Bone-related circulating microRNAs miR-29b-3p, miR-550a-3p and miR-324-3p and their Association to Bone Microstructure and Histomorphometry.
The assessment of bone quality and the prediction of fracture risk in idiopathic osteoporosis (IOP) are complex prospects as bone mineral density (BMD) and bone turnover markers (BTM) do not indicate fracture-risk. MicroRNAs (miRNAs) are promising new biomarkers for bone diseases, but the current understanding of the biological information contained in the variability of miRNAs is limited. Here, we investigated the association between serum-levels of 19 miRNA biomarkers of idiopathic osteoporosis to bone microstructure and bone histomorphometry based upon bone biopsies and µCT (9.3 μm) scans from 36 patients. Four miRNAs were found to be correlated to bone microarchitecture and seven miRNAs to dynamic histomorphometry (p<0.05). Three miRNAs, namely, miR-29b-3p, miR-324-3p, and miR-550a-3p showed significant correlations to histomorphometric parameters of bone formation as well as microstructure parameters. miR-29b-3p and miR-324-p were found to be reduced in patients undergoing anti-resorptive therapy. This is the first study to report that serum levels of bone-related miRNAs might be surrogates of dynamic histomorphometry and potentially reveal changes in bone microstructure. Although these findings enhance the potential value of circulating miRNAs as bone biomarkers, further experimental studies are required to qualify the clinical utility of miRNAs to reflect dynamic changes in bone formation and microstructure.
Feichtinger X, Muschitz C, Heimel P,Baierl A, Fahrleitner-Pammer A,Redl H, et al.
Scientific Reports, Vol 8 , Article number: 4867 (2018)
Altered MicroRNA Profile in Osteoporosis Caused by Impaired WNT Signaling
Context: WNT signaling is fundamental to bone health and its aberrant activation leads to skeletal pathologies. A heterozygous missense mutation p.C218G in WNT1, a key WNT pathway ligand, leads to severe early-onset and progressive osteoporosis with multiple peripheral and spinal fractures. Despite the severe skeletal manifestations, conventional bone turnover markers are normal in mutation-positive patients.
Objective:This study sought to explore the circulating miRNA pattern in patients with impaired WNT signaling.
Design and Setting: A cross-sectional cohort study at a University Hospital.
Participants: Altogether 12 mutation-positive (median age 39 years, range 11-76 years) and 12 mutation-negative (35 years, range 9-59 years) subjects from two Finnish families with WNT1 osteoporosis due to the heterozygous p.C218G WNT1 mutation.
Methods and main outcome measure: Serum samples were screened for 192 miRNAs using qPCR. Findings were compared between WNT1 mutation-positive and mutation-negative subjects.
Results: The pattern of circulating miRNAs was significantly different in the mutation-positive subjects as compared with the mutation-negative subjects with 2 upregulated (miR-18a-3p, miR-223-3p) and 6 downregulated miRNAs (miR-22-3p, miR-31-5p, miR-34a-5p, miR-143-5p miR-423-5p, miR-423-3p). Three of these (miR-22-3p, miR-34a-5p, and miR-31-5p) are known inhibitors of WNT signaling: miR-22-3p and miR-34a-5p target WNT1 mRNA and miR-31-5p is predicted to bind to WNT1 3'UTR.
Conclusions: The circulating miRNA pattern reflects WNT1 mutation status. The findings suggest that the WNT1 mutation disrupts a feed-back regulation between these miRNAs and WNT1, providing new insights into the pathogenesis of WNT-related bone disorders. These miRNAs could offer future potential in diagnosis and treatment of osteoporosis.
Mäkitie RE, Hackl M, Niinimäki R,Kakko S, Grillari J, Mäkitie O
J Clin Endocrinol Metab. 2018 Mar 1. doi: 10.1210/jc.2017-02585.
Cost-utility analysis of fracture risk assessment using microRNA compared with Standard Tools and no Monitoring in the Austrian female Population
Background: Osteoporosis poses an immense burden to the society in terms of morbidity, mortality and financial cost. To reduce this burden, it is essential to accurately assess the individual patient's fracture risk and, where indicated, to initiate appropriate treatment that reduces fracture probability. Current screening and monitoring approaches include utilization of FRAX®, a web-based country-specific fracture risk assessment tool, and bone mineral density measurement by Dual Energy X-ray Absorptiometry (DXA). Recently, microRNAs have been recognized as important regulators of bone physiology and potential biomarkers for fracture risk assessment and monitoring. A fracture risk assessment tool based on microRNAs (osteomiR™ test) is currently being developed. The aim of this study was to estimate the cost-effectiveness of fracture risk screening, monitoring, and resulting treatment decisions for the Austrian female population using the osteomiR™ test compared with DXA, with FRAX®, or with no screening/monitoring.
