Sodium citrate targeting Ca2+/CAMKK2 pathway exhibits anti-tumor activity through inducing apoptosis and ferroptosis in ovarian cancer.

INTRODUCTION: Ovarian cancer (OC) is known for its high mortality rate. Although sodium citrate has anti-tumor effects in various cancers, its effect and mechanism in OC remain unclear. OBJECTIVES: To analyze the inhibitory effect of sodium citrate on ovarian cancer cells and the underlying mechanism. METHODS: Cell apoptosis was examined by TUNEL staining, flow cytometry, and ferroptosis was examined intracellular Fe2+, MDA, LPO assays, respectively. Cell metabolism was examined by OCR and ECAR measurements. Immunoblotting and immunoprecipitation were used to elucidate the mechanism. RESULTS: This study suggested that sodium citrate not only promoted ovarian cancer cell apoptosis but also triggeredferroptosis, manifested as elevated levels of Fe2+, LPO, MDA andlipid ROS production. On one hand, sodium citrate treatment led to a decrease of Ca2+ content in the cytosol by chelatingCa2+, which further inhibited the Ca2+/CAMKK2/AKT/mTOR signaling, thereby suppressing HIF1α-dependent glycolysis pathway and inducing cell apoptosis. On the other hand, the chelation of Ca2+ by sodium citrate resulted in inactivation of CAMKK2 and AMPK, leading to increase of NCOA4-mediated ferritinophagy, causing increased intracellular Fe2+ levels. More importantly, the inhibition of Ca2+/CAMKK2/AMPK signaling pathway reduced the activity of the MCU and Ca2+ concentration within the mitochondria, resulting in an increase in mitochondrial ROS. Additionally, metabolomic analysis indicated that sodium citrate treatment significantly increased de novo lipid synthesis. Altogether, these factors contributed to ferroptosis. As expected, Ca2+ supplementation successfully reversed the cell death and decreased tumor growth induced by sodium citrate. Inspiringly, it was found that coadministration of sodium citrate increased the sensitivity of OC cells to chemo-drugs. CONCLUSION: These results revealed that the sodium citrate exerted its anti-cancer activity by inhibiting Ca2+/CAMKK2-dependent cell apoptosis and ferroptosis. Sodium citrate will hopefully serve as a prospective compound for OC treatment and for improvingthe efficacy of chemo-drugs.

Antitumor Effect of Anti-c-Myc Aptamer-Based PROTAC for Degradation of the c-Myc Protein.

Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c-Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate-based systematic evolution of ligands by exponential enrichment (microwell-SELEX), and identify the specific aptamer (MA9C1) against c-Myc. The multifunctional aptamer-based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c-Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c-Myc by the ubiquitin-proteasome system, but also reduces the Max protein, synergistically inhibiting c-Myc transcriptional activity. Combination of the artificial cyclization and anti-PD-L1 aptamer (PA1)-based delivery system, circular PA1-ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c-Myc degrader for the clinic. Therefore, this aptamer-based degrader provides an invaluable potential degrader in drug discovery and anti-tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets.

The opposite role of lactate dehydrogenase a (LDHA) in cervical cancer under energy stress conditions.

Due to insufficient and defective vascularization, the tumor microenvironment is often nutrient-depleted. LDHA has been demonstrated to play a tumor-promoting role by facilitating the glycolytic process. However, whether and how LDHA regulates cell survival in the nutrient-deficient tumor microenvironment is still unclear. Here, we sought to investigate the role and mechanism of LDHA in regulating cell survival and proliferation under energy stress conditions. Our results showed that the aerobic glycolysis levels, cell survival and proliferation of cervical cancer cells decreased significantly after inhibition of LDHA under normal culture condition while LDHA deficiency greatly inhibited glucose starvation-induced ferroptosis and promoted cell proliferation and tumor formation under energy stress conditions. Mechanistic studies suggested that glucose metabolism shifted from aerobic glycolysis to mitochondrial OXPHOS under energy stress conditions and LDHA knockdown increased accumulation of pyruvate in the cytosol, which entered the mitochondria and upregulated the level of oxaloacetate by phosphoenolpyruvate carboxylase (PC). Importantly, the increase in oxaloacetate production after absence of LDHA remarkably activated AMP-activated protein kinase (AMPK), which increased mitochondrial biogenesis and mitophagy, promoted mitochondrial homeostasis, thereby decreasing ROS level. Moreover, repression of lipogenesis by activation of AMPK led to elevated levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which effectively resisted ROS-induced cell ferroptosis and enhanced cell survival under energy stress conditions. These results suggested that LDHA played an opposing role in survival and proliferation of cervical cancer cells under energy stress conditions, and inhibition of LDHA may not be a suitable treatment strategy for cervical cancer.

