mRNA Microarray Analysis Research Articles

Regional heritability mapping identifies several novel loci (STAT4, ULK4, and KCNH5) for primary biliary cholangitis in the Japanese population

While the advent of GWAS more than a decade ago has ushered in remarkable advances in our understanding of complex traits, the limitations of single-SNP analysis have also led to the development of several other approaches. Simulation studies have shown that the regional heritability mapping (RHM) method, which makes use of multiple adjacent SNPs jointly to estimate the genetic effect of a given region of the genome, generally has higher detection power than single-SNP GWAS. However, thus far its use has been mostly limited to agricultural settings, and its potential for the discovery of new genes in human diseases is yet to be fully exploited. In this study, by applying the RHM method to primary biliary cholangitis (PBC) in the Japanese population, we identified three novel loci (STAT4, ULK4, and KCNH5) at the genome-wide significance level, two of which (ULK4 and KCNH5) have not been found associated with PBC in any population previously. Notably, these genes could not be detected by using conventional single-SNP GWAS, highlighting the potential of the RHM method for the detection of new susceptibility loci in human diseases. These findings thereby provide strong empirical evidence that RHM is an effective and practical complementary approach to GWAS in this context. Also, liver tissue mRNA microarray analysis revealed higher gene expression levels in ULK4 in PBC patients (P < 0.01). Lastly, we estimated the common SNP heritability of PBC in the Japanese population (0.210 ± 0.026).

SLC40A1 Mediates Ferroptosis and Cognitive Dysfunction in Type 1 Diabetes

Cognitive dysfunction often accompanies diabetes. Both hypoglycemia and hyperglycemia cause cognitive dysfunctions. However, the underlying pathophysiology remains unclear. Recent evidence show that ferroptosis primarily triggers nerve cell death, Alzheimer’s disease (AD), Huntington’s disease (HD), and Parkinson’s disease (PD). The present study aimed to investigate whether ferroptosis is a vital pathogenic pathway in diabetes-induced cognitive dysfunction. Type 1 diabetic rat model was created by intraperitoneal injection of streptozotocin (STZ). Significant cognitive dysfunction was observed in the diabetic rats as evidenced by increase in latency period to find a hidden platform and decreased cumulative time spent in the target quadrant (TQ) in the Morris water maze test. We detected the amplitude of low-frequency fluctuation (ALFF) of the BOLD (Blood Oxygenation Level-Dependent) signal using resting-state functional magnetic resonance imaging (rs-fMRI). Consequently, we found that the ALFF values, as well as the T2 relaxation time of the bilateral hippocampus, were reduced in Type 1 diabetic rats. We detected Fe2+ level and lipid peroxidation products (malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE)) in the hippocampus. Mitochondria and neuron injury in the STZ-induced diabetic rats were determined using a Transmission Electron Microscope and Nissl body staining. Iron overload and ferroptosis were detected in the hippocampus. Furthermore, mRNA microarray analysis revealed 201 dysregulated mRNAs in STZ-induced type 1 diabetes (T1D). Pathway enrichment analyses indicated that differentially expressed mRNAs associated-coding genes were associated with ferroptosis. Among ferroptosis signaling pathway genes, Slc40a1 gene (ferroportin) was downregulated. We show that ferroptosis is associated with diabetic cognitive dysfunction and Slc40a1 mediates ferroptosis in T1D.

MiR-34a-5p might have an important role for inducing apoptosis by down-regulation of SNAI1 in apigenin-treated lung cancer cells

Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells.

Gene Expression Analysis Identifies Cholesterol Metabolism Dysregulation in Hippocampus of Phenytoin-Resistant Pentylenetetrazol-Kindled Epileptic Mice.

Pharmaco-resistant Epilepsy has been a major challenge for medical interventions in controlling seizures. To date, up to 33% of the patients with epilepsy do not show adequate response to anti-epileptic drugs even after prolonged combinatorial drug usage. Using microarray, this study explores the changes in hippocampal gene expression in the phenytoin-resistant pentylenetetrazol (PTZ)-kindled mouse model of epilepsy. Our results from mRNA microarray analysis show distinct gene expression profiles in the hippocampus of phenytoin-resistant and sensitive mice. Pathway enrichment analysis showed differential expression of genes involved in cholesterol biosynthesis in phenytoin-resistant and sensitive mice.

