scholarly journals P089 IFNγ-macrophages could mediate EMT in Crohn’s disease

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S189-S189
Author(s):  
J Marín-Aracil ◽  
M D Barrachina ◽  
C Bauset ◽  
J Cosin-Roger ◽  
S Coll ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Epithelial Mesenchymal Transition ◽  
Macrophage Phenotype ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Ht29 Cells ◽  
Mrna And Protein Expression ◽  
Fold Induction ◽  
Emt Markers

Abstract Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT). Methods The mRNA and protein expression of IFN in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (2 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. IFNγ-U937 were coculture with HT29 cells for 2 days and the expression of EMT markers in HT29 cells were analyzed by RT-PCR and WB. Results are expressed as mean±SEM (n≥5). Statistical analysis was performed by ANOVA + Newman-Keuls. Results The mRNA and protein expression of IFNγ were significantly higher in intestinal samples from B2 CD patients (11.4±1.6 fold induction and 3.5±0.3 pg/mg, respectively) and B3 CD patients (14.2±1.8 fold induction and 3.1±0.1 pg/mg, respectively) than in controls (1,0±0,1 fold induction and 0.8±0,1 pg/mg, respectively). U937 cells treated with IFNγ increased significantly the mRNA expression of CD16 (1.9±0.2* vs vehicle) and CD86 (1.6±0.1* vs vehicle). IFNγ-U937 cocultured with HT29 increased significantly the mRNA and protein expression of EMT markers in HT29 cells (Vimentin: 3.0±0.5* vs vehicle-HT29; αSMA: 24.4±8.3* vs vehicle-HT29; SNAIL1: 2.7±0.5* vs vehicle-HT29) respect IFNγ-HT29 cells (Vimentin: 0.6±0.01 vs vehicle-HT29; αSMA: 1.1±0.4 vs vehicle-HT29; SNAIL1: 0.7±0.06 vs vehicle-HT29). Conclusion IFNγ could be responsible for the increase in the number of CD86/CD16 macrophages in CD patients. A macrophage phenotype expressing CD86/CD16 may act as a source of EMT mediators in intestinal tissue from CD patients.

WNT2b Activates Epithelial-mesenchymal Transition Through FZD4: Relevance in Penetrating Crohn´s Disease

2019 ◽  
Vol 14 (2) ◽  
pp. 230-239 ◽  
Author(s):  
Dolores Ortiz-Masià ◽  
Pedro Salvador ◽  
Dulce C Macias-Ceja ◽  
Laura Gisbert-Ferrándiz ◽  
Juan V Esplugues ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Fistula Formation ◽  
Rt Pcr ◽  
Intestinal Tissue ◽  
Mesenchymal Transition ◽  
Ht29 Cells ◽  
Wnt Ligands ◽  
Emt Markers

Abstract Background and Aims Epithelial-mesenchymal transition [EMT] has been related to fibrosis and fistula formation, common complications associated with Crohn´s disease [CD]. The WNT signalling pathway mediates EMT, and specific WNT/FZD interactions have been related to the activation of this process in several diseases. We aim to analyse the relevance of EMT and WNT ligands and receptors in the penetrating behaviour of CD. Methods Intestinal surgical resections were obtained from control and CD patients with a stenotic or penetrating behaviour. Fibrosis was determined by the histological analysis of collagen deposition and EMT by confocal microscopy. The expression of WNT ligands, inhibitors, and FZD receptors was analysed by RT-PCR, WB, IH, and IF studies. The effects of WNT2b and the role of FZD4 in EMT were analysed in HT29 epithelial cells. Results Fibrosis and expression of EMT markers were detected in samples from CD patients irrespective of the clinical behaviour. However, an increased colocalisation of E-CADHERIN and VIMENTIN, an increased number of cells expressing WNT2b, and a higher expression of FZD4 and WNT2b/FZD4 interaction, were detected in intestinal tissue from the penetrating compared with the stenotic CD behaviour. WNT2b induced EMT in HT29 cells through FZD4 activation. Conclusions An increased EMT, associated with increased WNT2b/FZD4 interaction, was detected in intestinal tissue from CD patients with a penetrating behaviour. WNT2b, through FZD4 activation, induces EMT in vitro which points to a novel pharmacological target to prevent intestinal penetrating complications of CD.