TSP50 Attenuates DSS-Induced Colitis by Regulating TGF-ß Signaling Mediated Maintenance of Intestinal Mucosal Barrier Integrity.

The integrity of the intestinal mucosal barrier is crucial for protecting the intestinal epithelium against invasion by commensal bacteria and pathogens, thereby combating colitis. The investigation revealed that the absence of TSP50 compromised the integrity of the intestinal mucosal barrier in murine subjects. This disruption facilitated direct contact between intestinal bacteria and the intestinal epithelium, thereby increasing susceptibility to colitis. Mechanistic analysis indicated that TSP50 deficiency in intestinal stem cells (ISCs) triggered aberrant activation of the TGF-ß signaling pathway and impeded the differentiation of goblet cells in mice, leading to impairment of mucosal permeability. By inhibiting the TGF-ß pathway, the functionality of the intestinal mucosal barrier is successfully restored and mitigated colitis in TSP50-deficient mice. In conclusion, TSP50 played a crucial role in maintaining the intestinal mucosal barrier function and exhibited the preventive effect against the development of colitis by regulating the TGF-ß signaling pathway.

Tectorigenin targets PKACα to promote GLUT4 expression in skeletal muscle and improve insulin resistance in vitro and in vivo.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle.

Adipose-derived mesenchymal stem cell-loaded ß-chitin nanofiber hydrogel activates the AldoA/HIF-1α pathway to promote diabetic wound healing.

OBJECTIVES: To identify the effect of adipose-derived mesenchymal stem cell-loaded ß-chitin nanofiber (ADSC-loaded ß-ChNF) hydrogel on diabetic wound healing and clarify its mechanism of action. METHODS: We prepared the ADSC-loaded ß-ChNF hydrogel to repair wounds of db/db diabetic mice. Wound healing rate, histopathology, enzyme-linked immunosorbent assay, and western blot were used to confirm its role and mechanism in promoting diabetic wound healing. RESULTS: The ADSC-loaded ß-ChNF hydrogel accelerated wound healing in db/db diabetic mice, as indicated by increased cell proliferation, epithelization, and tissue granulation in the skin. Moreover, expression of vascular endothelial growth factor (VEGF) and its receptor (VEGFR), matrix metalloproteinase 9 (MMP9), and TIMP metallopeptidase inhibitor 1 (TIMP1) were upregulated. These results demonstrate the beneficial effects of this ADSC-loaded ß-ChNF hydrogel on diabetic wound healing. Furthermore, we show that the ADSC-loaded ß-ChNF hydrogel activated aldolase A (AldoA)/hypoxia-inducible factor 1α (HIF-1α) signaling. An inhibitor of HIF-1α markedly decreased the promotive effects of the ADSC-loaded ß-ChNF hydrogel on wound healing and reduced expression of VEGF, VEGFR, MMP9, and TIMP1. CONCLUSIONS: Our findings suggest that the ADSC-loaded ß-ChNF hydrogel activated the HIF-1α/MMP9 axis through AldoA feedback to promote diabetic wound healing.

STS1 and STS2 Phosphatase Inhibitor Baicalein Enhances the Expansion of Hematopoietic and Progenitor Stem Cells and Alleviates 5-Fluorouracil-Induced Myelosuppression.