Expression and Diagnostic Value of NPTX1 in Thymoma Patients

背景与目的胸腺瘤是原发于前纵隔最常见的恶性肿瘤，但对于胸腺瘤的诊断没有特异性的实验室指标。本研究旨在通过mRNA微阵列分析技术筛选出对胸腺瘤诊断有提示作用的肿瘤标志物，并加以证实。方法前期对31份胸腺瘤和瘤旁胸腺组织进行mRNA微阵列分析，发现胸腺瘤中神经细胞正五聚体1（neuronal pentraxin 1, NPTX1）基因转录水平上调超过4倍。为进一步验证上述结果，采用实时荧光定量聚合酶链反应（polymerase chain reaction, PCR）法、免疫组化法和酶联免疫吸附测定法（enzyme-linked immunosorbent assay, ELISA）对60例胸腺瘤患者和30例胸腺囊肿患者NPTX1基因的转录和表达水平进行检测，并应用受试者工作特征曲线（receiver operating characteristic curve, ROC）分析其在胸腺瘤中的诊断价值。结果胸腺瘤组织中NPTX1 mRNA转录水平明显高于对照组胸腺组织[(2.88±1.02) vs (1.35±0.47), P < 0.001]；胸腺瘤组织中NPTX1蛋白的表达水平明显高于对照组胸腺组织（2 vs 1, P < 0.001）；术前胸腺瘤患者血清中NPTX1蛋白含量明显高于对照组胸腺囊肿患者[(1, 018.29±209.38) pg/mL vs (759.95±66.02) pg/mL, P < 0.001]；血清中NPTX1蛋白含量以842.22 pg/mL为诊断节点，诊断胸腺瘤的敏感性为85.00%，特异性为93.33%，ROC曲线下面积为0.902。结论NPTX1在胸腺瘤患者中高表达，且对胸腺瘤有诊断价值。

Identification of stemness and differentially expressed genes in human cementum-derived cells.

PurposePeriodontal treatment aims at complete regeneration of the periodontium, and developing strategies for periodontal regeneration requires a deep understanding of the tissues composing the periodontium. In the present study, the stemness characteristics and gene expression profiles of cementum-derived cells (CDCs) were investigated and compared with previously established human stem cells. Candidate marker proteins for CDCs were also explored.MethodsPeriodontal ligament stem cells (PDLSCs), pulp stem cells (PULPSCs), and CDCs were isolated and cultured from extracted human mandibular third molars. Human bone marrow stem cells (BMSCs) were used as a positive control. To identify the stemness of CDCs, cell differentiation (osteogenic, adipogenic, and chondrogenic) and surface antigens were evaluated through flow cytometry. The expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP) was investigated to explore marker proteins for CDCs through reverse-transcription polymerase chain reaction. To compare the gene expression profiles of the 4 cell types, mRNA and miRNA microarray analysis of 10 samples of BMSCs (n=1), PDLSCs (n=3), PULPSCs (n=3), and CDCs (n=3) were performed.ResultsThe expression of mesenchymal stem cell markers with a concomitant absence of hematopoietic markers was observed in PDLSCs, PULPSCs, CDCs and BMSCs. All 4 cell populations also showed differentiation into osteogenic, adipogenic, and chondrogenic lineages. CEMP1 was strongly expressed in CDCs, while it was weakly detected in the other 3 cell populations. Meanwhile, CAP was not found in any of the 4 cell populations. The mRNA and miRNA microarray analysis showed that 14 mRNA genes and 4 miRNA genes were differentially expressed in CDCs vs. PDLSCs and PULPSCs.ConclusionsWithin the limitations of the study, CDCs seem to have stemness and preferentially express CEMP1. Moreover, there were several up- or down-regulated genes in CDCs vs. PDLSCs, PULPSCs, and BMSCs and these genes could be candidate marker proteins of CDCs.

