Identification of novel miRNA targets in CHO cell lines and characterization of their impact on protein N-glycosylation

CHO cell lines are a workhorse for the production of pharmaceutical proteins, but show some limitations in the variability and stability of N-glycosylation profiles. One promising approach to addressing this at the required systems-level is miRNA, which can regulate a large number of genes and have predictable targets. Herein, we first identified de novo 656 potential miRNAs in the CHO genome based on a combination of literature, database searching, and miRNA sequencing. We further sequenced mRNA from the same cultures, and used a combination of mRNA-miRNA correlation analysis, target prediction and literature searches to find miRNAs potentially targeting N-glycosylation. Our ten best miRNA candidates were subjected to miRNA overexpression, knockdown, or knock-out in CHO cell lines. Out of the ten candidates, four (miR-128, miR-34c, miR-30b, and miR-449a) showed positive effects on N-glycosylation and could be applied directly for CHO cell engineering. The fact that 40% of the screened targets had a desired effect, and the prediction of 656 miRNAs illustrates the massive potential of miRNA engineering in CHO.

Macrophage-derived microRNA-223 Promotes Intestinal Mucosal Barrier Dysfunction via Exosomal Pathways in Inflammatory Bowel Disease

Background & Aim: Exosomes are effective mediators of cell-to-cell interactions and carry many regulatory molecules, including miRNAs, that can play crucial roles in diverse fundamental biological processes. However, to date, no study has reported macrophage exosomal involvement in the development of inflammatory bowel disease (IBD). This study investigated the specific miRNAs in macrophage-derived exosomes in IBD and the potential molecular mechanism. Methods: Dextran sulfate sodium (DSS) was used to generate IBD mice. The supernatants of murine bone marrow-derived macrophages (BMDMs) with or without lipopolysaccharide (LPS) were collected for exome isolation and miRNA sequencing. Lentiviruses were used to modify miRNA expression and further investigate the role of macrophage-derived exosomal miRNAs. In vitro, both mouse and human organoids were applied to a Transwell system in co-culture with BMDMs as a cellular IBD-related challenge.Results: Here, we show that LPS-induced macrophages can release exosomes containing various miRNAs, aggravating IBD. We analyzed miRNA sequencing of macrophage-derived exosomes, and miR-223 was selected for further study. In vivo, exosomes with high miR-223 expression contributed to the exacerbation of intestinal barrier dysfunction, which was further verified in both mouse and human colon organoids. Furthermore, time-dependent analysis of the mRNAs of DSS-induced colitis mouse tissue combined with miR-223 target gene prediction was performed to select the candidate gene, and the barrier-related factor TMIGD1 was identified.Conclusion: Collectively, these data indicated that macrophage-derived exosomal miR-223 played a novel role in intestinal barrier dysfunction by inhibiting TMIGD1 in the progression of DSS-induced colitis.

Circulating miRNAs as Potential Biomarkers for Celiac Disease Development

Background & AimsCeliac disease (CeD), an immune-mediated disease with enteropathy triggered by gluten, affects ~1% of the general European population. Currently, there are no biomarkers to predict CeD development. MicroRNAs (miRNAs) are short RNAs involved in post-transcriptional gene regulation, and certain disease- and stage-specific miRNA profiles have been found previously. We aimed to investigate whether circulating miRNAs can predict the development of CeD.MethodsUsing next-generation miRNA-sequencing, we determined miRNAs in >200 serum samples from 53 participants of the PreventCD study, of whom 33 developed CeD during follow-up. Following study inclusion at 3 months of age, samples were drawn at predefined ages, diagnosis (first anti-transglutaminase antibody (TGA) positivity or diagnostic biopsy) and after the start of a gluten-free diet (GFD). This allowed identification of circulating miRNAs that are deregulated before TGA positivity. For validation of the biomarkers for CeD and GFD response, two additional cohorts were included in subsequent meta-analyses. Additionally, miRNAs were measured in duodenal biopsies in a case-control cohort.Results53 circulating miRNAs were increased (27) or decreased (26) in CeD versus controls. We assessed specific trends in these individual miRNAs in the PreventCD cohort by grouping the pre-diagnostic samples of the CeD patients (all had negative TGA) by how close to seroconversion (first sample positive TGA) the samples were taken. 8/53 miRNAs differed significantly between controls and samples taken <1 year before TGA positivity: miR-21-3p, miR-374a-5p, 144-3p, miR-500a-3p, miR-486-3p let-7d-3p, let-7e-5p and miR-3605-3p. 6/26 downregulated miRNAs reconstituted upon GFD, including miR-150-5p/-3p, whereas no upregulated miRNAs were downregulated upon GFD. 15/53 biomarker candidates also differed between CeD biopsies and controls, with a concordant direction, indicating that these circulating miRNAs might originate from the intestine.ConclusionsWe identified 53 circulating miRNAs that are potential early biomarkers for CeD, of which several can be detected more than a year before TGA positivity and some start to normalize upon GFD.

