Identification of Parkinson's disease-related pathways and potential risk factors.

OBJECTIVE: To identify Parkinson's disease (PD)-associated deregulated pathways and genes, to further elucidate the pathogenesis of PD. METHODS: Dataset GSE100054 was downloaded from the Gene Expression Omnibus, and differentially expressed genes (DEGs) in PD samples were identified. Functional enrichment analyses were conducted for the DEGs. The top 10 hub genes in the protein-protein interaction (PPI) network were screened out and used to construct a support vector machine (SVM) model. The expression of the top 10 genes was then validated in another dataset, GSE46129, and a clinical patient cohort. RESULTS: A total of 333 DEGs were identified. The DEGs were clustered into two gene sets that were significantly enriched in 12 pathways, of which 8 were significantly deregulated in PD, including cytokine-cytokine receptor interaction, gap junction, and actin cytoskeleton regulation. The signature of the top 10 hub genes in the PPI network was used to construct the SVM model, which had high performance for predicting PD. Of the 10 genes, GP1BA, GP6, ITGB5, and P2RY12 were independent risk factors of PD. CONCLUSION: Genes such as GP1BA, GP6, P2RY12, and ITGB5 play critical roles in PD pathology through pathways including cytokine-cytokine receptor interaction, gap junctions, and actin cytoskeleton regulation.

MiR-181a inhibits vascular inflammation induced by ox-LDL via targeting TLR4 in human macrophages.

Atherosclerosis is a kind of chronic inflammation disease with lipid accumulation in in blood vessel linings. Increasing evidence has reported that microRNAs can exert crucial roles in atherosclerosis. In previous study, miR-181a has been implicated to be abnormally expressed in atherosclerosis mice, however its detailed function in atherosclerosis remains uninvestigated. Hence, in our current study, we focused on the biological role of miR-181a in atherosclerosis progression. Ox-LDL has been commonly identified as an important atherosclerosis regulator. We observed that ox-LDL induced THP-1 cell apoptosis dose-dependently and time- dependently. Meanwhile, 25 µg/ml ox-LDL can promote foam cell formation and increased miR-181a expression significantly. CD36 has been involved in atherosclerosis progression and it was found that overexpression of miR-181a inhibited its protein levels. Moreover, miR-181a mimics repressed foam cell formation, TC and TG levels induced by ox-LDL dramatically. In addition, miR-181a mimics were able to reverse THP-1 cell apoptosis, increased IL-6, IL-1ß, and TNF-α protein expression triggered by 25 µg/ml ox-LDL. TLR4 has been linked to various inflammation-associated diseases. In our present study, TLR4 was indicated as miR-181a target and the binding correlation between them was validated by dual-luciferase reporter assay. In conclusion, these results improves the understanding of atherosclerosis modulated by miR-181a/TLR4 and can contribute to development of new approaches for atherosclerosis.

MiR-370 inhibits vascular inflammation and oxidative stress triggered by oxidized low-density lipoprotein through targeting TLR4.

Atherosclerosis, as a chronic cardiovascular disease, still remains a serious threat to human health. Inflammation and oxidative stress are commonly involved in various stages of atherosclerosis development. MicroRNAs are reported to play important roles in macrophages, which can respond to inflammation and oxidative stress. In our current study, we focused on the biological roles of miR-370 in atherosclerosis. According to the previously research, miR-370 was downregulated in AS mice models. Oxidized low-density lipoprotein (Ox-LDL) is regarded as a crucial regulator of atherosclerosis and we observed that miR-370 was decreased by ox-LDL dose-dependently and time-dependently in THP-1 cells. Then, it was found that miR-370 overexpression was able to inhibit inflammation molecules including IL-6 and IL-1ß. Meanwhile, ROS levels, and malondialdehyde (MDA) were also restrained by miR-370 mimics in vitro. Toll-like receptor 4 (TLR4) has been implicated in many inflammation diseases. It can serve as a target of miR-370 and TLR4 expression was greatly increased in ox-LDL-incubated THP-1 cells in a time and dose dependent manner. The negative correlation was validated using a dual-luciferase reporter assay in our study. In conclusion, our present study revealed that miR-370 can reduce inflammatory reaction and inhibit the ROS production by targeting TLR4 in THP-1 cells.

