Hypomethylation-associated LINC00987 downregulation induced lung adenocarcinoma progression by inhibiting the phosphorylation-mediated degradation of SND1.

DNA methylation, an epigenetic regulatory mechanism dictating gene transcription, plays a critical role in the occurrence and development of cancer. However, the molecular underpinnings of LINC00987 methylation in the regulation of lung adenocarcinoma (LUAD) remain elusive. This study investigated LINC00987 expression in LUAD patients through analysis of The Cancer Genome Atlas data sets. Quantitative real-time polymerase chain reaction (RT-qPCR) and fluorescence in situ hybridization assays were used to assess LINC00987 expression in LUAD. The bisulfite genomic sequence PCR (BSP) assay was used to determine the methylation levels of the LINC00987 promoter. The interaction between LINC00987 and SND1 was elucidated via immunoprecipitation and RNA pull-down assays. The functional significance of LINC00987 and SND1 in Calu-3 and NCI-H1688 cells was evaluated in vitro through CCK-8, EdU, Transwell, flow cytometry, and vasculogenic mimicry (VM) tube formation assays. LINC00987 expression decreased in LUAD concomitant with hypermethylation of the promoter region, while hypomethylation of the LINC00987 promoter in LUAD tissues correlated with tumor progression. Treatment with 5-Aza-CdR augmented LINC00987 expression and inhibited tumor growth. Mechanistically, LINC00987 overexpression impeded LUAD progression and VM through direct binding with SND1, thereby facilitating its phosphorylation and subsequent degradation. Additionally, overexpression of SND1 counteracted the adverse effects of LINC00987 downregulation on cell proliferation, apoptosis, cell migration, invasion, and VM in LUAD in vitro. In conclusion, this pioneering study focuses on the expression and function of LINC00987 and reveals that hypermethylation of the LINC00987 gene may contribute to LUAD progression. LINC00987 has emerged as a potential tumor suppressor gene in tumorigenesis through its binding with SND1 to facilitate its phosphorylation and subsequent degradation.

Unravelling the impact of RNA methylation genetic and epigenetic machinery in the treatment of cardiomyopathy.

Cardiomyopathy (CM) represents a heterogeneous group of diseases primarily affecting cardiac structure and function, with genetic and epigenetic dysregulation playing a pivotal role in its pathogenesis. Emerging evidence from the burgeoning field of epitranscriptomics has brought to light the significant impact of various RNA modifications, notably N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N1-methyladenosine (m1A), 2'-O-methylation (Nm), and 6,2'-O-dimethyladenosine (m6Am), on cardiomyocyte function and the broader processes of cardiac and vascular remodelling. These modifications have been shown to influence key pathological mechanisms including mitochondrial dysfunction, oxidative stress, cardiomyocyte apoptosis, inflammation, immune response, and myocardial fibrosis. Importantly, aberrations in the RNA methylation machinery have been observed in human CM cases and animal models, highlighting the critical role of RNA methylating enzymes and their potential as therapeutic targets or biomarkers for CM. This review underscores the necessity for a deeper understanding of RNA methylation processes in the context of CM, to illuminate novel therapeutic avenues and diagnostic tools, thereby addressing a significant gap in the current management strategies for this complex disease.

Inflamma-miRs Profile in Myelodysplastic Syndrome Patients.

Etiological factors involved in myelodysplastic syndrome (MDS) include immunologic, oxidative stress and inflammatory factors, among others, and these are targets for microRNAs (miRNs). Here, we evaluated whether some miRNs may affect tumor development comparing untreated and 5-azacitidine (5-AZA) MDS-treated patients. Peripheral blood samples were collected from 20 controls and 24 MDS patients, and selected miRNs related to redox balance and inflammation (inflamma-miRs), including miR-18a, miR-21, miR-34a and miR-146a, were isolated and measured by quantitative real-time polymerase chain reaction (qRTPCR). A differential expression profile of miRNs was detected in untreated MDS patients and the 5-AZA group. Inflammation increases miRNs and, specifically, miR-18a, miR-21 and miR-34a were significantly overexpressed in untreated MDS, compared to controls. However, we did not observe any miRN profile alteration during the progression of the disease. On the other hand, 5-AZA treatment tends to restore miRN expression levels. Relating to prognostic risk factors, high-risk MDS groups (high Revised International Prognostic Scoring System (IPSS-R), high cytogenetic risk, high molecular risk (HMR) mutations) tended to be related with higher expression levels of miR-18a and miR-34a. Higher miRN expression is correlated with lower glutathione peroxidase activity, while they are related with a higher profile of pro-inflammatory cytokines (IL-2, IL-6, IL-8, TNF-α). Although our study was limited by the low number of MDS patients included, we identified miRN deregulation involved in MDS development that could regulate redox sensors and inflammatory responses. Finally, 5-AZA treatment is related with lower miRN expression levels in MDS patients.

