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Introduction: Human nasal inferior turbinate-derived stem cells (hNTSCs) are attractive sources of adult stem cells for medical application because they can be easily obtained and cultivated with a highly proliferative capacity. The ability of hNTSCs to differentiate into chondrocytes, osteocytes, and neural cells makes them potential replacement therapeutic candidates in intractable disease. Nevertheless, detailed expression pattern of genes associated with trilineage differentiation (osteogenesis, chondrogenesis, and neurogenesis) in hNTSCs has not been revealed yet. Methods: In this study, we aimed to evaluate gene expression patterns of various transcription factors and marker genes associated with a particular lineage (osteogenesis, chondrogenesis, and neurogenesis) of differentiation of hNTSCs by DNA microarrays. Results: In microarrays, 36 transcripts such as E2F transcription factor 1, activating transcription factor 5, and AKR1B10 were upregulated and 36 transcripts such as CA9, PPFIA4, HAS2, and COL4A4 were downregulated in osteogenically differentiated hNTSCs. In chondrogenically differentiated hNTSCs, 3 transcripts (NUDT14, CPA4, and heparin-binding epidermal growth factor-like growth factor) were upregulated and 82 transcripts such as PTGS1, CLEC2D, and TET1 were downregulated. In neurally differentiated hNTSCs, 61 transcripts such as insulin-like growth factor-binding protein-1, nerve growth factor receptor, FGF1, OLFML1, and EPGN were upregulated and 98 transcripts such as ACAN, RUNX2, and C21orf96 were downregulated. In gene ontology (GO) analysis, cell signal-related GO terms were highly expressed. By contrast, catalysis GO terms and GO terms related to oxidoreductase were overrepresented in chondrogenically differentiated hNTSCs and osteogenically differentiated hNTSCs, respectively. Conclusion: Considering overall results, hNTSCs-specific genetic information may promote further studies on intracellular mechanisms defining key features of these cells.

The airway mucosa is the first point of exposure to all inhalant materials. It is possible to produce microenvironments functionally similar to airway by using different types of extracellular matrices around mucosal cells, such as an extracellular matrix decellularized from trachea. In this study, we introduced a novel human respiratory mucosa-on-a-chip model that uses decellularized tracheal extracellular matrix to assess functional similarities. We also evaluated the effects of respiratory toxicant based on changes in cell viability, tight junction function, oxidative stress, expression of cell adhesion proteins in the vascular network, mucous secretion, and inflammatory cytokine secretion after treatment. This novel human respiratory mucosa-on-a-chip model is expected to be useful for assessing respiratory mucosa reactions to external bacteria or viruses and inhaled drugs.

BACKGROUND: Articular cartilage injury has a poor repair ability and limited regeneration capacity with therapy based on articular chondrocytes (ACs) implantation. Here, we validated the hypothesis that human nasal septum-derived chondrocytes (hNCs) are potent therapeutic agents for clinical use in cartilage tissue engineering using an injectable hydrogel, type I collagen (COL1). METHODS: We manufactured hNCs incorporated in clinical-grade soluble COL1 and investigated their clinical potential as agents in an articular defect model. RESULTS: The hNCs encapsulated in COL1 (hNC-collagen) were uniformly distributed throughout the collagen and showed much greater growth rate than hACs encapsulated in collagen for the 14 days of culture. Fluorescent staining of hNC-collagen showed high expression levels of chondrocyte-specific proteins under clinical conditions. Moreover, a negative mycoplasma screening result were obtained in culture of hNC-collagen. Notably, implantation of hNC-collagen increased the repair of osteochondral defects in rats compared with implantation of collagen only. Many human cells were detected within the cartilage defects. CONCLUSION: These results provide reliable evidences supporting for clinical applications of hNC-collagen in regenerative medicine for cartilage repair. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13770-020-00261-9) contains supplementary material, which is available to authorized users.

