome 5 for these AML and MDS subtypes. The top 100 differentially expressed probe sets between cases with and without 5q deletion represented 74 different annotated genes of which 23 are located on the long arm of chromosome 5. They are involved in a variety of different biological functions such as DNA repair (POLE, RAD21, RAD23B), regulation of transcription (ZNF75A, AF020591, MLLT3, HOXB6), protein biosynthesis (UPF2, TINP1, RPL12, RPL14, RPL15) cell cycle control (GMNN, CSPG6, PFDN1) and signal transduction (HINT1, STK24, APP, CAMLG). 10 of the top 74 genes associated with 5q deletion were involved in the CMYC-pathway with upregulation of RAD21, RAD23B, GMMN, CSPG6, APP, POLE STK24 and STAG2, and downregulation of ACTA2, and RPL12. Ten other genes out of the 74 top differentially expressed genes were involved in the TP53 pathway with upregulation of H1F0, PTPN11 and TAF2 and downregulation of DF, UBE2D2, EEF1A1, IGBP1, PPP2CA, EIF2S3, and NACA. In conclusion, loss of parts of the long arm of chromosome 5 leads to a lower expression of genes located on the long arm of chromosome 5. A specific pattern of functionally related genes was identified which shows a lower expression in AML and MDS subtypes with 5q deletion.

scholarly journals Global Transcriptome Profiling Identifies a Key Mir-185-PAK6 Axis That Promotes Survival of Leukemic Stem Cells and Drug-Insensitive Blasts in BCR-ABL+ Human Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 931-931
Author(s):  
Andrew Wu ◽  
Lin Hanyang ◽  
Katharina Rothe ◽  
Min Chen ◽  
Jens Ruschmann ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Transcriptome Profiling ◽  
Differentially Expressed ◽  
Leukemic Stem Cells ◽  
Cd34 Cells ◽  
Differentially Expressed Mirnas ◽  
Resistant Cells

Abstract Chronic myeloid leukemia (CML) stem/progenitor cells and BCR-ABL+ acute lymphoblastic leukemia (ALL) blast cells are insensitive to tyrosine kinase inhibitor (TKI) monotherapies. These cells rapidly generate therapy-resistant clones in vitro and in vivo and are often responsible for disease relapse. Therefore, identification of predictive biomarkers and novel treatments that target key molecular events active in leukemic stem cells (LSCs) are needed. MicroRNAs (miRNAs) are small molecules that regulate the gene expression network and are highly deregulated in many cancers. Through global transcriptome profiling, we have recently identified 66 differentially expressed miRNAs in pre-treatment CD34+ stem/progenitor cells from CML patients (n=6) compared to healthy bone marrow (NBM) controls (n=3, adjusted P<0.05); 26 differentially expressed miRNAs were identified between subsequent IM-nonresponders and IM-responders (P<0.05). 21 differentially expressed miRNAs were successfully validated in additional IM-responders (n=11), IM-nonresponders (n=11) and NBM (n=11). Interestingly, miR-185 was discovered to be one of the most highly deregulated miRNAs, with significant reduction in CD34+ cells from IM-nonresponders compared to IM-responders (p=0.0006). This significant change was further demonstrated in CD34+ cells from CML patients (n=60) before and after 3-month TKI nilotinib treatment in a clinical trial (p<0.05). We further demonstrated that miR-185 functions as a tumor suppressor; its restored expression by lentiviral transduction in CD34+ IM-nonresponder cells significantly impaired survival of these cells and sensitized them to TKI treatment in vitro. Restored miR-185 expression in BCR-ABL+ ALL blasts led to a profound decrease in leukemia burden and significantly enhanced survival compared to controls in vivo (median survival 65 vs. 47 days, P=0.0005). Strikingly, mice injected with miR-185-transduced cells and treated with dasatinib (DA) survived much longer than recipients of control cells treated with DA (median survival 83 vs. 60 days, P=0.0018). Moreover, restoration of miR-185 expression combined with DA treatment greatly reduced in vivo long-term regenerative activity of LSCs from IM-nonresponders as compared to control cells treated with DA in NRG mice (<0.2% vs. 5% GFP+ patient cells in the BM, 25 weeks post-transplantation). We observed not only a marked reduction in GFP+CD34+ cells, but also a near elimination of GFP+CD34+CD38- LSCs that were transduced with miR-185 and treated with DA compared to control cells treated with DA, indicating that restored miR-185 expression combined with DA preferentially prevents the growth of patient-derived long-term leukemia-initiating cells in vivo. Several miRNA target genes were further identified by integrating miRNA expression profiles with gene expression profiles from the same patient samples using strand-specific RNA-seq. Based on three out of six prediction algorithms (mirBase, TargetScan, miRanda, tarBase, mirTarget2, and PicTar), PAK6, a serine/threonine-protein kinase, was found to be highly expressed in CD34+ IM-nonresponder cells compared to IM-responders (p<0.003), which correlated with reduced expression of miR-185 in these cells (p=0.0002). PAK6 was confirmed as a target gene of miR-185 by a luciferase reporter assay. Western blot analysis showed that restored miR-185 expression caused a marked decrease in protein levels of PAK6 in miR-185-transduced cells and suppression of PAK6 reduced viability of these cells. These results indicate that PAK6 is a critical target of miR-185, and that loss of miR-185 expression in CML may lead to up-regulation of PAK6, which in turn contributes to disease progression and drug resistance. Indeed, the use of a pre-clinically validated pan PAK inhibitor (PF-3758309) significantly inhibited the growth of IM-resistant cells and CD34+ IM-nonresponder cells and these effects could be enhanced by TKIs (p<0.05). Mechanistically, we observed that p-ERK and p-AKT were significantly reduced in PAK6 knockdown or miR185-restored IM-resistant cells in response to IM treatment. Thus, we infer that downregulation of PAK6 may sensitize TKI-resistant cells to TKI therapy through inhibition of the RAS/MAPK pathway. Taken together, PAK6, a novel target of miR-185, emerges as an attractive druggable target for combination therapy of TKI-resistant patients. Disclosures No relevant conflicts of interest to declare.