MiR-135a represses oxidative stress and vascular inflammatory events via targeting toll-like receptor 4 in atherogenesis.

Plenty of microRNAs have been identified as critical mediators in atherosclerosis progression, which is still a great threat to human health. Oxidative stress and inflammation have been implicated to contribute a lot to atherosclerosis development. MiR-135a is abnormally expressed in various cancer types, however its function in atherosclerosis is largely unexplored. Ox-LDL is commonly recognized as a crucial atherosclerosis regulator. In our current study, we observed ox-LDL was able to induce RAW264.7 cell apoptosis and meanwhile miR-135a was restrained by ox-LDL both dose-dependently and time- dependently. CD36 has been reported to participate in atherosclerosis process and miR-135a mimics can inhibit its expression while miR-135a inhibitors exhibited a reverse phenomenon. Meanwhile, miR-135a overexpression can suppress foam cell formation, TC, TG levels, and cell apoptosis induced by 20 µg/mL ox-LDL. Subsequently, it was found that miR-135a overexpression can inhibit oxidative stress by decreasing ROS, MDA levels, and increasing SOD levels. Reversely, miR-135a inhibition demonstrated an inhibitory effect in vitro. Apart from these, miR-135a can also modulate inflammation molecules including IL-6, IL-1ß, and TNF-α. TLR4 was predicted as a target of miR-135a and the negative correlation between them was confirmed by dual-luciferase reporter assay in our study. This work improves our understanding of atherosclerosis events mediated by miR-135a/TLR4 and helps to develop new approaches for atherosclerosis.

MiR-21 attenuates apoptosis-triggered by amyloid-ß via modulating PDCD4/ PI3K/AKT/GSK-3ß pathway in SH-SY5Y cells.

Alzheimer's disease (AD) remains the most common neurodegenerative disease with amyloid beta (Aß) formatted and accumulated. Recently, microRNAs have been identified as significant regulators in neurogenesis of the central nervous system (CNS). However, the biological role of miR-21 in AD remains unclear. The purpose of our study was to investigate the mechanism of miR-21 in AD. AD model was established using 20 µM Aß1-42 in SH-SY5Y cells. Aß1-42 can induce cell apoptosis via increasing Bax and decreasing Bcl-2 protein levels. Meanwhile, we observed that miR-21 was remarkably elevated by indicated Aß1-42 in vitro. Subsequently, miR-21 mimics were transfected into SH-SY5Y cells and it was found that miR-21 can inhibit cell apoptosis induced by Aß1-42. Programmed cell death protein 4 (PDCD4), an important tumor suppressor in various cancers has been reported to prevent AKT activation. The phosphatidylinositol 3-kinase (PI3K)/AKT/GSK-3ß pathway can release a survival signal to protect from multiple injuries. Interestingly, it was found that PDCD4 was involved in miR-21-repressed cell apoptosis in AD models. miR-21 mimics can increase the PI3K, AKT and GSK-3ß activity while PDCD4 ovexexpression inhibited their activity respectively. Moreover, knockdown of PDCD4 can rescue PI3K/AKT/GSK-3ß pathway in SH-SY5Y cells. Taken these together, it was suggested by our data that miR-21 can exert protective roles in AD, which might be dependent on PDCD4/PI3K/AKT/GSK-3ß signaling pathway in vitro.

NEAT1 contributes to neuropathic pain development through targeting miR-381/HMGB1 axis in CCI rat models.

