Lower mRNA Expression Levels Research Articles

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy.

SummaryMultiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.

Asymmetric expression of H19 and ADIPOQ in concave/convex paravertebral muscles is associated with severe adolescent idiopathic scoliosis

BackgroundAdolescent idiopathic scoliosis (AIS) is the most common paediatric spinal deformity. The etiology and pathology of AIS remain unexplained, and have been reported to involve a combination of genetic and epigenetic factors. Since paravertebral muscle imbalance plays an important role in the onset and progression of scoliosis, we aimed to investigate transcriptomic differences by RNA-seq and identify significantly differentially expressed transcripts in two sides of paravertebral muscle in AIS.MethodsRNA-seq was performed on 5 pairs of paravertebral muscle from 5 AIS patients. Significantly differentially expressed transcripts were validated by quantitative reverse polymerase chain reaction. Gene expression difference was correlated to clinical characteristics.ResultsWe demonstrated that ADIPOQ mRNA and H19 is significantly differentially expressed between two sides of paravertebral muscle, relatively specific in the context of AIS. Relatively low H19 and high ADIPOQ mRNA expression levels in concave-sided muscle are associated with larger spinal curve and earlier age at initiation. We identified miR-675-5p encoded by H19 as a mechanistic regulator of ADIPOQ expression in AIS. We demonstrated that significantly reduced CCCTC-binding factor (CCTF) occupancy in the imprinting control region (ICR) of the H19 gene in the concave-sided muscle contributes to down-regulated H19 expression.ConclusionsRNA-seq revealed transcriptomic differences between two sides of paravertebral muscle in AIS patients. Our findings imply that transcriptomic differences caused by epigenetic factors in affected individuals may account for the structural and functional imbalance of paravertebral muscle, which can expand our etiologic understanding of this disease.

Histone deacetylase inhibitors sensitize 5-fluorouracil-resistant MDA-MB-468 breast cancer cells to 5-fluorouracil.

Resistance to 5-fluorouracil (5-FU) is a serious problem in cancer therapy and overcoming it is required in order to improve the efficacy of cancer chemotherapy. Histone deacetylase (HDAC) inhibitors are used in cancer treatments and, recently, it has been reported that HDAC inhibitors can overcome resistance to various anti-cancer drugs in vitro. In the present study, a 5-FU-resistant breast cancer cell line was established, and the effects of HDAC inhibitors in these cells were examined. The 5-FU-resistant cell line MDA-MB-468 (MDA468/FU) was established by continuous exposure of the parental cells to 5-FU. This subline was characterized by high resistance to 5-FU, higher mRNA expression levels of thymidylate synthetase and dihydropyrimidine dehydrogenase (DPD), and lower mRNA expression levels of uridine monophosphate synthetase (UMPS) than the parental cells. Gimeracil, a DPD inhibitor, did not affect the sensitivity of MDA468/FU cells to 5-FU. Oteracil, a UMPS inhibitor, decreased the cytotoxicity of 5-FU in MDA468 cells, but not in MDA468/FU cells. The HDAC inhibitors, valproic acid and suberanilohydroxamic acid sensitized the two cell lines to 5-FU in a concentration-dependent manner. In conclusion, the results of the present study revealed that HDAC inhibitors increase the sensitivity to 5-FU in 5-FU-sensitive and -resistant cells.

HDAC3 Expression Correlates with the Prognosis and Grade of Patients with Glioma: A Diversification Analysis Based on Transcriptome and Clinical Evidence