STS1 and STS2, as the protein phosphatases that dephosphorylate FLT3 and cKIT, negatively regulate the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs). To obtain the small molecule inhibitors of STS1/STS2 phosphatase activity used to expand HSPCs both in vitro and in vivo, we establish an in vitro phosphatase assay using the recombinant proteins of the STS1/STS2 histidine phosphatase (HP) domain, by which we screened out baicalein (BC) as one of the effective inhibitors targeting STS1 and STS2. Then, we further demonstrate the direct binding of BC with STS1/STS2 using molecular docking and capillary electrophoresis and verify that BC can restore the phosphorylation of FLT3 and cKIT from STS1/STS2 inhibition. In a short-term in vitro culture, BC promotes profound expansion and enhances the colony-forming capacity of both human and mouse HSPCs along with the elevation of phospho-FLT3 and phospho-cKIT levels. Likewise, in vivo administration with BC significantly increases the proportions of short-term hematopoietic stem cells (ST-HSCs), multipotent progenitors (MPPs) and especially long-term HSCs (LT-HSCs) in healthy mouse bone marrow and increases the numbers of colony-forming units (CFU) formed by HSPCs as well. More importantly, pre-administration of BC significantly enhances the survival of mice with lethal 5-fluorouracil (5-FU) injection due to the alleviation of 5-FU-induced myelosuppression, as evidenced by the recovery of bone marrow histologic injury, the increased proportions of LT-HSCs, ST-HSCs and MPPs, and enhanced colony-forming capacity. Collectively, our study not only suggests BC as one of the small molecule candidates to stimulate HSPC expansion both in vitro and in vivo when needed in either physiologic or pathologic conditions, but also supports STS1/STS2 as potential therapeutic drug targets for HSPC expansion and hematopoietic injury recovery.

FRMD3 inhibits the growth and metastasis of breast cancer through the ubiquitination-mediated degradation of vimentin and subsequent impairment of focal adhesion.

Recurrence and metastasis are the main causes of breast cancer (BRCA)-related death and remain a challenge for treatment. In-depth research on the molecular mechanisms underlying BRCA progression has been an important basis for developing precise biomarkers and therapy targets for early prediction and treatment of progressed BRCA. Herein, we identified FERM domain-containing protein 3 (FRMD3) as a novel potent BRCA tumor suppressor which is significantly downregulated in BRCA clinical tissue and cell lines, and low FRMD3 expression has been closely associated with progressive BRCA and shortened survival time in BRCA patients. Overexpression and knockdown experiments have revealed that FRMD3 significantly inhibits BRCA cell proliferation, migration, and invasion in vitro and suppresses BRCA xenograft growth and metastasis in vivo as well. Mechanistically, FRMD3 can interact with vimentin and ubiquitin protein ligase E3A(UBE3A) to induce the polyubiquitin-mediated proteasomal degradation of vimentin, which subsequently downregulates focal adhesion complex proteins and pro-cancerous signaling activation, thereby resulting in cytoskeletal rearrangement and defects in cell morphology and focal adhesion. Further evidence has confirmed that FRMD3-mediated vimentin degradation accounts for the anti-proliferation and anti-metastasis effects of FRMD3 on BRCA. Moreover, the N-terminal ubiquitin-like domain of FRMD3 has been identified as responsible for FRMD3-vimentin interaction through binding the head domain of vimentin and the truncated FRMD3 with the deletion of ubiquitin-like domain almost completely loses the anti-BRCA effects. Taken together, our study indicates significant potential for the use of FRMD3 as a novel prognosis biomarker and a therapeutic target of BRCA and provides an additional mechanism underlying the degradation of vimentin and BRCA progression.

Structural characterization and immunoregulatory activity of a neutral polysaccharide from the roots of Apocynum venetum L.

The structural characteristics and immunoregulatory activities of neutral heteropolysaccharide (AVRP-N) separated from the roots of Apocynum venetum L. were extensively investigated. The results showed that the weight average molecular mass (Mw) of AVRP-N was 6.430 × 103 Da. Moreover, the backbone is composed of natural acetylated (1 â†’ 4)-ß-D-Man and (1 â†’ 5)-α-L-Ara domains. The mannan is composed of →4)-ß-D-Manp-(1→, →4)-ß-D-Glcp-(1→, and the terminal group α-D-Galp-(1→ attached to →4,6)-ß-D-Manp-(1→ at O-6. Araban is composed of →5)-α-L-Araf-(1→; the terminal group α-L-Araf-(1→attached to→2,3,5)-α-L-Araf-(1→ at O-2, O-3 and →3,5)-α-L-Araf-(1→ at O-3. In addition, the senior structure shows that AVRP-N has a triple-helix conformation. Furthermore, AVRP-N exhibited immunomodulatory effects, which could significantly regulate the proliferation of mouse splenic lymphocytes by enhancing the secretion of the cytokines (IFN-γ, IL-2, IL-4, and IL-10). Our results provide new structural and immunoregulatory information for natural polysaccharides derived from Apocynum venetum L.

Application of adipose mesenchymal stem cell-derived exosomes-loaded ß-chitin nanofiber hydrogel for wound healing.

