The Antitumor Effect of Metformin Is Mediated by miR-26a in Breast Cancer

Metformin, a drug approved for diabetes type II treatment, has been associated with a reduction in the incidence of breast cancer and metastasis and increased survival in diabetic breast cancer patients. High levels of miR-26a expression have been proposed as one of the possible mechanisms for this effect; likewise, this miRNA has also been associated with survival/apoptosis processes in breast cancer. Our aim was to evaluate if miR-26a and some of its targets could mediate the effect of metformin in breast cancer. The viability of MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cell lines was evaluated with an MTT assay after ectopic overexpression and/or downregulation of miR-26a. Similarly, the expression levels of the miR-26a targets CASP3, CCNE2, ABL2, APAF1, XIAP, BCL-2, PTEN, p53, E2F3, CDC25A, BCL2L1, MCL-1, EZH2, and MTDH were assessed by quantitative polymerase chain reaction (PCR). The effect of metformin treatment on breast cancer cell viability and miR-26a, BCL-2, PTEN, MCL-1, EZH2, and MTDH modulation were evaluated. Wound healing experiments were performed to analyze the effect of miR-26a and metformin treatment on cell migration. MiR-26a overexpression resulted in a reduction in cell viability that was partially recovered by inhibiting it. E2F3, MCL-1, EZH2, MTDH, and PTEN were downregulated by miR-26a and the PTEN (phosphatase and tensin homolog) protein was also reduced after miR-26a overexpression. Metformin treatment reduced breast cancer cell viability, increased miR-26a expression, and led to a reduction in BCL-2, EZH2, and PTEN expression. miR-26a inhibition partly prevents the metformin viability effect and the PTEN and EZH2 expression reduction. Our results indicate that metformin effectively reduces breast cancer cell viability and suggests that the effects of the drug are mediated by an increase in miR-26a expression and a reduction of its targets, PTEN and EHZ2 Thus, the use of metformin in breast cancer treatment constitutes a promising potential breast cancer therapy.     

Identification of Tumor Suppressive Genes Regulated by miR-31-5p and miR-31-3p in Head and Neck Squamous Cell Carcinoma

We identified the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) tissues by RNA sequencing, in which 168 miRNAs were significantly upregulated, including both strands of the miR-31 duplex (miR-31-5p and miR-31-3p). The aims of this study were to identify networks of tumor suppressor genes regulated by miR-31-5p and miR-31-3p in HNSCC cells. Our functional assays showed that inhibition of miR-31-5p and miR-31-3p attenuated cancer cell malignant phenotypes (cell proliferation, migration, and invasion), suggesting that they had oncogenic potential in HNSCC cells. Our in silico analysis revealed 146 genes regulated by miR-31 in HNSCC cells. Among these targets, the low expression of seven genes (miR-31-5p targets: CACNB2 and IL34; miR-31-3p targets: CGNL1, CNTN3, GAS7, HOPX, and PBX1) was closely associated with poor prognosis in HNSCC. According to multivariate Cox regression analyses, the expression levels of five of those genes (CACNB2: p = 0.0189; IL34: p = 0.0425; CGNL1: p = 0.0014; CNTN3: p = 0.0304; and GAS7: p = 0.0412) were independent prognostic factors in patients with HNSCC. Our miRNA signature and miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.     

miR-22-3p and miR-30e-5p Are Associated with Prognosis in Cervical Squamous Cell Carcinoma