This study aimed to clarify the relationship between histone deacetylase 3 (HDAC3) expression and the prognosis as well as the grade of patients with glioma. The quantitative real-time polymerase chain reaction was profiled to examine the HDAC3 expression in glioma and normal glial cell lines. An Oncomine database analysis and prognosis analysis were performed. The correlation between World Health Organization (WHO) grade and HDAC3 was analyzed by Spearman rank correlation test. A meta-analysis was performed to confirm the conclusion. HDAC3 RNA overexpression in glioma cell lines was detected (P < 0.05). Four data sets were screened from the Oncomine database and showed that the expression level of HDAC3 was consistently higher in glioma than in normal tissue (P < 0.001). The prognostic analysis of 325 glioma samples from the Chinese Glioma Genome Atlas showed that patients with low HDAC3 expression had significantly better overall survival (OS) and progression-free survival (PFS) than did patients with high HDAC3 expression [hazard ratio [HR], 1.992; 95% confidence interval [CI], 1.490-2.662; P < 0.0001 and HR, 1.874; 95% CI, 1.412-2.487; P < 0.0001, respectively]. Both the WHO grade III group and the WHO grade IV group expressed significantly higher messenger RNA (mRNA) level than did the WHO grade II group (P < 0.05). Four cohort studies consisting of 490 patients were included in the meta-analysis. The pooled data of subgroup analysis showed significantly longer OS in low HDAC3 mRNA level [HR, 3.38; 95% CI, 1.80-6.37; P= 0.0002]. HDAC3 mRNA was expressed more in glioma than in the normal glial cell line. Low HDAC3 mRNA expression levels predicted better OS. HDAC3 expression could be a biomarker to discriminate glioma grade.

Abstract 4776: Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer

Abstract Purpose: Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-HER2 drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. Among HER2 targeting drug, afatinib and neratinib inhibit the activation of all HER family protein, and are called pan-HER inhibitors. In this study, we examined the effect of these pan-HER inhibitors to gastric cancer cells. Materials and Methods: We determined the molecular profiles of 12 gastric cancer cell lines. Protein level of HER2 and down-signal pathway molecules were analyzed by Western blotting, and copy number assay or gene expression assay were performed using qPCR. To detect HER2 mutation, we also performed direct-sequence of these cell lines. And next, we examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines, in vitro and in vivo. In addition, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. Results: HER2 was amplified in 5 out of 12 gastric cancer cell lines. Gene expression or protein level of HER2 were generally correlated with the copy number of HER2. HER2 mutation was found in one cell line, ECC10, at kinase domain (L755S). In the drug sensitivity analysis, both afatinib and neratinib showed an anti-tumor effect in all the HER2 amplified cell lines both in vitro and in vivo except MKN7 cell line. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene by inhibiting the activation of IGF-1R, were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, demonstrated a notable synergistic effect. Regarding HER2 alteration in 123 clinical samples, we found 19 cases of HER2 amplification and 3 cases of oncogenic mutations. Conclusion: Afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer. Citation Format: Takahiro Yoshioka, Kazuhiko Shien, Yuta Takahashi, Eisuke Kurihara, Kei Namba, Yusuke Ogoshi, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Toshiyoshi Fujiwara, Shinichi Toyooka. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4776.

Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma

BackgroundAberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC).MethodsMethyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5’azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11. Promoter reporter and EMSA assays were used to dissect the DNA region responsible for transcription activator binding and to confirm whether DNA methylation could affect activator’s binding, respectively. CLDN11 was transiently overexpressed in NPC cells followed by cell proliferation, migration, invasion assays to characterize its biological roles. Co-immunoprecipitation experiments and proteomic approach were carried out to identify novel interacting protein(s) and the binding domain of CLDN11. Anti-tumor activity of the CLDN11 was elucidated by in vitro functional assay.ResultsA tight junction gene, CLDN11, was identified as differentially hypermethylated gene in NPC. High methylation percentage of CLDN11 promoter in paired NPC clinical samples was correlated with low mRNA expression level. Immunohistochemistry staining of NPC paired samples tissue array demonstrated that CLDN11 protein expression was relatively low in NPC tumors. Transcription activator GATA1 bound to CLDN11 promoter region − 62 to − 53 and its DNA binding activity was inhibited by DNA methylation. Re-expression of CLDN11 reduced cell migration and invasion abilities in NPC cells. By co-immunoprecipitation and liquid chromatography-tandem mass spectrometry LC-MS/MS, tubulin alpha-1b (TUBA1B) and beta-3 (TUBB3), were identified as the novel CLDN11-interacting proteins. CLDN11 interacted with these two tubulins through its intracellular loop and C-terminus. Furthermore, these domains were required for CLDN11-mediated cell migration inhibition. Treatment with a tubulin polymerization inhibitor, nocodazole, blocked NPC cell migration.ConclusionsCLDN11 is a hypermethylated and downregulated gene in NPC. Through interacting with microtubules TUBA1B and TUBB3, CLDN11 blocks the polymerization of tubulins and cell migration activity. Thus, CLDN11 functions as a potential tumor suppressor gene and silencing of CLDN11 by DNA hypermethylation promotes NPC progression.