196 EMBRYO BIOPSY TRANSCRIPTOMICS: A POTENTIAL TOOL TO IDENTIFY TRANSCRIPTS DIRECTLY RELATED TO THE ABILITY OF THE EMBRYO TO INDUCE PREGNANCY AFTER TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 196
Author(s):  
D. Tesfaye ◽  
N. Ghanem ◽  
F. Rings ◽  
E. Tholen ◽  
C. Phatsara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pregnancy Outcome ◽  
Expression Profile ◽  
Expression Profiles ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Embryo Biopsy ◽  
Bovine Embryo

The incidence of pregnancy loss due to embryonic mortality in cattle is one of the major causes of reproductive failure. The early embryonic loss can be due to problems with the embryo itself, the uterine environment, or interactions between the embryo and the uterus. So, this study was conducted to investigate the gene expression profile of bovine embryo biopsies produced in vivo and in vitro that resulted in different pregnancy outcomes. For this, biopsies representing 30 to 40% of the intact in vitro and in vivo blastocysts were taken, and 60 to 70% part was allowed to re-expand prior to transfer to recipients. Based on the pregnancy outcome after transfer, biopsies (n = 10 per pool) were grouped into 3 distinct phenotypes: those that resulted in no pregnancy, those that resulted in resorption, and those that resulted in successful pregnancy and subsequent calf delivery. A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of 3 replicates from each embryo biopsy group. Array data analysis revealed a total of 50 and 52 genes to be differentially expressed between biopsies derived from in vivo blastocysts that resulted in no pregnancy v. calf delivery and resorption v. calf delivery, respectively. Similarly, a total of 52 and 58 transcripts were differentially expressed between biopsies derived from in vitro-produced blastocysts that resulted in no pregnancy v. calf delivery and resorption v. calf delivery, respectively. Quantitative real-time PCR has confirmed the expression profile of 6 selected candidate genes. A distinct set of genes were found to be commonly expressed between in vitro- and in vivo-derived blastocyst biopsies, which ended up with the same pregnancy outcome. Biopsies, which ended up with calf delivery, were found to be enriched with transcripts involved in nucleosome assembly (KRT8), translation (RPLPO), electron transport (COX-2), and placenta specific (PLAC8). On the other hand, transcripts regulating immune response (TNFa), response to stress (HSPD1), and cell adhesion (CD9) were up-regulated in embryos that resulted in no pregnancy or resorption. Differences in transcript abundance of some genes have been seen between biopsies derived from in vitro and in vivo blastocysts. Biopsies from in vivo-derived blastocysts and that ended up with resorption were found to be enriched with transcripts regulating calcium-binding protein (S100A10, S100A14). Transcription factor-related transcripts (CDX2, HOXB7) were up-regulated in vitro-derived blastocyst biopsies that resulted in no pregnancy. In conclusion, the results evidenced that embryos derived from either in vitro or in vivo have more similarities than differences in their transcript abundance with respect to the ability in initiating pregnancy.