Alteration in expression of miRNAs can cause various malignant changes and the metastatic process. Our aim was to identify the miRNAs involved in cervical squamous cell carcinoma (SqCC) and metastasis, and to test their utility as indicators of metastasis and survival. Using microarray technology, we performed miRNA expression profiling on primary cervical SqCC tissue (n = 6) compared with normal control (NC) tissue and compared SqCC that had (SqC-M; n = 3) and had not (SqC-NM; n = 3) metastasized. Four miRNAs were selected for validation by qRT-PCR on 29 SqC-NM and 27 SqC-M samples, and nine metastatic lesions (ML-SqC), from a total of 56 patients. Correlation of miRNA expression and clinicopathological parameters was analyzed to evaluate the clinical impact of candidate miRNAs. We found 40 miRNAs differentially altered in cervical SqCC tissue: 21 miRNAs were upregulated and 19 were downregulated (≥2-fold, p < 0.05). Eight were differentially altered in SqC-M compared with SqC-NM samples: four were upregulated (miR-494, miR-92a-3p, miR-205-5p, and miR-221-3p), and four were downregulated (miR-574-3p, miR-4769-3p, miR-1281, and miR-1825) (≥1.5-fold, p < 0.05). MiR-22-3p might be a metastamiR, which was gradually further downregulated in SqC-NM > SqC-M > ML-SqC. Downregulation of miR-30e-5p significantly correlated with high stage, lymph node metastasis, and low survival rate, suggesting an independent poor prognostic factor.     

GPC1 Regulated by miR-96-5p,Rather than miR-182-5p,in Inhibition of Pancreatic Carcinoma Cell Proliferation

To determine the relationships between miR-96-5p/-182-5p and GPC1 in pancreatic cancer (PC), we conducted the population and in vitro studies. We followed 38 pancreatic cancer patients, measured and compared the expression of miR-96-5p/-182-5p, GPC1, characteristics and patients’ survival time of different miR-96-5p/-182-5p expression levels in PC tissues. In an in vitro study, we investigated the proliferation, cycle and apotosis in cells transfected with mimics/inhibitors of the two miRNAs, and determine their effects on GPC1 by dual-luciferase assay. In the follow-up study, we found that the expressions of miR-96-5p/-182-5p were lower/higher in PC tissues; patients with lower/higher levels of miR-96-5p/-182-5p suffered poorer characteristics and decreased survival time. In the in vitro study, the expressions of miR-96-5p/-182-5p were different in cells. Proliferation of cells transfected with miR-96-5p mimics/inhibitors was lower/higher in Panc-1/BxPC-3; when transfected with miR-182-5p mimics/inhibitors, proliferation of cells were higher/lower in AsPC-1/Panc-1. In a cell cycle study, panc-1 cells transfected with miR-96-5p mimics was arrested at G0/G1; BxPC-3 cells transfected with miR-96-5p inhibitors showed a significantly decrease at G0/G1; AsPC-1 cells transfected with miR-182-5p mimics was arrested at S; Panc-1 cells transfected with miR-182-5p inhibitors showed a decrease at S. MiR-96-5p mimics increased the apoptosis rate in Panc-1 cells, and its inhibitors decreased the apoptosis rate in BxPC-3. Dual luciferase assay revealed that GPC1 was regulated by miR-96-5p, not -182-5p. We found that miR-96-5p/-182-5p as good markers for PC; miR-96-5p, rather than -182-5p, inhibits GPC1 to suppress proliferation of PC cells.     

Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.     

Serum miR-373-3p and miR-194-5p Are Associated with Early Tumor Progression during FOLFIRINOX Treatment in Pancreatic Cancer Patients: A Prospective Multicenter Study

In this study, we explored the predictive value of serum microRNA (miRNA) expression for early tumor progression during FOLFIRINOX chemotherapy and its association with overall survival (OS) in patients with pancreatic ductal adenocarcinoma (PDAC). A total of 132 PDAC patients of all disease stages were included in this study, of whom 25% showed progressive disease during FOLFIRINOX according to the RECIST criteria. MiRNA expression was analyzed in serum collected before the start and after one cycle of chemotherapy. In the discovery cohort (n = 12), a 352-miRNA RT-qPCR panel was used. In the validation cohorts (total n = 120), miRNA expression was detected using individual RT-qPCR miRNA primers. Before the start of FOLFIRINOX, serum miR-373-3p expression was higher in patients with progressive disease compared to patients with disease control after FOLFIRINOX (Log2 fold difference (FD) 0.88, p = 0.006). MiR-194-5p expression after one cycle of FOLFIRINOX was lower in patients with progressive disease (Log2 FD −0.29, p = 0.044). Both miRNAs were predictors of early tumor progression in a multivariable model including disease stage and baseline CA19-9 level (miR-373-3p odds ratio (OR) 3.99, 95% CI 1.10–14.49; miR-194-5p OR 0.91, 95% CI 0.83–0.99). MiR-373-3p and miR-194-5p did not show an association with OS after adjustment for disease stage, baseline CA19-9, and chemotherapy response. In conclusion, high serum miR-373-3p before the start and low serum miR-194-5p after one cycle are associated with early tumor progression during FOLFIRINOX.     

