scholarly journals Analysis of DNA methylation level and mRNA expression of Transient Receptor Ankyrin Member 1 (TRPA1) in endometriosis-associated pain

2021 ◽  
pp. 1-10
Author(s):  
Ocktariyana ◽  
Nurul Hikmawati ◽  
Andon Hestiantoro ◽  
Raden Muharam ◽  
Muhammad Luky Marwali ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Methylation Level ◽  
Rating Scale ◽  
Expression Level ◽  
Pain Level ◽  
Mrna Expression Level ◽  
Significant Difference ◽  
Peritoneal Endometriosis ◽  
Transient Receptor

Transient Receptor Ankyrin Member 1 (TRPA1) is an ion channel family protein that regulates pain sensation through sensory neurons' activity. This study's purpose to analyzes the DNA methylation and mRNA expression level of the TRPA1 gene in endometriosis and its correlation with pain level. Twenty samples of peritoneal endometriosis and endometrial samples were obtained from women with endometriosis, which was subsequently compared to 20 endometrial samples of women without endometriosis. The DNA methylation level of TRPA1 was analyzed using Methylation-specific PCR (MS-PCR) and ImageJ software, while the mRNA expression of TRPA1 was analyzed using qRT-PCR. Furthermore, the pain level was measured using the numeric rating scale (NRS) by interviewing all the women. This study showed that there was a significant difference in the mRNA expression of TRPA1 in peritoneal endometriosis. The TRPA1 was unmethylated in both peritoneal and endometrial samples in endometriosis. However, DNA Methylation level of TRPA1 in peritoneal and endometrial of endometriosis compared to normal endometrial were no significant difference. Additionally, there was no correlation between DNA methylation level and mRNA expression level of TRPA1 in all samples, along with the endometriosis-associated pain.

scholarly journals Analysis of mRNA expression and DNA methylation level of RAC1 gene encoding focal adhesion molecule in endometrial and peritoneal endometriosis

2020 ◽  
pp. 43-49
Author(s):  
Irwina Eka Deraya ◽  
Andon Hestiantoro ◽  
Raden Muharam ◽  
Muhammad Lucky Marwali ◽  
Agus Surur As'adi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Focal Adhesion ◽  
Methylation Level ◽  
Promoter Hypermethylation ◽  
Mrna Expression Level ◽  
Normal Endometrium ◽  
Significant Difference ◽  
Its Gene ◽  
Peritoneal Endometriosis

Focal adhesion molecules involve in cellular migration, attachment, and play a role in endometriosis pathomechanisms. Recent studies showed that the expression of RAC1, a gene encoded focal adhesion molecule, was predominantly found in endometriosis. As gene expression may be regulated by DNA methylation. Therefore, this study aimed to analyze promoter methylation level of RAC1 gene and mRNA expression in endometrial and peritoneal endometriosis tissues. This study using 20 endometrial and 9 peritoneal tissues from the same patients and 20 normal endometrial. The DNA and RNA from samples were isolated, DNA was converted using sodium bisulfite and amplified using Methyl Specific Polymerase Chain Reaction (MSP) method. The methylation level was determined by the intensity measurement of the bands that arose in gel electrophoresis using ImageJ software, whereas mRNA expression level was measured by Reverse Transcription-quantitative PCR (RT-qPCR) method. The mRNA expression level of RAC1 gene in peritoneal endometriosis increased compared to normal endometrium, as well as compared to endometrial endometriosis, but there was no significant difference in endometrial endometriosis compared to normal. Promoter hypermethylation level of RAC1 gene in peritoneal endometriosis was significantly different compared to normal endometrium, however not significant to endometrial endometriosis. Methylation level of its gene in endometrial endometriosis shown no significant difference compared to normal. There was association between promoter hypermethylation level and its mRNA expression in endometrial endometriosis (R= 0.014; p=0.952). The elevation of mRNA expression of RAC1 gene plays a role in endometrial cell migration to peritoneum, and associated with promoter hypermethylation level of its gene.

scholarly journals PSIV-B-38 Late-Breaking: Effect of Glutamate (Glu) on Developmental Block and the MZT Relative Genes Expression in Mouse embryo during in Vitro Culture