Methods: A cost-utility-model was developed to simulate long-term consequences of Austrian women from age 50 over lifetime or death with respect to osteoporosis. Markov-modelling techniques were used to calculate health state transitions of fracture incidence according to risk groups (high, intermediate, low). High-risk patients receive medical treatment. Probabilities were derived via systematic-literature-review; direct costs (2015, €) from published sources from the payer's perspective. Results evaluate the incremental cost-effectiveness ratios (ICER) for osteomiR™ against the comparators, gains or losses of fractures, life years (LYs), quality-adjusted life years (QALYs), and direct costs. QALYs, life years (LYs) and costs were discounted (3% p.a).
Results: Fracture risk assessment and monitoring using the osteomiR™ test reduces fracture incidence compared with no monitoring, DXA alone, or FRAX® alone. In the per-patient analysis, the ICER/QALY of osteomiR™ vs. no-monitoring was 13,103 €, vs. FRAX® 37,813 €, and vs. DXA -19,605 €, indicating that costs can be saved while gaining QALYs. Considering the total cohort over lifetime, the osteomiR™ test can avoid 57,919 fractures compared with DXA, 31,285 fractures compared with FRAX® and 133,394 fractures compared with no monitoring. Sensitivity analysis confirmed the robustness of these findings.
Conclusion: Fracture risk assessment and monitoring using the osteomiR™ test dominates DXA-strategy and constitutes a cost-effective alternative to FRAX®, and no-monitoring.
Walter E, Dellago H, Grillari J, Dimai HP, Hackl M
Bone. Volume 108, 2018; Pages 44-54.
MicroRNAs and toxicology: A love marriage
With the dawn of personalized medicine, secreted microRNAs (miRNAs) have come into the very focus of biomarker development for various diseases. MiRNAs fulfil key requirements of diagnostic tools such as i) non or minimally invasive accessibility, ii) robust, standardized and non-expensive quantitative analysis, iii) rapid turnaround of the test result and iv) most importantly because they provide a comprehensive snapshot of the ongoing physiologic processes in cells and tissues that package and release miRNAs into cell-free space. These characteristics have also established circulating miRNAs as promising biomarker candidates for toxicological studies, where they are used as biomarkers of drug-, or chemical-induced tissue injury for safety-assessment. The tissue-specificity and early release of circulating miRNAs upon tissue injury, when damage is still reversible, are main factors for their clinical utility in toxicology. Here we summarize in brief, current knowledge of this field.
Schraml E, Hackl M and Grillari J.
Toxicology Reports. Volume 4, 2017; Pages 634-636.
Clopidogrel in critically ill patients.
Only limited data are available regarding the treatment of critically ill patients with clopidogrel. This trial investigated the effects and the drug concentrations of the CYP450 activated pro-drug clopidogrel (n=43) and the half-life of the similarly metabolized pantoprazole (n=16) in critically ill patients. ADP-induced aggregometry in whole blood classified 74% (95% confidence intervals 59-87%) of critically ill patients as poor responders (n=43), and 65% (49-79%) responded poorly according to the VASP-P assay. Whilst the plasma levels of clopidogrel active metabolite normally exceed the inactive prodrug ∼30-fold, parent drug levels even exceeded those of the metabolite 2-fold in critically ill patients. The half-life of pantoprazole was several-fold longer in these patients compared to reference populations. The inverse ratio of prodrug/active metabolite indicates insufficient metabolization of clopidogrel, which is independently confirmed by the ∼5-fold increase in half-life of pantoprazole. Thus, high-risk patients may benefit from treatment with alternative platelet Inhibitors.
Schoergenhofer C, Hobl EL, Schellongowski P, Heinz G, Speidl W, et al
Clin Pharmacol Ther. 2017 Sep 15.
