Colony-forming Capacity Research Articles

Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression.

Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

Mechanism of METTL14 and m6A modification of lncRNA MALAT1 in the proliferation of oral squamous cell carcinoma cells.

Methyltransferase-like 14 (METTL14) plays an epigenetic role in various cancer through N6-methyladenosine (m6A) modification. This study sought to analyze the mechanism of METTL14 in oral squamous cell carcinoma (OSCC) cell proliferation. Expression levels of METTL14, lncRNA metastasis associated with lung adenocarcinoma transcript 1 (lncRNA MALAT1), microRNA (miR)-224-5p, and histone lysine demethylase 2A (KDM2A) in OSCC tissues (N=40), and cell lines (FaDu, SCC-25, CAL-27, and SCC-15) were detected. Cell viability and colony formation capacity were assessed. m6A level, stability, and subcellular localization of lncRNA MALAT1 were determined. Nude mouse xenograft tumor assay was performed to confirm the role of METTL14 in vivo. METTL14 and lncRNA MALAT1 were upregulated, and miR-224-5p was downregulated in OSCC tissues and cells. Silencing METTL14 repressed OSCC cell viability and colony formation. Overexpression of MALAT1 and KDM2A or miR-224-5p downregulation reversed the inhibition of silencing METTL14 on OSCC cell proliferation. METTL14 induced m6A modification of MALAT1 to upregulate MALAT1. MALAT1 is comparatively bound to miR-224-5p to promote KDM2A transcription. In vivo, METTL14promoted tumor growth via regulating MALAT1/miR-224-5p/ KDM2A. Overall, our findings verified the therapeutic role of silencing METTL14 in OSCC treatment through the MALAT1/miR-224-5p/KDM2A axis.

Honokiol Induces Ferroptosis by Upregulating HMOX1 in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is one of the malignant hematological cancers with high mortality. Finding a more effective and readily available treatment is of the utmost importance. Here, we aimed to identify the anti-leukemia effect of a natural small molecule compound honokiol on a panel of AML cell lines, including THP-1, U-937, and SKM-1, and explored honokiol’s potential biological pathways and mechanisms. The results showed that honokiol decreased the viability of the targeted AML cells, induced their cell cycle arrest at G0/G1 phase, and inhibited their colony-formation capacity. Honokiol also triggers a noncanonical ferroptosis pathway in THP-1 and U-937 cells by upregulating the level of intracellular lipid peroxide and HMOX1 significantly. Subsequent studies verified that HMOX1 was a critical target in honokiol-induced ferroptosis. These results reveal that honokiol is an effective anti-leukemia agent in AML cell lines and may be a potential ferroptosis activator in AML.

Inhibition of PLK‐1 by using a Small Molecule Inhibitor HMN‐214 is a Novel Therapeutic Approach for High‐risk Neuroblastoma

Polo‐like kinase1 (PLK‐1), a member of the polo‐like kinase family of serine/threonine protein kinases, plays an essential role in regulating the cell cycle. PLK‐1 has a strong relationship with numerous regulatory events progressing during G2/M transition, chromosomal segregation, spindle assembly maturation, and mitotic exit. In addition, PLK‐1 also regulates DNA damage response, DNA replication, transcription, translation, chromosomes dynamics, and checkpoint adoption. Elevated levels of PLK‐1 have been observed in a plethora of cancers, including neuroblastoma (NB). NB is the most prevalent solid tumor that develops during the early embryonic stage and accounts for 15% of pediatric cancer‐related deaths. Current cytotoxic treatment strategies for NB are limiting and failed to prevent relapse and metastasis. In the present study, we hypothesized that inhibition of cell cycle regulator PLK‐1 by a small molecule inhibitor HMN‐214 will inhibit NB growth. To determine the role of PLK‐1 in NB, we analyzed multiple NB patient datasets and observed that PLK‐1 expression is inversely correlated with overall patient survival. PLK‐1 also strongly correlates with MYCN amplification and overall NB disease progression in our patient dataset analysis. Further, we used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines, to evaluate the anti‐proliferative effects of HMN‐214 in NB. Results showed that HMN‐214 significantly and in a dose‐dependent manner inhibits NB proliferation and colony formation capacity. Additionally, we found that HMN‐214 significantly induces apoptosis and blocks cell cycle progression at the G2‐M phase in treatment groups compared to controls. Our molecular analysis further revealed that HMN‐214 significantly inhibits the mRNA and protein levels of PLK‐1 and CDK1 in contrast to control treatments. Further, we developed a 3D spheroid tumor model for NB to mimic in vivo tumor growth and utilized it to determine the effects of HMN‐214 on NB tumor growth. Results showed that HMN‐214 significantly and in a dose‐dependent manner inhibits NB 3D spheroidal tumor growth by specifically inducing tumor cell death. Overall, our data highlights the role of PLK‐1 in the oncogenic progression of NB and showed the efficacy of HMN‐214 in inhibiting NB growth. Our future efforts will be directed towards elucidating the role of PLK‐1 in NB and developing effective therapeutic strategies incorporating PLK‐1 inhibitor.