Circulating miRNAs Act as Diagnostic Biomarkers for Bladder Cancer in Urine

MicroRNAs (miRNAs) can be secreted into body fluids and have thus been reported as a new type of cancer biomarker. This study aimed to determine whether urinary miRNAs act as noninvasive biomarkers for diagnosing bladder cancer. Small RNA profiles from urine were generated for 10 patients with bladder cancer and 10 healthy controls by using next-generation sequencing. We identified 50 urinary miRNAs that were differentially expressed in bladder cancer compared with controls, comprising 44 upregulated and six downregulated miRNAs. Pathway enrichment analysis revealed that the biological role of these differentially expressed miRNAs might be involved in cancer-associated signaling pathways. Further analysis of the public database revealed that let-7b-5p, miR-149-5p, miR-146a-5p, miR-193a-5p, and miR-423-5p were significantly increased in bladder cancer compared with corresponding adjacent normal tissues. Furthermore, high miR-149-5p and miR-193a-5p expression was significantly correlated with poor overall survival in patients with bladder cancer. The qRT-PCR approach revealed that the expression levels of let-7b-5p, miR-149-5p, miR-146a-5p and miR-423-5p were significantly increased in the urine of patients with bladder cancer compared with those of controls. Although our results indicated that urinary miRNAs are promising biomarkers for diagnosing bladder cancer, this must be validated in larger cohorts in the future.     

Peritoneal Modulators of EZH2-miR-155 Cross-Talk in Endometriosis

Activation of trimethylation of histone 3 lysine 27 (H3K27me3) by EZH2, a component of the Polycomb repressive complex 2 (PRC2), is suggested to play a role in endometriosis. However, the mechanism by which this complex is dysregulated in endometriosis is not completely understood. Here, using eutopic and ectopic tissues, as well as peritoneal fluid (PF) from IRB-approved and consented patients with and without endometriosis, the expression of PRC2 complex components, JARID2, miR-155 (known regulators of EZH2), and a key inflammatory modulator, FOXP3, was measured. A higher expression of EZH2, H3K27me3, JARID2, and FOXP3 as well as miR-155 was noted in both the patient tissues and in endometrial PF treated cells. Gain-or-loss of function of miR-155 showed an effect on the PRC2 complex but had little effect on JARID2 expression, suggesting alternate pathways. Chromatin immunoprecipitation followed by qPCR showed differential expression of PRC2 complex proteins and its associated binding partners in JARID2 vs. EZH2 pull down assays. In particular, endometriotic PF treatment increased the expression of PHF19 (p = 0.0474), a gene silencer and co-factor that promotes PRC2 interaction with its targets. Thus, these studies have identified the potential novel crosstalk between miR-155-PRC2 complex-JARID2 and PHF19 in endometriosis, providing an opportunity to test other epigenetic targets in endometriosis.     