scholarly journals Transcriptome profiling and comparison of Rhinanthus major and Rhinanthus minor reciprocal F1 hybrids during seed stratification and germination

2021 ◽  
Author(s):  
Khaled Mirzaei ◽  
Renate A. Wesselingh
Keyword(s):  
Transcriptome Profiling ◽  
Differentially Expressed ◽  
F1 Hybrids ◽  
Rna Seq ◽  
Seed Formation ◽  
Maternal Parent ◽  
Expression Of Genes ◽  
Fitness Consequences ◽  
Hybrid Seeds ◽  
Rhinanthus Minor

Germination is a vital stage in a plants life cycle, and a different germination behavior of offspring in comparison to their parents can have fitness consequences. In studies on hybridization between Rhinanthus minor and R. major, low germination rates of F1 hybrids with R. major as the maternal parent have often been reported. In contrast, the F1m hybrid, with R. minor as the maternal parent, germinates readily and rapidly. In order to find the cause of this difference, we used RNA-Seq to obtain transcriptome profiles of F1a and F1m seeds during stratification at 4C and just after germination, after 40 days of stratification for the F1m seeds and 60 days for the F1a seeds. A comparison of the transcriptome of F1a seeds that had just germinated (60 days) with non-germinated F1a seeds after 40 and 60 days revealed 2918 and 1349 differentially expressed (DE) genes, respectively. For F1m seeds, 958 genes showed differential expression in germinated and non-germinated seeds after 40 days. The DE genes of F1a and F1m hybrids clustered into two separate groups, even though they had the same parents, and no differentially expression was found for plastid genes. Non-germinated F1a seeds had an abundance of enzymes and proteins associated with peroxidase activity, peroxiredoxin activity and nutrient reservoir activity. Expression of genes related to seed germination and seed development increased in non-germinated F1a hybrid seeds between 40 and 60 days of cold stratification. F1a seeds that had germinated showed an upregulation of genes related to the gibberellic acid-mediated signaling pathway and response to gibberellin, along with a low expression of DELLA superfamily. Although the results demonstrated strong differences in gene expression during stratification between the reciprocal hybrids, we could not identify its cause, since no plastid genes were differentially expressed. It is possible that differences in embryo development after seed formation and before stratification play a role, including epigenetic imprinting.

Dual role of EZH2 on megakaryocyte differentiation

Blood ◽  
2021 ◽  
Author(s):  
Stefania Mazzi ◽  
Philippe Dessen ◽  
Mathieu Vieira ◽  
Virginie Dufour ◽  
Marie Cambot ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Transcriptome Profiling ◽  
Loss Of Function ◽  
Expression Of Genes ◽  
Key Factor ◽  
Dna Replication And Repair ◽  
Proplatelet Formation

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss of function mutations have been found in myeloproliferative neoplasms, more particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and shRNA induces MK commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation, polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet level in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair, and an upregulation of CDK inhibitors, more particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and at a lesser extend of CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 ChIP assays revealed that only CDKN1A is a direct EZH2 target while CDKN2D expression is not directly regulated by EZH2 suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D.

