OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression.

MicroRNAs (miRNAs or miRs) are single-stranded, ∼22-nucleotide noncoding RNAs that regulate many cellular processes. While numerous miRNA quantification technologies are available, a recent analysis of 12 commercial platforms revealed high variations in reproducibility, sensitivity, accuracy, specificity and concordance within and/or between platforms. Here, we developed a universal hairpin primer (UHP) system that negates the use of miRNA-specific hairpin primers (MsHPs) for quantitative reverse transcription PCR (RT-qPCR)-based miRNA quantification. Specifically, we analyzed four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at 3'-ends (namely UHP2, UHP3, UHP4 and UHP6), and found that the four UHPs yielded robust RT products and quantified miRNAs with high efficiency. UHP-based RT-qPCR miRNA quantification was not affected by long transcripts. By analyzing 14 miRNAs, we demonstrated that UHP4 closely mimicked MsHPs in miRNA quantification. Fine-tuning experiments identified an optimized UHP (OUHP) mix with a molar composition of UHP2:UHP4:UHP6 = 8:1:1, which closely recapitulated MsHPs in miRNA quantification. Using synthetic LET7 isomiRs, we demonstrated that the OUHP-based qPCR system exhibited high specificity and sensitivity. Collectively, our results demonstrate that the OUHP system can serve as a reliable and cost-effective surrogate of MsHPs for RT-qPCR-based miRNA quantification for basic research and precision medicine.

BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/ß-catenin signalling activity.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/ß-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down ß-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down ß-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

Reversibly immortalized human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are responsive to BMP9-induced osteogenic and adipogenic differentiation.

Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.

Niclosamide Exhibits Potent Anticancer Activity and Synergizes with Sorafenib in Human Renal Cell Cancer Cells.

BACKGROUND/AIMS: As the most lethal urological cancers, renal cell carcinoma (RCC) comprises a heterogeneous group of cancer with diverse genetic and molecular alterations. There is an unmet clinical need to develop efficacious therapeutics for advanced, metastatic and/or relapsed RCC. Here, we investigate whether anthelmintic drug Niclosamide exhibits anticancer activity and synergizes with targeted therapy Sorafenib in suppressing RCC cell proliferation. METHODS: Cell proliferation and migration were assessed by Crystal violet staining, WST-1 assay, cell wounding and cell cycle analysis. Gene expression was assessed by qPCR. In vivo anticancer activity was assessed in xenograft tumor model. RESULTS: We find that Niclosamide effectively inhibits cell proliferation, cell migration and cell cycle progression, and induces apoptosis in human renal cancer cells. Mechanistically, Niclosamide inhibits the expression of C-MYC and E2F1 while inducing the expression of PTEN in RCC cells. Niclosamide is further shown to synergize with Sorafenib in suppressing RCC cell proliferation and survival. In the xenograft tumor model, Niclosamide is shown to effectively inhibit tumor growth and suppress RCC cell proliferation. CONCLUSIONS: Niclosamide may be repurposed as a potent anticancer agent, which can potentiate the anticancer activity of the other agents targeting different signaling pathways in the treatment of human RCC.

The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: implications in targeted cancer therapies.

The canonical WNT/ß-catenin signaling pathway governs a myriad of biological processes underlying the development and maintenance of adult tissue homeostasis, including regulation of stem cell self-renewal, cell proliferation, differentiation, and apoptosis. WNTs are secreted lipid-modified glycoproteins that act as short-range ligands to activate receptor-mediated signaling pathways. The hallmark of the canonical pathway is the activation of ß-catenin-mediated transcriptional activity. Canonical WNTs control the ß-catenin dynamics as the cytoplasmic level of ß-catenin is tightly regulated via phosphorylation by the 'destruction complex', consisting of glycogen synthase kinase 3ß (GSK3ß), casein kinase 1α (CK1α), the scaffold protein AXIN, and the tumor suppressor adenomatous polyposis coli (APC). Aberrant regulation of this signaling cascade is associated with varieties of human diseases, especially cancers. Over the past decade, significant progress has been made in understanding the mechanisms of canonical WNT signaling. In this review, we focus on the current understanding of WNT signaling at the extracellular, cytoplasmic membrane, and intracellular/nuclear levels, including the emerging knowledge of cross-talk with other pathways. Recent progresses in developing novel WNT pathway-targeted therapies will also be reviewed. Thus, this review is intended to serve as a refresher of the current understanding about the physiologic and pathogenic roles of WNT/ß-catenin signaling pathway, and to outline potential therapeutic opportunities by targeting the canonical WNT pathway.

