scholarly journals Epigenetic-Mediated Downregulation of μ-Protocadherin in Colorectal Tumours

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Bujko Mateusz ◽  
Kober Paulina ◽  
Statkiewicz Małgorzata ◽  
Mikula Michal ◽  
Ligaj Marcin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Normal Mucosa ◽  
Colorectal Carcinogenesis ◽  
Cellular Interaction ◽  
Mrna Levels ◽  
Aberrant Expression ◽  
Tissue Samples

Carcinogenesis involves altered cellular interaction and tissue morphology that partly arise from aberrant expression of cadherins. Mucin-like protocadherin is implicated in intercellular adhesion and its expression was found decreased in colorectal cancer (CRC). This study has compared MUPCDH (CDHR5) expression in three key types of colorectal tissue samples, for normal mucosa, adenoma, and carcinoma. A gradual decrease of mRNA levels and protein expression was observed in progressive stages of colorectal carcinogenesis which are consistent with reports of increasing MUPCDH 5′ promoter region DNA methylation. High MUPCDH methylation was also observed in HCT116 and SW480 CRC cell lines that revealed low gene expression levels compared to COLO205 and HT29 cell lines which lack DNA methylation at the MUPCDH locus. Furthermore, HCT116 and SW480 showed lower levels of RNA polymerase II and histone H3 lysine 4 trimethylation (H3K4me3) as well as higher levels of H3K27 trimethylation at the MUPCDH promoter. MUPCDH expression was however restored in HCT116 and SW480 cells in the presence of 5-Aza-2′-deoxycytidine (DNA methyltransferase inhibitor). Results indicate that μ-protocadherin downregulation occurs during early stages of tumourigenesis and progression into the adenoma-carcinoma sequence. Epigenetic mechanisms are involved in this silencing.

AML1/ETO-Mediated Lysozyme Repression Is Independently Relieved by Inhibitors of DNA Methylation and Histone Deacetylation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4310-4310
Author(s):  
Rainer Claus ◽  
Manfred Fliegauf ◽  
Michael Stock ◽  
Jesus Duque ◽  
Mateusz Kolanczyk ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Trichostatin A ◽  
Combined Treatment ◽  
Marker Gene ◽  
Dna Demethylation ◽  
Histone Deacetylation ◽  
Mrna Levels

Abstract The human lysozyme (LZM) gene, a marker gene for myeloid-specific development, is highly methylated in immature myeloid and in non-myeloid cells (all LZM-negative), and unmethylated in LZM-expressing mature phagocyte cells. Thus this gene provides an excellent model for investigating differentation-associated DNA methylation changes during myelopoiesis. There is now increasing evidence that LZM (containing five perfect consensus binding sites for AML1/RUNX1 in its 5′ region) is repressed by the AML1/ETO chimeric transcription factor (Fliegauf et al, Oncogene 23:9070–81, 2004), and this repression can be relieved by siRNA-mediated AML1/ETO depletion in AML1/ETO-positive Kasumi-1 cells (Dunne et al., Oncogene, 2006). Recently, AML1/ETO has also been implicated in gene-specific epigenetic repression of interleukin-3 (Liu et al, Cancer Res 65, 1277–84, 2005). By extensive methylation analyses of the LZM gene including bisulfite sequencing, we now demonstrate marked demethylation in both the CpG-poor 5′ region and the exonic CpG island after treatment of Kasumi-1 cells with non-cytotoxic concentrations of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-azaCdR), which was not associated with cellular differentiation. By Northern blot analysis, LZM mRNA levels in Kasumi-1 cells but not in AML1/ETO-negative HL-60 and U-937 cell lines were specifically and independently upregulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Combined treatment with subliminal concentrations of 5-azaCdR and TSA applied in different schedules did not reveal synergistic effects on LZM transcription. Relative chromatin accessibility of the LZM 5′ region, as detected by “MspI protection” assay, and associated with partial demethylation in several myeloid cell lines, was increased in Kasumi-1 with 5-azaCdR-induced further DNA demethylation, but not by TSA. As shown by chromatin immunoprecitation, TSA increased the acetylation of histones H3 and H4 both in the 5′ flanking region and exonic CpG island. In a U-937 inducible model, antagonization of AML1/ETO-mediated repression of LZM was achieved by TSA, implying that the histone deacetylation in this region of the human LZM gene is mediated by AML1/ETO protein. In conclusion, we demonstrate functional interactions between DNA methylation and histone modifications in mediating LZM gene repression which implicate AML1/ETO as one component involved in local chromatin remodelling. Interestingly, inhibitors of DNA methylation and histone deacetylation independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.