Molecular predictors for decitabine efficacy in meningiomas - a pilot study.

PURPOSE: Effective chemotherapeutical agents for the treatment of meningiomas are still lacking. Previous in-vitro analyses revealed efficacy of decitabine (DCT), a DNA methyltransferase (DNMT) inhibitor established in the treatment of leukemia, in a yet undefined subgroup of meningiomas. METHODS: Effects of DCT on proliferation and viability was analyzed in primary meningioma cells by immunofluorescence and MTT assays, and cases were classified as drug responders and non-responders. Molecular preconditions for efficacy were analyzed using immunofluorescence for Ki67, DNMT1, and five oncogenes (TRIM58, FAM84B, ELOVL2, MAL2, LMO3) previously found to be differentially methylated after DCT exposition, as well as by genome-wide DNA methylation analyses. RESULTS: Efficacy of DCT (10µM) was found in eight (62%) of 13 meningioma cell lines 48 h after drug exposition (p < .05). DCT significantly reduced DNMT1 expression in all but two cell lines, and median ΔDNMT1 reduction 48 h after drug exposition was lower in DCT-resistant (-11.1%) than in DCT-sensitive (-50.5%, p = .030) cells. Rates of cell lines responsive to DCT exposition distinctly decreased to 25% after 72 h. No significant correlation of the patients´ age, sex, histological subtype, location of the paternal tumor, expression of Ki67, DNMT1 or the analyzed oncogenes with treatment response was found (p > .05, each). DCT efficacy was further independent of the methylation class and global DNA methylation of the paternal tumor. CONCLUSION: Early effects of DCT in meningiomas are strongly related with DNMT1 expression, while clinical, histological, and molecular predictors for efficacy are sparse. Kinetics of drug efficacy might indicate necessity of repeated exposition and encourage further analyses.

Integrative analysis of the DNA methylome and transcriptome in uterine leiomyoma shows altered regulation of genes involved in metabolism, proliferation, extracellular matrix, and vesicles.

Uterine leiomyomas (ULs) are the most common benign tumors in women of reproductive age. Despite the high prevalence, tumor pathology remains unclear, which hampers the development of safe and effective treatments. Epigenetic mechanisms appear to be involved in UL development, particularly via DNA methylation that regulates gene expression. We aimed to determine the relationship between DNA methylation and gene expression in UL compared with adjacent myometrium (MM) to identify molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, and a different global transcriptome profile. Integration of DNA methylation and whole-transcriptome RNA-sequencing data identified 93 genes regulated by methylation, with 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes and molecular functions involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation, as well as pathways related to uterine biology and cancer. Cellular components such as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1, RIMS2, TFAP2C) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2), which are related to proliferation, invasion, altered metabolism, deposition of extracellular matrix, and Wnt/ß-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, inhibition of DNA methyltransferases by 5-aza-2'-deoxycitidine increased the expression of hypermethylated/downregulated genes in UL cells in vitro. In conclusion, gene regulation by DNA methylation is implicated in UL pathogenesis, and reversion of this methylation could offer a therapeutic option for UL. © 2022 The Pathological Society of Great Britain and Ireland.

Different mechanisms of drug resistance to hypomethylating agents in the treatment of myelodysplastic syndromes and acute myeloid leukemia.

Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) which are not suitable for hematopoietic stem cells transplantation. Approximately 50 % of patients do not respond to HMA treatment because of intrinsic (primary) resistance, while others could acquire drug resistance during the repeated cycles of the treatment. To prevent, delay or surmount resistance development, the molecular mechanisms underlying drug resistance must be first identified. This is crucial as no further standard therapeutic opportunities are available for these patients who failed hypomethylating agents-based treatment. The current review provides an updated information about the different mechanisms that may contribute to the development of resistance to HMAs. Despite the similar structure and mechanism of action of HMA, several studies did not report the expected development of cross-resistance. It is clear that in addition to the common modalities of chemoresistance, there must be some specific mechanisms of drug resistance. Changes in transport and metabolism of HMAs are among the most studied mechanisms of resistance. Drug uptake provided by two solute carrier (SLC) families: SLC28 and SLC29 (also known as the concentrative and equilibrative nucleoside transporter families, respectively), could represent one of the mechanisms of cross-resistance. Changes in the metabolism of these drugs are the most likely mechanism responsible for the unique mode of resistance to AZA and DAC. Deoxycytidine kinase and uridine-cytidine kinase due to their necessity for drug activation, each could represent one of the response markers to treatment with DAC and AZA, respectively. Other mechanisms involved in the development of resistance common for both drugs involved: i. increased DNA repair (caused for example by constitutive activation of the ATM/BRCA1 pathway and inhibition of p53-dependent apoptosis); ii. changes in the regulation of apoptosis/disrupted apoptotic pathways (specifically increased levels of the anti-apoptotic protein BCL2) and iii. increased resilience of leukemic stem cells to multiple drugs including HMAs. Despite intense research on the resistance of MDS and AML patients to HMAs, the mechanisms that may reduce the response of these cells to HMAs are not known in detail. We herein highlight the most important directions that future research should take.

Comparison the effects of 5-Aza-2'-deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos.

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.

Epigenetic Activation of TUSC3 Sensitizes Glioblastoma to Temozolomide Independent of MGMT Promoter Methylation Status.

Temozolomide (TMZ) is an important first-line treatment for glioblastoma (GBM), but there are limitations to TMZ response in terms of durability and dependence on the promoter methylation status of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT-promoter-hypermethylated (MGMT-M) GBMs are more sensitive to TMZ than MGMT-promoter-hypomethylated (MGMT-UM) GBMs. Moreover, TMZ resistance is inevitable even in TMZ-sensitive MGMT-M GBMs. Hence, epigenetic reprogramming strategies are desperately needed in order to enhance TMZ response in both MGMT-M and MGMT-UM GBMs. In this study, we present novel evidence that the epigenetic reactivation of Tumor Suppressor Candidate 3 (TUSC3) can reprogram sensitivity of GBM stem cells (GSCs) to TMZ irrespective of MGMT promoter methylation status. Interrogation of TCGA patient GBM datasets confirmed TUSC3 promoter regulation of TUSC3 expression and also revealed a strong positive correlation between TUSC3 expression and GBM patient survival. Using a combination of loss-of-function, gain-of-function and rescue studies, we demonstrate that TUSC3 reactivation is associated with enhanced TMZ response in both MGMT-M and MGMT-UM GSCs. Further, we provide novel evidence that the demethylating agent 5-Azacitidine (5-Aza) reactivates TUSC3 expression in MGMT-M GSCs, whereas the combination of 5-Aza and MGMT inhibitor Lomeguatrib is necessary for TUSC3 reactivation in MGMT-UM GSCs. Lastly, we propose a pharmacological epigenetic reactivation strategy involving TUSC3 that leads to significantly prolonged survival in MGMT-M and MGMT-UM orthotopic GSCs models. Collectively, our findings provide a framework and rationale to further explore TUSC3-mediated epigenetic reprogramming strategies that could enhance TMZ sensitivity and outcomes in GBM. Mechanistic and translational evidence gained from such studies could contribute towards optimal design of impactful trials for MGMT-UM GBMs that currently do not have good treatment options.

5-Aza-2'-Deoxycytidine Alters the Methylation Profile of Bortezomib-Resistant U266 Multiple Myeloma Cells and Affects Their Proliferative Potential.