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 330-331
Author(s):  
Yu Liu ◽  
An Gang Lou ◽  
Shuo Yang ◽  
Zhong Shu Li ◽  
Nan-Zhu Fang
Keyword(s):  
In Vitro Culture ◽  
Mrna Expression ◽  
Marker Gene ◽  
Treated Group ◽  
Mouse Embryos ◽  
Expression Level ◽  
Mrna Expression Level ◽  
Significant Difference ◽  
Developmental Block

Abstract The risk of developmental block in mammal’s embryos is high during in vitro as compare to in vivo environment because the in vitro embryo-culture systems are suboptimal. During in vitro-culture the balance between ROS production and elimination is disturbed and may lead to 2-cell block in mouse embryos [1]. In the current study, we investigated the effects of Glu as anti-developmental block during IVC on ZGA and MZT on mouse embryos. The mouse embryos were divided into control and different level of Glu treated group. The cleavage rate was determined, the ROS and GSH level was investigated using DCHF-DA and CMF2HC respectively. The mRNA expression level of ZGA marker gene such as Eif-1α, Muerv l, Zscan4d and Hsp70.1 was analyzed among the groups using RT-PCR. The transition rate from 2-cell to 4-cell was significantly higher in 6mmol/L Glu treated group as compare to control and others treated groups. No significant difference was recorded in the level of ROS and GSH during MZT stage among the different groups. The mRNA expression level of ZGA marker gene was significantly increased at middle and late stage in 6mmol/L Glu treated group as compare to control and others treated groups. In conclusion, this study shows that the concentration of 6mmol/L Glu could maintain the dynamic balance of GSH and ROS, increase the expression of ZGA marker gene and maintain its high expression pattern of time series, directly participate in the ZGA activated process; ultimately reduce the risk of developmental block to ensure the successful completion of MZT. Reference [1] Lee MT, Bonneau AR, Giraldez AJ.Zygotic Genome Activation during the Maternal-to-Zygotic Transition. Annual Rev Cell Dev Biol [J], 2014, 30:581–613.

195 EFFECTS OF GSK3 INHIBITOR ON THE PLURIPOTENCY MAINTENANCE OF BUFFALO EMBRYONIC STEM-CELL-LIKE CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 212 ◽  
Author(s):  
F. Lu ◽  
Y. Lao ◽  
H. Sun ◽  
C. Lei ◽  
Y. Deng ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Mrna Expression ◽  
Colony Formation ◽  
Embryonic Stem ◽  
Signalling Pathway ◽  
Expression Level ◽  
Control Group ◽  
Mrna Expression Level ◽  
Significant Difference

In this study, to explore the effects and mechanism of Wnt/β-catenin signalling pathway on the maintenance of pluripotency of buffalo embryonic stem-cell-like cells (buffalo ESC-like cells), the GSK3 inhibitors BIO and CHIR99021 were added throughout the experiment – i.e. from buffalo inner cell mass (ICM) culture to ESC-like line generation. The buffalo ICM were respectively cultured in the medium containing 0.5 μg mL–1 BIO and 5 mmol L–1 CHIR99021. The percentage of ICMs attachment and primary colony formation were observed, and found that there was no significant difference in the ICMs attachment rate among of the BIO, CHIR99021, and the control groups (91.18% and 92.98% v. 94.59%; P > 0.05). Treating ICMs with CHIR99021 resulted in more primary colony formation rate compared with the control group (77.71% v. 55.41%; P < 0.05). The proliferation rate of primary colonies of buffalo ESC-like cells was detected by bromodeoxyuridine immunofluorescence techniques. The results show that the proliferation rate of primary colonies in the group of buffalo ESC-like cells treated with CHIR99021 was significantly higher than that of the control group on Day 1, Day 3, Day 4, and Day 5 (P < 0.05), and it was also evidently higher than that of control group only on Day 1 (P < 0.05) in the group of BIO, but there was no significant difference in other days (P > 0.05). The mRNA expression level of proliferation marker PCNA of ESC-like cells was significantly up-regulated in both CHIR99021 and BIO treatment groups (P < 0.05), however, treating buffalo ESC-like cells with CHIR99021 significantly up-regulated the expression of pluripotent gene Oct4 and Sox2 (P < 0.05), but had no effect on pluripotent gene Nanog expression (P > 0.05). Oct4 expression was significantly increased (P < 0.05), and the expression of Sox2 and Nanog were significantly decreased (P < 0.05) in the group of BIO treatment. Furthermore, the relative protein level of β-catenin (the downstream effector of Wnt/β-catenin signalling pathway) and the mRNA expression level of c-Myc (the downstream target gene of Wnt/β-catenin signalling pathway) were significantly increased when buffalo ESC-like cells respectively treated with CHIR99021 and BIO (P < 0.05). In conclusion, treating buffalo ESC-like cells with GSK3 inhibitors CHIR99021 can promote proliferation of buffalo ESC-like cells, maintain their undifferentiated state, and up-regulate the expression levels of β-Catenin and c-Myc in buffalo ESC-like cells. These results indicate that Wnt/β-catenin signalling pathway plays an important role in regulation of self-renewal of buffalo ESC-like cells. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).