BACKGROUND: Cell-based therapies have been studied for articular cartilage regeneration. Articular cartilage defects have little treatments because articular cartilage was limited regenerative capacity. Damaged articular cartilage is difficult to obtain a successful therapeutic effect. In additionally these articular cartilage defects often cause osteoarthritis. Chondrocyte implantation is a widely available therapy used for regeneration of articular cartilage because this tissue has poor repair capacity after injury. Human nasal septum-drived chondrocytes (hNCs) from the septum show greater proliferation ability and chondrogenic capacity than human articular chondrocytes (hACs), even across different donors with different ages. Moreover, the chondrogenic properties of hNCs can be maintained after extensive culture expansion. METHODS: In this study, 2 dimensional (2D) monolayer cultured hNCs (hNCs-2D) and 3 dimensional (3D) spheroids cultured hNCs (hNCs-3D) were examined for chondrogenic capacity in vitro by PCR and immunofluorescence staining for chondrogenic marker, cell survival during cultured and for cartilage regeneration ability in vivo in a rat osteochondral defect model. RESULTS: hNCs-3D showed higher viability and more uniform morphology than 3D spheroids cultured hACs (hACs-3D) in culture. hNCs-3D also showed greater expression levels of the chondrocyte-specific marker Type II collagen (COL2A1) and sex-determining region Y (SRY)-box 9 (SOX9) than hNCs-2D. hNCs-3D also expressed chondrogenic markers in collagen. Specially, in the osteochondral defect model, implantation of hNCs-3D led to greater chondrogenic repair of focal cartilage defects in rats than implantation of hNCs-2D. CONCLUSION: These data suggest that hNCs-3D are valuable therapeutic agents for repair and regeneration of cartilage defects.

BACKGROUND: In this study, we manufactured a complex of human nasal septal cartilage (hNC) with polycaprolactone (PCL) for transplantation into cartilaginous skeletal defects and evaluated their characteristics. METHODS: Nasal septum tissue was obtained from five patients aged ≥ 20 years who were undergoing septoplasty. hNCs were isolated and subcultured for three passages in vitro. To formulate the cell–PCL complex, we used type I collagen as an adhesive between chondrocyte and PCL. Immunofluorescence staining, cell viability and growth in the hNC–PCL complex, and mycoplasma contamination were assessed. RESULTS: hNCs in PCL showed viability ≥ 70% and remained at these levels for 9 h of incubation at 4 °C. Immunostaining of the hNC–PCL complex also showed high expression levels of chondrocyte-specific protein, COL2A1, SOX9, and aggrecan during 24 h of clinically applicable conditions. CONCLUSION: The hNC–PCL complex may be a valuable therapeutic agent for implantation into injured cartilage tissue, and can be used clinically to repair cartilaginous skeletal defects. From a clinical perspective, it is important to set the short duration of the implantation process to achieve effective functional implantation.

Objective To produce alternate cell sources for tissue regeneration, human nasal septal cartilage-derived progenitor cells (NSPs) were tested to identify whether these cells meet the criteria of cartilage progenitor cells. We also evaluated the effects of prolonged cultivation on the characteristics of NSPs. Study Design In vitro study. Setting Academic research laboratory. Methods NSPs were isolated from discarded human nasal septal cartilage. NSPs were cultured for 10 passages. The expression of septal progenitor cell surface markers was assessed by fluorescence-activated cell sorting. Cell proliferation was measured with a cell-counting kit. Cytokine secretion was analyzed with multiplex immunoassays. Chondrogenic differentiation of NSPs without differentiation induction was analyzed with type II collagen immunohistochemistry. Cartilage-specific protein expression was evaluated by Western blotting. Under osteo- and adipodifferentiation media, 2 lineage differentiation potentials were evaluated by histology and gene expression analysis. Results Surface epitope analysis revealed that NSPs are positive for mesenchymal stem cells markers and negative for hematopoietic cell markers. Cultured NSPs showed sufficient cell expansion and chondrogenic potential, as demonstrated by immunostaining and expression of cartilage-specific protein. IL-6, IL-8, and transforming growth factor ß were secreted by over 200 pg/mL. The osteo- and adipodifferentiation potentials of NSPs were identified by histology and specific gene expression. The aforementioned characteristics were not influenced by prolonged cultivation. Conclusion NSPs represent an initial step toward creating a cell source from surgically discarded tissue that may prove useful in cartilage regeneration.