High Expression of Retinoic Acid Induced 14 (RAI14) in Gastric Cancer and Its Prognostic Value.

BackgroundTo explore the expression level of retinoic acid induced 14 (RAI14) in gastric cancer (GC) patients and its potentially clinical prognostic value.Material/MethodsInitially, The Cancer Genome Atlas (TCGA) and Oncomine databases were mined to examine the differential expression levels and clinical prognostic significance of RAI14 mRNA in GC patients. Subsequently, 68 cases of GC and paired adjacent normal tissues were collected retrospectively, and the expression level of RAI14 protein was detected by immunohistochemical staining. In addition, Kaplan-Meier univariate and Cox multivariate survival analyses were used to verify the correlation between RAI14 expression and clinicopathological parameters in GC patients and its clinical prognostic significance.ResultsTCGA and GEO (from Oncomine database) data mining results found that RAI14 mRNA level was remarkably higher in GC than normal gastric tissues (All P<0.05). Besides, immunohistochemical results detected that RAI14 protein level in GC was dramatically higher (P=0.004) compared to that in the matched normal tissues. Moreover, TCGA database and Kaplan-Meier Plotter mining results showed that compared to those with RAI14 low mRNA expression levels, GC patients with RAI14 high mRNA expression levels had remarkably lower time of both overall survival and disease-free survival (All P<0.05). Additionally, based on the immunohistochemical results, Kaplan-Meier univariate and Cox multivariate survival analyses indicated that high expression of RAI14 was the only independent predictor of unfavorable prognosis in patients with gastric cancer (P=0.000).ConclusionsRAI14 was highly expressed in GC, and the high expression of RAI14 could be an independent predictor of poor prognosis in GC patients.

Based on the Metabolomic Approach the Energy Metabolism Responses of Oriental River Prawn Macrobrachium nipponense Hepatopancreas to Acute Hypoxia and Reoxygenation.

Hypoxia represents a major physiological challenge for prawns and is a problem in aquaculture. Therefore, an understanding of the metabolic response mechanism of economically important prawn species to hypoxia and re-oxygenation is essential. However, little is known about the intrinsic mechanisms by which the oriental river prawn Macrobrachium nipponense copes with hypoxia at the metabolic level. In this study, we conducted gas chromatography-mass spectrometry-based metabolomics studies and assays of energy metabolism-related parameters to investigate the metabolic mechanisms in the hepatopancreas of M. nipponense in response to 2.0 O2/L hypoxia for 6 and 24 h, and reoxygenation for 6 h following hypoxia for 24 h. Prawns under hypoxic stress displayed higher glycolysis-related enzyme activities and lower mRNA expression levels of aerobic respiratory enzymes than those in the normoxic control group, and those parameters returned to control levels in the reoxygenated group. Our results showed that hypoxia induced significant metabolomic alterations in the prawn hepatopancreas within 24 h. The main metabolic alterations were depletion of amino acids and 2-hydroxybutanoic acid and accumulation of lactate. Further, the findings indicated that hypoxia disturbed energy metabolism and induced antioxidant defense regulation in prawns. Surprisingly, recovery from hypoxia (i.e., reoxygenation) significantly affected 25 metabolites. Some amino acids (valine, leucine, isoleucine, lysine, glutamate, and methionine) were markedly decreased compared to the control group, suggesting that increased degradation of amino acids occurred to provide energy in prawns at reoxygenation conditions. This study describes the acute metabolomic alterations that occur in prawns in response to hypoxia and demonstrates the potential of the altered metabolites as biomarkers of hypoxia.