Direct targeting of epigenetic regulator Menin inhibits neuroblastoma growth

Histone modifiers such as methyltransferases and demethylases have recently emerged as an attractive target to treat different cancers. Menin is a tumor suppressor protein and an essential oncogenic co‐factor of MLL1 COMPASS complex. Menin is a highly specific binding partner of the N‐terminus of MLL1 complex that have H3K4 methyltransferase activity. Menin‐MLL1 as an epigenetic regulator regulates the expression of target oncogenes, including HOXA, MEIS1, and AF9. Blocking menin‐MLL1 interaction destabilizes the COMPASS complex and hinders the oncogenic potential of cancer cells and cancer stem cells (CSCs). Neuroblastoma (NB) is an aggressive pediatric solid tumor develops during the early embryonic stage from extracranial sympathetic nervous system, and accounts for 10‐15% of all pediatric cancer‐related deaths. In the present study, we hypothesized that disrupting menin‐MLL1 interaction by using a specific small molecule inhibitor could disrupt COMPASS complex and overall inhibit NB growth, metastasis, and relapse. In the present study, we analyzed 1235 NB patients from different NB patient datasets and found that the upregulation of menin inversely correlate with overall survival of NB patients. We used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines and performed cytotoxic assays. Results showed that inhibition of menin‐MLL1 interaction by MI‐503 significantly inhibits NB proliferation and colony formation capacity in contract to control treatments. Furthermore, MI‐503 significantly and in a dose‐dependent manner blocked NB cell cycle progression at S phase and enhanced apoptosis in treatment groups compared to controls. Additionally, we developed a 3D spheroidal tumor model for NB to mimic in vivo tumor conditions and tested the ability of MI‐503 in inhibiting tumor growth. Results showed significant inhibition of spheroidal tumor growth by enhancing tumor cell death in response to MI‐503 treatment. To further determine the effect of MI‐503 on inhibition of H3K4me3 levels, a key function of menin‐MLL1 COMPASS complex, we performed Western blot assays and observed a significant and dose dependent inhibition of H3K4me3 levels in response to MI‐503 treatment. Overall, our study highlights the role of menin‐MLL1 interaction in NB. Inhibition of menin‐MLL1 interaction by a specific small‐molecule inhibitor MI‐503 leads to the inhibition of epigenetic regulator functions and inhibits NB growth. Our future efforts will focus on further elucidating the role of menin in NB and to develop effective therapeutic strategies incorporating epigenetic inhibitors for NB patients.

CD106/VCAM-1 distinguishes a fibroblast subpopulation with high colony-forming capacity and distinct protein expression from the uterosacral ligament.