Serum microRNAs Are Indicative of Skeletal Fractures in Postmenopausal Women with and without Type 2 Diabetes and Influence Osteogenic and Adipogenic Differentiation of Adipose-Tissue Derived Mesenchymal Stem Cells In Vitro.
Standard DXA measurements, including Fracture Risk Assessment Tool (FRAX) scores, have shown limitations in assessing fracture risk in Type 2 Diabetes (T2D), underscoring the need for novel biomarkers and suggesting that other pathomechanisms may drive diabetic bone fragility. MicroRNAs (miRNAs) are secreted into the circulation from cells of various tissues proportional to local disease severity and were recently found to be crucial to bone homeostasis and T2D. Here, we studied, if and which circulating miRNAs or combinations of miRNAs can discriminate best fracture status in a well-characterized study of diabetic bone disease and postmenopausal osteoporosis (n = 80 postmenopausal women). We then tested the most discriminative and most frequent miRNAs in vitro. Using miRNA-qPCR-arrays, we showed that 48 miRNAs can differentiate fracture status in T2D women and that several combinations of four miRNAs can discriminate diabetes-related fractures with high specificity and sensitivity (area under the receiver-operating characteristic curve values [AUCs], 0.92 to 0.96; 95% CI, 0.88 to 0.98). For the osteoporotic study arm, 23 miRNAs were fracture-indicative and potential combinations of four miRNAs showed AUCs from 0.97 to 1.00 (95% CI, 0.93 to 1.00). Because a role in bone homeostasis for those miRNAs that were most discriminative and most present among all miRNA combinations had not been described, we performed in vitro functional studies in human adipose tissue-derived mesenchymal stem cells to investigate the effect of miR-550a-5p, miR-188-3p, and miR-382-3p on osteogenesis, adipogenesis, and cell proliferation. We found that miR-382-3p significantly enhanced osteogenic differentiation (p < 0.001), whereas miR-550a-5p inhibited this process (p < 0.001). Both miRNAs, miR-382-3p and miR-550a-5p, impaired adipogenic differentiation, whereas miR-188-3p did not exert an effect on adipogenesis. None of the miRNAs affected significantly cell proliferation. Our data suggest for the first time that miRNAs are linked to fragility fractures in T2D postmenopausal women and should be further investigated for their diagnostic potential and their detailed function in diabetic bone. © 2016 American Society for Bone and Mineral Research.
Heilmeier U, Hackl M, Skalicky S, Weilner S, Schroeder F, Vierlinger K, et al.
J Bone Miner Res 2016. Dec;31(12):2173-2192
Circulating microRNA signatures in patients with idiopathic and postmenopausal osteoporosis and fragility fractures.
CONTEXT: Established bone turnover markers do not reflect fracture risk in idiopathic male and premenopausal osteoporosis and the role of microRNAs (miRNAs) in these patients is currently unclear. miRNAs are a class of small non-coding RNAs that regulate gene expression and bone tissue homeostasis. They are considered a new class of endocrine regulators with promising potential as biomarkers.
OBJECTIVE:Evaluation of circulating miRNA signatures in male and female subjects with idiopathic and postmenopausal osteoporotic low-traumatic fractures.
DESIGN, SETTING, AND PATIENTS: This was a case-control study of cross-sectional design of 36 patients with prevalent low-traumatic fractures and 39 control subjects Main Outcome Measures: One hundred eighty-seven miRNAs were quantified in serum by qPCR, compared between groups and correlated with established bone turnover markers.
RESULTS: Significant differences in serum levels of circulating miRNAs were identified in all three subgroups (46 in premenopausal, 52 in postmenopausal, 55 in male). A set of 19 miRNAs was consistently regulated in all three subgroups. Eight miRNAs [miR-152-3p, miR-30e-5p, miR-140-5p, miR-324-3p, miR-19b-3p, miR-335-5p, miR-19a-3p, miR-550a-3p] were excellent discriminators of patients with low-traumatic fractures, regardless of age and sex, with area under the curve values > 0.9. The 11 remaining miRNAs showed area under the curve values between 0.81 and 0.89. Correlation analysis identified significant correlations between miR-29b-3p and P1NP, and miR-365-5p and iPTH, TRAP5b, P1NP and Osteocalcin, as well as BMDL1-L4 and miR-19b-3p, miR-324-3p, miR-532-5p, and miR-93-5p.