Copy number changes at 8p11-12 predict adverse clinical outcome and chemo- and radiotherapy response in breast cancer

PurposeThe short arm of chromosome 8 (8p) is a frequent target of loss of heterozygosity (LOH) in cancer, and 8p LOH is commonly associated with a more aggressive tumor phenotype. The 8p11-12 region is a recurrent breakpoint area characterized by a sharp decrease in gains/amplifications and increase in allelic loss towards 8pter. However, the clustering of genomic aberrations in this region, even in the absence of proximal amplifications or distal LOH, suggests that the 8p11-12 region could play a pivotal role in oncogenesis.ResultsLoss in the FGFR1 and ZNF703-containing 8p11 region was seen in 25% of patients, correlated with lower mRNA expression levels and independently predicted poor survival, particularly in systemic treatment-naïve patients and even without adjacent 8p12 loss. Amplification of FGFR1 at 8p11 and loss of DUSP26 and UNC5D, located in the 8p12 breakpoint region, independently predicted worse event free survival. Gains in the 8p12 region encompassing WRN, NRG1, DUSP26 and UNC5D, seen in 20-30% of patients, were associated with higher mRNA expression and independently predicted chemotherapy sensitivity. Losses at 8p12 independently predicted radiotherapy resistance.Material and methodsMultiplex ligation-dependent probe amplification was used to investigate copy number aberrations at 8p11-12 in 234 female breast cancers. Alterations were correlated with clinicopathologic characteristics, survival and response to therapy. Results were validated using public METABRIC dataConclusionAllelic loss and amplification in the 8p11-12 breakpoint region predict poor survival and chemo- and radiotherapy response. Assessment of 8p11-12 gene copy number status seems to augment existing prognostic and predictive tools.

Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer.

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti‐human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti‐HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2‐positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan‐HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2‐amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin‐like growth factor‐1 receptor (IGF‐1R), were less sensitive to pan‐HER inhibitors. A combination therapy consisting of pan‐HER inhibitors and an IGF‐1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2‐amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF‐1R‐targeting therapy can overcome drug insensitiveness in HER2‐amplified gastric cancer.

Single nucleotide polymorphisms of casitas B-lineage lymphoma proto-oncogene-b predict outcomes of patients with advanced non-small cell lung cancer after first-line platinum based doublet chemotherapy.

Casitas B-lineage lymphoma proto-oncogene-b (CBLB) influences the threshold of T cell activation and controlling peripheral T cell tolerance. In the present study, we hypothesize that potentially functional single nucleotide polymorphisms (SNPs) in CBLB are associated with clinical outcomes in patients advanced non-small cell lung cancer (NSCLC) treated with the first-line chemotherapy. We genotyped three SNPs (rs2305035, rs3772534 and rs9657904) at CBLB in 116 advanced NSCLC patients with progression free survival (PFS) data and 133 advanced NSCLC patients with overall survival (OS) data, and we assessed their associations, 95% confidence interval (CI), with clinical outcomes by using Cox proportional hazards regression analyses. In silico functional analysis was also performed for the SNPs under investigation. We found that associations between the three SNPs and PFS/OS were not significant in the overall NSCLC patients. The rs2305035 AA genotype was associated with a worse PFS in female patients and those of non-smokers or light smokers (95% CI, 1.14-11.81, P=0.030; 95% CI, 1.42-10.24, P=0.008; and 95% CI, 1.39-9.93, P=0.009; respectively), compared with the GG+AA genotypes. We also found that the rs9657904 CC genotype was significantly associated with a worse OS than TT + TC genotypes in male advanced NSCLC patients. Further in silico functional analysis revealed that the rs965704 T allele was significantly associated with lower mRNA expression levels of the CBLB gene. Our findings identified two CBLB SNPs (rs2305035 and rs9657904) that were significantly associated with PFS and OS in several subgroups of Chinese advanced NSCLC patients after the first-line chemotherapy.