LncRNAs have been recognized as significant regulators in various diseases including neuropathic pain. Although the lncRNA NEAT1 has been reported to be involved in multiple cancers, its biological functions in neuropathic pain still remain unknown. In our present study, a chronic constriction injury (CCI) rat model was established and we found that NEAT1 was greatly upregulated in the spinal cord tissues of CCI rats. Knockdown of NEAT1 can repress neuropathic pain behaviors including mechanical and thermal hyperalgesia. In addition, NEAT1 downregulation inhibited neuroinflammation via inhibiting IL-6, IL-1ß, and tumor necrosis factor (TNF)-α in CCI rats. We also observed that miR-381 was decreased significantly in CCI rats. By using bioinformatics analysis, miR-381 was predicted to be a microRNA target of NEAT1, which indicated a negative correlation between miR-381 and NEAT1. Inhibition of NEAT1 can induce miR-381 expression in CCI rats, which indicated a negative correlation between NEAT1 and miR-381. HMGB1, as a downstream target gene of miR-381 was observed to be dramatically increased in CCI rats. miR-381 can modulate HMGB1 expression negatively and meanwhile, NEAT1 was able to regulate HMGB1 through sponging miR-381. Downregulation of HMGB1 can inhibit neuropathic pain behaviors which can be reversed by miR-381 inhibitors. Taken these together, it was indicated that NEAT1 can induce neuropathic pain development in CCI rats via regulating miR-381/HMGB1 axis.

MicroRNA expression data analysis to identify key miRNAs associated with Alzheimer's disease.

BACKGROUND: MicroRNAs (miRNAs) have become increasingly prevalent as a result of the association of their deregulation with neurodegenerative disorders, especially Alzheimer's disease (AD). However, the association between miRNAs and AD remains unclear. METHODS: In the present study, Nine representative miRNA datasets were selected for the identification of the critical miRNAs by analyzing the overlapping relationships among them. TargetScan software (http://www.targetscan.org) was used to predict the target genes of these miRNAs. In addition, the Database for Annotation Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov) and TfactS (http://www.tfacts.org) datasets were used for combined analysis of functional enrichment and transcription factor (TF) analysis. RESULTS: Thirteen key miRNAs were identified, of which four were significantly up-regulated (hsa-miR-101,hsa-miR-155, has-miR-34a, has-miR-9) and eight were found to be significantly down-regulated (hsa-let-7d-5p, hsa-let-7 g-5p, hsa-miR-15b, has-miR-191-5p, hsa-miR-125b, has-miR-26b-5p, hsa-miR-29b, hsa-miR-342-3p). The functional enrichment analysis indicated that up-regulated signature miRNA targets were associated with transcription from the RNA polymerase II promoter process and the chemical synaptic transmission process. Down-regulated signature miRNA targets were mostly enriched with respect to positive regulation of transcription from the RNA polymerase II promoter process, p53 signaling, and microRNAs in cancer pathways. TF analysis showed that 87 TFs were influenced by the up-regulated miRNAs, and 134 TFs were influenced by the down-regulated miRNAs. In total, 70 (45.5%) TFs were affected by both up-regulated and down-regulated miRNAs. CONCLUSIONS: In summary, 13 key miRNAs were found to have a vital function in the pathological progress of AD, as well as the target genes and TFs of these miRNAs. The potential functions of these miRNAs as diagnostic and therapeutic targets of the AD are revealed by the present study.

XIST accelerates neuropathic pain progression through regulation of miR-150 and ZEB1 in CCI rat models.

LncRNAs are reported to participate in neuropathic pain development. LncRNA X-inactive specific transcript (XIST) is involved in the progression of various cancers. However, the role of XIST in neuropathic pain remains unclear. In our present study, we established a chronic constriction injury (CCI) rat model and XIST was found to be greatly upregulated both in the spinal cord tissues and in the isolated microglias of CCI rats. Inhibition of XIST inhibited neuropathic pain behaviors including mechanical and thermal hyperalgesia. Moreover, decrease of XIST repressed neuroinflammation through inhibiting COX-2, tumor necrosis factor (TNF)-α and IL-6 and in CCI rats. Previously, miR-150 has been reported to restrain neuropathic pain by targeting TLR5. Currently, miR-150 was predicted to be a microRNA target of XIST, which indicated a negative correlation between miR-150 and XIST. miR-150 was remarkably decreased in CCI rats and overexpression of miR-150 can significantly suppress neuroinflammation-related cytokines. Furthermore, ZEB1 was exhibited to be a direct target of miR-150 and we found it was overexpressed in CCI rats. Silencing ZEB1 was able to inhibit neuropathic pain in vivo and downreguation of XIST decreased ZEB1, which can be reversed by miR-150 inhibitors. Taken these together, we indicated that XIST can induce neuropathic pain development in CCI rats via upregulating ZEB1 by acting as a sponge of miR-150. It was revealed that XIST/miR-150/ZEB1 axis can be provided as a therapeutic target in neuropathic pain.

