Profiling of Differentially Expressed MicroRNAs in Saliva of Parkinson's Disease Patients

Objective: This study aims to identify differentially expressed salivary miRNAs and validate the diagnostic potential for idiopathic Parkinson's disease (PD). Also, the disease specificity of candidate miRNAs was evaluated between PD, multiple system atrophy (MSA), and essential tremor (ET).Methods: We collected salivary samples from 50 PD, 20 ET, and 20 MSA patients, as well as 30 healthy controls (HCs). In the discovery phase, salivary miRNA microarray analysis was performed. In-silico analysis was used to investigate the target genes of differentially expressed miRNAs and clustered pathways. In validation phase, RT-qPCR was performed with samples from 30 PD patients and 30 HCs. Subsequently, we investigated candidate miRNAs in all recruited subjects. Receiver operating characteristic curve and Spearman correlation analysis was performed to determine diagnostic usefulness.Results: We identified 43 miRNAs that were differentially expressed between 5 PD patients and 5 HCs by miRNA microarray analysis. Computational analysis revealed the target genes were clustered in the pathways associated with ubiquitin protein ligase activity. The result of RT-qPCR showed that the miR-29a-3p and miR-29c-3p were found to be significantly downregulated (p = 0.004, p = 0.027), whereas the miR-6756-5p was significantly upregulated in 30 PD patients compared with 30 HCs (p = 0.032). The miR-29a-3p expression level in PD patients was significantly lower than ET patients (p = 0.035), but higher than MSA patients (p < 0.0001). The diagnostic efficacy reached a little higher when the combination of miR-29a-3p and miR-29c-3p.Conclusion: The miRNA combination of salivary miR-29a-3p and miR-29c-3p has potential to be a diagnostic biomarker for idiopathic PD.

Disruption of miRNA-mRNA Networks Defines Novel Molecular Signatures for Penile Carcinogenesis

Penile cancer (PeC) carcinogenesis is not fully understood, and no biomarkers are reported in clinical practice. We aimed to investigate molecular signatures based on miRNA and mRNA and perform an integrative analysis to identify molecular drivers and pathways for PeC development. Affymetrix miRNA microarray was used to identify differentially expressed miRNAs (DEmiRs) comparing 11 tumoral tissues (TT) paired with non-neoplastic tissues (NNT) with further validation in an independent cohort (n = 13). We also investigated the mRNA expression of 83 genes in the total sample. Experimentally validated targets of DEmiRs, miRNA-mRNA networks, and enriched pathways were evaluated in silico. Eight out of 69 DEmiRs identified by microarray analysis were validated by qRT-PCR (miR-145-5p, miR-432-5p, miR-487b-3p, miR-30a-5p, miR-200a-5p, miR-224-5p, miR-31-3p and miR-31-5p). Furthermore, 37 differentially expressed genes (DEGs) were identified when comparing TT and NNT. We identified four downregulated DEmiRs (miR-30a-5p, miR-432-5p, miR-487b-3p, and miR-145-5p) and six upregulated DEGs (IL1A, MCM2, MMP1, MMP12, SFN and VEGFA) as potential biomarkers in PeC by their capacity of discriminating TT and NNT with accuracy. The integration analysis showed eight dysregulated miRNA-mRNA pairs in penile carcinogenesis. Taken together, our findings contribute to a better understanding of the regulatory roles of miRNAs and altered transcripts levels in penile carcinogenesis.

MicroRNA-140-5p Suppresses The PD-L1 Level In Exosomes Derived From Hepatocellular Carcinoma Cells, Thereby Attenuating The Chemoresistance, Autophagy And Stemness Induced By Sorafenib In Hepatocellular Carcinoma