CONCLUSIONS: Specific serum miRNA profiles are strongly related to bone pathologies. Therefore miRNAs might be directly linked to bone tissue homeostasis. In particular, miR-29b-3p has previously been reported as regulator of osteogenic differentiation and could serve as a novel marker of bone turnover in osteoporotic patients as a member of a miRNA signature.
Kocijan R, Muschitz C, Geiger E, Skalicky S, Baierl A, Dormann R, et al.
J Clin Endocrinol Metab 2016:jc.2016-2365.
Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.
Aging results in a decline of physiological functions and in reduced repair capacities, in part due to impaired regenerative power of stem cells, influenced by the systemic environment. In particular osteogenic differentiation capacity (ODC) of mesenchymal stem cells (MSCs) has been shown to decrease with age, thereby contributing to reduced bone formation and an increased fracture risk. Searching for systemic factors that might contribute to this age related decline of regenerative capacity led us to investigate plasma-derived extracellular vesicles (EVs). EVs of the elderly were found to inhibit osteogenesis compared to those of young individuals. By analyzing the differences in the vesicular content Galectin-3 was shown to be reduced in elderly-derived vesicles. While overexpression of Galectin-3 resulted in an enhanced ODC of MSCs, siRNA against Galectin-3 reduced osteogenesis. Modulation of intravesicular Galectin-3 levels correlated with an altered osteo-inductive potential indicating that vesicular Galectin-3 contributes to the biological response of MSCs to EVs. By site-directed mutagenesis we identified a phosphorylation-site on Galectin-3 mediating this effect. Finally, we showed that cell penetrating peptides comprising this phosphorylation-site are sufficient to increase ODC in MSCs. Therefore, we suggest that decrease of Galectin-3 in the plasma of elderly contributes to the age-related loss of ODC.
Weilner S, Keider V, Winter M, Harreither E, Salzer B, Weiss F, Schraml E, et al.
Aging (Albany NY). 2016 Jan;8(1):16-33.
Urine is a novel source of autologous mesenchymal stem cells for patients with epidermolysis bullosa.
BACKGROUND: Regenerative medicine is strictly dependent on stem cells as a source for a high diversity of somatic cells. However, the isolation of such from individuals suffering from severe genetic skin blistering diseases like epidermolysis bullosa (EB) is often associated with further organ damage.
METHODS: Stem cells were isolated from 112 urine samples from 21 different healthy donors, as well as from 33 urine samples from 25 donors with EB. The cultivation of these cells was optimized by testing different media formulations and pre-coating of culture vessels with collagen. The identity of cells was confirmed by testing marker expression, differentiation potential and immune-modulatory properties.
RESULTS: We provide here an optimized protocol for the reproducible isolation of mesenchymal stem cells from urine, even from small volumes as obtained from patients with EB. Furthermore, we offer a basic characterization of those urine-derived stem cells (USCs) from healthy donors, as well as from patients with EB, and demonstrate their potential to differentiate into chondrocytes, osteoblasts and adipocytes, as well as their immune-modulatory properties.
CONCLUSIONS: Thus, USCs provide a novel and non-invasive source of stem cells, which might be applied for gene-therapeutic approaches to improve medical conditions of patients with EB.
Schosserer M, Reynoso R, Wally V, Jug B, Kantner V, Weilner S, et al.
BMC Res Notes. 2015 Dec 10;8(1):767.
Circulating microRNAs as novel biomarkers for bone diseases - Complex signatures for multifactorial diseases?
Biomarkers are essential tools in clinical research and practice. Useful biomarkers must combine good measurability, validated association with biological processes or outcomes, and should support clinical decision making if used in clinical practice. Several types of validated biomarkers have been reported in the context of bone diseases. However, because these biomarkers face certain limitations there is an interest in the identification of novel biomarkers for bone diseases, specifically in those that are tightly linked to the disease pathology leading to increased fracture-risk. MicroRNAs (miRNAs) are the most abundant RNA species to be found in cell-free blood. Encapsulated within microvesicles or bound to proteins, circulating miRNAs are remarkably stable analytes that can be measured using gold-standard technologies such as quantitative polymerase-chain-reaction (qPCR). Nevertheless, the analysis of circulating miRNAs faces several pre-analytical as well as analytical challenges. From a biological view, there is accumulating evidence that miRNAs play essential roles in the regulation of various biological processes including bone homeostasis. Moreover, specific changes in miRNA transcription levels or miRNA secretory levels have been linked to the development and progression of certain bone diseases. Only recently, results from circulating miRNAs analysis in patients with osteopenia, osteoporosis and fragility fractures have been reported. By comparing these findings to studies on circulating miRNAs in cellular senescence and aging or muscle physiology and sarcopenia, several overlaps were observed. This suggests that signatures observed during osteoporosis might not be specific to the pathophysiology in bone, but rather integrate information from several tissue types. Despite these promising first data, more work remains to be done until circulating miRNAs can serve as established and robust diagnostic tools for bone diseases in clinical research, clinical routine and in personalized medicine.