INTRODUCTION: Clarifying the role and mechanism of exosome gel in wound repair can provide a new effective strategy for wound treatment. MATERIALS AND METHODS: The cellular responses of adipose mesenchymal stem cell-derived exosomes (AMSC-exos) and the wound healing ability of AMSC-exos-loaded ß-chitin nanofiber (ß-ChNF) hydrogel were studied in vitro in mouse fibroblasts cells (L929) and in vivo in rat skin injury model. The transcriptome and proteome of rat skin were studied with the use of sequenator and LC-MS/MS, respectively. RESULTS: 80 and 160 µg/mL AMSC-exos could promote the proliferation and migration of mouse fibroblastic cells. Furthermore, AMSC-exos-loaded ß-ChNF hydrogel resulted in a significant acceleration rate of wound closure, notably, acceleration of re-epithelialization, and increased collagen expression based on the rat full-thickness skin injury model. The transcriptomics and proteomics studies revealed the changes of the expression of 18 genes, 516 transcripts and 250 proteins. The metabolic pathways, tight junction, NF-κB signaling pathways were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway. Complement factor D (CFD) and downstream Aldolase A (Aldoa) and Actn2 proteins in rats treated with AMSC-exos-loaded ß-ChNF hydrogel were noticed and further confirmed by ELISA and Western blot. CONCLUSION: These findings suggested that AMSC-exos-loaded ß-ChNF hydrogel could promote wound healing with the mechanism which is related to the effect of AMSC-exos on CFD and downstream proteins.

Adipose-derived mesenchymal stem cell-loaded ß-chitin nanofiber hydrogel promote wound healing in rats.

Because of stem cells are limited by the low efficiency of their cell homing and survival in vivo, cell delivery systems and scaffolds have attracted a great deal of attention for stem cells' successful clinical practice. ß-chitin nanofibers (ß-ChNF) were prepared from squid pens in this study. Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy proved that ß-ChNFs with the diameter of 5 to 10 nm were prepared. ß-ChNF dispersion became gelled upon the addition of cell culture medium. Cell culture experiments showed that ß-ChNFs exhibited negligible cytotoxicity towards ADSCs and L929 cells, and it was found that more exosomes were secreted by the globular ADSCs grown in the ß-ChNF hydrogel. The vivo experiments of rats showed that the ADSCs-loaded ß-ChNF hydrogel could directly cover the wound surface and significantly accelerate the wound healing and promote the generation of epithelization, granulation tissue and collagen. In addition, the ADSCs-loaded ß-ChNF hydrogel clearly regulated the expressions of VEGFR, α-SMA, collagen I and collagen III. Finally, we showed that ADSCs-loaded ß-ChNF hydrogel activated the TGFß/smad signaling. The neutralization of TGFß markedly reduced Smad phosphorylation and the expressions of TIMP1, VEGFR and α-SMA. Taken together, these findings suggest that ADSCs-loaded ß-ChNF hydrogel promises for treating wounds that are challenge to heal via conventional methods. Graphical abstract.

Discovery of Phenolic Glycoside from Hyssopus cuspidatus Attenuates LPS-Induced Inflammatory Responses by Inhibition of iNOS and COX-2 Expression through Suppression of NF-κB Activation.

It seems quite necessary to obtain effective substances from natural products against inflammatory response (IR) as there are presently clinical problems regarding accompanying side effects and lowered quality of life. This work aimed to investigate the abilities of hyssopuside (HY), a novel phenolic glycoside isolated from Hyssopus cuspidatus (H. cuspidatus), against IR in lipopolysaccharide (LPS)-induced RAW 264.7 cells and mouse peritoneal macrophages. The results indicated that HY could reduce nitric oxide (NO) production and inhibit the production and secretion of pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in LPS-stimulated macrophages. Moreover, data from the immunofluorescence study showed that HY suppressed nuclear translocation of nuclear factor-kappa B (NF-κB) upon LPS induction. The Western blot results suggested that HY reversed the LPS-induced degradation of IκB (inhibitor of NF-κB), which is normally required for the activation of NF-κB. Meanwhile, the overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) diminished significantly with the presence of HY in response to LPS stimulation. On the other hand, HY had a negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. Moreover, an in silico study of HY against four essential proteins/enzymes revealed that COX-2 was the most efficient enzyme for the interaction, and binding of residues Phe179, Asn351, and Ser424 with HY played crucial roles in the observed activity. The structure analysis indicated the typical characterizations with phenylethanoid glycoside contributed to the anti-inflammatory effects of HY. These results indicated that HY manipulated its anti-inflammatory effects mainly through blocking the NF-κB signal transduction pathways. Collectively, we believe that HY could be a potential alternative phenolic agent for alleviating excessive inflammation in many inflammation-associated diseases.