scholarly journals Thymine DNA Glycosylase Gene Knockdown Can Affect the Differentiation of Pig Preadipocytes

2016 ◽  
Vol 39 (3) ◽  
pp. 975-984 ◽  
Author(s):  
Lian-Jiang Zhang ◽  
Si-Yuan Liu ◽  
Ya-Nan Zhu ◽  
Yan Gao ◽  
Jian Chen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mrna Expression ◽  
Gene Knockdown ◽  
Expression Level ◽  
Dna Glycosylase ◽  
Peroxisome Proliferator ◽  
Mrna Expression Level ◽  
Promoter Regions ◽  
Fatty Acid Binding ◽  
Thymine Dna Glycosylase

Aims: To study the effect of thymine DNA glycosylase (TDG) gene knockdown on the differentiation of pig preadipocytes. Methods: Preadipocytes were obtained from subcutaneous adipose tissue from the neck of 1- to 7-day-old pigs. The TDG gene was knocked down using siRNA, and cell differentiation was induced. The mRNA expression level was measured using fluorescence quantitative PCR, and the protein expression level was determined using Western blot analysis. The DNA methylation levels in promoter regions of differentiation-related genes were also evaluated. Results: TDG gene knockdown decreased the mRNA expression levels of the peroxisome proliferator-activated receptorγ (PPARγ) and Fatty acid binding proteins 4(FABP4 Also known as aP2) genes (P<0.01), while the mRNA expression level of the CCAAT/enhancer binding protein alpha(C/EBPα) gene did not change significantly (P>0.05). In addition, after induced differentiation, the lipid droplet production significantly decreased, and the percentages of methylation in the promoter regions of C/EBPα, PPARγ, and aP2 genes were 0.9%, 80%, and 76%, respectively. In contrast, the percentages of methylation in the negative control groups were 0.5%, 67.5%, and 58%, respectively. Conclusion: TDG gene knockdown could inhibit the differentiation of pig preadipocytes and affect the DNA methylation levels of some transcription factors.

scholarly journals Role of FRG1 in predicting the overall survivability in cancers using multivariate based optimal model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rehan Khan ◽  
Ananya Palo ◽  
Manjusha Dixit
Keyword(s):  
Mrna Expression ◽  
Cancer Patients ◽  
Risk Score ◽  
Cox Regression ◽  
Expression Level ◽  
Risk Scores ◽  
Mrna Expression Level ◽  
Liver Cancers ◽  
Significant Difference

