Epigenome-wide association study of diabetic chronic kidney disease progression in the Korean population: the KNOW-CKD study.

Since the etiology of diabetic chronic kidney disease (CKD) is multifactorial, studies on DNA methylation for kidney function deterioration have rarely been performed despite the need for an epigenetic approach. Therefore, this study aimed to identify epigenetic markers associated with CKD progression based on the decline in the estimated glomerular filtration rate in diabetic CKD in Korea. An epigenome-wide association study was performed using whole blood samples from 180 CKD recruited from the KNOW-CKD cohort. Pyrosequencing was also performed on 133 CKD participants as an external replication analysis. Functional analyses, including the analysis of disease-gene networks, reactome pathways, and protein-protein interaction networks, were conducted to identify the biological mechanisms of CpG sites. A phenome-wide association study was performed to determine the associations between CpG sites and other phenotypes. Two epigenetic markers, cg10297223 on AGTR1 and cg02990553 on KRT28 indicated a potential association with diabetic CKD progression. Based on the functional analyses, other phenotypes (blood pressure and cardiac arrhythmia for AGTR1) and biological pathways (keratinization and cornified envelope for KRT28) related to CKD were also identified. This study suggests a potential association between the cg10297223 and cg02990553 and the progression of diabetic CKD in Koreans. Nevertheless, further validation is needed through additional studies.

Identification of a novel PARP4 gene promoter CpG locus associated with cisplatin chemoresistance.

The protein family of poly (ADP-ribose) polymerases (PARPs) is comprised of multifunctional nuclear enzymes. Several PARP inhibitors have been developed as new anticancer drugs to combat resistance to chemotherapy. Herein, we characterized PARP4 mRNA expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. PARP4 mRNA expression was significantly upregulated in cisplatin-resistant ovarian cancer cell lines, and this upregulation was associated with the hypomethylation of specific cytosine-phosphate-guanine (CpG) sites (cg18582260 and cg17117459) on its promoter. Reduced PARP4 expression was restored by treating cisplatin-sensitive cell lines with a demethylation agent, implicating the epigenetic regulation of PARP4 expression by promoter methylation. Depletion of PARP4 expression in cisplatin-resistant cell lines reduced cisplatin chemoresistance and promoted cisplatin-induced DNA fragmentation. The differential mRNA expression and DNA methylation status at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) according to cisplatin responses, was further validated in primary ovarian tumor tissues. The results showed significantly increased PARP4 mRNA expressions and decreased DNA methylation levels at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) in cisplatin-resistant patients. Additionally, the DNA methylation status at cg18582260 CpG sites in ovarian tumor tissues showed fairly clear discrimination between cisplatin-resistant patients and cisplatin-sensitive patients, with high accuracy (area under the curve = 0.86, P = 0.003845). Our findings suggest that the DNA methylation status of PARP4 at the specific promoter site (cg18582260) may be a useful diagnostic biomarker for predicting the response to cisplatin in ovarian cancer patients. [BMB Reports 2023; 56(6): 347-352].

Novel Genetic Variants Associated with Chronic Kidney Disease Progression.