scholarly journals Expression of microRNA during bovine adipogenesis

2010 ◽  
Vol 1 (1) ◽  
pp. 12 ◽  
Author(s):  
Scott L. Pratt ◽  
T. Ashley Burns ◽  
Erin Curry ◽  
Susan K. Duckett
Keyword(s):  
Messenger Rna ◽  
Differentially Expressed ◽  
Transcriptional Level ◽  
Messenger Rnas ◽  
Phosphatase And Tensin Homolog ◽  
Tensin Homolog ◽  
Expression Of Genes ◽  
Bovine Adipocytes

Studies have recently indicated that the adipogenic process and the expression of genes involved in lipid metabolism may be regulated in part at the post-transcriptional level by a class of small RNA called microRNA (miRNA). The objectives of this study were to i) determine if miRNAs are differentially expressed, and ii) evaluate expression of known miRNA targets in bovine adipocytes. Bovine adipose samples were collected from castrated males fattened on a high concentrate diet (C) or pasture (PA) and were frozen in liquid nitrogen and stored at -80°C, or used to generate primary stromal-vascular cells (SV). SV cells were cultured to confluence (Control) or differentiated at confluence and harvested 2 (D2), 6 (D6), or 12 (D12) days post-confluence. A 3x3 microarray analysis was performed comparing Control and differentiated samples. miR-21, -221, and -222 (P less than 0.05) were differentially expressed. qRT-PCR was conducted using the<em> in vitro</em> samples, and all three miRNAs were down regulated on D2 (P less than 0.05). miR-221 and -222 were decreased on D6 compared to Control (P less than 0.05), but only miR-222 expression was decreased at D12 (P less than 0.05) compared to Control. miR-21 increased in expression compared to Control on D12 (P less than 0.05). <em>In vivo</em>, only miR-21 expression was affected and it was reduced in PA compared to C fat samples (P less than 0.05). Two targets of miR-21 are Programmed Cell Death Protein 4 (PDCD4) and Phosphatase and Tensin Homolog (PTEN), and neither messenger RNA was differentially expressed<em> in vitro</em> (P greater than 0.05), but both messenger RNAs were elevated for PA compared to C (P less than 0.05). These data show that miRNAs are differentially expressed in adipose cells and tissue, and that miR-21 may be involved in adipocyte function by regulating the translation of PDCD4 and PTEN.

scholarly journals 216 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN BOVINE EMBRYOS CULTURED IN VIVO OR IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 259 ◽  
Author(s):  
D. Corcoran ◽  
T. Fair ◽  
D. Rizos ◽  
G.W. Smith ◽  
P.M. Coussens ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Differentially Expressed Genes ◽  
Intracellular Signalling ◽  
Differentially Expressed ◽  
Regulation Of Transcription ◽  
Gene Transcript ◽  
Bovine Embryo ◽  
Total Rna