Increased expression of PRKCB mRNA in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

The polymorphism of PRKCB has been proven to be associated with systemic lupus erythematosus (SLE) in our previous study. We aimed to investigate the relationship between expression of PRKCB mRNA and the Disease Activity Index (SLEDAI) and manifestations of SLE. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of PRKCB mRNA in peripheral blood mononuclear cells of 60 patients with SLE and 62 controls. The Sequenom MassArray System was used to detect genotype SNP rs16972959. The expression levels of PRKCB mRNA in SLE cases were significantly increased compared with those in healthy controls (P<0.001). In addition, PRKCB mRNA expression levels were negatively correlated with the SLEDAI (P<0.05, r=-0.322), with lower mRNA expression levels of PRKCB in patients found with higher SLEDAI, presence of a new rash (P<0.01), and proteinuria (P<0.05). No association evidence was observed between the genotype of the variant rs16972959 and PRKCB mRNA expression levels; however, SNP rs16972959 was found to be an expression quantitative trait loci for PRKCB with the SLE risk allele correlated with increased expression in naïve monocytes (PFDR =9.12×10-13 ) and stimulated monocytes (9.24×10-6 >PFDR >2.75×10-17 ). On the other hand, SNP rs16972959 of PRKCB was found to have suggestive significant associations with vasculitis (P=0.00718) of SLE. These results indicated that expression of PRKCB mRNA may be correlated with the pathogenesis of SLE; however, more investigation is still needed.

The morphological and molecular biological signs of impaired endometrial receptivity in infertility in women suffering from external genital endometriosis

To study endometrial receptivity in infertile women with external genital endometriosis (EGE). Clinical, morphological, immunohistochemical, and molecular genetic examinations of endometrial aspiration pipelle biopsy specimens obtained on days 22-24 of the menstrual cycle from 94 infertile women with endometriosis: 50 women with Stage I-II EGE and 44 women with ovarian endometrioid cysts (OEC). A control group consisted of 54 women with tubal peritoneal factor of infertility (TPFI) and a successful attempt at IVF. Hematoxylin and eosin-stained sections were found to contain a number of endometrial surface epithelial cells containing mature pinopods. The expression levels of leukemia inhibitory factor (LIF), HOXA-10, glycodelin A, avβ3 integrin, estrogen receptors (ER) and progesterone receptors (PR), aromatase in the superficial epithelium, glandular epithelium and endometrial stroma were immunohistochemically revealed. Forty-four patients, including 17 with Stage I-II EGE and 27 with TPFI, showed mRNA expression levels of leukemia inhibitory factor receptor (LIFR), LIF, ER1, PgR, HOXA-10, and PTEN by real-time quantitative polymerase chain reaction (PCR) with a preliminary reverse transcription PCR assay. It was established that in the infertile women with Stage I-II EGE and those with OEC, endometrial receptivity was impaired, which was manifested by a decline in the number of superficial epithelial cells containing mature pinopods, as well as a decrease in the endometrial level of the key receptivity markers: αvβ3 integrin, LIF, glycodelin A, and HOXA10 and increases in the synthesis of aromatase and in the imbalance of endometrial stromal expression of ER and PR detected by immunohistochemistry (IHC). Molecular genetic study showed lower mRNA expression levels of the HOXA-10, LIFR, and PgR genes, which confirms the data obtained by IHC.

LATS2 promoter hypermethylation and its effect on gene expression in human breast cancer.