Jun, Gal, Cd74, and C1qb as potential indicator for neuropathic pain.

Neuropathic pain is a kind of pain caused by primary or secondary impairment or dysfunction of peripheral or central nervous system. Patients with neuropathic pain were often with poor clinical outcome. We screened the differentially expressed genes between sciatic nerve injury and dorsal root ganglion gene in the sham operation model. Microarray and the spared nerve injury module were used to explore the molecular mechanism of neuropathic pain by injuries and the differentially expressed genes (DEGs) were identified out. Besides, the bioinformatics methods were used to figure out the signaling pathways and expression regulation pattern these DEGs were enriched in, which may provide a basis for the molecular research and medicine target of therapy. Besides, protein-protein interaction network analysis was performed on these selected intersection genes. A total of 40 DEGs were screened out and only pctp gene was down-regulated, the left 39 genes were all up-regulated. Then, GO and KEGG enrichment analysis were performed on these intersection genes by DAVID software. Furthermore, protein-protein interaction network analysis was used to analyze the critical genes of neuropathic pain. Finally, four genes, that is, Jun, Gal, Cd74, and C1qb were identified to have strong interactions with other genes, which may function as the prognostic and predictive genes of neuropathic pain caused by peripheral injuries. Our results suggested that four differentially expressed genes, Jun, Gal, Cd74, and C1qb, had the potential to serve as prognostic or predictive markers for neuropathic pain, suggesting a potential application in the improvement of prognostic tools and treatments.

MiR-150 alleviates neuropathic pain via inhibiting toll-like receptor 5.

MicroRNAs (miRNAs) are reported as vital participators in the pathophysiological course of neuropathic pain. However, the underlying mechanisms of the functional roles of miRNAs in neuropathic pain are largely unknown. This study was designed to explore the potential role of miR-150 in regulating the process of neuropathic pain in a rat model established by chronic sciatic nerve injury (CCI). Overexpression of miR-150 greatly alleviated neuropathic pain development and reduced inflammatory cytokine expression, including COX-2, interleukin IL-6, and tumor necrosis factor (TNF)-α in CCI rats. By bioinformatic analysis, 3'-untranslated region (UTR) of Toll-like receptor (TLR5) was predicted to be a target of miR-150. TLR5 commonly serves as an important regulator of inflammation. Overexpression of miR-150 significantly suppressed the expression of TLR5 in vitro and in vivo. Furthermore, upregulation of TLR5 decreased the miR-150 expression and downregulation of TLR5 increased miR-150, respectively. Overexpression of TLR5 significantly reversed the miR-150-induced suppressive effects on neuropathic pain. In conclusion, our current study indicates that miR-150 may inhibit neuropathic pain development of CCI rats through inhibiting TLR5-mediated neuroinflammation. Our findings suggest that miR-150 may provide a novel therapeutic target for neuropathic pain treatment.

MiR-182-5p inhibited oxidative stress and apoptosis triggered by oxidized low-density lipoprotein via targeting toll-like receptor 4.

MicroRNAs (miRNAs) exhibit various roles in multiple biological processes and abnormal expression of miR-182-5p has been involved in many diseases. However, the role miR-182-5p in Atherosclerosis (AS) remains poorly understood. In our current investigation, an AS model was established by using oxidized low-density lipoprotein (ox-LDL) in RAW264.7 cells. miR-182-5p was markedly decreased in AS model dose-dependently and time-dependently. Additionally, CD36, oil-red staining levels, TC, and TG were inhibited by miR-182-5p mimics, meanwhile ROS levels, MDA, and cell apoptosis were also restrained with an enhancement of SOD activity. Consistently, opposite results were exhibited when miR-182-5p inhibitors were transfected into RAW264.7 cells. It is well known that toll-like receptor 4 (TLR4) is responsible for many inflammation diseases. By using bioinformatics analysis, TLR4 was indicated as a potential target of miR-182-5p. We observed TLR4 was activated in AS models and miR-182-5p could repress AS progression by targeting TLR4 in vitro. In conclusion, we uncovered that miR-182-5p played significant roles in AS through inhibiting oxidative stress and apoptosis via inactivating TLR4 expression.

