Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Increased lentivirus titer using ultra expression vectors.

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers.

Sheep-derived cell immortalization through the expression of cell cycle regulators and biological characterization using transcriptomes.

Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.

Evaluation of Local Retinal Function in Light-Damaged Rats Using Multifocal Electroretinograms and Multifocal Visual Evoked Potentials.

Electroretinograms (ERGs) are often used to evaluate retinal function. However, assessing local retinal function can be challenging; therefore, photopic and scotopic ERGs are used to record whole-retinal function. This study evaluated focal retinal function in rats exposed to continuous light using a multifocal ERG (mfERG) system. The rats were exposed to 1000 lux of fluorescent light for 24 h to induce photoreceptor degeneration. After light exposure, the rats were reared under cyclic light conditions (12 h: 5 lux, 12 h: dark). Photopic and multifocal ERGs and single-flash and multifocal visual evoked potentials (mfVEPs) were recorded 7 days after light exposure. Fourteen days following light exposure, paraffin-embedded sections were prepared from the eyes for histological evaluation. The ERG and VEP responses dramatically decreased after 24 h of light exposure, and retinal area-dependent decreases were observed in mfERGs and mfVEPs. Histological assessment revealed severe damage to the superior retina and less damage to the inferior retina. Considering the recorded visual angles of mfERGs and mfVEPs, the degenerated area shown on the histological examinations correlates well with the responses from multifocal recordings.

Properties of a Single Amino Acid Residue in the Third Transmembrane Domain Determine the Kinetics of Ambient Light-Sensitive Channelrhodopsin.

Channelrhodopsins have been utilized in gene therapy to restore vision in patients with retinitis pigmentosa and their channel kinetics are an important factor to consider in such applications. We investigated the channel kinetics of ComV1 variants with different amino acid residues at the 172nd position. Patch clamp methods were used to record the photocurrents induced by stimuli from diodes in HEK293 cells transfected with plasmid vectors. The channel kinetics (τon and τoff) were considerably altered by the replacement of the 172nd amino acid and was dependent on the amino acid characteristics. The size of amino acids at this position correlated with τon and decay, whereas the solubility correlated with τon and τoff. Molecular dynamic simulation indicated that the ion tunnel constructed by H172, E121, and R306 widened due to H172A variant, whereas the interaction between A172 and the surrounding amino acids weakened compared with H172. The bottleneck radius of the ion gate constructed with the 172nd amino acid affected the photocurrent and channel kinetics. The 172nd amino acid in ComV1 is a key residue for determining channel kinetics as its properties alter the radius of the ion gate. Our findings can be used to improve the channel kinetics of channelrhodopsins.

SIG-1451, a Novel, Non-Steroidal Anti-Inflammatory Compound, Attenuates Light-Induced Photoreceptor Degeneration by Affecting the Inflammatory Process.

Age-related macular degeneration is a progressive retinal disease that is associated with factors such as oxidative stress and inflammation. In this study, we evaluated the protective effects of SIG-1451, a non-steroidal anti-inflammatory compound developed for treating atopic dermatitis and known to inhibit Toll-like receptor 4, in light-induced photoreceptor degeneration. SIG-1451 was intraperitoneally injected into rats once per day before exposure to 1000 lx light for 24 h; one day later, optical coherence tomography showed a decrease in retinal thickness, and electroretinogram (ERG) amplitude was also found to have decreased 3 d after light exposure. Moreover, SIG-1451 partially protected against this decrease in retinal thickness and increase in ERG amplitude. One day after light exposure, upregulation of inflammatory response-related genes was observed, and SIG-1451 was found to inhibit this upregulation. Iba-1, a microglial marker, was suppressed in SIG-1451-injected rats. To investigate the molecular mechanism underlying these effects, we used lipopolysaccharide (LPS)-stimulated rat immortalised Müller cells. The upregulation of C-C motif chemokine 2 by LPS stimulation was significantly inhibited by SIG-1451 treatment, and Western blot analysis revealed a decrease in phosphorylated I-κB levels. These results indicate that SIG-1451 indirectly protects photoreceptor cells by attenuating light damage progression, by affecting the inflammatory responses.

Generation of a new mouse line with conditionally activated signaling through the BMP receptor, ACVR1: A tool to characterize pleiotropic roles of BMP functions.

BMP signaling plays pleiotropic roles in various tissues during embryogenesis and after birth. We have previously generated a constitutively activated Acvr1(ca-Acvr1) transgenic mouse line (line L35) through pronuclei injection to investigate impacts of enhanced BMP signaling in a tissue specific manner. However, line L35 shows a restricted expression pattern of the transgene. Here, we generated another ca-Acvr1 transgenic line, line A11, using embryonic stem (ES) transgenesis. The generated line A11 shows distinctive phenotypes from line L35, along with very limited expression levels of the transgene. When the transgene is activated in the neural crest cells in a Cre-dependent manner, line A11 exhibits cleft palate and shorter jaws, while line L35 develops ectopic cartilages and highly hypomorphic facial structures. When activated in limb buds, line A11 develops organized but smaller limb skeletal structures, while line L35 forms disorganized limbs with little mineralization. Additionally, no heterotopic ossification (HO) is identified in line A11 when bred with NFATc1-Cre mice even after induction of tissue injury, which is an established protocol for HO for line L35. Therefore, the newly generated conditional ca-Acvr1 mouse line A11 provides an additional resource to dissect highly context dependent functions of BMP signaling in development and disease.