Samul-tang ameliorates oocyte damage due to cyclophosphamide-induced chronic ovarian dysfunction in mice

Samul-tang (SM), a traditional herbal medicine, has been used to treat menstrual irregularities and infertility in women. However, the cellular and molecular mechanisms underlying the effects of SM remain elusive. We investigated the potential protective effect of SM against chronic ovarian dysfunction and used bioinformatics analysis to identify its underlying mechanism in a mouse model of cyclophosphamide (CP)-induced diminished ovarian reserve. Female C57BL/6 mice were intraperitoneally injected with CP three times a week, followed by oral administration of distilled water (CP group) or SM (CP + SM group) for 4 weeks. Four weeks later, the effect of SM was assessed by ovarian tissue histological analysis, steroid hormone measurement, oocyte quality, and mRNA and microRNA microarray analysis in the ovaries. Although SM administration did not prevent CP-induced follicle loss in mice, the quality of oocytes was better in CP + SM mice than in CP mice. Gene expression analysis revealed that the expression of fertilisation- and ovarian follicle development-related genes was altered by CP treatment but normalized after SM administration. Further bioinformatics analysis showed possible interactions between differentially expressed mRNAs and microRNAs. Therefore, we demonstrated the protective effects of SM on ovarian function and oocyte maturation against CP-induced damage via multiple epigenetic mechanisms.

Radiosensitivity-Related Genes and Clinical Characteristics of Nasopharyngeal Carcinoma.

Materials and Methods Clinicopathological data of 185 patients with NPC treated at Nanfang Hospital of Southern Medical University between January 2013 and December 2014 were retrospectively analyzed. SPSS statistical software was used to analyze the clinicopathological data related to radiotherapy efficacy. Three patients who achieved complete remission and three with disease progression after CRT were selected. Differentially expressed genes (DEGs) were screened via mRNA microarray analysis of primary diagnostic endoscopy specimens. Results The peripheral blood leukocyte count, platelet count, and EBV-DNA copy number in NPC patients who were resistant to radiotherapy were higher than those in NPC patients who were sensitive to radiotherapy. The RobustRankAggreg (RRA) analysis method identified 392 DEGs, and the 66 most closely related genes among the DEGs were identified from the PPI network. Conclusion The results of this study indicate that screening for DEGs and pathways in NPC using integrated in silico analyses can help identify a series of genetic and clinical signatures for NPC patients treated with neoadjuvant chemotherapy followed by concurrent chemoradiotherapy.

Spinal neuronal excitability and neuroinflammation in a model of chemotherapeutic neuropathic pain: targeting the resolution pathways

BackgroundNeuroinflammation is a critical feature of sensitisation of spinal nociceptive processing in chronic pain states. We hypothesised that the resolvin pathways, a unique endogenous control system, may ameliorate aberrant spinal processing of somatosensory inputs associated with chemotherapy-induced neuropathic pain (CINP).MethodThe paclitaxel (PCX) model of CINP was established in male Sprague-Dawley rats and compared to control rats (n = 23 and 22, respectively). Behavioural pain responses were measured, and either single unit electrophysiological recordings of dorsal horn wide dynamic range (WDR) neurones were performed, or mRNA microarray analysis of the dorsal horn of the spinal cord was undertaken.ResultsPCX rats exhibited significant changes in behavioural responses to mechanical and cold stimuli. A higher proportion of WDR neurones in PCX rats were polymodal (generating post-discharge following a non-noxious mechanical stimulus, responding to non-noxious cold and exhibiting spontaneous activity) compared to control (p < 0.05). Microarray analysis revealed changes in proinflammatory pathways (Tlr, Tnfrsf1a, Nlrp1a, Cxcr1, Cxcr5, Ccr1, Cx3cr1) and anti-inflammatory lipid resolvin pathways (Alox5ap, Cyp2j4 and Ptgr1) compared to control (p < 0.05). Ingenuity pathway analysis predicted changes in glutamatergic and astrocyte signaling in the PCX group. Activation of the resolvin system via the spinal administration of aspirin-triggered resolvin D1 (AT-RvD1) markedly inhibited (73 ± 7% inhibition) normally non-noxious mechanically (8 g) evoked responses of WDR neurones only in PCX rats, whilst leaving responses to noxious mechanically induced stimuli intact. Inhibitory effects of AT-RvD1were comparable in magnitude to spinal morphine (84 ± 4% inhibition).ConclusionThe PCX model of CINP was associated with mechanical allodynia, altered neuronal responses and dysregulation of pro- and anti-inflammatory signalling in the spinal dorsal horn. The resolvin AT-RvD1 selectively inhibited low weight mechanical-evoked responses of WDR neurones in PCX rats, but not in controls. Our data support the targeting of spinal neuroinflammation via the activation of the resolvin system as a new therapeutic approach for CINP.