Background: Sorafenib (SO), inhibitor of receptor tyrosine kinase, possesses anti-tumor ability in hepatocellular carcinoma (HCC) by anti-angiogenic effects. However, patients with advanced HCC are resistant to SO. Dysregulated micro-RNA (miRNA) has been shown to be associated with the drug resistance of tumors. Little is known about the effect of miRNA-140-5p on SO-resistance of HCC.Methods: The human HCC cell line Hep3B and Huh-7 were used in this study. The miRNA microarray was used to sequence the transcripts of SO-chemoresistance HCC tissues and SO-chemosensitive tissues. Then, the HCC tissue chip in the GEO database was also analyzed to screen the target gene. Flow cytometry, in situ hybridisation (ISH), immunohistochemistry (IHC), Western blot, cell proliferation assay, transmission electron microscopy (TEM), real-time PCR analyses (qRT-PCR), and pull-down assay were used to explore the effects of miRNA-140-5p and programmed death ligand-1 (PD-L1) on the chemoresistance, autophagy, and stemness of SO-resistant HCC cells treated with SO. In vivo, the SO-resistant HCC cell lines treated with miRNA-140-5p overexpression were injected subcutaneously into mice to construct HCC subcutaneous xenograft tumor models.Results: Based on miRNA microarray, GEO database, and bioinformatics analysis, it was found that miRNA-140-5p and PD-L1 are the core molecules in the SO resistance regulatory network in HCC, and lower miRNA-140-5p and higher PD-L1 expression levels were observed in SO-resistant HCC tissues and cells. IHC results showed that compared with HCC tissues with high miRNA-140-5p expression, PD-L1 protein expression levels in HCC tissues with low miRNA-140-5p was significantly higher (P<0.01). Moreover, compared with SO-sensitive tissues, the levels of PD-L1 protein expression in SO-resistance tissues were significantly higher (P<0.01). In in vitro and in vivo experiments, the introduction of miRNA-140-5p can decreased PD-L1 expression. Mechanically, we found that the Hep3B/SO and Huh-7/SO cells pretreated with miRNA-140-5p inhibitor or exosomes containing PD-L1 have higher autophagy activity, higher stemness, and lower apoptosis rate, which could be abrogated by co-treating cells with anti-PD-L1 antibody or miRNA-140-5p mimic. Conclusions: MiRNA-140-5p can suppress the PD-L1 expression in HCC cells-derived exosomes, thereby attenuating the chemoresistance, autophagy and stemness induced by SO in HCC.

Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Analysis of MicroRNAs Associated With Carotid Atherosclerotic Plaque Rupture With Thrombosis

Atherosclerosis is a progressive vascular wall inflammatory disease, and the rupture of atherosclerotic vulnerable plaques is the leading cause of morbidity and mortality worldwide. This study intended to explore the potential mechanisms behind plaque rupture and thrombosis in ApoE knockout mice. The spontaneous plaque rupture models were established, and left carotid artery tissues at different time points (1-, 2-, 4-, 6-, 8-, 12-, and 16-week post-surgery) were collected. By the extent of plaque rupture, plaque was defined as (1) control groups, (2) atherosclerotic plaque group, and (3) plaque rupture group. Macrophage (CD68), MMP-8, and MMP-13 activities were measured by immunofluorescence. Cytokines and inflammatory markers were measured by ELISA. The left carotid artery sample tissue was collected to evaluate the miRNAs expression level by miRNA-microarray. Bioinformatic analyses were conducted at three levels: (2) vs. (1), (3) vs. (2), and again in seven time series analysis. The plaque rupture with thrombus and intraplaque hemorrhage results peaked at 8 weeks and decreased thereafter. Similar trends were seen in the number of plaque macrophages and lipids, the expression of matrix metalloproteinase, and the atherosclerotic and plasma cytokine levels. MiRNA-microarray showed that miR-322-5p and miR-206-3p were specifically upregulated in the atherosclerotic plaque group compared with those in the control group. Meanwhile, miR-466h-5p was specifically upregulated in the plaque rupture group compared with the atherosclerotic plaque group. The highest incidence of plaque rupture and thrombosis occurred at 8 weeks post-surgery. miR-322-5p and miR-206-3p may be associated with the formation of atherosclerotic plaques. miR-466h-5p may promote atherosclerotic plaque rupture via apoptosis-related pathways.