Identification and Validation of miR-222-3p and miR-409-3p as Plasma Biomarkers in Gestational Diabetes Mellitus Sharing Validated Target Genes Involved in Metabolic Homeostasis

Gestational diabetes mellitus (GDM) causes both maternal and fetal adverse outcomes. The deregulation of microRNAs (miRNAs) in GDM suggests their involvement in GDM pathogenesis and complications. Exosomes are extracellular vesicles (EVs) of endosomal origin, released via exocytosis into the extracellular compartment. Through EVs, miRNAs are delivered in distant target cells and are able to affect gene expression. In this study, miRNA expression was analyzed to find new miRNAs that could improve GDM classification and molecular characterization. MiRNA were profiled in total plasma and EVs in GDM patients and normal glucose tolerance (NGT) women. Samples were collected at third trimester of gestation from two diabetes centers. MiRNA expression was profiled in a discovery cohort using the multiplexed NanoString nCounter Human v3 miRNA. Validation analysis was performed in a second independent cohort using RT-qPCR. A set of miRNAs resulted to be differentially expressed (DE) in total plasma and EVs in GDM. Among them, total plasma miR-222-3p and miR-409-3p were validated in the independent cohort. MiR-222-3p levels correlated with fasting plasma glucose (FPG) (p < 0.001) and birth weight (p = 0.012), whereas miR-409-3p expression correlated with FPG (p < 0.001) and inversely with gestational age (p = 0.001). The major validated target genes of the deregulated miRNAs were consistently linked to type 2 diabetes and GDM pathophysiology. MiR-222-3p and miR-409-3p are two circulating biomarkers that could improve GDM classification power and act in the context of the molecular events leading to the metabolic alterations observed in GDM.     

Analysis of miR-9-5p,miR-124-3p,miR-21-5p,miR-138-5p,and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM’s diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.     

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.     

miR-21 Promotes Human Nucleus Pulposus Cell Proliferation through PTEN/AKT Signaling

The precise role of nucleus pulposus cell proliferation in the pathogenesis of intervertebral disc degeneration remains to be elucidated. Recent findings have revealed that microRNAs, a class of small noncoding RNAs, may regulate cell proliferation in many pathological conditions. Here, we showed that miR-21 was significantly upregulated in degenerative nucleus pulposus tissues when compared with nucleus pulposus tissues that were isolated from patients with idiopathic scoliosis and that miR-10b levels were associated with disc degeneration grade. Moreover, bioinformatics target prediction identified PTEN as a putative target of miR-21. miR-21 inhibited PTEN expression by directly targeting the 3′UTR, and this inhibition was abolished through miR-21 binding site mutations. miR-21 overexpression stimulated cell proliferation and AKT signaling pathway activation, which led to cyclin D1 translation. Additionally, the increase in proliferation and cyclin D1 expression induced by miR-21 overexpression was almost completely blocked by {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002, an AKT inhibitor. Taken together, aberrant miR-21 upregulation in intervertebral disc degeneration could target PTEN, which would contribute to abnormal nucleus pulposus cell proliferation through derepressing the Akt pathway. Our study also underscores the potential of miR-21 and the PTEN/Akt pathway as novel therapeutic targets in intervertebral disc degeneration.     

Identification of Tumor-Suppressive miR-30e-3p Targets: Involvement of SERPINE1 in the Molecular Pathogenesis of Head and Neck Squamous Cell Carcinoma

Recently, our studies revealed that some passenger strands of microRNAs (miRNAs) were closely involved in cancer pathogenesis. Analysis of miRNA expression signatures showed that the expression of miR-30e-3p (the passenger strand of pre-miR-30e) was significantly downregulated in cancer tissues. In this study, we focused on miR-30e-3p (the passenger strand of pre-miR-30e). We addressed target genes controlled by miR-30e-3p that were closely associated with the molecular pathogenesis of head and neck squamous cell carcinoma (HNSCC). Ectopic expression assays demonstrated that the expression of miR-30e-3p attenuated cancer cell malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities). Our analysis of miR-30e-3p targets revealed that 11 genes (ADA, CPNE8, C14orf126, ERGIC2, HMGA2, PLS3, PSMD10, RALB, SERPINE1, SFXN1, and TMEM87B) were expressed at high levels in HNSCC patients. Moreover, they significantly predicted the short survival of HNSCC patients based on 5-year overall survival rates (p < 0.05) in The Cancer Genome Atlas (TCGA). Among these targets, SERPINE1 was found to be an independent prognostic factor for patient survival (multivariate Cox regression; hazard ratio = 1.6078, p < 0.05). Aberrant expression of SERPINE1 was observed in HNSCC clinical samples by immunohistochemical analysis. Functional assays by targeting SERPINE1 expression revealed that the malignant phenotypes (e.g., proliferation, migration, and invasion abilities) of HNSCC cells were suppressed by the silencing of SERPINE1 expression. Our miRNA-based approach will accelerate our understanding of the molecular pathogenesis of HNSCC.     