Multiple myeloma (MM) is a plasma cell malignancy that accounts for 1% of all cancers and is the second-most-common hematological neoplasm. Bortezomib (BTZ) is a proteasome inhibitor widely implemented in the treatment of MM alone or in combination with other agents. The development of resistance to chemotherapy is one of the greatest challenges of modern oncology. Therefore, it is crucial to discover and implement new adjuvant therapies that can bypass therapeutic resistance. In this paper, we investigated the in vitro effect of methylation inhibitor 5-Aza-2'-deoxycytidine on the proliferative potential of MM cells and the development of resistance to BTZ. We demonstrate that alterations in the DNA methylation profile are associated with BTZ resistance. Moreover, the addition of methylation inhibitor 5-Aza-2'-deoxycytidine to BTZ-resistant MM cells led to a reduction in the proliferation of the BTZ-resistant phenotype, resulting in the restoration of sensitivity to BTZ. However, further in vitro and ex vivo studies are required before adjuvant therapy can be incorporated into existing treatment regimens.

The Methylation Analysis of the Glucose-Dependent Insulinotropic Polypeptide Receptor (GIPR) Locus in GH-Secreting Pituitary Adenomas.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is aberrantly expressed in about one-third of GH-secreting pituitary adenomas (GH-PAs) and has been associated with a paradoxical increase of GH after a glucose load. The reason for such an overexpression has not yet been clarified. In this work, we aimed to evaluate whether locus-specific changes in DNA methylation patterns could contribute to this phenomenon. By cloning bisulfite-sequencing PCR, we compared the methylation pattern of the GIPR locus in GIPR-positive (GIPR+) and GIPR-negative (GIPR-) GH-PAs. Then, to assess the correlation between Gipr expression and locus methylation, we induced global DNA methylation changes by treating the lactosomatotroph GH3 cells with 5-aza-2'-deoxycytidine. Differences in methylation levels were observed between GIPR+ and GIPR- GH-PAs, both within the promoter (31.9% vs. 68.2%, p < 0.05) and at two gene body regions (GB_1 20.7% vs. 9.1%; GB_2 51.2% vs. 65.8%, p < 0.05). GH3 cells treated with 5-aza-2'-deoxycytidine showed a ~75% reduction in Gipr steady-state level, possibly associated with the observed decrease in CpGs methylation. These results indicate that epigenetic regulation affects GIPR expression in GH-PAs, even though this possibly represents only a part of a much more complex regulatory mechanism.

Higher Expression Levels of SSX1 and SSX2 in Patients with Colon Cancer: Regulated In Vitro by the Inhibition of Methylation and Histone Deacetylation.

Background and Objectives: Colon cancer (CC) has a high mortality rate and is often diagnosed at an advanced stage in Saudi Arabia. Thus, the identification and characterization of potential new cancer-specific biomarkers are imperative for improving the diagnosis of CC by detecting it at an early stage. Cancer-testis (CT) genes have been identified as potential biomarkers for the early diagnosis of various cancers. Among the CT genes are those belonging to the SSX family. In order to assess the usefulness of SSX family genes as cancer biomarkers for the detection of early-stage CC, the goal of this research was to validate the expressions of these genes in patients with CC and in matched patients with normal colons (NCs). Materials and Methods: RT-PCR assays were used to analyze the SSX1, SSX2, and SSX3 family gene expression levels in 30 neighboring NC and CC tissue samples from male Saudi patients. Epigenetic alterations were also tested in vitro using qRT-PCR analysis to determine whether reduced DNA methyltransferase or histone deacetylation could stimulate SSX gene expression via 5-aza-2'-deoxycytidine and trichostatin treatments, respectively. Results: The RT-PCR results showed SSX1 and SSX2 gene expression in 10% and 20% of the CC tissue specimens, respectively, but not in any of the NC tissue specimens. However, no SSX3 expression was detected in any of the examined CC or NC tissue samples. In addition, the qRT-PCR results showed significantly higher SSX1 and SSX2 expression levels in the CC tissue samples than in the NC tissue samples. The 5-aza-2'-deoxycytidine and trichostatin treatments significantly induced the mRNA expression levels of the SSX1, SSX2, and SSX3 genes in the CC cells in vitro. Conclusions: These findings suggest that SSX1 and SSX2 are potentially suitable candidate biomarkers for CC. Their expressions can be regulated via hypomethylating and histone deacetylase treatments, subsequently providing a potential therapeutic target for CC.