SIGNIFICANCE STATEMENT: eGFR slope has been used as a surrogate outcome for progression of CKD. However, genetic markers associated with eGFR slope among patients with CKD were unknown. We aimed to identify genetic susceptibility loci associated with eGFR slope. A two-phase genome-wide association study identified single nucleotide polymorphisms (SNPs) in TPPP and FAT1-LINC02374 , and 22 of them were used to derive polygenic risk scores that mark the decline of eGFR by disrupting binding of nearby transcription factors. This work is the first to identify the impact of TPPP and FAT1-LINC02374 on CKD progression, providing predictive markers for the decline of eGFR in patients with CKD. BACKGROUND: The incidence of CKD is associated with genetic factors. However, genetic markers associated with the progression of CKD have not been fully elucidated. METHODS: We conducted a genome-wide association study among 1738 patients with CKD, mainly from the KoreaN cohort study for Outcomes in patients With CKD. The outcome was eGFR slope. We performed a replication study for discovered single nucleotide polymorphisms (SNPs) with P <10 -6 in 2498 patients with CKD from the Chronic Renal Insufficiency Cohort study. Several expression quantitative trait loci (eQTL) studies, pathway enrichment analyses, exploration of epigenetic architecture, and predicting disruption of transcription factor (TF) binding sites explored potential biological implications of the loci. We developed and evaluated the effect of polygenic risk scores (PRS) on incident CKD outcomes. RESULTS: SNPs in two novel loci, TPPP and FAT1-LINC02374 , were replicated (rs59402340 in TPPP , Pdiscovery =7.11×10 -7 , PCRIC =8.13×10 -4 , Pmeta =7.23×10 -8 ; rs28629773 in FAT1-LINC02374 , Pdiscovery =6.08×10 -7 , PCRIC =4.33×10 -2 , Pmeta =1.87×10 -7 ). The eQTL studies revealed that the replicated SNPs regulated the expression level of nearby genes associated with kidney function. Furthermore, these SNPs were near gene enhancer regions and predicted to disrupt the binding of TFs. PRS based on the independently significant top 22 SNPs were significantly associated with CKD outcomes. CONCLUSIONS: This study demonstrates that SNP markers in the TPPP and FAT1-LINC02374 loci could be predictive markers for the decline of eGFR in patients with CKD.

Increased Caveolin-2 Expression in Brain Endothelial Cells Promotes Age-Related Neuroinflammation.

Aging is a major risk factor for common neurodegenerative diseases. Although multiple molecular, cellular, structural, and functional changes occur in the brain during aging, the involvement of caveolin-2 (Cav-2) in brain ageing remains unknown. We investigated Cav-2 expression in brains of aged mice and its effects on endothelial cells. The human umbilical vein endothelial cells (HUVECs) showed decreased THP-1 adhesion and infiltration when treated with Cav-2 siRNA compared to control siRNA. In contrast, Cav-2 overexpression increased THP-1 adhesion and infiltration in HUVECs. Increased expression of Cav-2 and iba-1 was observed in brains of old mice. Moreover, there were fewer iba-1-positive cells in the brains of aged Cav-2 knockout (KO) mice than of wild-type aged mice. The levels of several chemokines were higher in brains of aged wild-type mice than in young wild-type mice; moreover, chemokine levels were significantly lower in brains of young mice as well as aged Cav-2 KO mice than in their wild-type counterparts. Expression of PECAM1 and VE-cadherin proteins increased in brains of old wild-type mice but was barely detected in brains of young wild-type and Cav-2 KO mice. Collectively, our results suggest that Cav-2 expression increases in the endothelial cells of aged brain, and promotes leukocyte infiltration and age-associated neuroinflammation.

Identification of Serum Metabolites for Predicting Chronic Kidney Disease Progression according to Chronic Kidney Disease Cause.

Early detection and proper management of chronic kidney disease (CKD) can delay progression to end-stage kidney disease. We applied metabolomics to discover novel biomarkers to predict the risk of deterioration in patients with different causes of CKD. We enrolled non-dialytic diabetic nephropathy (DMN, n = 124), hypertensive nephropathy (HTN, n = 118), and polycystic kidney disease (PKD, n = 124) patients from the KNOW-CKD cohort. Within each disease subgroup, subjects were categorized as progressors (P) or non-progressors (NP) based on the median eGFR slope. P and NP pairs were randomly selected after matching for age, sex, and baseline eGFR. Targeted metabolomics was performed to quantify 188 metabolites in the baseline serum samples. We selected ten progression-related biomarkers for DMN and nine biomarkers each for HTN and PKD. Clinical parameters showed good ability to predict DMN (AUC 0.734); however, this tendency was not evident for HTN (AUC 0.659) or PKD (AUC 0.560). Models constructed with selected metabolites and clinical parameters had better ability to predict CKD progression than clinical parameters only. When selected metabolites were used in combination with clinical indicators, random forest prediction models for CKD progression were constructed with AUCs of 0.826, 0.872, and 0.834 for DMN, HTN, and PKD, respectively. Select novel metabolites identified in this study can help identify high-risk CKD patients who may benefit from more aggressive medical treatment.

