Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype–phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype–phenotype correlations and improve prognostic outcomes.     

Novel Risk Associations between microRNA Polymorphisms and Gastric Cancer in a Chilean Population

Gastric cancer (GC) is the fifth leading cause of cancer deaths in the world, with variations across geographical regions and ethnicities. Emerging evidence indicates that miRNA expression is dysregulated in GC and its polymorphisms may contribute to these variations, which has yet to be explored in Latin American populations. In a case-control study of 310 GC patients and 311 healthy donors from Chile, we assessed the association of 279 polymorphisms in 242 miRNA genes. Two novel polymorphisms were found to be associated with GC: rs4822739:C>G (miR-548j) and rs701213:T>C (miR-4427). Additionally, rs1553867776:T>TCCCCA (miR-4274) and rs12416605:C>T (miR-938) were associated with intestinal-type GC, and rs4822739:C>G (miR-548j) and rs1439619:T>G (miR-3175) with TNM I-II stage. The polymorphisms rs6149511:T> TGAAGGGCTCCA (miR-6891), rs404337:G>A (miR-8084), and rs1439619:T>G (miR-3175) were identified among H.pylori-infected GC patients and rs7500280:T>C (miR-4719) and rs1439619:T>G (miR-3175) were found among H. pylori cagPAI+ infected GC cases. Prediction analysis suggests that seven polymorphisms could alter the secondary structure of the miRNA, and the other one is located in the seed region of miR-938. Targets of miRNAs are enriched in GC pathways, suggesting a possible biological effect. In this study, we identified seven novel associations and replicated one previously described in Caucasian population. These findings contribute to the understanding of miRNA genetic polymorphisms in the GC pathogenesis.     

Structural Characterization of the Interaction of Hypoxia Inducible Factor-1 with Its Hypoxia Responsive Element at the −964G > A Variation Site of the HLA-G Promoter Region

Human Antigen Leukocyte-G (HLA-G) gene encodes an immune checkpoint molecule that has restricted tissue expression in physiological conditions; however, the gene may be induced in hypoxic conditions by the interaction with the hypoxia inducible factor-1 (HIF1). Hypoxia regulatory elements (HRE) located at the HLA-G promoter region and at exon 2 are the major HIF1 target sites. Since the G allele of the −964G > A transversion induces higher HLA-G expression when compared to the A allele in hypoxic conditions, here we analyzed HIF1-HRE complex interaction at the pair-atom level considering both −964G > A polymorphism alleles. Mouse HIF2 dimer crystal (Protein Data Bank ID: 4ZPK) was used as template to perform homology modelling of human HIF1 quaternary structure using MODELLER v9.14. Two 3D DNA structures were built from 5′GCRTG’3 HRE sequence containing the −964G/A alleles using x3DNA. Protein-DNA docking was performed using the HADDOCK v2.4 server, and non-covalent bonds were computed by DNAproDB server. Molecular dynamic simulation was carried out per 200 ns, using Gromacs v.2019. HIF1 binding in the HRE containing −964G allele results in more hydrogen bonds and van der Waals contact formation than HRE with −964A allele. Protein-DNA complex trajectory analysis revealed that HIF1-HRE-964G complex is more stable. In conclusion, HIF1 binds in a more stable and specific manner at the HRE with G allele.     

Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease.     

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.     

Functional Characterization of FH Mutation c.557G>A Underlies Uterine Leiomyomas

The FH gene encodes the fumarate hydratase of the Krebs cycle and functions as a homotetramer to catalyze the hydration of fumarate to malate. Mutations in FH result in uterine leiomyomas, a rare autosomal dominant inherited metabolic disease. However, how FH mutations result in this disease is poorly understood. Here, the FH mutation c.557G>A (p.S186N) was identified in a family with uterine leiomyomas phenotype. A series of studies were performed to confirm the pathogenicity of this mutation. Results showed that the FH mutant exhibited significantly lower fumarase enzyme activity and increased the fumarates level compared with the wildtype, which might be due to the impaired homotetramer formation in the native gel electrophoresis. Interestingly, the immunofluorescence study revealed that the overexpressed FH mutant exhibited puncta structures compared with the evenly expressed FH wildtype in cytoplasm suggesting that the altered amino acid might result in dysfunctional proteins which were accumulated to reduce its cytotoxicity. Importantly, the cells overexpressing the FH mutant exhibited higher proliferation and extracellular acidification rate value (ECAR) which might be caused by the upregulated HIF-1α indicating the tumor phenotype. Notably, phospho-mTOR was significantly increased and autophagy was inhibited in the FH mutant overexpression cells compared with the wildtype. Our work provides new insight into the FH mutation c.557G>A (p.S186N) underlies uterine leiomyomas and important information for accurate genetic counseling and clinical diagnosis of the disease.     