scholarly journals Complement Factor C3 Methylation and mRNA Expression Is Associated to BMI and Insulin Resistance in Obesity

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 410 ◽  
Author(s):  
Daniel Castellano-Castillo ◽  
Isabel Moreno-Indias ◽  
Jose Carlos Fernandez-Garcia ◽  
Mercedes Clemente-Postigo ◽  
Manuel Castro-Cabezas ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Dna Methylation ◽  
Adipose Tissue ◽  
Messenger Rna ◽  
Density Lipoprotein ◽  
Complement Factor ◽  
Mrna Levels ◽  
Model Assessment ◽  
Tissue Samples ◽  
Obese Class

Epigenetic marks, and especially DNA methylation, are becoming an important factor in obesity, which could help to explain its etiology and associated comorbidities. Adipose tissue, now considered as an important endocrine organ, produces complement system factors. Complement component 3 (C3) turns out to be an important protein in metabolic disorders, via either inflammation or the C3 subproduct acylation stimulating protein (ASP) which directly stimulates lipid storage. In this study, we analyze C3 DNA methylation in adipose tissue from subjects with a different grade of obesity. Adipose tissue samples were collected from subjects with a different degree of obesity determined by their body mass index (BMI) as: Overweight subjects (BMI ≥ 25 and <30), obese class 1/2 subjects (BMI ≥ 30 and <40) and obese class 3 subjects (BMI ≥ 40). C3 DNA methylation was measured for 7 CpGs by pyrosequencition using the Pyromark technology (Qiagen, Madrid Spain). C3 messenger RNA (mRNA) levels were analyzed by pre-designed Taqman assays (Applied biosystems, Foster City, CA, USA) and ASP/C3a was measured using a ELISA kit. The data were analyzed using the statistic package SPSS. C3 DNA methylation levels were lower in the morbid obese group. Accordingly, C3 methylation correlated negatively with BMI and leptin. However, C3 mRNA levels were more associated with insulin resistance, and positive correlations with insulin, glucose and homeostasis model assessment-estimated insulin resistance (HOMA-IR) existed. ASP correlated negatively with high density lipoprotein (HDL) cholesterol. C3 methylation levels were associated to adiposity variables, such as BMI and leptin, while the C3 mRNA levels were associated to glucose metabolism.

Suppression of clonogenicity by mammalian Dnmt1 mediated by the PCNA-binding domain

2004 ◽  
Vol 82 (5) ◽  
pp. 589-596 ◽  
Author(s):  
Simeon Santourlidis ◽  
Fumihiro Kimura ◽  
Johannes Fischer ◽  
Wolfgang A Schulz
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Acid Domain ◽  
Safe Mechanism ◽  
Fail Safe ◽  
Proliferating Cell ◽  
Amino Acid Domain

Overexpression of the major DNA methyltransferase Dnmt1 is cytotoxic and has been hypothesized to result in aberrant hypermethylation of genes required for cell survival. Indeed, overexpression of mouse or human Dnmt1 in murine and human cell lines decreased clonogenicity. By frame-shift and deletion constructs, this effect of mouse Dnmt1 was localized at the N-terminal 124 amino acid domain, which mediates interaction with proliferating cell nuclear antigen (PCNA). Mutation of the PCNA-binding site restored normal cloning efficiencies. Overexpression of Dnmt3A or Dnmt3B, which do not interact with PCNA, yielded weaker effects on clonogenicity. Following introduction of the toxic domain, no significant effects on apoptosis, replication, or overall DNA methylation were observed for up to 3 d. Suppression of clonogenicity by Dnmt1 was also observed in cell lines lacking wild-type p53, p21CIP1, or p16INK4A. Suppression of clonogenicity by Dnmt1 overexpression may act as a fail-safe mechanism against carcinogenicity of sustained Dnmt1 overexpression.Key words: carcinogenesis, DNA methyltransferase, DNA methylation, p53, PCNA.