R-spondin2 Suppresses the Progression of Hepatocellular Carcinoma via MAPK Signaling Pathway.

The R-spondin family plays important roles in embryonic development, including in humans. However, information on the relationship between R-spondin2 and hepatocellular carcinoma (HCC) is lacking. This study aimed was to explore the mechanisms of R-spondin2 action in the progression of HCC. By analyzing R-spondin2 expression levels in HCC tissues by IHC and database, we identified that HCC tissues had lower expression levels of R-spondin2, correlated with a poor prognosis. We also established R-spondin2-overexpressing and knockdown cell lines and measured their viabilities and invasion abilities in vitro and their oncogenic capacity in vivo. Human mRNA microarray analysis was performed to screen for mRNAs that were differentially expressed between R-spondin2-overexpressing and control HCC cells. Microarray and Western blot analyses showed significant changes in the MAPK signaling pathway after transfection. Furthermore, in vivo experiments indicated that R-spondin2 knockdown increased the tumorigenicity of HCC cells after subcutaneous implantation in mice. Altogether, our results indicate that the R-spondin2, which might be a novel tumor suppressor gene, were responsible for inhibiting the proliferation and invasion of HCC via the MAPK signaling pathway. IMPLICATIONS: R-spondin2 gene might be a novel tumor suppressor gene providing new clues to clarify the biological behavior of HCC and thus reduce patient mortality and prolong survival.

EZH2-dependent epigenetic modulation of histone H3 lysine-27 contributes to psoriasis by promoting keratinocyte proliferation

Psoriasis is characterized by keratinocyte hyperproliferation. While significant progress has been made in understanding the molecular mechanism regulating the proliferation of keratinocytes, little is known about the epigenetic factors that control this process. EZH2 and EZH2 mediated trimethylation of histone H3 lysine 27 (H3K27me3) was previously shown ectopically expressed in carcinoma and mediated proliferation, thereby we sought to clarify the role of EZH2–H3K27me3 in the proliferation of psoriatic keratinocyte. Interestingly, we found that EZH2 and H3K27me3 were both overexpressed in the epidermis of psoriatic lesional skin compared to normal skin. In vitro, the expression of EZH2 and H3K27me3 was stimulated in human keratinocytes treated with mixture of psoriasis-related cytokines pool (TNF-α, IFN-γ, IL-17A, and IL-22). Knockdown of EZH2 significantly reduced keratinocyte proliferative activity. Results from mRNA microarray analysis suggested that Kallikrein-8 (KLK8) might be the target gene of EZH2 in psoriatic keratinocytes. Overexpression or knockdown KLK8 could partially reverse the abnormal proliferation of keratinocytes caused by knockdown or overexpression of EZH2. In vivo, the inhibitor of EZH2, GSK126 could ameliorate the imiquimod-induced psoriasiform lesion. These results suggest that EZH2 might be a therapeutic target for the treatment of psoriasis.