Hackl M, Heilmeier U, Weilner S, Grillari J.
Mol Cell Endocrinol. 2016 Sep 5;432:83-95.
Differentially circulating miRNAs after recent osteoporotic fractures can influence osteogenic Differentiation.
Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level and are known to take part in the control of bone formation and bone resorption. In addition, it is known that miRNAs are secreted by many cell types and can transfer "messages" to recipient cells. Thus, circulating miRNAs might not only be useful as surrogate biomarkers for the diagnosis or prognosis of pathological conditions, but could be actively modulating tissue physiology. Therefore, the aim of this study was to test whether circulating miRNAs that exhibit changes in recent osteoporotic fracture patients could be causally related to bone metabolism. In the first step we performed an explorative analysis of 175 miRNAs in serum samples obtained from 7 female patients with recent osteoporotic fractures at the femoral neck, and 7 age-matched female controls. Unsupervised cluster analysis revealed a high discriminatory power of the top 10 circulating miRNAs for patients with recent osteoporotic fractures. In total 6 miRNAs, miR-10a-5p, miR-10b-5p, miR-133b, miR-22-3p, miR-328-3p, and let-7g-5p exhibited significantly different serum levels in response to fracture (adjusted p-value<0.05). These miRNAs were subsequently analyzed in a validation cohort of 23 patients (11 control, 12 fracture), which confirmed significant regulation for miR-22-3p, miR-328-3p, and let-7g-5p. A set of these and of other miRNAs known to change in the context of osteoporotic fractures were subsequently tested for their effects on osteogenic differentiation of human mesenchymal stem cells (MSCs) in vitro. The results show that 5 out of 7 tested miRNAs can modulate osteogenic differentiation of MSCs in vitro. Overall, these data suggest that levels of specific circulating miRNAs change in the context of recent osteoporotic fractures and that such perturbations of "normal" levels might affect bone metabolism or bone healing processes.
Weilner S, Skalicky S, Salzer B, Keider V, Wagner M, Hildner F, et al.
Bone. 2015 May 28;79:43-51.
Annotation of additional evolutionary conserved microRNAs in CHO cells from updated genomic data.
MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from the Chinese Hamster (CGR) and Chinese hamster ovary (CHO) cells provide new tools for the discovery of novel miRNAs in this important production system. Version 20 of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences for CGR/CHO. We searched for evolutionary conserved miRNAs from miRBase v20 in recently published genomic data, derived from Chinese hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several hundred miRNA sequences in the genome. For several of these, the expression in CHO cells could be verified from multiple next-generation sequencing experiments. In addition, several hundred unexpressed miRNAs are awaiting further confirmation by testing for their transcription in different Chinese hamster tissues.
Diendorfer AB, Hackl M, Klanert G, Jadhav V, Reithofer M, Stiefel F, et al.
Biotechnol Bioeng. 2015 Jul;112(7):1488-93.
Molecular and cellular effects of in vitro shockwave treatment on lymphatic endothelial cells.
Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplaninhigh and podoplaninlow cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed differences in genes specific for signaling and vascular tissue. Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis in vivo.
Rohringer S, Holnthoner W, Hackl M, Weihs AM, Rünzler D, Skalicky S, et al.
PLoS One. 2014 Dec 11;9(12):e114806.
MicroRNAs differentially present in the plasma of HIV elite controllers reduce HIV infection in vitro.
Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.
Reynoso R, Laufer N, Hackl M, Skalicky S, Monteforte R, Turk G, Carobene M, et al.
Scientific Reports, 2014 Aug 1;4:5915.
Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation.
MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.
Maccani A, Hackl M, Leitner C, Steinfellner W, Graf AB, Tatto NE, et al.