Sensitivity of Diffuse Large B-Cell Lymphomas to DNA Methyltransferase Inhibitors Is Associated with a Specific Epigenetic Signature.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 831-831
Author(s):  
Megan Ryan ◽  
Leandro Cerchietti ◽  
Maria E. Figueroa ◽  
John Greally ◽  
Ari Melnick
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Serum Starvation ◽  
Gene Promoters ◽  
B Cell Lymphomas ◽  
Response Curves ◽  
Differentially Methylated Genes ◽  
Large B Cell

Abstract DNA methyltransferase inhibitor drugs (MTIs) such as decitabine can overcome gene silencing due to aberrant hypermethylation of gene promoters. Presumably, this effect is responsible for the therapeutic activity of MTIs as clinically demonstrated in myelodysplasias (MDS) and leukemias. Other tumors such as diffuse large B-cell lymphomas (DLBCLs) can also present with aberrant promoter hypermethylation. However, it is currently difficult to prospectively identify patients likely to respond to MTIs, since specific methylation markers or signatures have not yet been identified. We predicted that decitabine would have anti-lymphoma activity in a subset of DLBCLs, and that these cases would exhibit specific methylation signatures predictive of response to these drugs. To determine whether this is the case we first exposed a panel of 7 DLBCL cell lines (Ly1, Ly7, Ly10, SU-DHL6, Farage, Pfeiffer and Toledo) to increasing concentrations of decitabine (0.5, 1, 2.5, 5, 10, 50 and 100 μM) administered after synchronization by 12 hr serum starvation. Viability was assessed after 48 hr of culture by MTS-based assay and Trypan blue exclusion. The IC25 and IC50 were calculated for all cell lines by constructing dose-response curves. The IC25 was used to discriminate sensitive (6.3 ± 1.2 μM) vs. resistant (49.4 ± 5 μM, p &lt; 0.01) cell lines. Interestingly, there was no correlation between MTI sensitivity and DLBCL subtype as defined by recent gene expression profiling classification efforts (i.e. GCB vs. ABC, or BCR vs. OxPhos). To identify the methylation signatures of these DLBCL cells we used a method that we developed for genome-wide DNA methylation quantification called HELP (HpaII tiny fragment Enrichment by LM-PCR). HELP is based on comparative Msp1 and HpaII digestion of genomic DNA, followed by size specific amplification and co-hybridization to custom high-density oligonucleotide arrays designed to provide uniform data collection over 25,000 promoters. HELP compares favorably to other high throughput methods in that it is highly reproducible (R &gt; 0.98) and has an extremely robust signal-to-noise ratio. DNA was collected from the DLBCL cells for HELP prior to drug treatment. Most significantly we found that unsupervised (i.e. unbiased) clustering of DNA methylation profiles could readily segregate decitabine resistant vs. sensitive DLBCL cell lines. Correspondence analysis clearly identified a methylation signature consisting of 133 differentially methylated genes that distinguishes between decitabine sensitive and resistant cells. Most of these appeared to be functionally relevant including such genes as Caspase-9, RARB, JUNB, and ELK1. Biological assays to determine the contribution of these genes to the phenotype are underway. Taken together, our data suggest that MTIs might be effective in a cohort of DLBCL cases that exhibit the specific methylation signature that we have identified. Prospective evaluation of the predictive value of this signature may allow optimal selection of patients for clinical trials with these agents.