Phototoxicities Caused by Continuous Light Exposure Were Not Induced in Retinal Ganglion Cells Transduced by an Optogenetic Gene.

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.

Primary and immortalized cell lines derived from the Amami rabbit (Pentalagus furnessi) and evolutionally conserved cell cycle control with CDK4 and Cyclin D1.

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.

Generation of mouse iPS cells using an inducible expression of transgenes via the cumate gene-switch.

We applied an inducible gene expression system that utilizes the p-cmt operon, the cumate gene-switch, to generate mouse induced pluripotent stem (iPS) cells. Mouse embryonic fibroblast (MEF) E6E7-MEF cells were transfected with a single cumate gene-switch vector enabling concomitant expression of Oct4, Sox2, c-Myc, Klf4, and Gfp. Then, the cells were cultured with cumate, a monoterpene. An increase in colonies positive for alkaline phosphatase activity was observed dose-dependently with cumate. In the absence of cumate, the expression of GFP, a marker for transgene expression, was undetectable in tightly aggregated iPS cell-like colonies with endogenous expression of NANOG and OCT4. From primary MEFs using the cumate gene-switch, we also isolated iPS cells expressing endogenous NANOG, OCT4, SOX2, KLF4, and SSEA1 with hypo-methylated genomic promoter regions of endogenous Nanog and Oct4. In embryoid bodies with the progression of differentiation, expression of markers for all three germ layers was detected, and contracting cardiomyocytes were observed. Overall, we suggest that the cumate gene-switch is applicable for the generation of mouse iPS cells. The cumate gene-switch in combination with other inducible systems, such as the tet system, may provide useful approaches for analyzing the roles of transgenes underlying the establishment of iPS cells.

ADAMTSL6ß promotes fibrillin-1 microfibril assembly, which is possibly mediated via binding through the third thrombospondin type I domain to fibrillin-1.

Fibrillin-1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs-like) 6ß is one of the fibrillin-1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6ß in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6ß and examined their interactions with fibrillin-1 assembly. Pull-down assay of the ADAMTSL6ß deletion mutants and fibrillin-1 protein revealed that ADAMTSL6ß binds to fibrillin-1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin-1 matrix assembly was enhanced in MG63 cells, expressing full-length ADAMTSL6ß, when compared with that of wild type MG63 cells. While MG63 cells expressing Δ TSP3-ADAMTSL6ß form showed enhanced assembly formation, Δ TSP2-ADAMTSL6ß form did not enhance that, indicating the difference between Δ TSP2-Δ TSP3 has a critical role for fibrillin-1 assembly. As the difference of Δ TSP2-Δ TSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6ß influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6ß in the process of microfibril formation.

Establishment of porcine nuclear transfer-derived embryonic stem cells using induced pluripotent stem cells as donor nuclei.

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

Oxytocin induced labor causes region and sex-specific transient oligodendrocyte cell death in neonatal mouse brain.

AIM: Previous reports showed associations between oxytocin induced labor and mental disorders in offspring. However, those reports are restricted in epidemiological analyses and its mechanism remains unclear. In this study, we hypothesized that induced labor directly causes brain damage in newborns and results in the development of mental disorders. Therefore we aimed to investigate this hypothesis with animal model. METHODS: The animal model of induced labor was established by subcutaneous oxytocin administration to term-pregnant C57BL/6J mice. We investigated the neonatal brain damage with evaluating immediate early gene expression (c-Fos, c-Jun and JunB) by quantitative polymerase reaction and TdT-mediated dUTP nick end labeling staining. To investigate the injured brain cell types, we performed double-immunostaining with TdT-mediated dUTP nick end labeling staining and each brain component specific protein, such as Oligo2, NeuN, GFAP and Iba1. RESULTS: Brain damage during induced labor led to cell death in specific brain regions, which are implicated in mental disorders, in only male offspring at P0. Furthermore, oligodendrocyte precursors were selectively vulnerable compared to the other cell types. This oligodendrocyte-specific impairment during the perinatal period led to an increased numbers of Olig2-positive cells at P5. Expression levels of oxytocin and Oxtr in the fetal brain were not affected by the oxytocin administered to mothers during induced labor. CONCLUSION: Oligodendrocyte cell death in specific brain regions, which was unrelated to the oxytocin itself, was caused by induced labor in only male offspring. This may be an underlying mechanism explaining the human epidemiological data suggesting an association between induced labor and mental disorders.