Microarray Analysis of lncRNA and mRNA Reveals Enhanced Lipolysis Along With Metabolic Remodeling in Mice Infected With Larval Echinococcus granulosus

Parasitic infection improves metabolic homeostasis in “western diet”-induced obesity through the regulation of adipogenesis. However, the underlying mechanism is not yet fully understood. Using microarray analysis, this study investigated the long non-coding RNA (lncRNA) and messenger RNA (mRNA) profiles of subcutaneous adipose tissues from mice infected with Echinococcus granulosus protoscoleces. A total of 1052 mRNA (541 upregulated, 511 downregulated) and 220 lncRNA (126 upregulated, 94 downregulated) transcripts were differentially expressed (fold change ≥2, P < 0.05) in the infected subcutaneous adipose tissues. When compared with the control group, the infected mice showed a decrease in adipose tissue mass and a reduction in adipocyte size. Indirect calorimetry revealed the change in the energy metabolism after infection, characterized by a lower CO2 production and O2 consumption, a sharp decline in carbohydrate oxidation, and a slight increase in fat oxidation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the parasitic infection reprogrammed a complex metabolic network. Notably, “lipoxygenase” and “arginine and proline metabolism” pathways were significantly upregulated while “glycolysis,” “tricarboxylic acid cycle,” “de novo lipogenesis,” and “lipid droplet” pathways were dramatically downregulated. In addition, several key lncRNAs were associated with insulin resistance and adipocyte differentiation. Overall, the present study suggested that E. granulosus infection could enhance lipolysis. Thus, our findings provide novel insights into parasite-mediated metabolic homeostasis, and into the mechanism of hypertrophic adipocytes in obesity.

1H NMR metabolomic and transcriptomic analyses reveal urinary metabolites as biomarker candidates in response to protein undernutrition in adult rats.

Protein undernutrition contributes to the development of various diseases in broad generations. Urinary metabolites may serve as non-invasive biomarkers of protein undernutrition; however, this requires further investigation. We aimed to identify novel urinary metabolites as biomarker candidates responsive to protein undernutrition. Adult rats were fed control (CT; 14 % casein) or isoenergetic low-protein (LP; 5 % casein) diets for 4 weeks. 1H NMR metabolomics was applied to urine, plasma and liver samples to identify metabolites responsive to protein undernutrition. Liver samples were subjected to mRNA microarray and quantitative PCR analyses to elucidate the mechanisms causing fluctuations in identified metabolites. Urinary taurine levels were significantly lower in the LP group than in the CT group at week 1 and remained constant until week 4. Hepatic taurine level and gene expression level of cysteine dioxygenase type 1 were also significantly lower in the LP group than in the CT group. Urinary trimethylamine N-oxide (TMAO) levels were significantly higher in the LP group than in the CT group at week 2 and remained constant until week 4. Hepatic TMAO level and gene expression levels of flavin-containing mono-oxygenase 1 and 5 were also significantly higher in the LP group than in the CT group. In conclusion, urinary taurine and TMAO levels substantially responded to protein undernutrition. Furthermore, changes in hepatic levels of these metabolites and gene expressions associated with their metabolic pathways were also reflected in their fluctuating urinary levels. Thus, taurine and TMAO could act as non-invasive urinary biomarker candidates to detect protein undernutrition.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Polychlorinated Biphenyl Coexposure Alters the Expression Profile of MicroRNAs in the Liver Associated with Atherosclerosis.

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) are persistent organic pollutants that exist as complex mixtures in vivo. When humans are simultaneously exposed to these compounds, the development of atherosclerosis is known to be enhanced. However, the roles of miRNA in TCDD- and PCB-induced atherosclerosis are largely unknown. Therefore, the present study is aimed at elucidating the possible dysregulation of miRNAs in atherogenesis induced by coexposure to TCDD and PCBs. Eight-week-old male ApoE−/− mice were coexposed to TCDD (15 μg/kg) and Aroclor1254 (55 mg/kg, a representative mixture of PCBs) by intraperitoneal injection four times over a 6-week period. Microarray analysis of miRNAs and mRNAs in the liver of ApoE−/− mice with or without TCDD and Aroclor1254 coexposure was performed. We discovered that 68 miRNAs and 1312 mRNAs exhibited significant expression changes in response to TCDD and PCB coexposure and revealed that both changed miRNAs and mRNAs are involved in cardiovascular disease processes. An integrated miRNA-mRNA approach indicated that miRNA-26a-5p, miRNA-193a-3p, and miRNA-30c-5p participated in specific TCDD and Aroclor1254 coresponsive networks which are relevant to the cardiovascular system development and function network. Furthermore, our results also indicated that miRNA-130a-3p and miRNA-376a-3p were novel players in the regulation of TCDD- and Aroclor1254-induced atherosclerosis pathways. In summary, our finding provided new insights into the mechanism of atherosclerosis in response to TCDD and PCB coexposure.