A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti-Proliferative and Anticancer Activities.

BACKGROUND/AIMS: Ovarian cancer is the most lethal gynecologic malignancy, and there is an unmet clinical need to develop new therapies. Although showing promising anticancer activity, Niclosamide may not be used as a monotherapy. We seek to investigate whether inhibiting IGF signaling potentiates Niclosamide's anticancer efficacy in human ovarian cancer cells. METHODS: Cell proliferation and migration are assessed. Cell cycle progression and apoptosis are analyzed by flow cytometry. Inhibition of IGF signaling is accomplished by adenovirus-mediated expression of siRNAs targeting IGF-1R. Cancer-associated pathways are assessed using pathway-specific reporters. Subcutaneous xenograft model is used to determine anticancer activity. RESULTS: We find that Niclosamide is highly effective on inhibiting cell proliferation, cell migration, and cell cycle progression, and inducing apoptosis in human ovarian cancer cells, possibly by targeting multiple signaling pathways involved in ELK1/SRF, AP-1, MYC/MAX and NFkB. Silencing IGF-1R exert a similar but weaker effect than that of Niclosamide's. However, silencing IGF-1R significantly sensitizes ovarian cancer cells to Niclosamide-induced anti-proliferative and anticancer activities both in vitro and in vivo. CONCLUSION: Niclosamide as a repurposed anticancer agent may be more efficacious when combined with agents that target other signaling pathways such as IGF signaling in the treatment of human cancers including ovarian cancer.

BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells.

Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically, HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

The Calcium-Binding Protein S100A6 Accelerates Human Osteosarcoma Growth by Promoting Cell Proliferation and Inhibiting Osteogenic Differentiation.

BACKGROUND/AIMS: Although osteosarcoma (OS) is the most common primary malignancy of bone, its molecular pathogenesis remains to be fully understood. We previously found the calcium-binding protein S100A6 was expressed in ∼80% of the analyzed OS primary and/or metastatic tumor samples. Here, we investigate the role of S100A6 in OS growth and progression. METHODS: S100A6 expression was assessed by qPCR and Western blotting. Overexpression or knockdown of S100A6 was carried out to determine S100A6's effect on proliferation, cell cycle, apoptosis, tumor growth, and osteogenic differentiation. RESULTS: S100A6 expression was readily detected in human OS cell lines. Exogenous S100A6 expression promoted cell proliferation in vitro and tumor growth in an orthotopic xenograft model of human OS. S100A6 overexpression reduced the numbers of OS cells in G1 phase and increased viable cells under serum starvation condition. Conversely, silencing S100A6 expression induced the production of cleaved caspase 3, and increased early stage apoptosis. S100A6 knockdown increased osteogenic differentiation activity of mesenchymal stem cells, while S100A6 overexpression inhibited osteogenic differentiation. BMP9-induced bone formation was augmented by S100A6 knockdown. CONCLUSION: Our findings strongly suggest that S100A6 may promote OS cell proliferation and OS tumor growth at least in part by facilitating cell cycle progression, preventing apoptosis, and inhibiting osteogenic differentiation. Thus, it is conceivable that targeting S100A6 may be exploited as a novel anti-OS therapy.

Functional characteristics of reversibly immortalized hepatic progenitor cells derived from mouse embryonic liver.

BACKGROUND/AIMS: Liver is a vital organ and retains its regeneration capability throughout adulthood, which requires contributions from different cell populations, including liver precursors and intrahepatic stem cells. To overcome the mortality of hepatic progenitors (iHPs) in vitro, we aim to establish reversibly immortalized hepatic progenitor cells from mouse embryonic liver. METHODS AND RESULTS: Using retroviral system to stably express SV40 T antigen flanked with Cre/LoxP sites, we establish a repertoire of iHP clones with varied differentiation potential. The iHP cells maintain long-term proliferative activity and express varied levels of progenitor markers (Pou5f1/Oct4 and Dlk) and hepatocyte markers (AFP, Alb and ApoB). Five representative iHP clones express hepatic/pancreatic transcription factors HNF3α/Foxa1, HNF3ß/Foxa2, and HNF4α/MODY1. Dexamethasone is shown to promote the expression of hepatocyte markers AFP and TAT, along with ICG-uptake and glycogen storage functions in the iHP clones. Cre-mediated removal of SV40 T antigen reverses the proliferative activity of iHP cells. When iHP cells are subcutaneously implanted in athymic nude mice, no tumor formation is observed for up to 8 weeks. CONCLUSIONS: We demonstrate that the established iHP cells are stable, reversible, and non-tumorigenic hepatic progenitor-like cells, which should be valuable for studying liver organogenesis, metabolic regulations, and hepatic lineage-specific differentiation.