5-Aza-2'-deoxycytidine advances the epithelial-mesenchymal transition of breast cancer cells by demethylating Sipa1 promoter-proximal elements.

Human breast cancer cells exhibit considerable diversity in the methylation status of genomic DNA CpGs that regulate metastatic transcriptome networks. In this study, we identified human Sipa1 promoter-proximal elements that contained a CpG island and demonstrated that the methylation status of the CpG island was inversely correlated with SIPA1 protein expression in cancer cells. 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, promoted the expression of Sipa1 in the MCF7 breast cancer cells with a low level of SIPA1 expression. On the contrary, in MDA-MB-231 breast cancer cells with high SIPA1 expression levels, hypermethylation of the CpG island negatively regulated the transcription of Sipa1 In addition, the epithelial-mesenchymal transition (EMT) was reversed after knocking down Sipa1 in MDA-MB-231 cells. However, the EMT was promoted in MCF7 cells with over-expression of SIPA1 or treated with 5-Aza-CdR. Taken together, hypomethylation of the CpG island in Sipa1 promoter-proximal elements could enhance SIPA1 expression in breast cancer cells, which could facilitate EMT of cancer cells, possibly increasing a risk of cancer cell metastasis in individuals treated with 5-Aza-CdR.

Epigenetic Anti-Cancer Treatment With a Stabilized Carbocyclic Decitabine Analogue.

5-Aza-2'-deoxycytidine (Decitabine, AzadC) is a nucleoside analogue, which is in clinical use to treat patients with myelodysplastic syndrome or acute myeloid leukemia. Its mode of action is unusual because the compound is one of the few drugs that act at the epigenetic level of the genetic code. AzadC is incorporated as an antimetabolite into the genome and creates covalent, inhibitory links to DNA methyltransferases (DNMTs) that methylate 2'-deoxycytidine (dC) to 5-methyl-dC (mdC). Consequently, AzadC treatment leads to a global loss of mdC, which presumably results in a reactivation of silenced genes, among them tumor suppressor and DNA damage response genes. Because AzadC suffers from severe instability, which limits its use in the clinic, a more sophisticated AzadC derivative would be highly valuable. Here, we report that a recently developed carbocyclic AzadC analogue (cAzadC) blocks DNMT1 in the AML cell line MOLM-13 as efficient as AzadC. Moreover, cAzadC has a surprisingly strong anti-proliferative effect and leads to a significantly higher number of double strand breaks compared to AzadC, while showing less off-target toxicity. These results show that cAzadC triggers more deleterious repair and apoptotic pathways in cancer cells than AzadC, which makes cAzadC a promising next generation epigenetic drug.

Iodine-125 seed represses the growth and facilitates the apoptosis of colorectal cancer cells by suppressing the methylation of miR-615 promoter.

BACKGROUND: Colorectal cancer (CRC) represents a common malignancy in gastrointestinal tract. Iodine-125 (125I) seed implantation is an emerging treatment technology for unresectable tumors. This study investigated the mechanism of 125I seed in the function of CRC cells. METHODS: The CRC cells were irradiated with different doses of 125I seed (0.4, 0.6 and 0.8 mCi). miR-615 expression in CRC tissues and adjacent tissues was detected by RT-qPCR. miR-615 expression was intervened with miR-615 mimic or miR-615 inhibitor, and then the CRC cells were treated with 5-AZA (methylation inhibitor). The CRC cell growth, invasion and apoptosis were measured. The methylation level of miR-615 promoter region was detected. The xenograft tumor model irradiated by 125I seed was established in nude mice. The methylation of miR-615, Ki67 expression and CRC cell apoptosis were detected. RESULTS: 125I seed irradiation repressed the growth and facilitated apoptosis of CRC cells in a dose-dependent manner. Compared with adjacent tissues, miR-615 expression in CRC tissues was downregulated and miR-615 was poorly expressed in CRC cells. Overexpression of miR-615 suppressed the growth of CRC cells. 125I seed-irradiated CRC cells showed increased miR-615 expression, reduced growth rate and enhanced apoptosis. The methylation level of miR-615 promoter region in CRC cells was decreased after 125I seed treatment. In vivo experiments confirmed that 125I seed-irradiated xenograft tumors showed reduced methylation of the miR-615 promoter and increased miR-615 expression, as well as decreased Ki67 expression and enhanced apoptosis. The target genes of miR-615 and its regulatory downstream pathway were further predicted by bioinformatics analysis. CONCLUSIONS: 125I seed repressed the growth and facilitated the apoptosis of CRC cells by suppressing the methylation of the miR-615 promoter and thus activating miR-615 expression. The possible mechanism was that miR-615-5p targeted MAPK13, thus affecting the MAPK pathway and the progression of CRC.