Tumor-specific promoter hypermethylation of large tumor suppressor, homolog 2 (LATS2), a tumor suppressor gene, has been investigated using methylation-specific polymerase chain reaction (MSP) assays in different types of human cancer producing conflicting results. The aim of the present study was to evaluate the methylation status of the LATS2 promoter region using bisulfite sequencing with a next generation sequencer for breast cancer. In the 11 patients enrolled in the present study, the LATS2 promoter methylation index (MI) was uniformly high in tumor and normal tissues of the breast (median, 84.0 and 87.4%, respectively). The presence of LATS2 promoter hypermethylation was confirmed in isolated tumor cells and normal epithelial cells using the magnetic-activated cell sorting method. In situ hybridization for LATS2 messenger RNA (mRNA) revealed that the mRNA expression of LATS2 was higher in normal epithelial cells, compared with tumor cells, however, it was not significantly associated with LATS2 MI. In 12 breast cancer cell (BCC) lines and two normal breast cell lines, the LATS2 promoter was uniformly hypermethylated with no correlation between the mRNA expression of LATS2 and the LATS2 MI. In addition, treatment of the BCC lines with a demethylating reagent had minimal effect on the mRNA expression of LATS2 in any of these cell lines. These results demonstrated that LATS2 hypermethylation was not involved in silencing the mRNA expression of LATS2 mRNA. The lower mRNA expression level of LATS2 in tumor cells, compared with normal epithelial cells, suggested the possible involvement of downregulation in the mRNA expression of LATS2 in the pathogenesis of breast cancer. Therefore, the conflicting results previously reported for LATS2 promoter methylation in different types of cancer, detected using MSP assays may be attributable to the low fidelity of the MSP assay.

Epigenetic regulation of AXL and risk of childhood asthma symptoms

BackgroundAXL is one of the TAM (TYRO3, AXL and MERTK) receptor tyrosine kinases and may affect numerous immune-related health conditions. However, the role for AXL in asthma, including its epigenetic regulation, has not been extensively studied.MethodsWe investigated the association between AXL DNA methylation at birth and risk of childhood asthma symptoms at age 6 years. DNA methylation of multiple CpG loci across the regulatory regions of AXL was measured in newborn bloodspots using the Illumina HumanMethylation450 array on a subset of 246 children from the Children’s Health Study (CHS). Logistic regression models were fitted to assess the association between asthma symptoms and DNA methylation. Findings were evaluated for replication in a separate population of 1038 CHS subjects using Pyrosequencing on newborn bloodspot samples. AXL genotypes were extracted from genome-wide data.ResultsHigher average methylation of CpGs in the AXL gene at birth was associated with higher risk of parent-reported wheezing, and the association was stronger in girls than in boys. This relationship reflected the methylation status of the gene-body region near the 5′ end, for which a 1% higher methylation level was significantly associated with a 72% increased risk of ever having wheezed by 6 years. The association of one CpG locus, cg00360107 was replicated using Pyrosequencing. Increased AXL methylation was also associated with lower mRNA expression level of this gene in lung tissue from the Cancer Genome Atlas (TCGA) dataset. Furthermore, AXL DNA methylation was strongly linked to underlying genetic polymorphisms.ConclusionsAXL DNA methylation at birth was associated with higher risk for asthma-related symptoms in early childhood.

Mitochondrial haplotypes influence metabolic traits across bovine inter- and intra-species cybrids