Objectives Polycaprolactone (PCL) is an U.S. Food and Drug Administration-approved synthetic biodegradable polymer and is easily fabricated into three-dimensional (3D) structures. In this study, the 3D-printed PCL implant for nasal augmentation was further evaluated for its suitability for nasal surgeries such as septoplasty and rhinoplasty. Methods Ten New Zealand White rabbits were included and divided into study and sham groups (7 and 3, respectively). A lateral incision was made on the nasal dorsum and a pocket formed in the subperichondrial plane between the upper lateral cartilage and nasal septum. Polycaprolactone was fabricated based on 3D printing technology into a 0.8 × 0.8-cm rectangular shape for use as a nasal implant. The material was inserted as a septal extension graft and sutured with alar cartilage for nasal reshaping. The implants were harvested 4, 8, and 12 weeks after implantation and evaluated by gross morphological assessment and histological examination. Results The initial shape of the implant was unchanged in all cases, and no definitive postoperative complications were seen over the 3-month period. Gross morphological evaluation confirmed that implants remained in their initial location without migration or extrusion. Histologic evaluations showed that the implant architectures were maintained with excellent fibrovascular ingrowth and minimal inflammatory reactions. Conclusion Polycaprolactone can be used for nasal reconstruction such as nasal augmentation. Polycaprolactone is easy to work with and will avoid the increased operative time and morbidity associated with autograft harvesting. Therefore, PCL implants designed by 3D printing can serve as clinically biocompatible materials in craniofacial reconstruction in the future. Level of Evidence NA. Laryngoscope, 127:1036–1043, 2017

Background and Objective: Human nasal inferior turbinate-derived stem cells (hNTSCs) have been considered as a potent and useful source for regenerative medicine. To most effectively mimic the native environment of inferior turbinate could be very effective to hNTSCs biology. Thus, the purpose of this study was to evaluate partial pressure of oxygen (ppO2) and temperature in inferior turbinate. Methods: Ten patients were enrolled who underwent endoscopic endonasal transsphenoidal skull base tumor surgery between January 2014 and December 2015. The commercially available OxyLab pO2 monitor gauges the ppO2 and temperature using a fluorescence quenching technique. Also, hNTSCs were isolated from 10 patients and cultivated under hypercapnic condition (5, 10, and 15%) to mimic hypoxic intranasal conditions. Results: The measured oxygen concentration in submucosa tissue was higher than that at the surface of the inferior turbinate and the temperature in submucosa tissue was higher than the value at the surface of inferior turbinate. The patterns of proliferation were significantly different according to hypercapnic cultivation conditions and there were statistically significant decreased proliferation rates after the exposure of higher CO2 over a period of 5 days. Conclusions: Intranasal turbinate tissue showed the hypoxia state in concordance with the result of the other tissues or organs. However, indirectly induced hypoxia influenced the influence on the hNTSCs proliferation negatively. Further study is needed to mimic the real hypoxic state, but our results could be used to optimize the culture environment of hNTSCs, thereby producing the stem cells for regenerative therapies.

Objective: Though keratoplasty is used to treat corneal blindness, donor shortage, tendency of stimulated keratocyte transformed to fibroblast and immunological rejection are still big problems. As a solution, cornea tissue engineering using non-corneal tissue sourced cells become emerging issue. Thus, this study was designed to find novel material for keratoplasty. Methods: Human turbinate-derived mesenchymal stem cells (hTMSCs) were obtained from patients and cultured with differentiation medium for 14 days. The keratocyte markers, stem cell markers, early corneal stromal stem cell (CSSC) markers, were measured by real time-PCR. The MSC markers were detected by FACS. Results: After 14 days of differentiation medium exposure, hTMSCs expressed markers of keratocyte such as keratocan sulfate proteoglycan (KERA) and aldehydrogenase (ALDH). As the hTMSCs became keratocytes, the expression of embryonic ocular precursor markers ABCG2 and PAX6 decreased but were still measurable. Early CSSC markers including SIX2, SIX3, BMI expression was elevated after 7 d and reduced after 14 d of KDM treatment. The stem cell markers such as SOX2, Notch were decreased. After 14 d of differentiation, the hTMSCs expressed MSC markers CD73, CD90, and CD105, but not hematopoietic markers CD14, CD19, CD34, HLA-DR; these changes indicate development of a characteristic MSC phenotype. hTMSCs inhibited the tube-formation ability of human microvessel endothelial cells. hTMSCs derived from neural crest could differentiate into keratocyte progenitors. Conclusions: This study first reveals that the hTMSCs have potential to be differentiated into keratocyte progenitor- like cells. Use of hTMSCs derived from neural crest in cell-based therapeutics as source for corneal tissue engineering may overcome the problems of keratoplasty such as immunological rejection and limiting supply of human donor corneas.