Protective role of ABCA1 in ischemic preconditioning is mediated by downregulation of miR-33-5p and miR-135-5p.

Ischemic preconditioning (IPC) significantly reduces ischemia-reperfusion injury in the brain by inducing ischemic tolerance. Although emerging evidence suggests that microRNAs (miRNAs) contribute to the pathogenesis of brain ischemia and IPC-induced neuroprotection, the role of miRNAs and their underlying mechanisms are still unclear. IPC was induced in male C57BL/6 mice by brief bilateral common carotid artery occlusion. After 24 h, mice underwent transient middle cerebral artery occlusion followed by 3 h of reperfusion. Expression levels of messenger RNAs (mRNAs) and proteins were examined in the ipsilateral cortex, and mimics and inhibitors of selective miRNAs were transfected into Neuro-2a cells before oxygen-glucose deprivation (OGD). Post-IPC miRNA expression profiling identified neuroprotection-associated changes in miRNA expression in the ipsilateral cortex after ischemic stroke. Among them, miR-33-5p and miR-135b-5p were significantly downregulated by IPC. Inhibition of miR-33-5p and miR-135b-5p expression protected Neuro-2a cells from OGD-induced apoptosis. Inhibition of these two miRNAs significantly increased mRNA and protein levels of ATP-binding cassette subfamily A member 1 (ABCA1), and a binding assay showed that these two miRNAs showed specificity for Abca1 mRNA. Overexpression of ABCA1 decreased the Bax/Bcl2 mRNA ratio and activation of caspase-9 and caspase-3, whereas knockdown of ABCA1 expression increased the Bax/Bcl2 mRNA ratio and the percentage of Neuro-2a cells with a loss of mitochondrial membrane potential after OGD-treatment. In conclusion, ABCA1 expression is regulated by miR-33-5p and miR-135b-5p. Increased ABCA1 expression following IPC exerts a protective influence against cerebral ischemia via suppression of a mitochondria-dependent apoptosis pathway.

Enhanced repair processes and iron uptake by ischemic preconditioning in the brain during the recovery phase after ischemic stroke.

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood-brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.

Ahnak scaffolds p11/Anxa2 complex and L-type voltage-gated calcium channel and modulates depressive behavior.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the ß subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

SPSB1 enhances ovarian cancer cell survival by destabilizing p21.

SPRY domain-containing SOCS box protein 1 (SPSB1) is an E3 ligase adaptor protein with unknown functions in cancer cells. In this study, we found that SPSB1 knockdown markedly decreased the viability and migration of ovarian cancer cells, while ectopic SPSB1 overexpression in IL-3-dependent Ba/F3 cells significantly increased their proliferation rate compared with empty vector-transfected cells. SPSB1 knockdown significantly elevated p21 protein and mRNA levels and induced apoptosis in ovarian cancer cells, as evidenced by increased levels of cleaved PARP and decreased levels of Bcl-2. Notably, mechanistic investigations revealed that SPSB1 accelerated p21 destabilization by directly interacting with p21 and promoting its ubiquitin-mediated proteasomal degradation. Taken together, our findings provide novel insights into the role of SPSB1 in ovarian cancer cells.

Anti-inflammatory and antioxidant mechanisms of urolithin B in activated microglia.