Prognostic value of DNA repair genes based on stratification of glioblastomas.

Abnormal expression of DNA repair genes is frequently associated with cancerogenesis of many tumors, however, the role DNA repair genes play in the progression of glioblastoma remains unclear. In this study, taking advantage of large scale of RNA-seq data, as well as clinical data, the function and prognosis value of key DNA repair genes in glioblastoma were analyzed by systematically bioinformatic approaches. Clustering was performed to screen potentially abnormal DNA repair genes related to the prognosis of glioblastoma, followed by unsupervised clustering to identify molecular subtypes of glioblastomas. Characteristics and prognosis differences were analyzed among these molecular subtypes, and modular driver genes in molecular subtypes were identified based on changes in expression correlation. Multifactor Cox proportional hazard analysis was used to find the independent prognostic factor. A total of 15 key genes, which were significantly related to prognosis, were identified and four molecular subtypes of disease were obtained through unsupervised clustering, based on these 15 genes. By analyzing the clinical features of these 4 molecular subtypes, Cluster 4 was found to be different from others in terms of age and prognosis level. A total of 5 key DNA repair genes, CDK7, DDB2, RNH1, RFC2 and FAH, were screened to be significantly related to the prognosis of glioblastomas (p = 9.74e-05). In summary, the DNA repair genes which can predict the prognosis of patients with Glioblastoma multiforme (GBM) were identified and validated. The expression level of DNA repair genes shows the potential of predicting the prognosis and therapy design in targeting GBM.

Integrated Cox's model for predicting survival time of glioblastoma multiforme.

Glioblastoma multiforme is the most common primary brain tumor and is highly lethal. This study aims to figure out signatures for predicting the survival time of patients with glioblastoma multiforme. Clinical information, messenger RNA expression, microRNA expression, and single-nucleotide polymorphism array data of patients with glioblastoma multiforme were retrieved from The Cancer Genome Atlas. Patients were separated into two groups by using 1 year as a cutoff, and a logistic regression model was used to figure out any variables that can predict whether the patient was able to live longer than 1 year. Furthermore, Cox's model was used to find out features that were correlated with the survival time. Finally, a Cox model integrated the significant clinical variables, messenger RNA expression, microRNA expression, and single-nucleotide polymorphism was built. Although the classification method failed, signatures of clinical features, messenger RNA expression levels, and microRNA expression levels were figured out by using Cox's model. However, no single-nucleotide polymorphisms related to prognosis were found. The selected clinical features were age at initial diagnosis, Karnofsky score, and race, all of which had been suggested to correlate with survival time. Both of the two significant microRNAs, microRNA-221 and microRNA-222, were targeted to p27Kip1 protein, which implied the important role of p27Kip1 on the prognosis of glioblastoma multiforme patients. Our results suggested that survival modeling was more suitable than classification to figure out prognostic biomarkers for patients with glioblastoma multiforme. An integrated model containing clinical features, messenger RNA levels, and microRNA expression levels was built, which has the potential to be used in clinics and thus to improve the survival status of glioblastoma multiforme patients.

MicroRNA-93 alleviates neuropathic pain through targeting signal transducer and activator of transcription 3.

Emerging evidence suggests that microRNAs (miRNAs) play a critical role in the pathogenesis of neuropathic pain. However, the exact role of miRNAs in regulating neuropathic pain remains largely unknown. In this study, we aimed to investigate the potential role of miR-93 in a rat model of neuropathic pain induced by chronic constriction sciatic nerve injury (CCI). We found a significant decrease of miR-93 in the spinal cord of CCI rats compared with sham rats. Overexpression of miR-93 significantly alleviated neuropathic pain development and reduced inflammatory cytokine expression, including interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 in CCI rats. By bioinformatic analysis and dual-luciferase reporter assay, we found that miR-93 directly targeted the 3'-untranslated region (UTR) of signal transducer and activator of transcription 3 (STAT3), an important regulator of inflammation. Overexpression of miR-93 markedly suppressed the expression of STAT3 in vitro and in vivo. Furthermore, overexpression of STAT3 significantly reversed the miR-93 overexpression-induced suppressive effects on neuropathic pain development and neuroinflammation. Taken together, our study suggests that miR-93 inhibits neuropathic pain development of CCI rats possibly through inhibiting STAT3-mediated neuroinflammation. Our findings indicate that miR-93 may serve as a novel therapeutic target for neuropathic pain intervention.