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3, has been implicated in anti-stress and anti-apoptotic pathways. In a previous study, we observed a significant induction of Bis in reactive astrocytes of the rat hippocampus after transient forebrain ischemia. To investigate the significance of this induction in ischemic injury, the expression of Bis was reduced with siRNA in C6 glioma cells and exposed to oxygen-glucose deprivation (OGD) conditions. Bis knock-down resulted in an increase in the cell death rate of the C6 cells after OGD, accompanied by accumulation of reactive oxygen species. Among the cellular antioxidants, the induction of superoxide dismutase (SOD) activity was significantly interfered within the cells treated with bis siRNA treated cells (bis-kd C6). A Western blot assay revealed that SOD1 expression gradually increased in control cells, which was not observed in bis-kd cells upon OGD treatment. A quantitative analysis of Sod1 and Sod2 transcripts indicated that the induction of Sod1 was more evidently suppressed by the reduction of Bis. As a transcription factor candidate for the Sod1 gene, the activity of NF-kappaB was determined the nuclear translocation of p65, showing that the activation of NF-kappaB was attenuated in bis-kd C6. Supporting this, an overexpression of Bis augments the activation of NF-kappaB and Sod1 mRNA with an increased cell survival under OGD conditions. These results suggest that one of physiological significances of Bis induction in reactive astrocytes after ischemia in vivo is to protect glial cells from oxidative stress, probably via the induction of SOD1, which is related to the activation of NF-kappaB.

We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). A super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.

The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs.

We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.

Objectives To determine whether low-intensity ultrasound (US) can reduce red blood cell (RBC) edema and, if so, whether the US activity is associated with aquaporin 1 (AQP-1), a water channel in the cell membrane. Methods Red blood cell edema was induced by gramicidin D treatment at 40 ng/mL for 20 minutes and evaluated by a hematocrit assay. Low-intensity continuous wave US at 1 MHz was applied to RBCs for the last 10 minutes of gramicidin D treatment. To determine whether US activity was associated with AQP-1, RBCs were treated with 40 μM mercuric chloride (HgCl2), an AQP-1 inhibitor, for 20 minutes at the time of gramicidin D treatment. Posttreatment morphologic changes in RBCs were observed by actin staining with phalloidin. Results Red blood cell edema increased significantly with gramicidin D at 20 (1.8%), 40 (6.7%), 60 (16.7%), and 80 (11.3%) ng/mL, reaching a peak at 60 ng/mL, compared to the control group (20 ng/mL, P = .019; 40, 60, and 80 ng/mL, P < .001). No significant RBC hemolysis was observed in any group. Edema induced by gramicidin D at 40 ng/mL was significantly reduced by US at 30 (3.4%; P = .003), 70 (4.4%; P = .001), and 100 (2.9%; P = .001) mW/cm2. Subsequent experiments showed that edema reduction by US ranged from 7% to 10%. Cotreatment with HgCl2 partially reversed the US effect and showed a significantly different level of edema compared to gramicidin D-alone and US-cotreated groups (P = .001). These results were confirmed by microscopic observation of RBC morphologic changes. Conclusions Low-intensity US could reduce gramicidin D–induced RBC edema, and its effect appeared to at least partly involve regulation of AQP-1 activity. These results suggest that low-intensity US can be used as an alternative treatment to control edema and related disorders.