Appl Microbiol Biotechnol. 2014 Sep;98(17):7535-48.
Stable overexpression of miR-17 enhances recombinant protein production of CHO cells.
miRNAs negatively regulate gene expression at post-transcriptional level, and consequently play an important role in the control of many cellular pathways. The use of miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve recombinant protein production. Here, we describe the effect of transient and stable miRNA overexpression on CHO cell phenotype. Using an established transient miRNA screening protocol, the effects of miR-17, miR-92a and cluster miR17-92a on CHO growth and protein productivity were studied and followed by analysis of cell pools with stable overexpression of these miRNAs. CHO cells stably engineered with miR-17 exhibited both enhanced growth performance and a 2-fold increase in specific productivity, which resulted in a 3-fold overall increase in EpoFc titer. While further studies of miRNA-mRNA interactions will be necessary to understand the molecular basis of this effect, these data provide valuable evidence for miR-17 as a cell engineering target to enhance CHO cell productivity.
Jadhav V, Hackl M, Klanert G, Hernandez Bort JA, Kunert R, Grillari J, Borth N.
J Biotechnol. 2014 Apr 10;175:38-44.
Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell Proliferation.
CHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation.
Hackl M, Jadhav V, Klanert G, Karbiener M, Scheideler M, Grillari J, Borth N.
J Biotechnol. 2014 Nov 20;190:76-84.
Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells.
MicroRNAs (miRNAs) are small non-coding RNAs (∼22 nucleotides) which regulate gene expression by silencing mRNA translation. MiRNAs are transcribed as long primary transcripts, which are enzymatically processed by Drosha/Dgcr8, in the nucleus, and by Dicer in the cytoplasm, into mature miRNAs. The importance of miRNAs for coordinated gene expression is commonly accepted. Consequentially, there is a growing interest in the application of miRNAs to improve phenotypes of mammalian cell factories such as Chinese hamster ovary (CHO) cells. Few studies have reported the targeted over-expression of miRNAs in CHO cells using vector-based systems. These approaches were hampered by limited sequence availability, and required the design of "chimeric" miRNA genes, consisting of the mature CHO miRNA sequence encompassed by murine flanking and loop sequences. Here we show that the substitution of chimeric sequences with CHO-specific sequences for expression of miRNA clusters yields significantly higher expression levels of the mature miRNA in the case of miR-221/222 and miR-15b/16. Our data suggest that the Drosha/Dgcr8-mediated excision from primary transcripts is reduced for chimeric miRNA sequences compared to the endogenous sequence. Overall, this study provides important guidelines for the targeted over-expression of clustered miRNAs in CHO cells.
Klanert G, Jadhav V, Chanoumidou K, Grillari J, Borth N, Hackl M.
Biotechnol J. 2014 Apr;9(4):538-44.
Review of the importance of microRNAs in mammalian cell culture technology
Jadhav V, Hackl M, Druz A, Shridhar S, Chung CY, Heffner KM, Kreil DP, Betenbaugh M, Shiloach J, Barron N, Grillari J, Borth N. CHO microRNA engineering is growing up: recent successes and future challenges (2013). Biotechnol Adv. 31(8):1501-13. Review.
PubMed PMID: 23916872
Review of the importance of secreted microRNAs during aging of cells, tissues and entire organisms.
Weilner, S., Schraml, E., Redl, H., Grillari-Voglauer, R., Grillari, J. Secretion of microvesicular miRNAs in cellular and organismal aging (2013) Experimental Gerontology, 48 (7), pp. 626-633.
Original research article on the role of miR-21 in cellular lifespan
Dellago, H., Preschitz-Kammerhofer, B., Terlecki-Zaniewicz, L., Schreiner, C., Fortschegger, K., Chang, M.W.-F., Hackl, M., Monteforte, R., Kühnel, H., Schosserer, M., Gruber, F., Tschachler, E., Scheideler, M., Grillari-Voglauer, R., Grillari, J., Wieser, M. High levels of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan (2013) Aging Cell, 12 (3), pp. 446-458.
PubMed PMID: 23496142
Bioinformatic prediction of piRNAs – a new class of small non-coding RNAs – in a common recombinant therapeutic production cell line
Gerstl, M.P., Hackl, M., Graf, A.B., Borth, N., Grillari, J. Prediction of transcribed PIWI-interacting RNAs from CHO RNAseq data (2013) Journal of Biotechnology, 166 (1-2), pp. 51-57.