Identification of epigenetic silencing of GCNT2 expression by comprehensive real-time PCR screening in colorectal cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 506-506
Author(s):  
Kazunorii Nakamura ◽  
Horomichi Sawaki ◽  
Keishi Yamashita ◽  
Masahiko Watanabe ◽  
Hisashi Narimatsu
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Critical Role ◽  
Normal Mucosa ◽  
Primary Cancer ◽  
Normal Tissues ◽  
Cancer Tissues

506 Background: Glycoprotein expression profile has been proved to be dramatically altered in human cancers, however specific glycogenes which are aberrant in expression in cancer cells has not been fully identified. Recent accumulated evidence supported notion that the reduced expression of tumor suppressor genes is explained by DNA promoter methylation in human cancer. Methods: We used Comprehensive Real time PCR system (CRPS) for glycogenes (189 genes) to identify genes aberrantly expressed in colorectal cancer tissues (CRC) as compared to the corresponding normal mucosa tissues. GCNT2 was of particular interest among the identified genes in CRC. Results: (1) GCNT2 harbors 3 isoforms which have different promoter regions. (2) All of the 3 isoforms of GCNT2 genes were remarkably decreased in CRC as compared to the corresponding normal mucosa, and each isoform expression was strongly associated with other 2 isoforms in primary cancer tissues by TaqMan real time PCR (R = 0.99-995, p < 0.0001). (3) Among the 5 CRC cell lines (DLD1, HCT116, CACO2, LOVO), those which were silenced in expression were reactivated by demethylating agents such as 5-aza-2’ deoxycytidine and trichostatin A. (4) Promoter region of the variant 2 of GCNT2 was consistent with its silenced expression in CRC cell lines by cloned sequence, so we examined DNA methylation status of the promoter of the GCNT2 variant 2 in 50 primary cancer tissues and the corresponding normal tissues. Quantitative MSP revealed that almost half of normal tissues have methylation as high as tumor tissues, while, in the primary CRC with less methylation in the corresponding normal tissues, DNA methylation was higher in primary CRC tissues than in the corresponding normal tissues. Finally, GCNT2 variant 2 stable transfection induced expression of other 2 isoform variants. Conclusions: We identified novel methylation gene GCNT2 among the glycoenes. Glycoenes that were altered in genomic or epigenetic manner have been few, so GCNT2 may play a critical role in cancer progression through glycan change.

scholarly journals Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors

2008 ◽  
Vol 200 (2) ◽  
pp. 167-175 ◽  
Author(s):  
Marcia Helena Soares Costa ◽  
Ana Claudia Latronico ◽  
Regina Matsunaga Martin ◽  
Angela S Barbosa ◽  
Madson Q Almeida ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Mrna Levels ◽  
Aberrant Expression ◽  
Tissue Samples ◽  
Peptide Receptor ◽  
Adrenocortical Tumors ◽  
Adrenocortical Carcinomas ◽  
Insulinotropic Peptide

Glucose-dependent insulinotropic peptide receptor (GIPR) and LHCGR are G-protein-coupled receptors with a wide tissue expression pattern. Aberrant expression of these receptors has rarely been demonstrated in adult sporadic adrenocortical tumors with a lack of data on pediatric tumors. We quantified the GIPR and LHCGR expression in a large cohort of 55 patients (25 children and 30 adults) with functioning and non-functioning sporadic adrenocortical tumors. Thirty-eight tumors were classified as adenomas whereas 17 were carcinomas. GIPR and LHCGR expression were analyzed by real-time PCR and normal human pancreatic and testicular tissue samples were used as positive controls. Mean expression values were determined by fold increase in comparison with a normal adrenal pool. GIPR mRNA levels were significantly higher in adrenocortical carcinomas than in adenomas from both pediatric and adult groups. LHCGR expression was similar in both carcinomas and adenomas from the pediatric group but significantly lower in carcinomas than in adenomas from the adult group (median 0.06 and 2.3 respectively, P<0.001). GIPR was detected by immunohistochemistry in both pediatric and adult tumors. Staining and real-time PCR results correlated positively only when GIPR mRNA levels were increased at least two-fold in comparison with normal adrenal expression levels. In conclusion, GIPR overexpression was observed in pediatric and adult adrenocortical tumors and very low levels of LHCGR expression were found in all adult adrenocortical carcinomas.