BackgroundPelvic organ prolapse (POP) is a common degenerative disease in women which may diminish quality of life. Investigating the pathological changes of the uterosacral ligament, including the functional changes of fibroblasts, is critical to understanding the pathophysiology of POP. This study was designed to isolate CD106-positive (CD106+) fibroblasts from the human uterosacral ligament and assess the function and expression of this subpopulation.MethodsWe separated CD106+ fibroblasts and CD106 negative (CD106−) fibroblasts by fluorescence-activated cell sorting (FACS) and cultured them for subsequent experiments. Flow cytometric analysis was used to test the sorting efficiency, CD106 expression, and typical mesenchymal stem cell (MSC) phenotype marker expression. A colony-forming unit (CFU) assay was applied to evaluate the colony-forming ability of the fibroblasts. Trilineage differentiation capacities were assessed after in vitro induction. The protein levels of vimentin, fibroblast specific protein-1 (FSP-1), collagen I (COL 1), matrix metallopeptidase-1 (MMP-1), and α-smooth muscle actin (α-SMA) were detected by western blot analysis. The expression of CD106 was verified by flow cytometric analysis and immunohistochemistry (IHC) in the POP and non-POP groups.ResultsThe CD106+ fibroblasts were isolated with a purity of (93.50±3.91)%. The CD106+ fibroblasts exhibited higher colony-forming capacity than that of CD106− fibroblasts, but neither of them showed adipogenic or osteogenic differentiation similar to that of MSCs. The protein levels of MMP-1 and α-SMA were lower, and the level of COL 1 was higher in the CD106+ fibroblasts than in the CD106− fibroblasts. In addition, we observed a decreased expression of CD106 in the POP group compared with the non-POP group.ConclusionsOur results suggest that CD106+ fibroblasts possess a high colony-forming capacity and distinct protein expression, and this subpopulation is reduced in POP.

Effects of interferon regulatory factor 9 on the biological phenotypes in PML-RARα-induced promyelocytic leukemia

目的研究急性早幼粒细胞白血病（APL）中干扰素调节因子9（IRF9）的表达特征、预后意义和生物学功能，探索IRF9作为潜在治疗靶点的临床转化意义。方法挖掘TCGA公共数据分析IRF9在APL中的表达水平及其表达高低对患者生存的影响；利用Dox诱导的慢病毒载体系统构建可诱导表达PML-RARα（PR）融合基因的U937细胞系，诱导PR融合基因早期表达并加入全反式维甲酸（ATRA）检测IRF9表达变化；构建可诱导表达IRF9的NB4细胞系，进行细胞分化和集落形成实验，初步探讨IRF9诱导表达对NB4白血病细胞生物学功能的影响。结果①TCGA数据库分析显示，在各种类型的AML中，IRF9在APL中表达水平最低且与预后不良相关。②成功构建可诱导表达PR融合基因的U937细胞系；PR表达引起IRF9蛋白水平下调，在NB4细胞系中IRF9表达缺失，ATRA处理后可表达上调。③成功构建可诱导表达IRF9的NB4细胞系，IRF9诱导表达促进NB4细胞的分化，且与较低剂量ATRA有协同促分化作用；IRF9诱导表达显著抑制了NB4细胞的集落形成能力。结论IRF9在APL中低表达且与预后不良相关，提示存在特异性调控机制；上调IRF9表达可发挥抗白血病效应，为其成为临床潜在治疗靶点奠定生物学基础。

Targeting the NLRP3 inflammasome in Rare hereditary blood disorder Fanconi Anemia