BACKGROUND: Urolithin B is one of the gut microbial metabolites of ellagitannins and is found in diverse plant foods, including pomegranates, berries, walnuts, tropical fruits, and medicinal herbs. Although a number of biological activities of urolithin B have been reported, the anti-inflammatory and antioxidant effects of urolithin B in neuroinflammation have not been clearly demonstrated. PURPOSE: The present study aimed to investigate the anti-inflammatory and antioxidant effects of urolithin B in activated microglia and define its underlying molecular mechanisms. STUDY DESIGN: The effects of urolithin B on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and cytokines were examined in BV2 microglial cells using enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Microglial activation in the lipopolysaccharide (LPS)-injected mouse brain was assessed using immunohistochemistry. The detailed molecular mechanisms underlying the anti-inflammatory and antioxidant effects of urolithin B were analyzed using an electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR. RESULTS: Urolithin B inhibited the production of NO and pro-inflammatory cytokines, while increased anti-inflammatory cytokine IL-10 in LPS-stimulated BV2 microglial cells. In addition, urolithin B inhibited NO, TNF-α, and IL-6 production in lipoteichoic acid (LTA) or polyinosinic-polycytidylic acid (poly(I:C))-stimulated BV2 cells, suggesting that the anti-inflammatory effect of urolithin B is not confined to LPS stimulation. Urolithin B also showed an antioxidant effect by reducing intracellular reactive oxygen species (ROS) production and NADPH oxidase subunit expression, and by upregulating the antioxidant hemeoxygenase-1 expression via Nrf2/ARE signaling. More detailed mechanistic studies showed that urolithin B inhibited NF-κB activity by reducing the phosphorylation and degradation of IκBα. In addition, urolithin B suppressed the phosphorylation of JNK, ERK, and Akt, and enhanced the phosphorylation of AMPK, which is associated with anti-inflammatory and antioxidant processes. Finally, we demonstrated that urolithin B suppressed microglia activation in LPS-injected mouse brains. CONCLUSIONS: The strong anti-inflammatory and antioxidant effects of urolithin B may provide therapeutic potential for neuroinflammatory disorders that are associated with oxidative stress and microglial activation.

Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker.

BACKGROUND AND PURPOSE: The pathogenesis of moyamoya disease (MMD) remains poorly understood, and no reliable molecular biomarkers for MMD have been identified to date. The present study aimed to identify epigenetic biomarkers for use in the diagnosis of MMD. METHODS: We performed integrated analyses of gene expression profiles and DNA methylation profiles in endothelial colony forming cells (ECFCs) from three patients with MMD and two healthy individuals. Candidate gene mRNA expression and DNA methylation status were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and pyrosequencing analysis of an expanded ECFC sample set from nine patients with MMD and ten controls. We evaluated the diagnostic accuracy of the potential biomarkers identified here using receiver operating characteristic curve analyses and further measured major angiogenic factor expression levels using a tube formation assay and RT-qPCR. RESULTS: Five candidate genes were selected via integrated analysis; all five were upregulated by hypomethylation of specific promoter CpG sites. After further validation in an expanded sample set, we identified a candidate biomarker gene, sortilin 1 (SORT1). DNA methylation status at a specific SORT1 promoter CpG site in ECFCs readily distinguished patients with MMD from the normal controls with high accuracy (area under the curve 0.98, sensitivity 83.33%, specificity 100%). Furthermore, SORT1 overexpression suppressed endothelial cell tube formation and modulated major angiogenic factor and matrix metalloproteinase-9 expression, implying SORT1 involvement in MMD pathogenesis. CONCLUSION: s Our findings suggest that DNA methylation status at the SORT1 promoter CpG site may be a potential biomarker for MMD.

C16­ceramide and sphingosine 1­phosphate/S1PR2 have opposite effects on cell growth through mTOR signaling pathway regulation.

Recently, sphingolipid derivatives, such as ceramide and sphingosine­1­phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF­7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal­regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF­7 cell proliferation. This effect was partially reversed by co­treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6­mediated decrease in MCF­7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF­7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16­ceramide, which is generated by CerS6.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer.