MicroRNA-103 Promotes Colorectal Cancer by Targeting Tumor Suppressor DICER and PTEN

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that act as key regulators in various physiological and pathological processes. However, the regulatory mechanisms for miRNAs in colorectal cancer remain largely unknown. Here, we found that miR-103 is up-regulated in colorectal cancer and its overexpression is closely associated with tumor proliferation and migration. In addition, repressing the expression of miR-103 apparently inhibits colorectal cancer cell proliferation and migration in vitro and HCT-116 xenograft tumor growth in vivo. Subsequent software analysis and dual-luciferase reporter assay identified two tumor suppressor genes DICER and PTEN as direct targets of miR-103, and up-regulation of DICER and PTEN obtained similar results to that occurred in the silencing of miR-103. In addition, restoration of DICER and PTEN can inhibit miR-103-induced colorectal cancer cell proliferation and migration. Our data collectively demonstrate that miR-103 is an oncogene miRNA that promotes colorectal cancer proliferation and migration through down-regulation of the tumor suppressor genes DICER and PTEN. Thus, miR-103 may represent a new potential diagnostic and therapeutic target for colorectal cancer treatment.     

RNA-Sequencing Based microRNA Expression Signature of Colorectal Cancer: The Impact of Oncogenic Targets Regulated by miR-490-3p

To elucidate novel aspects of the molecular pathogenesis of colorectal cancer (CRC), we have created a new microRNA (miRNA) expression signature based on RNA-sequencing. Analysis of the signature showed that 84 miRNAs were upregulated, and 70 were downregulated in CRC tissues. Interestingly, our signature indicated that both guide and passenger strands of some miRNAs were significantly dysregulated in CRC tissues. These findings support our earlier data demonstrating the involvement of miRNA passenger strands in cancer pathogenesis. Our study focused on downregulated miR-490-3p and investigated its tumor-suppressive function in CRC cells. We successfully identified a total of 38 putative oncogenic targets regulated by miR-490-3p in CRC cells. Among these targets, the expression of three genes (IRAK1: p = 0.0427, FUT1: p = 0.0468, and GPRIN2: p = 0.0080) significantly predicted 5-year overall survival of CRC patients. Moreover, we analyzed the direct regulation of IRAK1 by miR-490-3p, and its resultant oncogenic function in CRC cells. Thus, we have clarified a part of the molecular pathway of CRC based on the action of tumor-suppressive miR-490-3p. This new miRNA expression signature of CRC will be a useful tool for elucidating new molecular pathogenesis in this disease.     

Imatinib Regulates miR-483-3p and Mitochondrial Respiratory Complexes in Gastrointestinal Stromal Tumors

Metabolic adaptation to increased oxidative phosphorylation (OXPHOS) has been found in gastrointestinal stromal tumor (GIST) upon imatinib treatment. However, the underlying mechanism of imatinib-induced OXPHOS is unknown. Discovering molecules that mediate imatinib-induced OXPHOS may lead to the development of therapeutic strategies synergizing the efficacy of imatinib. In this study, we explored the role of microRNAs in regulating OXPHOS in GIST upon imatinib treatment. Using a microarray approach, we found that miR-483-3p was one of the most downregulated miRNAs in imatinib-treated tumors compared to untreated tumors. Using an extended series of GIST samples, we further validated the downregulation of miR-483-3p in imatinib-treated GIST samples by RT-qPCR. Using both gain- and loss-of-function experiments, we showed that miR-483-3p could regulate mitochondrial respiratory Complex II expression, suggesting its role in OXPHOS regulation. Functionally, miR-483-3p overexpression could rescue imatinib-induced cell death. These findings provide the molecular link for imatinib-induced OXPHOS expression and the biological role of miR-483-3p in regulating cell viability upon imatinib treatment.     