Abstract Fanconi anemia (FA) patients are at higher risk of developing bone marrow failure (BMF) or cancer upon exposure to potential threats like viral infections, bacterial infections, carcinogens, and mutagens. Deleterious mutations in FA genes cause multiple cellular defects which include impaired ability to repair DNA interstrand crosslink lesions in the nucleus and impaired removal of damaged mitochondria via mitophagy in the cytoplasm. Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to lipopolysaccharide-induced septic shock as a result of hematopoietic suppression. Considering the role of inflammation in pathogenesis of Fanconi anemia and bone marrow failure diseases in general, we asked whether inflammasome inhibition could be a promising therapeutic for the treatment of FA. Our studies demonstrate that MCC950, a small molecule inhibitor of the NLRP3 inflammasome, can protect Fancc null mice from LPS-induced hemopoietic suppression and septic shock as evidenced by increased survival rate of Fancc−/− mice. MCC950 partially rescues the thrombocytopenia in peripheral blood and partially restores the loss of megakaryocyte-erythroid progenitor (MEP) and granulocyte-macrophage progenitor (GMP) pools upon LPS challenge. Further, Treatment with MCC950 significantly improved the bone marrow colony-forming capacity of Fancc−/− bone marrow cells in vitro. Our studies demonstrate that MCC950, a small molecule inhibitor of the NLRP3 inflammasome, can protect Fancc null mice from LPS-induced hemopoietic suppression and septic shock and warrants further evaluation as a potential therapeutic strategy in FA.

MiR-133b regulates the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.

Bladder cancer is one of the most common urothelial tumors with high incidence and mortality rates. Although it has been reported that microRNA (miR)-133b can regulate tumorigenesis of bladder cancer, the mechanism remains unclear. Sex-determining region Y-box transcription factor 4 (SOX4) exhibits an important role in tumorigenesis, but it is unclear whether SOX4 and miR-133b are associated with regulation of pathogenesis of bladder cancer. This study aims to determine the expressions of SOX4 and miR-133b in bladder cancer tissues and cells, investigate their effects on the proliferation, colony formation, and invasion of bladder cancer cells, and to explore the association between miR-133b and SOX4 in regulating biological featurss of bladder cancer cells. The bladder cancer and adjacent tissue samples of 10 patients who underwent surgical resection in the Second Xiangya Hospital of Central South Universty from Januray to June 2015 were obtained. The levels of miR-133b were tested by real-time PCR, and the protein levels of SOX4 were evaluated using Western blotting in bladder cancer tissues, matched adjacent tissues, and cell lines. The correlation between miR-133b expression and SOX4 expression in bladder cancer tissues was analyzed. Using the online database TargetScan, the relationship between SOX4 and miR-133b was predicted. MiR-133b mimics, miR-133b inhibitor, and short hairpin RNA (shRNA)-SOX4 were transfected into T24 cells by Lipofectamine 2000. The relationship between miR-133b and SOX4 was also verified by a dual-luciferase reporter assay. The proliferation of T24 cells cultured for 0, 12, 48, 72, and 96 h was evaluated by cell counting kit-8 (CCK-8) assay. The colony formation capacity of bladder cancer cells was tested after 14-day culture, and cell invasion capacity was evaluated with Transwell invasion assay. Bladder cancer tissue and bladder cancer cells had low level of miR-133b but high level of SOX4, compared with matched adjacent tissues and normal bladder epithelial cells. A negative correlation between miR-133b mRNA and SOX4 protein levels in bladder cancer tissues was also found (r=-0.84). The results of online database TargetScan showed that miR-133b targets at SOX4, and overexpression of miR-133b significantly attenuated the expression of SOX4 in T24 cells. Both overexpression of miR-133b and knockdown of SOX4 significantly inhibited the proliferation, colony formation, and invasion capacity of bladder cancer cells in vitro. SOX4 down-regulation restored the effects of miR-133b inhibitor on the proliferation, colony formation, and invasion capacity of T24 cells. The up-regulation of SOX4 contributes to the progression of bladder cancer, and miR-133b can regulate the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.

A Single-Cell Raman Spectroscopy Analysis of Bone Marrow Mesenchymal Stem/Stromal Cells to Identify Inter-Individual Diversity.