Immunomodulatory effects of flazin from Crassostrea sikamea on splenic lymphocytes of Sprague-Dawley rats.

Crassostrea sikamea (C.sikamea) is an important edible and medicinal seafood in China. In the present study, a compound named flazin was separated and identified from the ethyl acetate extract of C.sikamea (EAECs) for the first time. In addition, the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra zolium (MTS) assay revealed that EAECs and flazin inhibited the transformation of splenic lymphocytes in vitro. Moreover, flazin (20 µg·mL-1) altered the populations of splenic lymphocyte subtypes. Real-time quantitative PCR (RT-qPCR) analysis and enzyme-linked immunosorbent assay (ELISA) showed that flazin suppressed the mRNA expression and secretion of TNF-α and IL-2, and reversed Concanavalin A (ConA)-induced mRNA up-regulation and protein secretion of TNF-α and IL-2. Western blot results showed that flazin reversed ConA-induced increases in p-ERK1/2 and p-p38 in splenocytes. In conclusion, flazin exhibits effective immunomodulatory function and may be useful for treating immune-related disorders, which indicates the application potential of C.sikamea as a functional food or immunomodulator.

Discovery and Verification of Key Liver Cancer Genes and Alternative Splicing Events Based on Second-Generation Sequencing Data Analysis.

Hepatocellular carcinoma (HCC) is the most common malignant liver disease in the world. Existing screening and early diagnosis methods are not highly sensitive for HCC, and patients are likely to develop the disease to the middle and advanced stages before being diagnosed. Therefore, finding new and efficient diagnosis and treatment methods has become an urgent problem. We aimed at finding and verifying new liver cancer markers by combining informatics analysis with experimental exploration to provide new ideas and methods for the diagnosis and treatment of clinical liver cancer. We used two different bioinformatic pipelines to analyze sequencing data of clinical liver cancer samples and identify differentially expressed genes and key variants after combining them with The Cancer Genome Atlas sequencing data. Then, we explored the functions and mechanisms of the key variants to identify potential liver cancer markers. Through bioinformatic analysis of sequencing data, 139 differentially expressed genes were found, including 53 upregulated genes and 86 downregulated genes. Through enrichment and alternative splicing event analysis of sequencing data, we found nine key variants with exon skipping events. Metallothionein 1E (MT1E)-203 was found to be a key variant that influenced cell proliferation through the p53 cell cycle pathway through cell viability and proliferation assays, and MT1E-203 lost the ability to bind two zinc ions due to exon skipping according to the structure prediction of MT1E-203. MT1E-203 is a potential biomarker for HCC and may play an important role in the diagnosis and treatment of HCC.

miR-3929 Inhibits Proliferation and Promotes Apoptosis by Downregulating Cripto-1 Expression in Cervical Cancer Cells.

Cripto-1 is highly expressed in many cancers, and downregulating its expression may become a promising approach for cancer treatment. However, the regulation of Cripto-1 expression is not well characterized. In this study, we focused on the post-transcriptional regulation of Cripto-1 expression and analyzed the potential miRNAs that bind to the 3'UTR of Cripto-1 mRNA. miR-3929 was found to be able to bind to the 3'UTR and downregulate the expression of Cripto-1 in cervical cancer cells. Then, we analyzed the effect of miR-3929 on the biological behavior of cervical cancer cells, finding that miR-3929 could reduce cell viability, DNA synthesis, and Ki67 expression and induce cell cycle arrest in the G2/M phase; overexpression of Cripto-1 reversed the inhibitory effect of miR-3929 on proliferation. Moreover, DAPI staining and flow cytometry revealed that miR-3929-induced cell apoptosis is dependent on the mitochondrial pathway; the overexpression of Cripto-1 reversed the proapoptotic effect of miR-3929. Finally, the in vivo results showed that miR-3929 significantly inhibits the growth of HeLa xenograft tumors in nude mice. Therefore, our findings suggest that miR-3929 inhibits the proliferation and induces the apoptosis of cervical cancer cells by downregulating Cripto-1 via specifically targeting the 3'UTR of its mRNA.

Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways.