215 SNAIL AND SLUG, MARKERS OF EPITHELIAL MESENCHYMAL TRANSITION, APPEARED TO BE ALTERED BY ALKYL-PHENOLS, BISPHENOL A AND NONYL-PHENOL, IN OVARIAN CANCER CELLS EXPRESSING ESTROGEN RECEPTORS

2015 ◽  
Vol 27 (1) ◽  
pp. 198 ◽  
Author(s):  
Y.-S. Kim ◽  
K.-C. Choi
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Epithelial Mesenchymal Transition ◽  
Ovarian Cancer Cells ◽  
Rt Pcr ◽  
Nonyl Phenol ◽  
Mesenchymal Transition ◽  
And Migration ◽  
Emt Markers ◽  
E Cadherin

The ovary is the important organ to produce oocytes. Any disorder will affect embryo production. Ovarian cancer is one of gynecologic cancers in women which can affect ovarian functions. Oestradiol (E2) may be involved in ovarian cell growth and epithelial-mesenchymal transition (EMT) for diverse functions. EMT is an important process in embryo development and tumour migration or progression. Bis-phenol A (BPA) and nonyl-phenol (NP) have an estrogenic property, which can be suspected as endocrine disrupting chemicals (EDC). In this study, it has been examined whether BPA and NP can cause EMT process and migration in BG-1 ovarian cancer cells. To confirm the effect of these EDCs, BG-1 ovarian cancer cells were cultured and treated with DMSO (0.1%), E2 (10–7 M), BPA (10–6 M) and NP (10–6 M) for 0, 6, and 24 h. The mRNAs were extracted to perform reverse-transcription (RT)-PCR and the changes in the mRNA expressions were analysed by ANOVA test. Following treatments with BPA and NP, alterations of EMT markers; that is, vimentin and E-cadherin, were examined at mRNA levels by RT-PCR. The levels of vimentin were up-regulated by E2, BPA, or NP in a time-dependent manner. In addition, transcriptional factors of EMT response, i.e. snail and slug, were enhanced by these treatments more than 2 times. BG-1 cells were exposed to these EDCs for 0, 24, and 48 h. Vimentin and snail proteins were induced by E2, BPA, or NP, while the expression of E-cadherin was decreased by them. To reveal that this EMT response is affected by oestrogen receptor (ER), the cells were treated with these EDCs in the presence of an ER antagonist, ICI 182 780 (10–6 M). Treatment with ICI 182 780 reversed EDC-induced alteration of these EMT markers, E-cadherin, vimentin, and snail. Since EMT response can cause metastasis, a scratch assay was performed to show migration caused by BPA or NP. BPA or E2 enhanced migratory capability of these BG-1 cells. Taken together, these results indicate that BPA and NP, potential EDC, may have an ability to influence ovarian cancer metastasis via regulating snail and slug genes in ER-positive ovarian cancers. In a future study, their effects in inducing EMT and migration will be tested in a xenograft mouse model.This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ009599), Rural Development Administration, Republic of Korea.

scholarly journals Combination of quercetin and 2-methoxyestradiol inhibits epithelial–mesenchymal transition in PC-3 cell line via Wnt signaling pathway

2021 ◽  
pp. FSO747
Author(s):  
Neeti Sharma ◽  
Piyush W Raut ◽  
Meghna M Baruah ◽  
Akshay Sharma
Keyword(s):  
Wnt Signaling ◽  
Epithelial Mesenchymal Transition ◽  
Wnt Signaling Pathway ◽  
Signaling Proteins ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Promising Therapeutic Approach ◽  
Signaling Components ◽  
Emt Markers