mRNA and miRNA Expression Analyses of the MYC/E2F/miR-17-92 Network in the Most Common Pediatric Brain Tumors

Numerous molecular factors disrupt the correctness of the cell cycle process leading to the development of cancer due to increased cell proliferation. Among known causative factors of such process is abnormal gene expression. Nowadays in the light of current knowledge such alterations are frequently considered in the context of mRNA–miRNA correlation. One of the molecular factors with potential value in tumorigenesis is the feedback loop between MYC and E2F genes in which miR-17-5p and miR-20a from the miR-17-92 cluster are involved. The current literature shows that overexpression of the members of the OncomiR-1 are involved in the development of many solid tumors. In the present work, we investigated the expression of components of the MYC/E2F/miR-17-92 network and their closely related elements including members of MYC and E2F families and miRNAs from two paralogs of miR-17-92: miR-106b-25 and miR-106a-363, in the most common brain tumors of childhood, pilocytic astrocytoma (PA), WHO grade 1; ependymoma (EP), WHO grade 2; and medulloblastoma (MB), WHO grade 4. We showed that the highest gene expression was observed in the MYC family for MYCN and in the E2F family for E2F2. Positive correlation was observed between the gene expression and tumor grade and type, with the highest expression being noted for medulloblastomas, followed by ependymomas, and the lowest for pilocytic astrocytomas. Most members of miR-17-92, miR-106a-363 and miR-106b-25 clusters were upregulated and the highest expression was noted for miR-18a and miR-18b. The rest of the miRNAs, including miR-19a, miR-92a, miR-106a, miR-93, or miR-25 also showed high values. miR-17-5p, miR-20a obtained a high level of expression in medulloblastomas and ependymomas, while close to the control in the pilocytic astrocytoma samples. miRNA expression also depended on tumor grade and histology.     

Circulating miRNAs as Biomarkers for Mitochondrial Neuro-Gastrointestinal Encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease for which there are currently no validated outcome measures for assessing therapeutic intervention efficacy. The aim of this study was to identify a plasma and/or serum microRNA (miRNA) biomarker panel for MNGIE. Sixty-five patients and 65 age and sex matched healthy controls were recruited and assigned to one of four study phases: (i) discovery for sample size determination; (ii) candidate screening; (iii) candidate validation; and (iv) verifying the performance of the validated miRNA panel in four patients treated with erythrocyte-encapsulated thymidine phosphorylase (EE-TP), an enzyme replacement under development for MNGIE. Quantitative PCR (qPCR) was used to profile miRNAs in serum and/or plasma samples collected for the discovery, validation and performance phases, and next generation sequencing (NGS) analysis was applied to serum samples assigned to the candidate screening phase. Forty-one differentially expressed candidate miRNAs were identified in the sera of patients (p < 0.05, log₂ fold change > 1). The validation cohort revealed that of those, 27 miRNAs were upregulated in plasma and three miRNAs were upregulated in sera (p < 0.05). Through binary logistic regression analyses, five plasma miRNAs (miR-192-5p, miR-193a-5p, miR-194-5p, miR-215-5p and miR-34a-5p) and three serum miRNAs (miR-192-5p, miR-194-5p and miR-34a-5p) were shown to robustly distinguish MNGIE from healthy controls. Reduced longitudinal miRNA expression of miR-34a-5p was observed in all four patients treated with EE-TP and coincided with biochemical and clinical improvements. We recommend the inclusion of the plasma exploratory miRNA biomarker panel in future clinical trials of investigational therapies for MNGIE; it may have prognostic value for assessing clinical status.