The heterogeneity of stem cells represents the main challenge in regenerative medicine development. This issue is particularly pronounced when it comes to the use of primary mesenchymal stem/stromal cells (MSCs) due to a lack of identification markers. Considering the need for additional approaches in MSCs characterization, we applied Raman spectroscopy to investigate inter-individual differences between bone marrow MSCs (BM-MSCs). Based on standard biological tests, BM-MSCs of analyzed donors fulfill all conditions for their characterization, while no donor-related specifics were observed in terms of BM-MSCs morphology, phenotype, multilineage differentiation potential, colony-forming capacity, expression of pluripotency-associated markers or proliferative capacity. However, examination of BM-MSCs at a single-cell level by Raman spectroscopy revealed that despite similar biochemical background, fine differences in the Raman spectra of BM-MSCs of each donor can be detected. After extensive principal component analysis (PCA) of Raman spectra, our study revealed the possibility of this method to diversify BM-MSCs populations, whereby the grouping of cell populations was most prominent when cell populations were analyzed in pairs. These results indicate that Raman spectroscopy, as a label-free assay, could have a huge potential in understanding stem cell heterogeneity and sorting cell populations with a similar biochemical background that can be significant for the development of personalized therapy approaches.

Lactate Induces the Expressions of MCT1 and HCAR1 to Promote Tumor Growth and Progression in Glioblastoma.

The tumor microenvironment (TME) plays a pivotal role in establishing malignancy, and it is associated with high glycolytic metabolism and lactate release through monocarboxylate transporters (MCTs). Several lines of evidence suggest that lactate also serves as a signaling molecule through its receptor hydroxycarboxylic acid receptor 1 (HCAR1/GPR81), thus functioning as a paracrine and autocrine signaling molecule. The aim of the present study was to investigate the role of lactate in glioblastoma (GBM) progression and metabolic reprogramming in an in vitro and in vivo model. The cell proliferation, migration, and clonogenicity were tested in vitro in three different human GBM cell lines. The expressions of MCT1, MCT4, and HCAR1 were evaluated both in vitro and in a zebrafish GBM model. The results were further validated in patient-derived GBM biopsies. Our results showed that lactate significantly increased the cell proliferation, migration, and colony formation capacity of GBM cells, both in vitro and in vivo. We also showed that lactate increased the expressions of MCT1 and HCAR1. Moreover, lactate modulated the epithelial–mesenchymal transition protein markers E-cadherin and β-catenin. Interestingly, lactate induced mitochondrial mass and the OXPHOS gene, suggesting improved mitochondrial fitness. Similar effects were observed after treatment with 3,5-dihydroxybenzoic acid, a known agonist of HCAR1. Consistently, the GBM zebrafish model exhibited an altered metabolism and increased expressions of MCT1 and HCAR1, leading to high levels of extracellular lactate and, thus, supporting tumor cell proliferation. Our data from human GBM biopsies also showed that, in high proliferative GBM biopsies, Ki67-positive cells expressed significantly higher levels of MCT1 compared to low proliferative GBM cells. In conclusion, our data suggest that lactate and its transporter and receptor play a major role in GBM proliferation and migration, thus representing a potential target for new therapeutic strategies to counteract tumor progression and recurrence.

Effect of cucurbitacin on malignant biological behavior of breast cancer cells, and its possible underlying mechanism

Purpose: To study the influence of cucurbitacin on malignant biological behavior of mammary carcinoma cells, and the likely mechanism involved.Methods: Human mammary carcinoma cell line MDA-MB-436 was selected for cell culture and treated with different concentrations of cucurbitacin. The effect of cucurbitacin on cell activity, cell colonyformation capacity, cell invasion, migration potential, matrix metalloproteinase-9 (MMP-9) activity, and levels of vascular endothelial growth factor A (VEGFA), epithelial calcium adhesion (E-cadherin), and neurogenic calcium adhesion (N-cadherin) were measured. Moreover, levels of wave protein (vimentin), phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated signaling transduction, and transcription activation factor 3 (p-STAT3) and phosphorylated protein kinase B (p-Akt) were determined.Results: With increase in cucurbitacin dose, there was significant decrease in cell viability, cell colony ratio, cell invasion and migration capacity, and expression levels of MMP-9, VEGFA, e-cadherin, ncadherin, vimentin, P-EGFR, P-STAT3 and p-Akt (p < 0.05).Conclusion: Cucurbitacin inhibits the proliferation, invasion, and migration of breast cancer cells by down-regulating the expressions of EGFR/STAT3/Akt signaling-related proteins, and inhibiting epithelial-mesenchymal transition transformation.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG—a human glioblastoma-derived cell lineage—reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/β-catenin signaling pathways, in addition to upregulation of genes related to epithelial–mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