miR-181a is a novel player in the STAT3-mediated survival network of TCRαβ+ CD8+ T large granular lymphocyte leukemia

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.

CSF MicroRNAs Reveal Impairment of Angiogenesis and Autophagy in Parkinson Disease

Background and ObjectivesWe assessed longitudinal changes in CSF microRNAs (miRNAs) in patients with moderately severe Parkinson disease.MethodsWe used next-generation whole-genome miRNA sequencing to determine CSF miRNA expression in 75 patients with Parkinson disease after single random ascending doses of nilotinib and longitudinal miRNA expression after daily nilotinib, 150 and 300 mg, vs placebo for 1 year.ResultsSignificant changes in the expression of miRNAs that control genes and pathways that regulate angiogenesis, autophagy, and the blood-brain-barrier components, primarily collagen, were observed over 1 year, suggesting impairment of these pathways in Parkinson progression in these patients. Different miRNAs that indicate activation of genes associated with autophagy flux and clearance and angiogenesis were significantly altered in the nilotinib, 300 mg vs 150 mg, or placebo group, and these changes correlated with clinical outcomes. No changes were observed in miRNAs after a single dose of nilotinib vs placebo.DiscussionThis study suggests vascular and autophagy defects in Parkinson progression. Nilotinib, 300 mg, reverses these effects via alteration of miRNA expression, suggesting epigenomic changes that may underlie long-term disease-modifying effects.Trial Registration InformationClinical trial registration number: NCT02954978.

Y/X-Chromosome-Bearing Sperm Shows Elevated Ratio in the Left but Not the Right Testes in Healthy Mice

The sex chromosomes play central roles in determining the sex of almost all of the multicellular organisms. It is well known that meiosis in mammalian spermatogenesis produces ~50% Y- and ~50% X-chromosome-bearing sperm, a 1:1 ratio. Here we first reveal that the X-chromosome-encoded miRNAs show lower expression levels in the left testis than in the right testis in healthy mice using bioinformatics modeling of miRNA-sequencing data, suggesting that the Y:X ratio could be unbalanced between the left testis and the right testis. We further reveal that the Y:X ratio is significantly elevated in the left testis but balanced in the right testis using flow cytometry. This study represents the first time the biased Y:X ratio in the left testis but not in the right testis is revealed.

The Identification of MiRNA and MRNA Expression Profiles Associated with Pediatric Atypical Teratoid/rhabdoid Tumor

Background: Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant pediatric tumor of the central nervous system (CNS) with high recurrence and low survival rates that is often misdiagnosed. MicroRNAs (miRNAs) are involved in the tumorigenesis of numerous pediatric cancers, but their roles in AT/RT remain unclear.Methods: In this study, we used miRNA sequencing and gene expression microarrays from patient tissue to study both the miRNAome and transcriptome traits of AT/RT.Results: Our findings demonstrate that 5 miRNAs were up-regulated, 16 miRNAs were down-regulated, 179 mRNAs were up-regulated and 402 mRNAs were down-regulated in AT/RT. The expressions of hsa-miR-17-5p and MAP7 mRNA showed the most significant differences in AT/RT tissues as assayed by qPCR, and analyses using the miRTarBase database identified MAP7 mRNA as a target gene of hsa-miR-17-5p. Conclusions: Our findings suggest that the dysregulation of hsa-miR-17-5p may be a pivotal event in AT/RT and MAP7 miRNAs that may represent potential therapeutic targets and diagnostic biomarkers.