The post-fertilization culture environment of the bovine embryo is known to influence the quality of the resulting blastocyst, manifested in terms of morphology, cryotolerance, and the relative gene transcript abundance of several candidate genes. This may have consequences for the pregnancy rate following embryo transfer. The objective of the current study was to take a broader approach toward identifying differentially expressed genes in bovine blastocysts derived from either in vivo or in vitro culture. Presumptive zygotes, produced by in vitro maturation and fertilization, were randomly assigned to one of two groups and cultured for 6 days, either in vitro in SOF medium, or in vivo in the ewe oviduct following transfer by mid-ventral laparotomy. Blastocysts were recovered from both systems on Day 7 after insemination, snap frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from 50 blastocysts for each culture group from each of four replicates using the PicoPureTM RNA Isolation Kit (ARCTURUS, Mountain View, CA 94043, USA). The RiboAmpTM RNA Amplification Kit (ARCTURUS) was used to linearly amplify the mRNA fraction of total RNA using double-stranded cDNA as template in a T7 RNA polymerase-catalyzed amplification. Samples from both culture environments were differentially labelled using N-hydroxysuccinimide (NHS)-activated fluorescent Cy3 or Cy5 dyes (Amersham Pharmacia Ltd., Piscataway, NJ, USA) and were hybridized onto a cDNA microarray. Each microarray contained 3888 total spots, with 932 bovine EST clone inserts developed from a normalized bovine total leukocyte (BOTL) cDNA library and an additional 459 amplicons representing additional genes including cytokines, receptors, signal transduction molecules, transcription and growth factors, enzymes, cell cycle regulators, and cellular components. Data were normalized and an expression ratio calculated between the two groups. This was compared to 1 within each of the 4 replicates by Student's t-test. Microarray analysis identified 15 gene transcripts that were differentially expressed P ≤ 0.05) between blastocysts produced in vivo or in vitro. Among these, four genes involved in transcription (nuclear receptor co-repressor 1, zinc finger protein 22, CCR4-NOT transcription complex, DOT1) and two genes involved in intracellular signalling (proteasome 26S subunit non-ATPase 13 and guanine nucleotide binding protein) had a higher mRNA expression level in blastocysts produced in vivo compared to those produced in vitro. In addition, Connexin 43, a gene involved in gap junction formation, was down regulated following in vitro culture which is consistent with our previous studies. Among the genes up-regulated following in vitro culture were WNT2B wingless-type MMTV integration site, CD103 integrin, and tumor necrosis factor superfamily member 8. In conclusion, we have identified previously uncharacterized, differentially expressed genes involved in cell communication, intracellular signalling, and regulation of transcription in bovine blastocysts cultured in vivo or in vitro. This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78.

scholarly journals DRES-04. LONG NON-CODING RNAS IN GLIOBLASTOMA TUMOR RECURRENCE AND THERAPY RESISTANCE

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi72-vi72
Author(s):  
Christian Stackhouse ◽  
G Yancey Gillespie ◽  
Christopher Willey
Keyword(s):  
Overall Survival ◽  
Radiation Resistance ◽  
Dna Methyltransferase ◽  
Therapy Resistance ◽  
Differentially Expressed ◽  
Age At Diagnosis ◽  
Regulation Of Transcription ◽  
Specific Expression ◽  
Non Coding Rnas

Abstract Recurrence of therapy resistant Glioblastoma multiforme (GBM) is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We are modeling acquired radiation resistance using 8 radiation-sensitive GBM patient-derived xenolines (PDX) made resistant by six irradiation series (6x2Gy each) in vivo. In 4 resistance-induced PDX, MGMT (O6-methylguanine–DNA methyltransferase) protein expression increased over original isogenic PDX. Paradoxically, temozolomide screening of orthotopic radiation-resistant PDX with increased MGMT expression revealed increased, not decreased chemo-sensitivity. This suggests an unanticipated mechanism associating acquired radiation resistance and alkylating chemotherapy sensitivity. RNA-seq data of long non-coding RNAs (lncRNAs) has revealed associations with patient overall survival and age at diagnosis. Out of 24,076 lncRNAs, 5 are significantly differentially expressed with regard to patient overall survival and age at diagnosis. The functions of lnc-ZNF117-1, lnc-DCUN1D4-1, and LINC01397 are unknown, but they are enriched in brain over other tissues. Tissue specific expression and function are unknown for lnc-TBL1XR1-5, but it is highly expressed in HepG2 (hepatocellular carcinoma) as well as in non-functioning pituitary adenomas (NFPAs). The lnc-CDH17-1 transcript is also highly expressed in NFPAs. All 5 of these lncRNAs have complex secondary and tertiary structures, but their physiologic or pathologic functions are unknown. LncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. Deep sequencing of total RNA isolated from intracranial radiation–sensitive/–resistant PDX to relate transcriptional programs and therapy resistance is underway. These data will be paired with kinomic profiling of matched tumors to elucidate basal signaling modality changes between radiation–sensitive/–resistant tumors. Analysis of differentially expressed lncRNA will be used to predict lncRNA structure/function, elucidating druggable mechanisms mediated by lncRNAs.

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