Aim: We have previously reported that quercetin (Qu) regulates epithelial–mesenchymal transition (EMT) by modulating Wnt signaling components. In this study, we investigated the synergistic effect of Qu and 2-methoxyestradiol (2-ME) and the role of Wnt signaling components in regulating EMT in PC-3 cells. Materials & methods: EMT was induced by treating PC-3 cells with TGF-β, followed by evaluation of expression of EMT markers and Wnt signaling proteins in naive, induced and after exposing induced cells to Qu and 2-ME at both gene and protein level by real-time PCR (RT-PCR) and western blot, respectively. Results: Qu and 2-ME synergistically downregulated mesenchymal markers with simultaneous upregulation of epithelial markers. Wnt signaling proteins expression was also downregulated by Qu and 2-ME in TGF-β-induced EMT in PC-3 cells. Conclusion: Thus, combination therapy of Qu and 2-ME could be a new promising therapeutic approach for the treatment of prostate cancer.

scholarly journals P017 C86/CD16 macrophages accumulate in the mucosa of B3 patients and could mediate EMT in Crohn’s disease

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S138-S138
Author(s):  
A Boronat-Muñoz ◽  
A Cejudo-Garces ◽  
P Lledo-Gil ◽  
J Cosín-Roger ◽  
S Coll ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Statistical Analysis ◽  
Crohn's Disease ◽  
Mrna Expression ◽  
U937 Cells ◽  
Macrophage Phenotype ◽  
Rt Pcr ◽  
Intestinal Tissue ◽  
Healthy Donors ◽  
Emt Markers

Abstract Background Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their phenotype. Methods The aim of the present study is to analyse the pattern of expression of macrophages, of EMT-related genes and cytokines in surgical resections from Crohn’s disease (CD, n = 43) patients which were categorised according to Montreal classification (B2 or B3); unaffected mucosa of patients with ileocecal cancer was used as control (n = 20). mRNA was isolated from intestinal samples and the expression of macrophage, EMT markers and cytokines were analysed by RT-PCR. PBMCS were isolated from healthy donors and treated during 5 days with secretomes, from control, B2 or B3 surgical resections; the mRNA expression of macrophage markers was determined by RT-PCR. U937 cells were differentiated to macrophages and then treated with IFNγ (20 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. Results are expressed as mean ± SEM (n ≥ 5). Statistical analysis was performed by ANOVA + Newman–Keuls or t-test. Correlations between data were analysed using Pearson’s correlation coefficient (*p < 0.05). Results The expression of CD16 and CD86 was significantly higher in intestinal samples from B3 CD patients (7.2 ± 1.1 and 7.7 ± 1.3, respectively) than in controls (1.4 ± 0.2 and 2.5 ± 0.4, respectively) or B2 CD patients (4.8 ± 0.9 and 4.5 ± 0.6, respectively). The mRNA expression of CD16 and CD86 were significantly higher in PBMCS treated with B3-secretomes than in those treated with B2- or control secretomes. The expression of CD16 and CD86 significantly correlated with FSP1 (r = 0.74, p = 0.002* and r = 0.66, p = 0.003*, respectively), VIMENTIN (r = 0.60, p = 0.02* and r = 0.82, p = 0.001*, respectively), SNAIL1 (r = 0.61, p < 0.01* r = 0.52, p = 0.04*, respectively), IL4 (r = 0.63, p = 0.01* and r = 0.60, p = 0.02*, respectively) and IFNγ (r = 0.56, p = 0.001* and r = 0.58, p = 0.01*, respectively) in intestinal tissue from the fistulising CD group. U937 cells treated with IFNγ increased significantly the mRNA expression of CD16 (1.94 ± 0.24* vs. vehicle) and CD86 (1.60 ± 0.17* vs. vehicle). Conclusion A macrophage phenotype expressing CD86/CD16 may act as a source of EMT mediators in intestinal tissue from CD patients with a penetrating (B3) behaviour. IFNγ could be responsible for the increase in the number of CD86/CD16 macrophages in the B3 behaviour.