Combination effect of poly (ADP-ribose) polymerase inhibitor and DNA demethylating agents for treatment of epithelial ovarian cancer.

OBJECTIVE: Poly (ADP)-ribose polymerase inhibitors (PARPi) are effective clinical agents for treatment of epithelial ovarian cancer (EOC) harboring BRCA mutations as well as those without BRCA mutations. In this study, we evaluate the efficacy of combined PARPi and DNA methyltransferase inhibitor (DNMTi) in EOCs. METHODS: Expression levels of DNMT1 and PARP1 proteins in EOC cells were assessed using western blot analysis and immunohistochemistry. To evaluate the effects of co-treatment of PARPi (olaparib) and DNMTi (5-azacitidine, 5-AZA), we performed cell proliferation, apoptosis, and wound-healing assays in EOC cells. In addition, we performed in vivo experiments using both cell-line and patient-derived xenograft (PDX) models of EOC. RESULTS: The combination of olaparib and 5-AZA significantly inhibited cell proliferation and migration and induced apoptosis compared with olaparib or 5-AZA alone in EOC cell lines including A2780, HeyA8, A2780-CP20, and HeyA8-MDR. Moreover, in vivo experiments with this combination showed significantly decreased weight and nodule numbers of tumors in cell-line xenograft models with A2780 cells and a PDX model compared with control, olaparib, and 5-AZA groups. As a potential mechanism, the expression of intracellular reactive oxygen species (ROS) and its related proteins, including p-ERK, NRF2, p-p38, HO-1, and γH2AX, was affected in EOC cells. CONCLUSIONS: Co-treatment with PARPi and DNMTi had a significant anti-tumor effect in EOC cells. This combination might be a potential therapeutic strategy for EOCs.

The effect of shear stress on cardiac differentiation of mesenchymal stem cells.

BACKGROUND: Stem cell therapy is developing as a valuable therapeutic trend for heart diseases. Most recent studies are aimed to find the most appropriate types of stem cells for the treatment of myocardial infarction (MI). The animal models have shown that bone marrow-derived mesenchymal stem cells (BMSCs) are a possible, safe, and efficient type of stem cell used in MI. The previous study demonstrated that 5-Azacytidine (5-Aza) could promote cardiac differentiation in stem cells. METHODS: This study used 5-Aza to induce cardiomyocyte differentiation in BMSCs both in static and microfluidic cell culture systems. For this purpose, we investigated the differentiation by using real-time PCR and Immunocytochemistry (ICC) Analysis. RESULTS: Our results showed that 5-Aza could cause to express cardiac markers in BMSCs as indicated by real-time PCR and immunocytochemistry (ICC). However, BMSCs are exposed to both 5-Aza and shear stress, and their synergistic effects could significantly induce cardiac gene expressions in BMSCs. This level of gene expression was observed neither in 5-Aza nor in shear stress groups only. CONCLUSIONS: These results demonstrate that when BMSCs expose to 5-Aza as well as mechanical cues such as shear stress, the cardiac gene expression can be increased dramatically.

Trichostatin A-Mediated Epigenetic Modulation Predominantly Triggers Transcriptomic Alterations in the Ex Vivo Expanded Equine Chondrocytes.