Ewing's sarcoma of bone.

Ewing's sarcoma of bone is a primary bone sarcoma found predominantly in patients during their second decade of life. It is a high-grade aggressive small round blue cell tumor that is part of the Ewing's family of tumors. Its exact eitiology is unknown but it commonly demonstrates reproducible staining of CD99 and translocations of the EWS gene. Historically, this diagnosis was associated with near certain metastasis and subsequent mortality. However, current management consists of extensive chemotherapy in addition to local control with surgical resection and/or radiation. As a result, survival has improved to the 55-75% range in those patients who present without known metastases. Current research aims to continue this improvement by looking further into the associated gene abnormalities and possibly targeted therapies.

The evolving roles of Wnt signaling in stem cell proliferation and differentiation, the development of human diseases, and therapeutic opportunities.

The evolutionarily conserved Wnt signaling pathway plays a central role in development and adult tissue homeostasis across species. Wnt proteins are secreted, lipid-modified signaling molecules that activate the canonical (ß-catenin dependent) and non-canonical (ß-catenin independent) Wnt signaling pathways. Cellular behaviors such as proliferation, differentiation, maturation, and proper body-axis specification are carried out by the canonical pathway, which is the best characterized of the known Wnt signaling paths. Wnt signaling has emerged as an important factor in stem cell biology and is known to affect the self-renewal of stem cells in various tissues. This includes but is not limited to embryonic, hematopoietic, mesenchymal, gut, neural, and epidermal stem cells. Wnt signaling has also been implicated in tumor cells that exhibit stem cell-like properties. Wnt signaling is crucial for bone formation and presents a potential target for the development of therapeutics for bone disorders. Not surprisingly, aberrant Wnt signaling is also associated with a wide variety of diseases, including cancer. Mutations of Wnt pathway members in cancer can lead to unchecked cell proliferation, epithelial-mesenchymal transition, and metastasis. Altogether, advances in the understanding of dysregulated Wnt signaling in disease have paved the way for the development of novel therapeutics that target components of the Wnt pathway. Beginning with a brief overview of the mechanisms of canonical and non-canonical Wnt, this review aims to summarize the current knowledge of Wnt signaling in stem cells, aberrations to the Wnt pathway associated with diseases, and novel therapeutics targeting the Wnt pathway in preclinical and clinical studies.

Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.

Inhibition of histone deacetylases potentiates BMP9-induced osteogenic signaling in mouse mesenchymal stem cells.

BACKGROUND/AIMS: We have demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent BMPs in regulating osteoblast differentiation of mesenchymal stem cells (MSCs) although the molecular mechanism underlying BMP9-induced osteogenesis remains to be fully elucidated. It is known that epigenetic regulations play an important role in regulating the stem cell potency and lineage commitment. Here, we investigate if the inhibition of histone deacetylases (Hdacs) affects BMP9-induced osteogenic differentiation of MSCs. METHODS: Using the Hdac inhibitor trichostatin A (TSA), we assess that TSA enhances BMP9-mediated osteogenic markers and matrix mineralization in MSCs, and bone formation in mouse embryonic limb explants. RESULTS: We find that the endogenous expression of most of the 11 Hdacs is readily detectable in MSCs. BMP9 is shown to induce most Hdacs in MSCs. We demonstrate that TSA potentiates BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs, as well as late osteogenic markers osteopontin (OPN) and osteocalcin (OCN) and matrix mineralization. Fetal limb explant culture studies reveal that TSA potentiates BMP9-induced endochondral bone formation, possibly by expanding hypertrophic chondrocyte zone of growth plate. CONCLUSION: Our findings strongly suggest histone deacetylases may play an important role in fine-tuning BMP9-mediated osteogenic signaling through a negative feedback network in MSCs. Thus, Hdac inhibitors may be used as novel therapeutics for bone fracture healing.

Destabilization of heterologous proteins mediated by the GSK3ß phosphorylation domain of the ß-catenin protein.