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Effect of Necrosis on the miRNA-mRNA Regulatory Network in CRT-MG Human Astroglioma Cells.

PURPOSE: Glioblastoma multiforme (GBM) is the most common adult primary intracranial tumor. The remarkable features of GBM include central necrosis. MicroRNAs (miRNAs) have been considered as diagnostic/prognostic biomarkers for many cancers, including glioblastoma. However, the effect of necrosis on the miRNA expression profile and predicted miRNA-mRNA regulatory information remain unclear. The purpose of this study is to examine the effect of necrotic cells on the modulation of miRNA and mRNA expression profiles and miRNA-mRNA network in CRT-MG cells. MATERIALS AND METHODS: We used human astroglioma cells, CRT-MG, treated with necrotic CRT-MG cells to examine the effect of necrosis on the modulation of miRNA and mRNA by next-generation sequencing. For preparation of necrotic cells, CRT-MGcellswere frozen and thawed through cycle of liquid nitrogen-water bath. The putative miRNA-mRNA regulatory relationshipwas inferred through target information, using miRDB. RESULTS: The necrotic cells induced dysregulation of 106 miRNAs and 887 mRNAs. Among them, 11 miRNAs that had a negative correlation value of p < 0.05 by the hypergeometric test were screened, and their target mRNAs were analyzed by Gene Ontology enrichment analysis. Using the Kyoto Encyclopedia of Genes and Genomes database, we also found several necrotic cell treatment-activated pathways that were modulated by relevant gene targets of differentially expressed miRNAs. CONCLUSION: Our result demonstrated that dysregulation of miRNA and mRNA expression profiles occurs when GBM cells are exposed to necrotic cells, suggesting that several miRNAs may have the potential to be used as biomarkers for predicting GBM progression and pathogenesis.

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate-induced colitis in mice due to increased intestinal permeability.

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long-chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very-long acyl chain ceramides with concomitant increase of long chain bases and C16-ceramides, were more susceptible to dextran sodium sulphate-induced colitis, and their survival rate was markedly decreased compared with that of wild-type littermates. Using mixed bone-marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule-A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco-2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2-knockdown via CRISPR-Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long-chain bases and C16-ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC-dextran levels, indicating that altered SLs including deficiency of very-long-chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.

Aberrant epigenetic regulation of GABRP associates with aggressive phenotype of ovarian cancer.

Metastasis is a major cause of therapeutic failure in ovarian cancer. To elucidate molecular mechanisms of ovarian cancer metastasis, we previously established a metastatic xenograft mouse model using human ovarian carcinoma SK-OV-3 cells. Using gene expression profiling, we found that γ-aminobutyric acid (GABA)A receptor π subunit (GABRP) expression was upregulated (>4-fold) in metastatic tissues from our xenograft mice compared with SK-OV-3 cells. Importantly, GABRP knockdown diminished the migration and invasion of SK-OV-3 cells, and reduced extracellular signal-regulated kinase (ERK) activation while overexpression of GABRP exhibited significantly increased cell migration, invasion and ERK activation. Moreover, treatment with the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 similarly suppressed the migration and invasion of SK-OV-3 cells, implying that GABRP promotes these cellular behaviors by activating the MAPK/ERK pathway. Using genome-wide DNA methylation profiling, we identified hypomethylated CpG sites in the GABRP promoter in metastatic tissues from the xenograft mice compared with SK-OV-3 cells. Treatment with a DNA methyltransferase inhibitor demonstrated that methylation at -963 bp from the GABRP transcription start site (-963 CpG site) was critical for the epigenetic regulation of GABRP. Finally, we analyzed human ovarian cancer patient samples and showed DNA hypomethylation at the GABRP -963 CpG site in advanced stage, but not early-stage, primary tumors compared with their paired normal tissues. These findings suggest that GABRP enhances the aggressive phenotype of ovarian cancer cells, and that the DNA methylation status of the GABRP -963 CpG site may be useful for predicting the metastatic potential in ovarian cancer patients.