36 EFFECT OF DNA METHYLTRANSFERASE INHIBITOR, RG108, ON IN VITRO DEVELOPMENT AND ntES ESTABLISHMENT RATE IN CLONED MOUSE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 130 ◽  
Author(s):  
C. Li ◽  
Y. Terashita ◽  
M. Tokoro ◽  
S. Wakayama ◽  
T. Wakayama
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Control Group ◽  
Cloning Efficiency ◽  
Dna Methyltransferase Inhibitor

Somatic cell nuclear transfer technique increased expectations among many for its potential to advance the regenerative therapy field. Cloned embryos, however, exhibit several epigenetic abnormalities, such as low histone acetylation or high DNA methylation levels compared with normal fertilized embryos. Therefore, increasing histone acetylation or reducing DNA methylation levels in cloned embryos using chemical treatments may improve cloning efficiency. We recently succeeded in improving the success rate of mouse cloning by using class IIb histone deacetylase inhibitors, such as trichostatin A (TSA), scriptaid and suberoylanilide hydroxamic acid. It has also been reported that 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor that is a chemical analogue of cytidine, inhibits the potential of embryos to develop into blastocysts and later to fetuses. In the present study, another DNA methyltransferase inhibitor RG108, which is thought to strongly interact with the DNMT1 active site to inhibit DNMT1 activity, was used to examine whether it could improve cloning efficiency. To determine the effects of RG108, cloned embryos were treated with 100 to 500 μM RG108. When cloned embryos were treated at the 1-cell stage (from artificial activation to 10 h, n = 219), the cloning efficiency was similar to the control group (8.2 vs 10.8%). On the other hand, when 500 μM RG108 was added to the culture medium from the 2-cell to morula/blastocyst stage (n = 113), although the developmental rate to blastocyst stage did not change significantly (79.6% vs 72.3%), higher Oct3/4 expression and more ICM cells were observed compared with non-treated, control cloned embryos. Moreover, we tried to establish ES cell lines from those cloned embryos and 11 ntES lines were generated from 21 blastocysts, which was higher than that of control (6 ntES cell lines from 20 blastocysts). All ntES lines showed AP staining positively. This finding showed that the quality of cloned mouse blastocysts increased when treated with a DNA methyltransferase inhibitor, suggesting a possible means for improving cloning efficiency in the future.

Oncogenic NK-Like Homeodomain Proteins Inhibit Etoposide-Induced Apoptosis in T-ALL Cell Lines by Activation of the Polycistronic miR-17-92 Transcript.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2787-2787
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Corinna Meyer ◽  
Hans G. Drexler ◽  
Roderick A.F. MacLeod ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Mrna Translation ◽  
Mature Mirnas ◽  
Mrna Levels ◽  
Homeodomain Proteins ◽  
Aberrant Expression ◽  
Protein Levels ◽  
Micro Rnas