BACKGROUND AND AIMS: Wnt/ß-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of ß-catenin protein in cytoplasm and/or nucleus. The stability of ß-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of ß-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3ßmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3ß phosphorylation site-containing degradation domain of ß-catenin is sufficient to destabilize heterologous proteins. METHODS AND RESULTS: We engineer chimeric proteins by fusing ß-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. CONCLUSION: Our findings strongly suggest that the ß-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of ß-catenin signaling, and to identify potential anticancer drugs that target ß-catenin signaling.

The E-F hand calcium-binding protein S100A4 regulates the proliferation, survival and differentiation potential of human osteosarcoma cells.

BACKGROUND/AIMS: Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. Molecular mechanisms underlying the pathogenesis of OS remain to be fully understood. Several members of the E-F hand calcium-binding S100 protein family are differentially expressed in human cancers. We previously showed that S100A6 is highly expressed in OS tumors. In this study, we investigated the role of S100A4 in regulating OS proliferation and osteogenic differentiation. METHODS/RESULTS: Endogenous S100 expression was examined by semi-quantitative PCR in human OS lines. Adenoviral vector-mediated overexpression and RNAi knockdown of S100A4 were used to assess S100A4's effects on cell proliferation, migration and invasion and osteogenic differentiation. Apoptosis was assessed by using anti-caspase-3 immunostaining and flow cytometry with annexin V staining. Early osteogenic marker alkaline phosphatase (ALP) and late markers osteocalcin (OCN) and osteopontin (OPN) were assessed to determine the status of osteogenic differentiation. We found that S100A4 was elevated in metastatic MG63.2 cells. S100A4 knockdown inhibited cell proliferation, prolonged cell doubling time, and induced significant apoptosis. Silencing S100A4 expression in OS cells delayed cell wounding closure and diminished the numbers of migrated OS cells in transwell invasion assay. Furthermore, silencing S100A4 expression stimulated ALP activity, as well as late markers OPN and OCN, in both OS cells and mesenchymal stem cells. CONCLUSION: Our results strongly suggest that S100A4 may promote OS tumor growth by regulating the cell cycle, reducing apoptosis, and inhibiting osteogenic differentiation. Thus, S100A4 may serve as a marker for tumorigenic potential, as well as a therapeutic target.

Modulation of ß-catenin signaling by the inhibitors of MAP kinase, tyrosine kinase, and PI3-kinase pathways.

Aberrant activation of ß-catenin signaling plays an important role in human tumorigenesis. However, molecular mechanisms behind the ß-catenin signaling deregulation are mostly unknown because genetic alterations in this pathway only account for a small fraction of tumors. Here, we investigator if other major pathways can regulate ß-catenin signaling activity. By employing a panel of chemical activators and/or inhibitors of several cellular signaling pathways, we assess these modulators' effects on luciferase reporter driven by ß-catenin/TCF4-responsive elements. We find that lithium-stimulated ß-catenin activity is synergistically enhanced by protein kinase C activator PMA. However, ß-catenin-regulated transcriptional (CRT) activity is significantly inhibited by casein kinase II inhibitor DRB, MEK inhibitor PD98059, G-proteins and their receptor uncoupling agent suramin, protein tyrosine kinase inhibitor genistein, and PI-3 kinase inhibitor wortmannin, suggesting that these cellular pathways may participate in regulating ß-catenin signaling. Interestingly, the Ca⁺⁺/calmodulin kinase II inhibitor HDBA is shown to activate ß-catenin activity at low doses. Furthermore, Wnt3A-stimulated and constitutively activated CRT activities, as well as the intracellular accumulation of ß-catenin protein in human colon cancer cells, are effectively suppressed by PD98059, genistein, and wortmannin. We further demonstrate that EGF can activate TCF4/ß-catenin activity and induce the tyrosine phosphorylation of ß-catenin protein. Thus, our results should provide important insights into the molecular mechanisms underlying Wnt/ß-catenin activation. This knowledge should facilitate our efforts to develop efficacious and novel therapeutics by targeting these pathways.

Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors.