BACKGROUND: Stem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells. METHODS: In the present study, we used the previously constructed luciferase reporters driven by the promoter and 3'-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways. RESULTS: We screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways. CONCLUSIONS: These findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

SUVA: splicing site usage variation analysis from RNA-seq data reveals highly conserved complex splicing biomarkers in liver cancer.

Most of the current alternative splicing (AS) analysis tools are powerless to analyse complex splicing. To address this, we developed SUVA (Splice sites Usage Variation Analysis) that decomposes complex splicing events into five types of splice junction pairs. By analysing real and simulated data, SUVA showed higher sensitivity and accuracy in detecting AS events than the compared methods. Notably, SUVA detected extensive complex AS events and screened out 69 highly conserved and dominant AS events associated with cancer. The cancer-associated complex AS events in FN1 and the co-regulated RNA-binding proteins were significantly correlated with patient survival.

miR-4709-3p Inhibits Cell Proliferation by Downregulating TSP50 Expression in Breast Cancer Cells.

Breast cancer is a serious threat to the physical and mental health of women all over the world. Our previous results have shown that Serine protease 50 (TSP50), an oncogene overexpressed in breast cancer, can promote proliferation, migration, and invasion of breast cancer cells. Mechanistic studies have revealed that TSP50 promoted tumorigenesis mainly by activating NF-kappa B (NF-κB) and inhibiting activin signaling pathway, indicating that TSP50 played a critical role in the occurrence and development of breast cancer. However, there are few reports on the regulation of TSP50 expression in breast cancer. MicroRNAs (miRNAs) have emerged as an essential posttranscriptional regulator in gene expression and they played a significant role in breast cancer regulation. In the present study, bioinformatics software miRBase and TargetScan were first used to predict and analyze miRNAs that could target TSP50 mRNA 3'UTR and six miRNAs were found. Results from quantitative real-time PCR (qRT-PCR) and western blot suggested that miR-4709-3p could bind to TSP50 mRNA 3'UTR and significantly inhibit the expression of TSP50 protein. Moreover, the effects of miR-4709-3p on the proliferation of breast cancer cells and mammary epithelial cells were detected in vitro. Our data suggested that overexpression of miR-4709-3p mimic greatly inhibited the proliferation of breast cancer cells, whereas overexpression of miR-4709-3p inhibitors significantly promoted the proliferation of breast epithelial cells. Furthermore, the effect of miR-4709-3p on the tumorigenicity of breast cancer cells in vivo was tested, and the results showed that miR-4709-3p significantly reduced the volume and weight of tumor in nude mice. All these results suggested that miR-4709-3p could inhibit the tumorigenesis of breast cancer cells by targeting TSP50. Finally, the underlying molecular mechanisms were investigated and we found that both NF-κB and activin signaling were involved in miR-4709-3p-related tumor inhibitory effect.

TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation.

Metabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC-MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.

TSP50 promotes hepatocyte proliferation and tumour formation by activating glucose-6-phosphate dehydrogenase (G6PD).

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a common malignant tumour with high morbidity and mortality. Metabolic regulation by oncogenes is necessary for tumour growth. Testes-specific protease 50 (TSP50) has been found to promote cell proliferation in multiple tumour types. However, the mechanism that TSP50 promotes HCC progression are not known. METHODS: Hepatocyte proliferation was analysed by MTT and BrdU incorporation after TSP50 transfection. Furthermore, LC-MS/MS, co-immunoprecipitation and GST pull-down assays were performed to analyse protein(s) binding to TSP50. Moreover, the site-specific mutation of G6PD was used to reveal the key site critical for G6PD acetylation mediated by TSP50. Finally, the role of G6PD K171 acetylation regulated by TSP50 in cell proliferation and tumour formation was investigated. RESULTS: Our data suggest that the overexpression of TSP50 accelerates hepatocyte proliferation. In addition, G6PD is an important protein that binds to TSP50 in the cytoplasm. TSP50 activates G6PD activity by inhibiting the acetylation of G6PD at the K171 site. In addition, TSP50 promotes the binding of G6PD to SIRT2. Furthermore, the K171ac of G6PD regulated by TSP50 is required for TSP50-induced cell proliferation in vitro and tumour formation in vivo. Additionally, according to The Cancer Genome Atlas (TCGA) programme, TSP50 and G6PD are negatively correlated with the survival of HCC patients. CONCLUSIONS: Collectively, our findings demonstrate that TSP50-induced cell proliferation and tumour formation are mediated by G6PD K171 acetylation.