The low survival rate of graft stem cells after transplantation into recipient tissue is a major obstacle for successful stem cell therapy. After transplantation into the site of spinal cord injury, the stem cells face not only hypoxia due to low oxygen conditions, but also a lack of nutrients caused by damaged tissues and poor vascular supply. To improve the survival of therapeutic stem cells after grafting into the injured spinal cord, we examined the effects of cotransplanting mouse neural stem cells (mNSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on mNSC viability. The viability of mNSCs in coculture with AT-MSCs was significantly increased compared to mNSCs alone in an in vitro injury model using serum deprivation (SD), hydrogen peroxide (H2O2), and combined (SD + H2O2) injury mimicking the ischemic environment of the injured spinal cord. We demonstrated that AT-MSCs inhibited the apoptosis of mNSCs in SD, H2O2, and combined injury models. Consistent with these in vitro results, mNSCs transplanted into rat spinal cords with AT-MSCs showed better survival rates than mNSCs transplanted alone. These findings suggest that cotransplantation of mNSCs with AT-MSCs may be a more effective transplantation protocol to improve the survival of cells transplanted into the injured spinal cord.

OBJECTIVE In this study, the authors assessed the ability of rat bone marrow derived mesenchymal stem cells (BMDMSCs), in the presence of a growth factor, fibroblast growth factor-4 (FGF-4) and hydroxyapatite, to act as a scaffold for posterolateral spinal fusion in a rat model. METHODS Using a rat posterolateral spine fusion model, the experimental study comprised 3 groups. Group 1 was composed of 6 animals that were implanted with 0.08 gram hydroxyapatite only. Group 2 was composed of 6 animals that were implanted with 0.08 gram hydroxyapatite containing 1 x 10(6)/ 60 microL rat of BMDMSCs. Group 3 was composed of 6 animals that were implanted with 0.08 gram hydroxyapatite containing 1 x 10(6)/ 60 microL of rat BMDMSCs and FGF-4 1 microG to induce the bony differentiation of the BMDMSCs. Rats were assessed using radiographs obtained at 4, 6, and 8 weeks postoperatively. After sacrifice, spines were explanted and assessed by manual palpation, high-resolution microcomputerized tomography, and histological analysis. RESULTS Radiographic, high-resolution microcomputerized tomographic, and manual palpation revealed spinal fusion in five rats (83%) in Group 2 at 8 weeks. However, in Group 1, three (60%) rats developed fusion at L4-L5 by radiography and two (40%) by manual palpation in radiographic examination. In addition, in Group 3, bone fusion was observed in only 50% of rats by manual palpation and radiographic examination at this time. CONCLUSION The present study demonstrates that 0.08 gram of hydroxyapatite with 1 x 10(6)/ 60 microL rat of BMDMSCs induced bone fusion. FGF-4, added to differentiate primitive 1 x 10(6)/ 60 microL rat of BMDMSCs did not induce fusion. Based on histologic data, FGF-4 appears to induce fibrotic change rather than differentiation to bone by 1 x 10(6)/ 60 microL rat of BMDMSCs.

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis−/− mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and β-oxidation are activated in the liver of bis−/− mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.

Abstract 4926 Signal transducers and activators of transcription 3 (STAT3) is highly activated in multiple myeloma triggered by IL-6. Although DC vaccine can induce antitumor immune response, DCs are faced with dysfunction after loading of tumor antigen. Here we show that the dysfunction of DCs from antigen loading can be recovered by treating JSI-124 and bortezomib on myeloma cells. Treatment of combination JSI-124 and bortezomib induced high expression of heat shock protein 90 (Hsp90) on the surface of dying myeloma cells, DCs loaded these myeloma cells showed low expression of p-STAT3 and reduced IL-10, IL-6 and IL-23 production without any effect on IL-12p70. Also DCs loaded with dying myeloma cells treated by JSI-124 and bortezomib combination could generate the most potent myeloma-specific CTLs targeting to myeloma cells. Our data suggested that treatment of myeloma cells with combination JSI-124 and bortezomib can recover the dying myeloma cells-loaded DC dysfunction through the highly expressed Hsp90 on tumor cells, down-regulated p-STAT3 and inhibitory cytokines, and these DCs can generate to potent myeloma-specific CTLs for obtaining better immunotherapeutic method in multiple myeloma. Disclosures: Lee: Vaxcell-Bio therapeutics Co.: Employment. Kim:Vaxcell-Bio therapeutics Co.: Employment. Lim:Vaxcell-Bio therapeutics Co.: Employment. Lee:Vaxcell-Bio therapeutics Co.: Employment.

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.