Epigenetic mechanisms of gene regulation are important for the proper differentiation of cells used for therapeutic and regenerative purposes. The primary goal of the present study was to investigate the impacts of 5-aza-2' deoxycytidine (5-AZA-dc)- and/or trichostatin A (TSA)-mediated approaches applied to epigenomically modulate the ex vivo expanded equine chondrocytes maintained in monolayer culture on the status of chondrogenic cytodifferentiation at the transcriptome level. The results of next-generation sequencing of 3' mRNA-seq libraries on stimulated and unstimulated chondrocytes of the third passage showed no significant influence of 5-AZA-dc treatment. Chondrocytes stimulated with TSA or with a combination of 5-AZA-dc+TSA revealed significant expressional decline, mainly for genes encoding histone and DNA methyltransferases, but also for other genes, many of which are enriched in canonical pathways that are important for chondrocyte biology. The TSA- or 5-AZA-dc+TSA-induced upregulation of expanded chondrocytes included genes that are involved in histone hyperacetylation and also genes relevant to rheumatoid arthritis and inflammation. Chondrocyte stimulation experiments including a TSA modifier also led to the unexpected expression incrementation of genes encoding HDAC3, SIRT2, and SIRT5 histone deacetylases and the MBD1 CpG-binding domain protein, pointing to another function of the TSA agent besides its epigenetic-like properties. Based on the transcriptomic data, TSA stimulation seems to be undesirable for chondrogenic differentiation of passaged cartilaginous cells in a monolayer culture. Nonetheless, obtained transcriptomic results of TSA-dependent epigenomic modification of the ex vivo expanded equine chondrocytes provide a new source of data important for the potential application of epigenetically altered cells for transplantation purposes in tissue engineering of the equine skeletal system.

5-aza-2'-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/ß-catenin pathway in human uterine leiomyomas.

BACKGROUND: Uterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2'-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option. METHODS: Prospective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR. RESULTS: DNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 µM (85.3%). Treatment with 10 µM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/ß-catenin pathway final targets, including WISP1 protein expression (10 µM, FC = 0.699), c-MYC gene expression (2 µM, FC = 0.745 and 10 µM, FC = 0.728), and MMP7 gene expression (5 µM, FC = 0.520 and 10 µM, FC = 0.577). CONCLUSIONS: 5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/ß-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

Methylation of RILP in lung cancer promotes tumor cell proliferation and invasion.

Rab-interacting lysosomal protein (RILP) has been suggested to perform as a tumor suppressor in breast and prostate cancer cell lines. However, its expression profile and functional role in lung cancer have never been investigated. We applied the well-established cancer genomic database-The Cancer Genome Atlas to compare the RILP expression and methylation between lung cancer tissues and normal tissues. The potential correlation of RILP with clinical characteristics of lung cancer patients (e.g., stages, smoking, TP53, and methylation) was also be explored. Our results showed that the downregulation of RILP and upregulation of RILP methylation were identified in lung cancer tissues compared to normal healthy tissues. Downregulation of RILP was positively associated with lung cancer later stage (N3), smoking history, TP53 mutation, and poor prognosis, as well as inversely correlated with DNA (cytosine-5)-methyltransferase 1 (DNMT1) expression. Demethylation treatment enhanced RILP expression in lung cancer cells, suggesting hypermethylation is responsible for RILP silencing in lung cancer. We further found that RILP depletion promoted lung cancer cell proliferation, migration, and invasion. We concluded that RILP acts as a tumor suppressor in lung cancer cells. Our results provided the theoretical basis for developing RILP-targeting or demethylating agents for lung cancer treatment.

Role of epigenetic regulation in myocardial ischemia/reperfusion injury.

Nowadays acute myocardial infarction (AMI) is a serious cardiovascular disease threatening the human life and health worldwide. The most effective treatment is to quickly restore coronary blood flow through revascularization. However, timely revascularization may lead to reperfusion injury, thereby reducing the clinical benefits of revascularization. At present, no effective treatment is available for myocardial ischemia/reperfusion injury. Emerging evidence indicates that epigenetic regulation is closely related to the pathogenesis of myocardial ischemia/reperfusion injury, indicating that epigenetics may serve as a novel therapeutic target to ameliorate or prevent ischemia/reperfusion injury. This review aimed to briefly summarize the role of histone modification, DNA methylation, noncoding RNAs, and N6-methyladenosine (m6A) methylation in myocardial ischemia/reperfusion injury, with a view to providing new methods and ideas for the research and treatment of myocardial ischemia/reperfusion injury.