Abstract Homeobox genes of the NK-like familiy, including TLX1, TLX3 and NKX2-5, are ectopically activated in T-cell acute lymphoblastic leukemia (T-ALL) cells mostly via chromosomal aberrations. The pathologic function of these closely related genes is still unclear. Here we analyzed their effect on the C13ORF25 gene, containing the miR-17-92 cluster. Micro RNAs (miRNAs) are a class of small non-coding RNAs which are part of an evolutionarily highly conserved intracellular mechanism, regulating gene expression by hybridization to complementary sequences usually located in the 3′untranslated region of coding transcripts. The primary transcripts (pri-mRNA) are processed to short mature miRNAs, mediating either inhibition of mRNA translation or mRNA cleavage. Aberrant expression of specific miRNAs is involved in oncogenesis as recently described for several human malignancies. The miR-17-92 polycistron encodes miRNAs which decrease E2F1 protein expression. Transcription of both E2F1 and miR-17-92 is induced by MYC, itself a target of E2F1, generating a highly regulated interactive network. Depending on the cellular context, E2F1 performs conflicting tasks by triggering proliferation or inducing apoptosis. We investigated the expression of the miR-17-92 cluster in T-ALL cell lines. Real-time RT-PCR analysis of both miR-17-92 pri-mRNA and mature miRNAs revealed different expression levels in these cells, suggesting a possible implication of the NK-like homeodomain proteins in the regulation of the miR-17-92 cluster in T-ALL. HELA cells transfected with TLX1 or NKX2-5 expression constructs showed elevated miR-17-92 pri-mRNA expression, demonstrating an activating effect. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently showed increased miR-17-92 pri-mRNA levels and decreased E2F1 protein amounts. For functional analysis of these downstream targets, another T-ALL cell line (PEER) was lentivirally transduced with expression constructs for either miR-17-92 or E2F1, resulting in reduced or elevated E2F1 protein levels, respectively. Overexpression of miR-17-92 or E2F1 did not significantly influenced the cell proliferation. However, induction of apoptosis by treating these cells with etoposide, an inhibitor of topoisomerase II, indicated that overexpression of miR-17-92 and E2F1 resulted in enhanced and reduced cell viability, respectively, as analyzed by MTT assay. In summary, these data indicate an activatory effect of oncogenic NK-like homeodomain proteins on miR-17-92 expression, reducing E2F1 protein levels and thereby enhancing survival of leukemic T-cells.

MicroRNA (miRNA)-29b Targets DNMT3A and B and Induces Re-Expression of the Hypermethylated ESR1 and p15 Genes in Acute Myeloid Leukemia (AML).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 718-718
Author(s):  
Ramiro Garzon ◽  
Shujun Liu ◽  
Muller Fabbri ◽  
Zhongfa Liu ◽  
Jiuxia Pang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Cell Lines ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Aberrant Methylation ◽  
Mrna Levels ◽  
Down Regulation ◽  
Functional Link ◽  
Qrt Pcr

Abstract Aberrant DNA methylation has been shown to play an important role in AML by silencing structurally normal genes important for hematopoiesis. MiRNAs are small non-coding RNAs that down-regulate gene expression. Although aberrant methylation of miRNAs has been reported, it is not known whether miRNAs themselves can modulate DNA methylation. To address this question, we tested the functional role of miRNAs that are predicted to target both DNA methyltransferase 3A and 3B (DNMT3A and B), in AML cells. We cloned the 3′UTR of both DNMT3s into a luciferase reporter and co-transfected K562 cells with scrambled or sense sequences of each of the miRNAs predicted to interact with DNMT3A and B. We found that miR-29b significantly repressed luciferase activity of both DNMT3A and B constructs (42% and 67%, respectively) compared with the controls. Transfection of the pre-miR-29b, but not of the scrambled, resulted in a marked reduction of the DNMT3A and B mRNA levels measured by qRT-PCR in MV4-11 cells (80% and 90%, respectively) and in K562 cells (23% and 67% respectively).We also observed a significant down-regulation of DNMT3s protein by immunoblotting in K562 cells transfected with miR-29b relative to the controls. These results were then validated in AML patients. Both miR-29b and DNMT3B expression levels were measured by qRT-PCR in bone marrow samples obtained from 12 AML patients with primary disease. We found that miR-29b expression was inversely correlated to DNMT3B mRNA levels (Pearson coefficient R=−0.66; P=0.03). Functionally, we showed that the mRNA expression of p15 and ESR1, that are silenced through promoter hypermethylation in AML, were increased in K562 and MV4-11 cells transfected with miR-29b compared with the controls. ESR1 was 1.9 and 1000 fold upregulated in miR-29b-tranfected K562 and MV4-11cells, respectively and p15 was 11.6 fold upregulated in miR-29b-tranfected MV 4–11 cells. Interestingly, we observed that the expression of miR-29b-1, which is silenced in K562 cell lines, was 6.5 fold up-regulated following treatment with 2.5 uM decitabine, a DNA hypomethylating nucleoside compared with untreated controls. Consistent with these results, we also showed 5.6 fold increase in miR-29b-1 expression in post-treatment CD34+ selected bone marrow blasts from AML patients (n=3) who received decitabine (20 mg/m2/day x 10 days) on a clinical protocol (OSU 0336) compared with pretreatment baselines. These results were consistent with the methylation analysis of CpGs up to 30kb upstream from the 5′ encoding regions of the miR-29b-1 precursors in the same patients. Using MassArray (sequenom) technology, we showed that all the CpGs were highly methylated (95 to 100%) in pre-treatment baseline samples and these methylation levels decreased by 20% after decitabine treatment. These preliminary data provide a functional link between miRNAs and aberrant epigenetics by suggesting that: miR-29b targets DNMT3A and B; miR-29b is silenced in AML cell lines and patient primary blasts and its expression is restored in vitro and in vivo by ypomethylating agents; 3) increase of miR-29b levels results in DNMTs down-regulation and re-expression of otherwise hypermethylated genes. Altogether, there data support a rationale for developing novel miRNA-based therapeutic strategies that alone and in combination with other hypomethylating agents may target aberrant epigenetics in AML.