AbstractFRG1 has a role in tumorigenesis and angiogenesis. Our preliminary analysis showed that FRG1 mRNA expression is associated with overall survival (OS) in certain cancers, but the effect varies. In cervix and gastric cancers, we found a clear difference in the OS between the low and high FRG1 mRNA expression groups, but the difference was not prominent in breast, lung, and liver cancers. We hypothesized that FRG1 expression level could affect the functionality of the correlated genes or vice versa, which might mask the effect of a single gene on the OS analysis in cancer patients. We used the multivariate Cox regression, risk score, and Kaplan Meier analyses to determine OS in a multigene model. STRING, Cytoscape, HIPPIE, Gene Ontology, and DAVID (KEGG) were used to deduce FRG1 associated pathways. In breast, lung, and liver cancers, we found a distinct difference in the OS between the low and high FRG1 mRNA expression groups in the multigene model, suggesting an independent role of FRG1 in survival. Risk scores were calculated based upon regression coefficients in the multigene model. Low and high-risk score groups showed a significant difference in the FRG1 mRNA expression level and OS. HPF1, RPL34, and EXOSC9 were the most common genes present in FRG1 associated pathways across the cancer types. Validation of the effect of FRG1 mRNA expression level on these genes by qRT-PCR supports that FRG1 might be an upstream regulator of their expression. These genes may have multiple regulators, which also affect their expression, leading to the masking effect in the survival analysis. In conclusion, our study highlights the role of FRG1 in the survivability of cancer patients in tissue-specific manner and the use of multigene models in prognosis.

scholarly journals VDR Pathway-Specific Dose-Related Effects of 1,25(OH)2D3 on Salmonella typhimurium-induced Mouse Colitis

2020 ◽  
Author(s):  
Fubin Qiu ◽  
Lijuan Zhang ◽  
Chunyan Li ◽  
Ying Zhang ◽  
Xiaoyan Zhang
Keyword(s):  
Mrna Expression ◽  
Current Data ◽  
Expression Level ◽  
Control Group ◽  
High Dose ◽  
Mrna Expression Level ◽  
Significant Difference ◽  
Dose Limits ◽  
Group A ◽  
Cervical Dislocation

Abstract Background: Whether and how 1,25(OH)2D3 supplementation influences VDRs in experimental mice with colitis remains to be seen. To explore the effect of 1,25(OH)2D3 on S. typhimurium colitis through the VDR pathway and to discover the role of VDR in its action. Methods: We established a mouse UC model induced by S. typhimurium. After streptococcal typhus infection, the mice were fasted for 12 hours. Blood was collected by the eyeball extraction method, and then sacrificed by cervical dislocation, specimens were collected for corresponding indicators. Results: Mice exposed to S. typhimurium infection developed signs of acute colitis. After HE staining were performed on the diseased colons from the mice. high dose VD supplementation, the pathological colonic damage did not improve in the mice, and there was no statistical difference between the groups with VD deficiency (P>0.05). VDR expression in the UC group treated with Salmonella was higher than that in the control group, a statistically significant difference (P<0.01). Compared with the VDD+UC group, VDR expression rose in both the LVDS+UC group and the HVDS+UC group, with VDR protein expression being highest after high dose VD supplementation (P<0.01). Compared with the control and the UC groups, the VDR mRNA expression level in the VDD+UC group was significantly higher, and the colon VDR mRNA expression level decreased after active VD supplementation (Fig.7C). Conclusions: Our data suggest the need for defining the accurate 1,25(OH)2D3 dose limits that induce an anti-inflammatory effect as current data indicate that higher doses would produce an inflammatory response.

ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders

2020 ◽  
Vol 20 (18) ◽  
pp. 2274-2284
Author(s):  
Faroogh Marofi ◽  
Jalal Choupani ◽  
Saeed Solali ◽  
Ghasem Vahedi ◽  
Ali Hassanzadeh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zoledronic Acid ◽  
Mrna Expression ◽  
Promoter Methylation ◽  
Methylation Level ◽  
Target Genes ◽  
Osteoblastic Differentiation ◽  
Therapeutic Effects ◽  
Significant Difference ◽  
Scheduled Time

Objective: Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation (OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood. It seems that the methylation of the promoter region of key genes might be an important factor involved in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment with ZA. Materials and Methods: MSCs were induced to be differentiated into the osteoblastic cell lineage using routine protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and real.time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the related markers to this stage. Results: Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes, while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result for FRA1 was not significant in both groups at the same scheduled time (days 0-21). Conclusion: The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1 may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined that promoter-methylation is an important process in the regulation of OSD. However, yet there was no significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation- independent pathway might be involved in the regulation of target genes during OSD of MSCs.