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.     

Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p < 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population.     

Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.     

Different Role of Tumor Necrosis Factor-α Polymorphism in Non-Hodgkin Lymphomas among Caucasian and Asian Populations: A Meta-Analysis

Tumor necrosis factor-α (TNF-α) is an immunoregulatory cytokine involved in B- and T-cell function, and also plays an important role in inflammation and cancer. TNF-α-308G>A has been associated with constitutively elevated TNF-α expression. Several studies have reported the association between the TNF-α-308G>A polymorphism and non-Hodgkin lymphomas (NHL) risk, however, results are still inconsistent. To solve these conflicts, we conducted the first meta-analysis to assess the effect of TNF-α-308G>A polymorphism on the risk of NHL and various subtypes (additive model) including 10,619 cases and 12,977 controls in Caucasian and Asian populations. Our meta-analysis indicated that TNF-α-308G>A polymorphism is not associated with NHL risk when pooling all studies together (OR = 1.06, 95% CI: 0.92–1.23, p = 0.413). In stratified analyses, we found TNF-α-308A allele was significantly associated with higher risk of NHL, B-cell lymphomas (BCL), T-cell lymphomas (TCL) and diffuse large B-cell lymphomas (DLBCL) in Caucasians (OR = 1.22, 95% CI: 1.06–1.40, p = 0.007; OR = 1.18, 95% CI: 1.03–1.34, p = 0.014; OR = 1.20, 95% CI: 1.01–1.42, p = 0.040; OR = 1.21, 95% CI: 1.11–1.32, p < 0.001, respectively). Interestingly, it was associated with decreased risk of NHL, BCL and DLBCL in Asians (OR = 0.75, 95% CI: 0.66–0.86, p < 0.001; OR = 0.70, 95% CI: 0.52–0.94, p = 0.018; OR = 0.70, 95% CI: 0.57–0.86, p = 0.001). These findings also suggest TNF-α might play a distinct role in pathogenesis of NHL in different populations.     

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan^® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)^® microarrays from Agilent^® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.     

The Role of Circulating MicroRNA-126 (miR-126): A Novel Biomarker for Screening Prediabetes and Newly Diagnosed Type 2 Diabetes Mellitus

Recent studies suggested an association of endothelial microRNA-126 (miR-126) with type 2 diabetes mellitus (T2DM). In the current study, we examined whether circulating miR-126 is associated with T2DM and pre-diabetic syndrome. The study included 82 subjects with impaired glucose tolerance (IGT), 75 subjects with impaired fasting glucose (IFG), 160 patients with newly diagnosed T2DM, and 138 healthy individuals. Quantitative polymerase chain reaction (qPCR) was used to examine serum miR-126. Serum miR-126 was significantly lower in IGT/IFG subjects and T2DM patients than in healthy controls (p < 0.05). After six months of treatment (diet control and exercise in IGT/IFG subjects, insulin plus diet control and exercise in T2DM patients), serum miR-126 increased significantly (p < 0.05). An analysis based on serum miR-126 in the sample revealed a significantly higher odds ratio (OR) for the subjects with the lowest 1/3 of serum miR-126 for T2DM (OR: 3.500, 95% confidence interval: 1.901–6.445, p < 0.05) than subjects within the highest 1/3 of serum miR-126. Such an association was still apparent after adjusting for other major risk factors. The area under the curve (AUC) for the receiver-operating characteristic (ROC) analysis was 0.792 (95% confidence interval: 0.707–0.877, p < 0.001). These results encourage the use of serum miR-126 as a biomarker for pre-diabetes and diabetes mellitus, as well as therapeutic response.     

miR-9-5p,miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation,Differentiation, and Adhesion

This study was designed to evaluate the effects of strontium on the expression levels of microRNAs (miRNAs) and to explore their effects on skeletal cell proliferation, differentiation, adhesion, and apoptosis. The targets of these miRNAs were also studied. Molecular cloning, cell proliferation assay, cell apoptosis assay, quantitative real-time PCR, and luciferase reporter assay were used. Strontium altered the expression levels of miRNAs in vitro and in vivo. miR-9-5p, miR-675-5p, and miR-138-5p impaired skeletal cell proliferation, cell differentiation and cell adhesion. miR-9-5p and miR-675-5p induced MC3T3-E1 cell apoptosis more specifically than miR-138-5p. miR-9-5p, miR-675-5p, and miR-138-5p targeted glycogen synthase kinase 3 β (GSK3β), ATPase Aminophospholipid Transporter Class I Type 8A Member 2 (ATP8A2), and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1), respectively. Low-density lipoprotein receptor-related protein 5 (LRP5) played a positive role in skeletal development. miR-9-5p, miR-675-5p, and miR-138-5p damage strontium and LRP5-mediated skeletal cell proliferation, differentiation, and adhesion, and induce cell apoptosis by targeting GSK3β, ATP8A2, and EIF4EBP1, respectively.     

CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts

Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.     

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.     

miR-378a-3p Participates in Metformin’s Mechanism of Action on C2C12 Cells under Hyperglycemia

Metformin is the most used biguanide drug for the treatment of type 2 diabetes mellitus. Despite being mostly known for its hepatic anti-gluconeogenic effect, it is also known to modulate microRNAs (miRNAs, miRs) associated with metabolic diseases. The latter mechanism could be relevant for better understanding metformin’s mechanisms underlying its biological effects. In the current work, we found that metformin increases miR-378a-3p expression (p < 0.002) in C2C12 myoblasts previously exposed to hyperglycemic conditions. While the inhibition of miR-378a-3p was shown to impair metformin’s effect in ATP production, PEPCK activity and the expression of Tfam. Finally, mitophagy, an autophagic process responsible for the selective degradation of mitochondria, was found to be induced by miR-378a-3p (p < 0.04). miR-378a-3p stimulated mitophagy through a process independent of sestrin-2 (SESN2), a stress-responsible protein that has been recently demonstrated to positively modulate mitophagy. Our findings provide novel insights into an alternative mechanism of action of metformin involving miR-378a-3, which can be used in the future for the development of improved therapeutic strategies against metabolic diseases.     

Smad8 Is Increased in Duchenne Muscular Dystrophy and Suppresses miR-1, miR-133a,and miR-133b

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor β (TGFβ) signaling. In this report, we investigated the major transducers of TGFβ signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx^5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.     

miR-196b-5p and miR-107 Expression Differentiates Ocular Sebaceous Carcinoma from Squamous Cell Carcinoma of the Conjunctiva

An Ocular Sebaceous Carcinoma (OSC) is a rare malignant tumor for which initial clinical and pathological diagnosis is often incorrect. OSCs can mimic Squamous Cell Carcinomas of the Conjunctiva (SCCC). The aim of this study was to find microRNA biomarkers to distinguish OSCs and SCCCs from normal tissue and from each other. Clinical OSC and SCCC case files and the corresponding histopathological slides were collected and reviewed. Micro dissected formalin-fixed paraffin-embedded tumor and control tissues were subjected to semi-high throughput microRNA profiling. MicroRNA expression distinguishes OSCs and SCCCs from corresponding control tissues. Selected differentially expressed miRNAs were validated using single RT-PCR assays. No prognostic miRNAs could be identified that reliably predict SCCC metastasis or OSC recurrence. A comparison between OSCs (n = 14) and SCCCs (n = 18) revealed 38 differentially expressed microRNAs (p < 0.05). Differentially expressed miRNAs were selected for validation in the discovery cohort and an independent validation cohort (OSCs, n = 11; SCCCs, n = 12). At least two miRNAs, miR-196b-5p (p ≤ 0.05) and miR-107 (p ≤ 0.001), displayed a statistically significant differential expression between OSCs and SCCCs with miR-196b-5p upregulated in SCCCs and miR-107 upregulated in OSCs. In the validation cohort, microRNA miR-493-3p also showed significant upregulation in SCCCs when compared to OSCs (p ≤ 0.05). ROC analyses indicated that the combined miR-196b-5p and miR-107 expression levels predicted OSCs with 90.0% sensitivity and 83.3% specificity. In conclusion, the combined testing of miR-196b-5p and miR-107, can be of additional use in routine diagnostics to discriminate OSCs from SCCCs.     

Novel Single Nucleotide Polymorphisms of the Insulin-Like Growth Factor-I Gene and Their Associations with Growth Traits in Common Carp (Cyprinus carpio L.)

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of vertebrates. To study polymorphisms of IGF-I, we screened a total of 4555 bp of genomic sequences in four exons and partial introns for the discovery of single nucleotide polymorphism (SNP) in common carp (Cyprinus carpio). Three SNPs (g.3759T>G, g.7627T>A and g.7722T>C) in intron 2 and a nonsynonymous SNP (g.7892C>T) in exon 3 were identified in a pilot population including random parents and their progenies. 289 progenies were further genotyped for studying possible associations between genotypes or combined genotypes and growth traits. The results showed that the locus g.7627T>A was significantly associated with body weight and body length, and fish with genotype AA had a mean body weight 5.9% higher than those with genotype TT. No significant associations were observed between genotypes of other loci and growth traits. However, when both g.7627T>A and g.7722T>C were considered, the combined genotype TT/TT was extremely associated with the lowest values of body length and body weight and the highest K value in comparison with other diplotypes (p < 0.01). These results suggest that genotype AA at g.7627T>A and its combined genotypes with alleles from another locus have positive effects on growth traits, which would be a candidate molecular marker for further studies in marker-assisted selection in common carp.