OBJECTIVE: Progenitor cell-based cardiomyocyte regeneration holds great promise of repairing an injured heart. Although cardiomyogenic differentiation has been reported for a variety of progenitor cell types, the biological factors that regulate effective cardiomyogenesis remain largely undefined. Primary cardiomyogenic progenitors (CPs) have a limited life span in culture, hampering the CPs' in vitro and in vivo studies. The objective of this study is to investigate if primary CPs isolated from fetal mouse heart can be reversibly immortalized with SV40 large T and maintain long-term cell proliferation without compromising cardiomyogenic differentiation potential. METHODS: Primary cardiomyocytes were isolated from mouse E15.5 fetal heart, and immortalized retrovirally with the expression of SV40 large T antigen flanked with loxP sites. Expression of cardiomyogenic markers were determined by quantitative RT-PCR and immunofluorescence staining. The immortalization phenotype was reversed by using an adenovirus-mediated expression of the Cre reconbinase. Cardiomyogenic differentiation induced by retinoids or dexamethasone was assessed by an α-myosin heavy chain (MyHC) promoter-driven reporter. RESULTS: We demonstrate that the CPs derived from mouse E15.5 fetal heart can be efficiently immortalized by SV40 T antigen. The conditionally immortalized CPs (iCP15 clones) exhibit an increased proliferative activity and are able to maintain long-term proliferation, which can be reversed by Cre recombinase. The iCP15 cells express cardiomyogenic markers and retain differentiation potential as they can undergo terminal differentiate into cardiomyctes under appropriate differentiation conditions although the iCP15 clones represent a large repertoire of CPs at various differentiation stages. The removal of SV40 large T increases the iCPs' differentiation potential. Thus, the iCPs not only maintain long-term cell proliferative activity but also retain cardiomyogenic differentiation potential. CONCLUSIONS: Our results suggest that the reported reversible SV40 T antigen-mediated immortalization represents an efficient approach for establishing long-term culture of primary cardiomyogenic progenitors for basic and translational research.

Niclosamide (NA) overcomes cisplatin resistance in human ovarian cancer.

Ovarian cancer (OC) is one of the most lethal malignancies of the female reproductive system. OC patients are usually diagnosed at advanced stages due to the lack of early diagnosis. The standard treatment for OC includes a combination of debulking surgery and platinum-taxane chemotherapy, while several targeted therapies have recently been approved for maintenance treatment. The vast majority of OC patients relapse with chemoresistant tumors after an initial response. Thus, there is an unmet clinical need to develop new therapeutic agents to overcome the chemoresistance of OC. The anti-parasite agent niclosamide (NA) has been repurposed as an anti-cancer agent and exerts potent anti-cancer activities in human cancers including OC. Here, we investigated whether NA could be repurposed as a therapeutic agent to overcome cisplatin-resistant (CR) in human OC cells. To this end, we first established two CR lines SKOV3CR and OVCAR8CR that exhibit the essential biological characteristics of cisplatin resistance in human cancer. We showed that NA inhibited cell proliferation, suppressed cell migration, and induced cell apoptosis in both CR lines at a low micromole range. Mechanistically, NA inhibited multiple cancer-related pathways including AP1, ELK/SRF, HIF1, and TCF/LEF, in SKOV3CR and OVCAR8CR cells. NA was further shown to effectively inhibit xenograft tumor growth of SKOV3CR cells. Collectively, our findings strongly suggest that NA may be repurposed as an efficacious agent to combat cisplatin resistance in chemoresistant human OC, and further clinical trials are highly warranted.

Long noncoding RNA (lncRNA) H19: An essential developmental regulator with expanding roles in cancer, stem cell differentiation, and metabolic diseases.

Recent advances in deep sequencing technologies have revealed that, while less than 2% of the human genome is transcribed into mRNA for protein synthesis, over 80% of the genome is transcribed, leading to the production of large amounts of noncoding RNAs (ncRNAs). It has been shown that ncRNAs, especially long non-coding RNAs (lncRNAs), may play crucial regulatory roles in gene expression. As one of the first isolated and reported lncRNAs, H19 has gained much attention due to its essential roles in regulating many physiological and/or pathological processes including embryogenesis, development, tumorigenesis, osteogenesis, and metabolism. Mechanistically, H19 mediates diverse regulatory functions by serving as competing endogenous RNAs (CeRNAs), Igf2/H19 imprinted tandem gene, modular scaffold, cooperating with H19 antisense, and acting directly with other mRNAs or lncRNAs. Here, we summarized the current understanding of H19 in embryogenesis and development, cancer development and progression, mesenchymal stem cell lineage-specific differentiation, and metabolic diseases. We discussed the potential regulatory mechanisms underlying H19's functions in those processes although more in-depth studies are warranted to delineate the exact molecular, cellular, epigenetic, and genomic regulatory mechanisms underlying the physiological and pathological roles of H19. Ultimately, these lines of investigation may lead to the development of novel therapeutics for human diseases by exploiting H19 functions.