scholarly journals Cytokine and Chemokine mRNA Expressions after Mycobacterium tuberculosis-Specific Antigen Stimulation in Whole Blood from Hemodialysis Patients with Latent Tuberculosis Infection

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 595
Author(s):  
Ji Young Park ◽  
Sung-Bae Park ◽  
Heechul Park ◽  
Jungho Kim ◽  
Ye Na Kim ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mrna Expression ◽  
Whole Blood ◽  
Latent Tuberculosis Infection ◽  
Latent Tuberculosis ◽  
Expression Level ◽  
Mrna Expression Level ◽  
Healthy Individuals ◽  
Relative Mrna Expression ◽  
Mrna Expression Levels

There have been few reports on the kinetics of hemodialyzed (HD) patients’ immune responses in latent tuberculosis infection (LTBI). Therefore, in the present study, messenger ribonucleic acid (mRNA) expression levels of nine immune markers were analyzed to discriminate between HD patients with LTBI and healthy individuals. Nine cytokines and chemokines were screened through relative mRNA expression levels in whole blood samples after stimulation with Mycobacterium tuberculosis (MTB)-specific antigens from HD patients with LTBI (HD/LTBI), HD patients without LTBI, and healthy individuals, and results were compared with the QuantiFERON-TB Gold In-Tube (QFT-GIT) test. We confirmed that the C-C motif chemokine 11 (CCL11) mRNA expression level of the HD/LTBI group was significantly higher than the other two groups. Especially, the CCL11 mRNA expression level of the >0.7 IU/mL group in the QFT-GIT test was significantly higher than the <0.2 IU/mL group in the QFT-GIT test and the 0.2–0.7 IU/mL group in the QFT-GIT test (p = 0.0043). The present study reveals that the relative mRNA expression of CCL11 was statistically different in LTBI based on the current cut-off value (i.e., ≥0.35 IU/mL) and in the >0.7 IU/mL group. These results suggest that CCL11 mRNA expression might be an alternative biomarker for LTBI diagnosis in HD patients.

scholarly journals DNA methylation mediated RSPO2 to promote follicular development in mammals

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Xiaofeng Zhou ◽  
Yingting He ◽  
Nian Li ◽  
Guofeng Bai ◽  
Xiangchun Pan ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Methylation Level ◽  
Molecular Mechanisms ◽  
Cpg Island ◽  
Wnt Signaling Pathway ◽  
Follicular Development ◽  
Expression Level

AbstractIn female mammals, the proliferation, apoptosis, and estradiol-17β (E2) secretion of granulosa cells (GCs) have come to decide the fate of follicles. DNA methylation and RSPO2 gene of Wnt signaling pathway have been reported to involve in the survival of GCs and follicular development. However, the molecular mechanisms for how DNA methylation regulates the expression of RSPO2 and participates in the follicular development are not clear. In this study, we found that the mRNA and protein levels of RSPO2 significantly increased during follicular development, but the DNA methylation level of RSPO2 promoter decreased gradually. Inhibition of DNA methylation or DNMT1 knockdown could decrease the methylation level of CpG island (CGI) in RSPO2 promoter and upregulate the expression level of RSPO2 in porcine GCs. The hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of transcription factor E2F1 and promoted the transcriptional activity of RSPO2. Moreover, RSPO2 promoted the proliferation of GCs with increasing the expression level of PCNA, CDK1, and CCND1 and promoted the E2 secretion of GCs with increasing the expression level of CYP19A1 and HSD17B1 and inhibited the apoptosis of GCs with decreasing the expression level of Caspase3, cleaved Caspase3, cleaved Caspase8, cleaved Caspase9, cleaved PARP, and BAX. In addition, RSPO2 knockdown promoted the apoptosis of GCs, blocked the development of follicles, and delayed the onset of puberty with decreasing the expression level of Wnt signaling pathway-related genes (LGR4 and CTNNB1) in vivo. Taken together, the hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of E2F1 and enhanced the transcription of RSPO2, which further promoted the proliferation and E2 secretion of GCs, inhibited the apoptosis of GCs, and ultimately ameliorated the development of follicles through Wnt signaling pathway. This study will provide useful information for further exploration on DNA-methylation-mediated RSPO2 pathway during follicular development.

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