Identification of differentially expressed miRNA 48 h after cerebral ischemia–reperfusion injury in mice by the technique of miRNA microarray

The objective was to identify the differential expressed miRNA during cerebral ischemia–reperfusion injury (CIRI) process, thereby assisting in elucidating the mechanism of CIRI development and providing a potential target for CIRI prevention and treatment. Six mice were randomly assigned to two groups: control group and CIRI model group. A global cerebral IR model by four-vessel occlusion was prepared among the CIRI model group. Brain tissues were collected 48 h after reperfusion. Total RNA was extracted for each sample. miRNA microarrays were employed to detect the differentially expressed miRNA between the CIRI group and the control group. One differentially expressed miRNA was selected for verification by PCR. Compared with the control group, 69 miRNAs were significantly differential expressed in samples of the CIRI group, among which 50 miRNAs were upregulated and 19 miRNAs were downregulated. The real-time qPCR results indicated that the results of the miRNA microarray were reliable. A number of miRNAs were significantly regulated in the CIRI model, which suggested that miRNA was closely associated with the pathological alterations after ischemia. These identified miRNAs may provide directions and targets for the future pathological research of CIRI.

Circulating microRNAs Is a Potential Prognostic Biomarker in Primary Central Nervous System Lymphoma

Backgrounds and purposes Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin's lymphoma. The prognosis of PCNSL is poor and heterogeneous. MicroRNA is associated with prognosis of various cancers. Our study is the first time to compare circulating microRNAs of PCNSL patients with different prognosis using miRNA microarray scanning and find novel microRNAs associated with prognosis of PCNSL by further large-scale validation. Materials and methods From a retrospective cohort, we collected the clinical data of patients who were diagnosed as PCNSL in Huashan Hospital between January 2007 and June 2016. We collected clinical data and blood samples from residual blood routine test of PCNSLs. First, we compared circulating microRNAs between patients with different overall survival (OS) using miRNA microarray scanning. Second, PCR assay was used for miRNAs quantification in blood sample from 6 patients that had long overall survival and other 6 patients that had short overall survival. Further miRNA validation was made by PCR assay in blood sample from 94 patients to validate prognostic value of miRNAs in PCNSLs. The Kruskal-Wallis and Mann-Whitney U tests were used for quantitative parameters and the χ2 square test was used for non-quantitative parameters. Univariate analysis was performed using log-rank test, and survival distributions were analyzed by the Kaplan-Meier curve and log-rank test. Multivariate analysis was done by Cox regression model. Results A total of 90 differentially expressed miRNAs were identified, including 24 up-regulated miRNAs and 66 down-regulated miRNAs using miRNA microarray scanning. Then PCR assay was used for 10 differentially expressed miRNAs quantification in blood sample from 6 patients that had long overall survival and 6 patients that had short overall survival. Mir-21, mir-129-5p, mir-144-5, mir-363-5p, mir-409-3p, mir-1246, mir-1299 and mir-1825 showed no differences between 2 groups, while the expression of mir-455-3P and mir-940 between 2 groups are significantly different (P&lt;0.05). Further validation in 94 patients showed median OS in miR-940 overexpression group was 91 months and in miR-940 low-expression group was 28 months (P=0.0005), while median PFS in miR-940 overexpression group was 25 months and in miR-940 low-expression group was 16 months (P =0.0292). Multivariate analysis of COX model showed miR-940 was independent factors for OS and PFS. Conclusion Circulating mir-940 had prognostic value in PCNSL and was an independent prognostic factor for PCNSL. Figure 1 Disclosures No relevant conflicts of interest to declare.

532 SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of early-stage myeloid-derived suppressor cells via the miR-155/C/EBPß/Wnt axis