PubMed PMID: 23639388
Original research article on the relevance of microRNAs in mediating the effect of UVB-irradiation on fibroblasts.
Greussing, R., Hackl, M., Charoentong, P., Pauck, A., Monteforte, R., Cavinato, M., Hofer, E., Scheideler, M., Neuhaus, M., Micutkova, L., Mueck, C., Trajanoski, Z., Grillari, J., Jansen-Dürr, P. Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts (2013) BMC Genomics, 2013 Apr 4;14:224.
PubMed PMID: 23557329
Protocol describing the isolation and subsequent reprogramming of urine cell into pluripotent stem cells.
Zhou, T., Benda, C., Dunzinger, S., Huang, Y., Ho, J.C., Yang, J., Wang, Y., Zhang, Y., Zhuang, Q., Li, Y., Bao, X., Tse, H.-F., Grillari, J., Grillari-Voglauer, R., Pei, D., Esteban, M.A. Generation of human induced pluripotent stem cells from urine samples (2012) Nature Protocols, 7 (12), pp. 2080-2089. PubMed PMID: 23138349
Opinion Article on the use of microRNAs in engineering cell lines to become better tools for the production of recombinant therapeutic proteins
Hackl, M., Borth, N., Grillari, J. MiRNAs - pathway engineering of CHO cell factories that avoids translational burdening (2012) Trends in Biotechnology, 30 (8), pp. 405-406. PubMed PMID:22673691
Orignial research article on a microRNA that controls DNA Damage in T-Cells in response to chemotherapeutic treatment with Etoposide
Brunner, S., Herndler-Brandstetter, D., Arnold, C.R., Wiegers, G.J., Villunger, A., Hackl, M., Grillari, J., Moreno-Villanueva, M., Bürkle, A., Grubeck-Loebenstein, B. Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8 + T cells sensitizing them to apoptotic cell death (2012) Aging Cell, 11 (4), pp. 579-587. PubMed PMID: 22435726
Original research article on microRNAs that control proliferation of CHO cell lines, which are the most frequently mammalian cell line for recombinant therapeutic protein production.
Jadhav, V., Hackl, M., Hernandez Bort, J.A., Wieser, M., Harreither, E., Kunert, R., Borth, N., Grillari, J. A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells (2012) Biotechnology and Bioengineering, 109 (6), pp. 1376-1385. PubMed PMID: 22407745
Bioinformatic analysis of microRNA sequences and genomic organization in CHO cell lines.
Hackl, M., Jadhav, V., Jakobi, T., Rupp, O., Brinkrolf, K., Goesmann, A., Pühler, A., Noll, T., Borth, N., Grillari, J. Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines (2012) Journal of Biotechnology, 158 (3), pp. 151-155. PubMed PMID: 22306111
Original research article describing the changes in microRNA expression in vitro and ex vivo during aging.
Hackl, M., Brunner, S., Fortschegger, K., Schreiner, C., Micutkova, L., Mück, C., Laschober, G.T., Lepperdinger, G., Sampson, N., Berger, P., Herndler-Brandstetter, D., Wieser, M., Kühnel, H., Strasser, A., Rinnerthaler, M., Breitenbach, M., Mildner, M., Eckhart, L., Tschachler, E., Trost, A., Bauer, J.W., Papak, C., Trajanoski, Z., Scheideler, M., Grillari-Voglauer, R., Grubeck-Loebenstein, B., Jansen-Dürr, P., Grillari, J. miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging (2010) Aging Cell, 9 (2), pp. 291-296.
Opinion article on the potential of microRNAs for engineering the phenotype of cells to better fit to the needs in modern biotechnological processes.
Müller, D., Katinger, H., Grillari, J. MicroRNAs as targets for engineering of CHO cell factories (2008) Trends in Biotechnology, 26 (7), pp. 359-365.
Lepperdinger, G., Berger, P., Breitenbach, M., Frohlich, K.-U., Grillari, J., Grubeck-Loebenstein, B., Madeo, F., Minois, N., Zwerschke, W., Jansen-Durr, P. The use of genetically engineered model systems for research on human aging (2008) Frontiers in Bioscience, 13 (18), pp. 7022-7031.