MET overexpression as a hallmark of the epithelial-mesenchymal transition (EMT) phenotype in colorectal cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3529-3529 ◽  
Author(s):  
Kanwal Pratap Singh Raghav ◽  
Hesham M. Amin ◽  
Wenting Wang ◽  
Ganiraju C. Manyam ◽  
Bradley Broom ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Overall Survival ◽  
Protein Expression ◽  
Protein Gene ◽  
Epithelial Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Gene Signatures ◽  
Protein Levels ◽  
Emt Markers

3529 Background: Epithelial-mesenchymal transition (EMT) has been identified as a dominant molecular subtype of colorectal cancer (CRC). This EMT phenotype as recognized by complex gene signatures is prognostic and associated with chemoresistance, but a biomarker for EMT suitable for clinical utilization has not yet been validated. The purpose of this study was to compare MET protein expression with protein/gene expression of EMT markers and to evaluate its impact on overall survival (OS). Methods: We performed an exploratory analysis of 139 untreated primary CRC samples using data from The Cancer Genome Atlas. Protein and gene expressions were measured using reverse-phase protein array (RPPA) and RNA-sequencing, respectively. MET high/overexpressed group was defined by protein level in the highest quartile. Mann-Whitney U-test and Spearman rank correlation was used to determine association between MET protein expression and protein/gene expression of EMT markers and EMT gene signature scores. Regression tree method and Kaplan-Meier estimates were used to assess overall survival (OS). Results: The MET protein distribution is right skewed, demonstrating a unique population of MET high expressing tumors (P < 0.01). Colon tumors had higher MET protein levels compared to rectal tumors (P < 0.01). MET overexpression was associated with decreased OS (HR 2.92; 95% CI: 1.45 - 5.92). MET protein expression correlated strongly with protein expressions of SLUG (transcription factor for EMT) (r = 0.6) and ERCC1 (a marker for oxaliplatin chemo-resistance) (r = 0.6) (P < 0.01). Higher MET protein levels were associated with higher gene expression of 28 EMT markers including AXL, VIM, ZEB1, ZEB2, FGF1, TGFB1I1 and MMP11 (P < 0.05). Higher MET protein levels were also associated with higher gene scores derived from three published EMT gene signatures (P < 0.05). MET protein expression did not correlate with MET gene expression (r = 0.16). Conclusions: Increased MET protein expression strongly correlates with a molecular EMT phenotype and poor survival in patients with CRC. MET protein expression may be used as a surrogate biomarker to represent and select for this unique molecular subset of CRC driven by EMT biology.

scholarly journals A D-vitamin metabolizmusa humán hepatocellularis carcinomában és az azt körülvevő tumormentes májszövetben

2016 ◽  
Vol 157 (48) ◽  
pp. 1910-1918 ◽  
Author(s):  
Evelin Horváth ◽  
Bernadett Balla ◽  
János Kósa ◽  
Péter András Lakatos ◽  
Áron Lazáry ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Vitamin D ◽  
Protein Synthesis ◽  
Protein Expression ◽  
Mrna Expression ◽  
Gene Activation ◽  
Human Hepatocellular Carcinoma ◽  
Rt Pcr ◽  
Antitumor Effects ◽  
Mrna And Protein Expression

Introduction: 1,25-Dihydroxy vitamin D3 mediates antitumor effects in hepatocellular carcinoma. Aim: We examined mRNA and protein expression differences in 1,25-Dihydroxy vitamin D3-inactivating CYP24A1, mRNA of activating CYP27B1 enzymes, and that of VDR between human hepatocellular carcinoma and surrounding non-tumorous liver. Methods: Snap-frozen tissues from 13 patients were studied for mRNA and protein expression of CYP24A1. Paraffin-embedded tissues from 36 patients were used to study mRNA of VDR and CYP27B1. mRNA expression was measured by RT-PCR, CYP24A1 protein was detected by immunohistochemistry. Results: Expression of VDR and CYP27B1 was significantly lower in hepatocellular carcinoma compared with non-tumorous liver (p<0.05). The majority of the HCC samples expressed CYP24A1 mRNA, but neither of the non-tumorous liver. The gene activation was followed by CYP24A1 protein synthesis. Conclusions: The presence of CYP24A1 mRNA and the reduced expression of VDR and CYP27B1 mRNA in human hepatocellular carcinoma samples indicate decreased bioavailability of 1,25-Dihydroxy vitamin D3, providing an escape mechanism from the anti-tumor effect. Orv. Hetil., 2016, 157(48), 1910–1918.