An oriented-collagen scaffold including Wnt5a promotes osteochondral regeneration and cartilage interface integration in a rabbit model.

Articular cartilage regeneration remains a major challenge in orthopedics. Noncanonical Wnt5a is a particularly attractive growth factor in this context; Wnt5a inhibits chondrocyte hypertrophy but maintains chondrogenesis. We designed a novel, vertically oriented-collagen scaffold. The effect of Wnt5a on MSCs and chondrocytes and the therapeutic effects of the Wnt5a/oriented-collagen scaffold in terms of osteochondral repair and cartilage integration were evaluated. In vitro, the proliferation, migration, and differentiation of MSCs and chondrocytes treated with Wnt5a, and the mechanisms thereof, were assessed. mRNA microarray analysis was performed to compare the expression profiles of MSCs before and after Wnt5a treatment. In vivo, full-thickness cylindrical osteochondral defects (4mm in diameter, 3mm in depth) were created in the patellar grooves of 24 New Zealand white rabbits and implanted with oriented-collagen scaffolds (n=8), Wnt5a/oriented-collagen scaffolds (n=8), or nothing (n=8). After 6 and 12weeks, integration and tissue responses were evaluated. The proliferation, migration, chondrogenic differentiation, and extracellular matrix formation of/by MSCs and chondrocytes improved greatly after treatment with Wnt5a. Western blotting showed that the PI3K/AKT/JNK signaling pathway was activated. Microarray analysis revealed that the Wnt5a group exhibited a significant upregulation of the PI3K pathway. Reactome GSEA pathway interaction analysis revealed that such upregulation was associated with collagen and extracellular matrix formation. In vivo, the Wnt5a/oriented-collagen scaffold group exhibited optimal interface integration, cartilage regeneration, and collagenous fiber arrangement, accompanied by significantly increased glycosaminoglycan and collagen accumulations in the zones of regeneration and integration, compared to the other groups. Gene expression analysis showed that the levels of mRNAs encoding genes involved in cartilage formation were significantly increased in the Wnt5a/oriented, collagen scaffold group (all P<.05). Wnt5a promoted the proliferation, migration, and chondrogenic differentiation of MSCs and chondrocytes via the activation of the PI3K/AKT/JNK signaling pathway. The Wnt5a/oriented-collagen constructs enhanced the structure-specific regeneration of hyaline cartilage in a rabbit model and may be a promising treatment for the repair of human cartilage defects.

Analysis of the Differences in the Expression of mRNAs and miRNAs Associated with Drug Resistance in Endometrial Cancer Cells Treated with Salinomycin.