Extended proliferation of chicken- and Okinawa rail-derived fibroblasts by expression of cell cycle regulators.

Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species.

The poly-cistronic expression of four transcriptional factors (CRX, RAX, NEURO-D, OTX2) in fibroblasts via retro- or lentivirus causes partial reprogramming into photoreceptor cells.

The introduction of four key transcriptional factors (CRX, RAX, NEURO-D, OTX2) allows the direct differentiation of fibroblasts to retinal photoreceptor cells. This reprogramming was achieved with a combination of mono-cistronic viruses. Although the combination of mono-cistronic viruses was useful, a relatively high titer of recombinant viruses was necessary because co-infections are required. To overcome this issue, we established a poly-cistronic expression system for direct reprogramming and analyzed the biological characteristics of introduced cells after the exogenous introduction. The coding region of four reprogramming factors and EGFP (CRX, RAX, NEURO-D, OTX2, and EGFP; CNROE) was inserted into multiple sites of the pMYs-IP retrovirus or CSII-CMV lentivirus vector. The recombinant viruses were exposed to HE16 human embryonic fibroblasts. The expression levels of cone related genes were detected with real-time PCR. We detected the activation of two of the photoreceptor-related genes after the poly-cistronic expression of CRX, RAX, NEURO-D, and OTX2, but the rest of the genes did not exhibit transcriptional elevation. We concluded that the poly-cistronic expression of CNROE induced partial reprogramming into photoreceptor cells. We hypothesize that the direct reprogramming into photoreceptor cells might require relatively high protein expression levels of transcriptional factors.

A basic fibroblast growth factor slow-release system combined to a biodegradable nerve conduit improves endothelial cell and Schwann cell proliferation: A preliminary study in a rat model.

BACKGROUND: A basic fibroblast growth factor (bFGF) slow-release system was combined to a biodegradable nerve conduit with the hypothesis this slow-release system would increase the capacity to promote nerve vascularization and Schwann cell proliferation in a rat model. MATERIALS AND METHODS: Slow-release of bFGF was determined using Enzyme-Linked ImmunoSorbent Assay (ELISA). A total of 60 rats were used to create a 10 mm gap in the sciatic nerve. A polyglycolic acid-based nerve conduit was used to bridge the gap, either without or with a bFGF slow-release incorporated around the conduit (n = 30 in each group). At 2 (n = 6), 4 (n = 6), 8 (n = 6), and 20 (n = 12) weeks after surgery, samples were resected and subjected to histological, immunohistochemical, and transmission electron microscopic evaluation for nerve regeneration. RESULTS: Continuous release of bFGF was found during the observation period of 2 weeks. After in vivo implantation of the nerve conduit, greater endothelial cell migration and vascularization resulted at 2 weeks (proximal: 20.0 ± 2.0 vs. 12.7 ± 2.1, P = .01, middle: 17.3 ± 3.5 vs. 8.7 ± 3.2, P = .03). Schwann cells showed a trend toward greater proliferation and axonal growth had significant elongation (4.9 ± 1.1 mm vs. 2.8 ± 1.5 mm, P = .04) at 4 weeks after implantation. The number of myelinated nerve fibers, indicating nerve maturation, were increased 20 weeks after implantation (proximal: 83.3 ± 7.5 vs. 53.3 ± 5.5, P = .06, distal: 71.0 ± 12.5 vs. 44.0 ± 11.1, P = .04). CONCLUSIONS: These findings suggest that the bFGF slow-release system improves nerve vascularization and Schwann cell proliferation through the biodegradable nerve conduit.

Specific and spatial labeling of P0-Cre versus Wnt1-Cre in cranial neural crest in early mouse embryos.

P0-Cre and Wnt1-Cre mouse lines have been widely used in combination with loxP-flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1-Cre has been regarded as the gold standard and there have been concerns about the specificity of P0-Cre because it is not clear about the timing and spatial distribution of the P0-Cre transgene in labeling NC cells at early embryonic stages. We re-visited P0-Cre and Wnt1-Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26-lacZ Cre reporter responded to Cre activity more reliably than CAAG-lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0-Cre and reporter (lacZ and RFP) activity in P0-Cre/R26-lacZ and P0-Cre/R26-RFP embryos was detected in the cranial NC and notochord regions in E8.0-9.5 (4-19 somites) embryos. P0-Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0-Cre and Wnt1-Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1-Cre and in the hindbrain of P0-Cre embryos. The difference between P0-Cre and Wnt1-Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre-driven genetic modifications.

Expression of Six Proteins Causes Reprogramming of Porcine Fibroblasts Into Induced Pluripotent Stem Cells With Both Active X Chromosomes.

In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. © 2016 Wiley Periodicals, Inc.

miR-663a regulates growth of colon cancer cells, after administration of antimicrobial peptides, by targeting CXCR4-p21 pathway.

BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown. METHODS: miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis. RESULTS: miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation. CONCLUSIONS: This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.