scholarly journals LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4

2021 ◽  
Vol 14 (3) ◽  
pp. 349-355
Author(s):  
Li Zhou ◽  
◽  
Wen-Juan Chu ◽  
Ling-Ling Yang ◽  
Hai-Feng Xu ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Protein Expression ◽  
Epithelial Mesenchymal Transition ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Down Regulation ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Mrna And Protein Expression ◽  
Tgf Β1

AIM: To investigate the effect of leucine-rich-alpha-2-glycoprotein 1 (LRG1) on epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, and to explore the role of NADPH oxidase 4 (NOX4). METHODS: RPE cells (ARPE-19 cell line) were treated with transforming growth factor-β1 (TGF-β1) to induce EMT. Changes of the mRNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells. The recombinant human LRG1 protein (rLRG1) and siRNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively, and to detect EMT-related markers including fibronectin, α-smooth muscle actin (α-SMA) and zonula occludens-1 (ZO-1). The mRNA and protein expression level of NOX4 were measured according to the above treatments. VAS2870 was used as a NOX4 inhibitor in rLRG1-treated cells. EMT-related markers were detected to verify the effect of NOX4 in the process of EMT. RESULTS: TGF-β1 promoted the expression of LRG1 at both the mRNA and protein levels during the process of EMT which showed the up-regulation of fibronectin and α-SMA, as well as the down-regulation of ZO-1. Furthermore, the rLRG1 promoted EMT of ARPE-19 cells, which manifested high levels of fibronectin and α-SMA and low level of ZO-1, whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin and α-SMA and increasing the expression of ZO-1 in ARPE-19 cells. Besides, the rLRG1 activated and LRG1 siRNA suppressed NOX4 expression. EMT was inhibited when VAS2870 was used in the rLRG1-treated cells. CONCLUSION: These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4, which may provide a novel direction to explore the mechanisms of subretinal fibrosis.

scholarly journals miR-22 Suppresses Epithelial-Mesenchymal Transition by Modulating Snail and MAPK1 in Hepatocellular Carcinoma

2020 ◽  
Author(s):  
Cheng-Gong Liao ◽  
Zhi Qu ◽  
Zao-Xia Guo ◽  
Xiao-Hua Liang ◽  
Ling-Min Kong
Keyword(s):  
Real Time ◽  
Epithelial Mesenchymal Transition ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Normal Tissues ◽  
Mitogen Activated Protein ◽  
Emt Markers

Abstract Background: Recently studies have reported that miR-22 plays an important role in epithelial-mesenchymal transition (EMT) of many human cancers. However, the involvement of miR-22 in hepatocellular carcinomas (HCC) EMT progression has not been investigated. Methods: We measured miR-22 expression level in 38 paired of HCC and matched normal tissues by real-time quantitative RT-PCR. Then, we performed morphological analysis and immunofluorescence to observe the role of miR-22 in HCC EMT progression. The expression of EMT markers were detected by real-time RT-PCR and western blot. The regulation role of miR-22 on Snail, mitogen-activated protein kinase 1(MAPK1) and slug were determined by luciferase reporter assay. The expression of Snail and MAPK1 were also detected by real-time quantitative RT-PCR in HCC and normal tissues. Results: We found that the expression of miR-22 in HCC tissues were much lower than that in normal control. The expression of miR-22 was inversely correlated with HCC metastatic ability. Then, we found that overexpression of miR-22 could inhibit HCC EMT. Importantly, miR-22 is found to inhibit cell motility by directly targeting both Snail and MAPK1. Furthermore, the suppression role of miR-22 in HCC EMT could be blocked by Snail and MAPK1 overexpression. Additionally, the expression of Snail and MAPK1 were inversely correlated with miR-22 expression in HCC tissues. Conclusion: Our results suggested that miR-22 was downexpressed in HCC tissues and inhibited HCC EMT through downregulating Snail and MAPK1 which may provide a new bio-target for HCC therapy.