scholarly journals Overexpression of long noncoding RNA LINC01419 in esophageal squamous cell carcinoma and its relation to the sensitivity to 5-fluorouracil by mediating GSTP1 methylation

2019 ◽  
Vol 11 ◽  
pp. 175883591983895 ◽  
Author(s):  
Jian-Liang Chen ◽  
Zhi-Xiong Lin ◽  
Yun-Sheng Qin ◽  
Yu-Qi She ◽  
Yun Chen ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Esophageal Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Noncoding Rna ◽  
Dna Methyltransferase ◽  
The Cancer Genome Atlas ◽  
Aberrant Expression ◽  
Tissue Samples ◽  
Enhanced Sensitivity

Background: Genome-wide sequencing investigations have identified numerous long noncoding RNAs (lncRNAs) among mammals, many of which exhibit aberrant expression in cancers, including esophageal squamous cell carcinoma (ESCC). Herein, this study elucidates the role and mechanism by which LINC01419 regulates the DNA methylation of glutathione S-transferase pi 1 (GSTP1) in relation to ESCC progression and the sensitivity of ESCC cells to 5-fluorouracil (5-FU). Methods: LINC01419 and GSTP1 levels were quantified among 38 paired ESCC and adjacent tissue samples collected from patients with ESCC. To ascertain the contributory role of LINC01419 in the progression of ESCC and identify the interaction between LINC01419 and GSTP1 promoter methylation, LINC01419 was overexpressed or silenced, and the DNA methyltransferase inhibitor 5-Aza-CdR was treated. Results: Data from the GEO database (GSE21362) and the Cancer Genome Atlas displayed elevated levels of LINC01419 and downregulated levels of GSTP1 in the ESCC tissues and cells. The silencing of LINC01419 led to decreased proliferation, increased apoptosis, and enhanced sensitivity to 5-FU in ESCC cells. Notably, LINC01419 could bind to the promoter region of the GSTP1 gene, resulting in elevated GSTP1 methylation and reduced GSTP1 levels via the recruitment of DNA methyltransferase among ESCC cells, whereby ESCC progression was stimulated accompanied by reduced ESCC cell sensitivity to 5-FU. GSTP1 demethylation by 5-Aza-CdR was observed to reverse the effects of LINC01419 overexpression in ESCC cells and the response to 5-FU. Conclusion: Highly expressed LINC01419 in ESCC promotes GSTP1 methylation, which ultimately acts to promote the event of ESCC and diminish the sensitivity of ESCC cells to 5-FU, highlighting a novel potential strategy to improve 5-FU-based chemotherapy in ESCC.

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