BackgroundEarly-stage myeloid-derived suppressor cells (eMDSCs) are a newly defined subset of myeloid-derived suppressor cells (MDSCs) that accumulate densely in tumors and potently promote tumor growth and metastasis by suppressing antitumor immune responses in vitro and in vivo. We previously identified a subset of eMDSCs in human breast cancer with a characteristic phenotype of Lin-HLA-DR-CD33+. We also found that SOCS3 deficiency and sustained activation of the JAK/STAT signaling pathway are critical molecular events coordinating the differentiation of eMDSCs, although the distinct molecular regulation has not been fully elucidated.MethodsHerein, we genetically constructed conditional SOCS3 knockout mice with SOCS3 deficiency specifically in the myeloid linage (SOCS3MyeKO). We analyzed the number of eMDSCs in SOCS3MyeKO mice (eMDSCsSOCS3KO). To explore which pathways participated in dysfunctional eMDSC differentiation, we performed whole-genome RNA sequencing and miRNA microarray on CD11b+Gr-1+ cells, eMDSCsfl/fl and eMDSCsSOCS3KO to screen the potential regulatory ceRNA network in eMDSCsSOCS3KO. CD11b+Gr-1+ cells isolated from SOCS3fl/fl mouse spleens were used as mature myeloid cell controls. Furthermore, we applied a specific miR-155 antagonist and the autophagy agonist rapamycin to suppress tumor growth and eMDSC infiltration.ResultsThe transcriptome results and corresponding intervention experiment revealed that the differentiation block in eMDSCsSOCS3KO was caused by SOCS3 deficiency-mediated limited autophagy activation in an AMPK-independent manner. The results of miRNA microarray and RNA sequencing demonstrated that miR-155 overexpression and Wnt/ß-catenin pathway activation were involved in the SOCS3 knockout-mediated myeloid differentiation block and autophagy repression. Further experiments revealed that miR-155 was induced by activation of the STAT3/NK-?B pathway upon SOCS3 deficiency, which consequently activated the Wnt/ß-catenin pathway via targeting C/EBPß. Furthermore, applying a specific miR-155 antagonist or the autophagy agonist rapamycin efficiently suppressed tumor growth and eMDSC infiltration in vivo.ConclusionsOverall, these findings indicated that SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of e-MDSCs via the miR-155/C/EBPß/Wnt axis, and thus targeted therapy against this pathway could be a potential therapeutic target in breast cancer.

Differences in MicroRNA Expression in Chronic Hepatitis B Patients with Early Liver Fibrosis Based on Traditional Chinese Medicine Syndromes

The aim of this study was to determine if microRNA (miRNA) expression is different among chronic hepatitis B (CHB) patients with early liver fibrosis classified according to traditional Chinese medicine (TCM) syndromes. Eighteen CHB-fibrosis patients and 12 CHB patients without fibrosis were enrolled. The CHB-fibrosis group included 9 patients with the TCM syndrome of Ganyu Pixu Xueyu (GYPXXY), characterized by liver stagnation, spleen deficiency, and blood stasis, and 9 patients with the TCM syndrome of Qixu Xueyu (QXXY), characterized by deficiency of qi, blood, and blood stasis. Agilent miRNA microarray was performed first in liver specimens to determine whether miRNA expression is different in patients with these two TCM syndromes of CHB-fibrosis. Gene Ontology (GO) analysis and KEGG analysis were applied to determine the roles of the differentially expressed miRNAs. QRT-PCR was performed to validate the Agilent miRNA microarray results. Compared with GYPXXY patients, 6 differentially expressed miRNAs were upregulated (miR-144-5p, miR-18a-5p, miR-148b-3p, miR-654-3p, miR-139-3p, and miR-24-1-5p) and 1 was downregulated (miR-6834-3p) in QXXY patients. According to qRT-PCR data, miR-144-5p and miR-654-3p were confirmed as upregulated in CHB-liver fibrosis patients compared to CHB patients without fibrosis, whereas the other 4 miRNAs were not significantly different. More importantly, miR-654-3p was confirmed to be significantly upregulated in QXXY patients compared with values in GYPXXY patients, whereas no significant difference was found in miR-144-5p. Moreover, the pathways of central carbon metabolism in cancer and cell cycle related to miR-654-3p and the target genes of PTEN and ATM were found to be different between QXXY patients and GYPXXY patients. These results indicate that there are different miRNAs, pathways, and target genes between QXXY patients and GYPXXY patients. However, due to the limited sample, whether miR-654-3p and the target genes PTEN and ATM could be molecular markers to differentiate TCM syndromes could not be established.