It is important to understand the molecular mechanisms involved in cancer drug resistance and to study the activity of new drugs, e.g. salinomycin. The purpose of the study was to analyze changes in the expression of genes associated with drug resistance in the Ishikawa endometrial cancer cell line when treated with salinomycin. In addition, changes in the level of miRNA potentially regulating these mRNAs were evaluated. Endometrial cancer cells were treated with 1 μM of salinomycin for 12, 24 and 48 hours periods. Untreated cells were a control culture. The molecular analysis consists of mRNA and miRNA microarray analysis and the RTqPCR technique. The following was observed about the number of mRNAs differentiating the cell culture exposed to the drug compared to a control culture: H-12 vs. C - 9 mRNAs, H_24 vs. C - 6 mRNAs, and H_48 vs. C - 1 mRNA. It was noted that 4 of the 9 differentiating mRNAs were characteristic for 12 hours of exposure to salinomycin and they correspond to the following genes: TUFT1, ABCB1, MTMR11, and MX2. After 24 hours, 2 mRNAs were characteristic for this time of incubation cells with salinomycin: TUFT1 and MYD88 and after 48 hours, SLC30A5 could also be observed. The highest differences in expression were indicated for TUFT1, MTMR11, and SLC30A5. The highest influence probability was determined between TUFT1 and hsa- miR-3188 (FC + 2.48), MTMR11and has-miR-16 (FC -1.74), and between SLC30A5 and hsa-miR-30d (FC -2.01). Salinomycin induces changes in the activity of mRNA and miRNA participating in drug resistance; however, the observed changes in character are the expected result of anti-cancer treatment.

TAK1-AMPK Pathway in Macrophages Regulates Hypothyroid Atherosclerosis.

Hypothyroidism (HT) is associated with accelerated atherosclerosis (AS). The efficacy of traditional strategies of hypothyroid AS remains controversial. Here, we aimed to deepen the understanding of the HT-induced acceleration of AS, to decrease the residual risk of coronary artery disease (CAD) and to find a new therapeutic target. We collected peripheral venous blood samples from 20 patients and divided them into 4 groups, namely, the normal group, the HT group, the CAD group and the HT + CAD group. Then we performed mRNA microarray analysis and bioinformatics analysis to screen the differentially expressed genes and pathways, and we also conducted validations on ApoE knockout mice models and Raw264.7 cell models. In short, (1) in the analysis between the CAD group and the HT + CAD group, we found a total of 1218 differentially expressed genes, 11 upregulated pathways and 40 downregulated pathways. (2) We validated that patients with HT and CAD had a significantly decreased expression of MAP3K7 (encoding transforming growth factor-β-activated kinase 1, TAK1) gene than normal subjects. (3) In animal and cell experiments, we found the decreased expression of TAK1 and the reduced phosphorylation of AMP-activated protein kinase (AMPK) under the hypothyroid and atherosclerotic condition. (4) Changes in the expressions of TAK1 may affect the progression of AS. Taken together, these data suggest that the accelerated AS in hypothyroid patients may be due to the suppression of TAK1-AMPK pathway in macrophages. This new finding may become a novel therapeutic target in hypothyroid AS.

Upregulated Plant Homeodomain Finger Protein 6 Promotes Extracellular Matrix Degradation in Intervertebral Disc Degeneration Based on Microarray Analysis.

mRNA analysis. The aim of this study was to identify differentially expressed genes (DEGs) in disc degeneration, analyze the potential biological functions of DEGs, and screen for a new target to prevent the degeneration. Intervertebral disc degeneration (IDD) is an irreversible process and causes long-term heavy socioeconomic burdens. Existing and therapies under development are unable to prevent disc degeneration in a safe and effective manner. Therefore, elucidating the potential mechanism underlying degeneration and the development of new targets for IDD therapy are urgently required. Nucleus pulposus (NP) cells from mild and severe IDD (Ctrl and IDD groups) were separated, and DEGs of the two groups were identified with mRNA microarray analysis, followed by bioinformatics analysis.Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to verify the microarray results. Gene over-expression and silencing technologies were used to study the role of plant homeodomain finger protein 6 (PHF6). qRT-PCR and western blot analyses were used to detect the expressions of collagen II (COL2), matrix metalloproteinases 13 (MMP13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS4). The study identified 377 up- and 116 downregulated DEGs in NP cells from two groups. These DEGs were mainly involved in cellular and metabolic processes and enriched in immune system and nucleotide metabolism pathways. Upregulated PHF6, with the highest verified fold change, was significantly increased in the IDD group. Over-expressing PHF6 in Ctrl NP cells significantly inhibited the expression of COL2 and enhanced the expressions of MMP13 and ADAMTS4, whereas silencing PHF6 in IDD NP cells reversed such expression alterations. Upregulated PHF6 caused IDD by promoting extracellular matrix degradation; therefore, PHF6 could be developed as a potential novel target to prevent the degeneration. Our DEG profiling of NP cells from IDD patients provided a database to identify the key genes involved in IDD. N/A.