Knockdown of E2F3 Inhibits Proliferation, Migration, and Invasion and Increases Apoptosis in Glioma Cells.

E2F3a, as a member of the E2F family, is essential for cell division associated with the progression of many cancers. However, the biological effect of E2F3a on glioma is not understood as well. To investigate the functional mechanism of E2F3a in glioma, we examined the expression of E2F3a in glioma tissue and cell lines. We found that E2F3a was upregulated in glioma tissue compared with adjacent tissue, and this was associated with a poor survival rate. E2F3a was highly expressed in glioma cell lines compared with normal HEB cell lines. Knockdown of E2F3a significantly inhibited cell proliferation, promoted G0/G1 phase arrest, elevated apoptosis rates, and suppressed cell migration and invasion. However, overexpression of E2F3a markedly promoted cell proliferation, migration, and invasion and inhibited apoptosis. Moreover, in vivo studies showed that knockdown of E2F3a expression dramatically inhibited U373 tumor growth in a nude mouse model. Results of real-time PCR and Western blot showed that the depletion of E2F3a upregulated the expression levels of cell apoptosis-related proteins and downregulated migration-related proteins. Conversely, E2F3a overexpression downregulated the expression levels of cell apoptosis-related proteins and upregulated migration-related proteins. In conclusion, our results highlight the importance of E2F3a in glioma and provide new insights into the diagnostics and therapeutics of gliomas.

MicroRNA biomarkers of Parkinson's disease in serum exosome-like microvesicles.

Parkinson's disease (PD) is a progressive age-related debilitating motor disorder and the second most common neurodegenerative disease after Alzheimer's disease. In this study, we aimed to investigate the expression of 24 candidate miRNAs in PD and to assess their diagnostic value in patients with PD. We collected serum samples from 109 patients with PD and 40 age- and sex-matched healthy volunteers (control group). RNAs encapsulated in exosome-like microvesicles in serum were extracted and reverse transcribed. Serum miRNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the ability of the miRNAs to accurately discriminate PD was analyzed by receiver operating characteristic curves. Based on our analysis, we further validated the downregulation of miR-19b and the upregulation of miR-195 and miR-24 in patients with PD. When compared with the control group, the area under the curve (AUC) values for miR-19b, miR-24, and miR-195 were 0.753, 0.908, and 0.697, respectively. Therefore, analysis of the expression levels of miR-19b, miR-24, and miR-195 in serum may be useful for the diagnosis of PD.

Methylated of genes behaving as potential biomarkers in evaluating malignant degree of glioblastoma.

Abnormal methylation genes usually act as oncogenes or anti-oncogenes in the occurrence and development of tumor, indicating their potential role as biomarkers in the evaluation of malignant tumor. However, the research on methylation's association with glioblastoma was rare. We attempted to figure out whether the methylation of genes could serve as the biomarker in evaluating the malignant degree of GBM. Methylation microarray data of 275 GBM patients have been downloaded from The Cancer Genome Atlas (TCGA) dataset. Logistic regression was used to find the methylated genes associated with the malignant degree of patients with the tumor. Functional enrichment analysis and network analysis were further performed on these selected genes. A total of 668, 412, 470, and 620 genes relevant with the methylation or demethylation were found to be associated with the malignant degree, Grades 1-4 of tumor. The higher the degree of malignant tumor, the higher of its methylation degree of its corresponding genes. GO and KEGG analysis results showed that these methylated genes were enriched in many functions as cell adhesion, abnormal transcription, and cell cycle disorder, etc. Of note, CCL11 and LCN11 were found to be significantly related to the progression of GBM. Critical genes associated with cell cycle as CCL11 and LCN1 may play essential roles in the occurrence, development, and transition of glioblastoma. More research was needed to explore its potential molecular mechanism.