Fatty acid oxidation enzyme Δ3, Δ2-enoyl-CoA isomerase 1 (ECI1) drives aggressive tumor phenotype and predicts poor clinical outcome in prostate cancer patients.

Prostate cancer (PCa) metastases are highly enriched with genomic alterations including a gain at the 16p13.3 locus, recently shown to be associated with disease progression and poor clinical outcome. ECI1, residing at the 16p13.3 gain region, encodes Δ3, Δ2-Enoyl-CoA Delta Isomerase 1 (ECI1), a key mitochondrial fatty acid β-oxidation enzyme. Although deregulated mitochondrial fatty acid β-oxidation is known to drive PCa pathogenesis, the role of ECI1 in PCa is still unknown. We investigated the impacts of ECI1 on PCa phenotype in vitro and in vivo by modulating its expression in cell lines and assessed the clinical implications of its expression in human prostate tissue samples. In vitro, ECI1 overexpression increased PCa cell growth while ECI1 deficiency reduced its growth. ECI1 also enhanced colony formation, cell motility, and maximal mitochondrial respiratory capacity. In vivo, PCa cells stably overexpressing ECI1 injected orthotopically in nude mice formed larger prostate tumors with higher number of metastases. Immunohistochemistry analysis of the human tissue microarray representing 332 radical prostatectomy cases revealed a stronger ECI1 staining in prostate tumors compared to corresponding benign tissues. ECI1 expression varied amongst tumors and was higher in cases with 16p13.3 gain, high Gleason grade, and advanced tumor stage. ECI1 overexpression was a strong independent predictor of biochemical recurrence after adjusting for known clinicopathologic parameters (hazard ratio: 3.65, P < 0.001) or the established CAPRA-S score (hazard ratio: 3.95, P < 0.001). ECI1 overexpression was also associated with significant increased risk of distant metastasis and reduced overall survival. Overall, this study demonstrates the functional capacity of ECI1 in PCa progression and highlights the clinical implication of ECI1 as a potential target for the management of PCa.

MiR-3150a-3p, miR-6883-3p and miR-627-5p participate in the phycocyanin-mediated growth diminishment of A549 cells, via regulating a common target toll/interleukin 1 receptor domain-containing adaptor protein

• Phycocyanin diminishes the viability of A549 cells. • Phycocyanin significantly promotes expressions of miR-3150a-3p, miR-6883-3p and miR-627-5p. • TIRAP is a shared directed target of miR-3150a-3p, miR-6883-3p and miR-627-5p. • Knockdown TIRAP expression resulted in similar cell phenotype as miRNAs overexpression. • Overexpression TIRAP restores the diminished A549 viability caused by miRNAs and phycocyanin. As a type of functional food additives, phycocyanin is well-known for its extraordinary antineoplastic activity in non-small cell lung cancer (NSCLC). Previously, a high-throughput miRNA sequencing was employed to excavate miRNAs altered by phycocyanin in A549 cells, while the in-depth regulatory mechanism was inadequate. In this work, three significant up-regulated miRNAs in phycocyanin-treated cells are identified for the first time, including miR-3150a-3p, miR-6883-3p and miR-627-5p. Overexpression of miRNAs obviously diminished the cell growth and colony formation capacity, respectively. Dual-luciferase reporter assay verified that toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) was a direct shared target of three miRNAs. Knockdown the expression of TIRAP resulted in similar cell growth inhibition phenotype as miRNAs overexpression. Strikingly, overexpression of TIRAP could significantly restore the diminished cell viability caused by miRNAs up-regulation, as well as phycocyanin treatment. Consequently, this study proposes that phycocyanin plays an inhibitory effect of NSCLC cells via a miR-3150a-3p/miR-6883-3p/miR-627-5p-TIRAP axis.