Integrated GWAS and mRNA Microarray Analysis Identified IFNG and CD40L as the Central Upstream Regulators in Primary Biliary Cholangitis.

Genome‐wide association studies (GWASs) in European and East Asian populations have identified more than 40 disease‐susceptibility genes in primary biliary cholangitis (PBC). The aim of this study is to computationally identify disease pathways, upstream regulators, and therapeutic targets in PBC through integrated GWAS and messenger RNA (mRNA) microarray analysis. Disease pathways and upstream regulators were analyzed with ingenuity pathway analysis in data set 1 for GWASs (1,920 patients with PBC and 1,770 controls), which included 261 annotated genes derived from 6,760 single‐nucleotide polymorphisms (P < 0.00001), and data set 2 for mRNA microarray analysis of liver biopsy specimens (36 patients with PBC and 5 normal controls), which included 1,574 genes with fold change >2 versus controls (P < 0.05). Hierarchical cluster analysis and categorization of cell type–specific genes were performed for data set 2. There were 27 genes, 10 pathways, and 149 upstream regulators that overlapped between data sets 1 and 2. All 10 pathways were immune‐related. The most significant common upstream regulators associated with PBC disease susceptibility identified were interferon‐gamma (IFNG) and CD40 ligand (CD40L). Hierarchical cluster analysis of data set 2 revealed two distinct groups of patients with PBC by disease activity. The most significant upstream regulators associated with disease activity were IFNG and CD40L. Several molecules expressed in B cells, T cells, Kupffer cells, and natural killer–like cells were identified as potential therapeutic targets in PBC with reference to a recently reported list of cell type–specific gene expression in the liver. Conclusion: Our integrated analysis using GWAS and mRNA microarray data sets predicted that IFNG and CD40L are the central upstream regulators in both disease susceptibility and activity of PBC and identified potential downstream therapeutic targets.

Long Noncoding RNA SNHG6 Promotes Carcinogenesis by Enhancing YBX1-Mediated Translation of HIF1α in Clear Cell Renal Cell Carcinoma

Background: Dysregulated expression of long noncoding RNA SNHG6 plays important roles in the oncogenesis of many human cancers while the function of SNHG6 remains unclear in clear cell renal cell carcinoma (ccRCC). In this study, we aimed to assess the role of SNHG6 in the progression of ccRCC. Methods: RNA-Seq data from The Cancer Genome Atlas (TCGA) were analyzed by bioinformatic methods to identify lncRNA dysregulated in ccRCC. The CCK-8, colony formation, Transwell and wound-healing assays were used to assess the biological function of SNHG6 in vitro, and an in vivo xenograft model in nude mice was established. Quantitative real-time PCR and immunohistochemistry (IHC) were used to analyze the expression of SNHG6 in ccRCC tissues. The underlying mechanisms of SNHG6 were investigated using mRNA microarray analyses, Western blotting, RNA pull-down, and RNA immunoprecipitation. Findings: SNHG6 expression was significantly upregulated in ccRCC tissues compared with paired normal renal tissues. High SNHG6 expression was correlated with advanced T stage and poor survival. Further functional experiments demonstrated that knockdown of SNHG6 in ccRCC cells inhibited cell proliferation, migration and invasion in vitro as well as tumor growth and migration in vivo. Mechanistically, SNHG6 bound to YBX1 to act as a complex, thereby enhancing HIF1α translation. Interpretation: SNHG6 promotes ccRCC progression by binding YBX1 and may serve as a novel molecular target for ccRCC therapy. Funding Statement: This work was supported in part by grants from the National Natural Science Foundation of China (No.81903050) and the Science and Technology Commission of Shanghai Municipality (17ZR1417500). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: The study was approved by the Human Ethics Committee of the Fudan University Shanghai Cancer Center.