scholarly journals Long non-coding (lnc)RNA profiling and the role of a key regulator lnc-PNRC2-1 in the transforming growth factor-β1-induced epithelial–mesenchymal transition of CNE1 nasopharyngeal carcinoma cells

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199651
Author(s):  
Jie Yang ◽  
Enzi Feng ◽  
Yanxin Ren ◽  
Shun Qiu ◽  
Liufang Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Nasopharyngeal Carcinoma ◽  
Rna Sequencing ◽  
Western Blotting ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Carcinoma Cells ◽  
Mesenchymal Transition ◽  
Emt Markers ◽  
Tgf Β1

Objectives To identify key long non-coding (lnc)RNAs responsible for the epithelial–mesenchymal transition (EMT) of CNE1 nasopharyngeal carcinoma cells and to investigate possible regulatory mechanisms in EMT. Methods CNE1 cells were divided into transforming growth factor (TGF)-β1-induced EMT and control groups. The mRNA and protein expression of EMT markers was determined by real-time quantitative PCR and western blotting. Differentially expressed genes (DEGs) between the two groups were identified by RNA sequencing analysis, and DEG functions were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. EMT marker expression was re-evaluated by western blotting after knockdown of a selected lncRNA. Results TGF-β1-induced EMT was characterized by decreased E-cadherin and increased vimentin, N-cadherin, and Twist expression at both mRNA and protein levels. Sixty lncRNA genes were clustered in a heatmap, and mRNA expression of 14 dysregulated lncRNAs was consistent with RNA sequencing. Knockdown of lnc-PNRC2-1 increased expression of its antisense gene MYOM3 and reduced expression of EMT markers, resembling treatment with the TGF-β1 receptor inhibitor LY2109761. Conclusion Various lncRNAs participated indirectly in the TGF-β1-induced EMT of CNE1 cells. Lnc-PNRC2-1 may be a key regulator of this and is a potential target to alleviate CNE1 cell EMT.

scholarly journals Cloning and Expression Analysis of Two Kdm Lysine Demethylases in the Testes of Mature Yaks and Their Sterile Hybrids

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 521
Author(s):  
Zhenhua Shen ◽  
Lin Huang ◽  
Suyu Jin ◽  
Yucai Zheng
Keyword(s):  
Amino Acids ◽  
Protein Expression ◽  
Male Sterility ◽  
Histone Methylation ◽  
Real Time Pcr ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Coding Sequences ◽  
Mrna And Protein Expression ◽  
Sterile Hybrids

The objective of this study was to explore the molecular mechanism for male sterility of yak hybrids based on two demethylases. Total RNA was extracted from the testes of adult yaks (n = 10) and yak hybrids (cattle–yaks, n = 10). The coding sequences (CDS) of two lysine demethylases (KDMs), KDM1A and KDM4B, were cloned by RT-PCR. The levels of KDM1A and KDM4B in yaks and cattle–yaks testes were detected using Real-time PCR and Western blotting for mRNA and protein, respectively. In addition, the histone methylation modifications of H3K36me3 and H3K27me3 were compared between testes of yaks and cattle–yaks using ELISA. The CDS of KDM1A and KDM4B were obtained from yak testes. The results showed that the CDS of KDM1A exhibited two variants: variant 1 has a CDS of 2622 bp, encoding 873 amino acids, while variant 2 has a CDS of 2562 bp, encoding 853 amino acids. The CDS of the KDM4B gene was 3351 bp in length, encoding 1116 amino acids. The mRNA and protein expression of KDM1A and KDM4B, as well as the level of H3K36me3, were dramatically decreased in the testes of cattle–yaks compared with yaks. The present results suggest that the male sterility of cattle–yaks might be associated with reduced histone methylation modifications.

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