In bovine species, mitochondrial DNA polymorphisms and their correlation to productive or reproductive performances have been widely reported across breeds and individuals. However, experimental evidence of this correlation has never been provided. In order to identify differences among bovine mtDNA haplotypes, transmitochondrial cybrids were generated, with the nucleus from MAC-T cell line, derived from a Holstein dairy cow (Bos taurus) and mitochondria from either primary cell line derived from a domestic Chinese native beef Luxi cattle breed or central Asian domestic yak (Bos grunniens). Yak primary cells illustrated a stronger metabolic capacity than that of Luxi. However, all yak cybrid parameters illustrated a drop in relative yak mtDNA compared to Luxi mtDNA, in line with a mitonuclear imbalance in yak interspecies cybrid. Luxi has 250 divergent variations relative to the mitogenome of Holsteins. In cybrids there were generally higher rates of oxygen consumption (OCR) and extracellular acidification (ECAR), and lower mRNA expression levels of nuclear-encoded mitochondrial genes, potentially reflecting active energy metabolism and cellular stress resistance. The results demonstrate that functional differences exist between bovine cybrid cells. While cybrid viability was similar between Holstein and Luxi breeds, the mitonuclear mismatch caused a marked metabolic dysfunction in cattle:yak cybrid species.

Patterns of PD-1, PD-L1, and PD-L2 expression in pediatric solid tumors.

Significant antitumor effects have been observed in a variety of malignancies via blockade of immune checkpoints. Interaction of programmed death 1 (PD-1) with its ligands PD-L1 and PD-L2 suppresses T-cell function and restricts immune-mediated tumor killing. We examined expression of these proteins in children with solid tumors, as expression may serve as biomarkers of response to this class of drugs. Sections cut from formalin-fixed paraffin-embedded (FFPE) tissue blocks were processed and evaluated for PD-1, PD-L1, and PD-L2 by immunohistochemistry (IHC) as well as by mRNA expression. A semiquantitative 0-5 IHC scoring system (0 = negative to 5 = very high) was applied, with scores incorporating combined prevalence of tumor cell and nontumor cell labeling. Expression profiling was performed using the NanoString nCounter™ system. Data analysis was performed using quantile normalization. All quantile-normalized data underwent subsequent log10 transformation. One hundred twenty-four FFPE blocks were included in the analysis. PD-1, PD-L1, and PD-L2 IHC were not evaluable in 8, 0, and 12 blocks, respectively. PD-1, PDL-1, and PDL-2 expression was negative to moderate by both IHC (range 0-3) and mRNA expression (range 0-2.62). Correlation between IHC score and mRNA expression was poor for all three tested proteins (PD-1, r2 = 0.06; PDL-1, r2 = 0.007; and PDL-2, r2 = 0.15). Expression of PD-1, PD-L1, and PD-L2 is low in pediatric solid tumors. At low levels of expression, IHC score and mRNA expression correlate poorly. Current and planned clinical trials will determine whether this low level of expression predicts limited response to immune checkpoint inhibitors.

Differential regulation by IFN‑γ on TNF‑α‑induced chemokine expression in synovial fibroblasts from temporomandibular joint.

Tumor necrosis factor(TNF)‑α and interferon(IFN)‑γ, are inflammatory cytokines in the synovial fluid of patients with temporomandibular joint disorder(TMD). However, it remains unknown whether they participate in the regulation of various chemokine expression levels associated with TMD. The effects of TNF‑α and IFN‑γ on the expression of several different inflammatory chemokines, including interleukin(IL)‑8, C‑X‑C motif chemokine ligand (CXCL)1, C‑C motif chemokine ligand (CCL)20, CXCL9, CXCL10, and CXCL11 in synovial fibroblasts obtained from the temporomandibular joint (TMJ) were examined. The results demonstrated that TNF‑α increased the mRNA levels of all examined chemokines in synovial fibroblasts obtained from the TMJ. IFN‑γ treatment alone increased the mRNA expression levels of CXCR3 chemokines, including CXCL10, while they were significantly enhanced when administered in combination with TNF‑α compared with either treatment alone. However, the combination of IFN‑γ and TNF‑α resulted in lower mRNA expression levels of IL‑8 and CXCL1 as compared with those induced by TNF‑α alone. The nuclear factor‑κB inhibitor, Bay 11‑7082, decreased the TNF‑α‑mediated expression of IL‑8 and CXCL10 in the absence, and presence of IFN‑γ. In addition, the JAK2 inhibitor, AG490, decreased CXCL10 expression when administered with TNF‑α and IFN‑γ. Finally, the decrease in TNF‑α‑induced IL‑8 caused by IFN‑γ was recovered by AG490. The results of the present study suggest that TNF‑α and IFN‑γ function in a cooperative manner to regulate inflammatory chemokine expression in synovial fibroblasts, which may contribute to the pathological condition of the TMJ.