Autophagy induction in the skeletal myogenic differentiation of human tonsil-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.

Aberrant Hypomethylation of Solute Carrier Family 6 Member 12 Promoter Induces Metastasis of Ovarian Cancer.

PURPOSE: Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. MATERIALS AND METHODS: Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. RESULTS: SLC6A12 expression was 8.1-14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41-62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. CONCLUSION: Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.

Synaptotagmin-like protein 2 gene promotes the metastatic potential in ovarian cancer.

Ovarian cancer (OC) metastasis has unique biological behavior and most commonly occurs via the transcoelomic route. Previously, we established a mouse xenograft model of human ovarian carcinoma and analyzed alterations in gene expression during metastasis. Among the genes that were differentially expressed more than 2-fold in the xenografts compared with the SK-OV-3 cells, we selected synaptotagmin-like protein 2 (SYTL2) and investigated the mechanisms regulating its expression and its gene function in OC. The mRNA expression of SYTL2 was significantly upregulated and the methylation of specific CpG sites within the SYTL2 promoter was decreased in the metastatic implants from the ovarian carcinoma xenografts compared to wild-type SK-OV-3 cells. Treatment with the demethylating agent 5-aza­2'-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A induced upregulation of SYTL2 in SK-OV-3 cells, implying that a DNA methylation-dependent epigenetic mechanism is involved in the regulation of SYTL2 expression. We also found that overexpression of SYTL2 promoted metastatic potential, including increased migration and invasiveness in the ovarian carcinoma cells. Furthermore, we utilized publicly available gene expression data to confirm the correlation between SYTL2 expression and poor prognosis in serous-type OC patients. Our findings provide novel evidence for the direct association of SYTL2 with the metastatic potential of ovarian carcinoma cells and its influence on metastatic recurrence of OC.

Amyloid Beta-Mediated Hypomethylation of Heme Oxygenase 1 Correlates with Cognitive Impairment in Alzheimer's Disease.

To identify epigenetically regulated genes involved in the pathogenesis of Alzheimer's disease (AD) we analyzed global mRNA expression and methylation profiles in amyloid precursor protein (APP)-Swedish mutant-expressing AD model cells, H4-sw and selected heme oxygenase-1 (HMOX1), which is associated with pathological features of AD such as neurofibrillary tangles and senile plaques. We examined the epigenetic regulatory mechanism of HMOX1 and its application as a diagnostic and prognostic biomarker for AD. Our results show that HMOX1 mRNA and protein expression was approximately 12.2-fold and 7.9-fold increased in H4-sw cells, respectively. Increased HMOX1 expression was also detected in the brain, particularly the hippocampus, of AD model transgenic mice. However, the methylation of specific CpG sites within its promoter, particularly at CpG located -374 was significantly decreased in H4-sw cells. Treatment of neuroglioma cells with the demethylating agent 5-aza-2'-deoxycytidine resulted in reduced methylation of HMOX1 promoter accompanied by enhanced HMOX1 expression strongly supporting DNA methylation-dependent transcriptional regulation of HMOX1. Toxic Aß-induced aberrant hypomethylation of HMOX1 at -374 promoter CpG site was correlated with increased HMOX1 expression. In addition to neuroglioma cells, we also found Aß-induced epigenetic regulation of HMOX1 in human T lymphocyte Jurkat cells. We evaluated DNA methylation status of HMOX1 at -374 promoter CpG site in blood samples from AD patients, patients with mild cognitive impairment (MCI), and control individuals using quantitative methylation-specific polymerase chain reaction. We observed lower methylation of HMOX1 at the -374 promoter CpG site in AD patients compared to MCI and control individuals, and a correlation between Mini-Mental State Examination score and demethylation level. Receiver operating characteristics analysis revealed good discrimination of AD patients from MCI patients and control individuals. Our findings suggest that the methylation status of HMOX1 at a specific promoter CpG site is related to AD progression.