Disturbances in iron homeostasis result in accelerated rejection after experimental heart transplantation

Clinical data suggest that iron disturbances deleteriously affect graft survival after heart transplantation (HTx), but immunological mechanisms underlying this phenomenon have not yet been elucidated. To identify the mechanistic influence of iron in a murine model of HTx, fully allogeneic BALB/c donor organs were transplanted into iron-overloaded or iron-deficient C57BL/6 mice, and recipients were analyzed for functional and immunological parameters. After HTx, iron overload accelerated acute rejection as observed by shortened graft survival (HTx vs HTx + iron; p = 0.01), elevated rejection score (p < 0.01), and induction of troponin T (p < 0.01). Compared with controls, allografts and recipient spleens derived from iron-overloaded recipients were characterized by a pronounced graft infiltration of CD4+ T cells (p < 0.01), CD3-NKp46+ natural killer cells (p < 0.05), and reduced frequencies of regulatory T cells (p < 0.01). This was accompanied by lower mRNA expression levels of anti-inflammatory cytokines, including interleukin-10, transforming graft factor-β, and Foxp3. Cardiac allograft survival was further tested under co-stimulation blockade (CTLA4-Ig) showing that naïve grafts transplanted into iron-overloaded recipients illustrated restricted graft outcome compared with wild types (p = 0.0051), which was rescued after treatment with the iron chelator deferoxamine. Iron deficiency (ID) also resulted in enhanced intragraft infiltration of inflammatory cells and accelerated rejection in the acute setting (HTx vs HTx + ID; p = 0.02) and after co-stimulation blockade (p = 0.0059). We provide novel insights into the understanding of disturbances in iron homeostasis and their consequences after HTX, allowing novel insights regarding improvements in personalized immunosuppression to prolong allograft survival.

Effects of SOST Gene Silencing on Proliferation, Apoptosis, Invasion, and Migration of Human Osteosarcoma Cells Through the Wnt/β-Catenin Signaling Pathway.

Our study explored the effects of SOST gene silencing on the proliferation, apoptosis, invasion, and migration of human osteosarcoma cells through Wnt/β-catenin signaling pathway. Fresh tissues were obtained from 108 patients with osteosarcoma and 46 patients with osteochondroma. Human osteosarcoma cells (MG-63, U2-OS, HOS, and Saos-2) and normal osteoblast (hFoB1.19) were selected and cultured. Osteosarcoma cells were grouped randomly into the blank group, the scrambled control group, and the SOST-siRNA group. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis were tested by flow cytometry. Transwell and scratch test were performed to determine cell invasion and migration. The qRT-PCR and Western blotting were used to detect mRNA and protein expression level of sclerostin, Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7. The activity of caspase-3 was assessed by immunocytochemistry. Alkaline phosphatase (ALP) activity was measured using P-nitrophenylphosphate as a substrate. Low SOST mRNA and sclerostin protein expression levels were observed in osteosarcoma tissues and cells. Compared with the blank and scrambled control groups, sclerostin expression, apoptotic cells, ALP activity, and caspase-3 activity were down-regulated, while the proliferation, invasion, and migration abilities of osteosarcoma cells were evidently enhanced in the SOST-siRNA group. After SOST gene silencing, the mRNA and protein expression levels of Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7 in osteosarcoma cells and β-catenin protein expression levels in the nucleus and cytoplasm were significantly elevated. SOST gene silencing promotes the proliferation, invasion, and migration, and inhibits apoptosis of osteosarcoma cells by activating Wnt/β-catenin signaling pathway.