Polymer-Stabilized Elemental Boron Nanoparticles for Boron Neutron Capture Therapy: Initial Irradiation Experiments.

Sufficient boron-10 isotope (10B) accumulation by tumor cells is one of the main requirements for successful boron neutron capture therapy (BNCT). The inability of the clinically registered 10B-containing borophenylalanine (BPA) to maintain a high boron tumor concentration during neutron irradiation after a single injection has been partially solved by its continuous infusion; however, its lack of persistence has driven the development of new compounds that overcome the imperfections of BPA. We propose using elemental boron nanoparticles (eBNPs) synthesized by cascade ultrasonic dispersion and destruction of elemental boron microparticles and stabilized with hydroxyethylcellulose (HEC) as a core component of a novel boron drug for BNCT. These HEC particles are stable in aqueous media and show no apparent influence on U251, U87, and T98G human glioma cell proliferation without neutron beam irradiation. In BNCT experiments, cells incubated with eBNPs or BPA at an equivalent concentration of 40 µg 10B/mL for 24 h or control cells without boron were irradiated at an accelerator-based neutron source with a total fluence of thermal and epithermal neutrons of 2.685, 5.370, or 8.055 × 1012/cm2. The eBNPs significantly reduced colony-forming capacity in all studied cells during BNCT compared to BPA, verified by cell-survival curves fit to the linear-quadratic model and calculated radiobiological parameters, though the effect of both compounds differed depending on the cell line. The results of our study warrant further tumor targeting-oriented modifications of synthesized nanoparticles and subsequent in vivo BNCT experiments.

Age-dependent effects of Igf2bp2 on gene regulation, function, and aging of hematopoietic stem cells in mice

AbstractIncreasing evidence links metabolism, protein synthesis, and growth signaling to impairments in the function of hematopoietic stem and progenitor cells (HSPCs) during aging. The Lin28b/Hmga2 pathway controls tissue development, and the postnatal downregulation of this pathway limits the self-renewal of adult vs fetal hematopoietic stem cells (HSCs). Igf2bp2 is an RNA binding protein downstream of Lin28b/Hmga2, which regulates messenger RNA stability and translation. The role of Igf2bp2 in HSC aging is unknown. In this study, an analysis of wild-type and Igf2bp2 knockout mice showed that Igf2bp2 regulates oxidative metabolism in HSPCs and the expression of metabolism, protein synthesis, and stemness-related genes in HSCs of young mice. Interestingly, Igf2bp2 expression and function strongly declined in aging HSCs. In young mice, Igf2bp2 deletion mimicked aging-related changes in HSCs, including changes in Igf2bp2 target gene expression and impairment of colony formation and repopulation capacity. In aged mice, Igf2bp2 gene status had no effect on these parameters in HSCs. Unexpectedly, Igf2bp2-deficient mice exhibited an amelioration of the aging-associated increase in HSCs and myeloid-skewed differentiation. The results suggest that Igf2bp2 controls mitochondrial metabolism, protein synthesis, growth, and stemness of young HSCs, which is necessary for full HSC function during young adult age. However, Igf2bp2 gene function is lost during aging, and it appears to contribute to HSC aging in 2 ways: the aging-related loss of Igf2bp2 gene function impairs the growth and repopulation capacity of aging HSCs, and the activity of Igf2bp2 at a young age contributes to aging-associated HSC expansion and myeloid skewing.

Polystyrene micro-/nanoplastics induced hematopoietic damages via the crosstalk of gut microbiota, metabolites, and cytokines.

Micro-/nanoplastics (MNPLs), novel environmental pollutants, widely exist in the environment and life and bring health risks. Previous studies have shown that NMPLs can penetrate bone marrow, but whether they cause hematopoietic damage remains uncertain. In this study, C57BL/6J mice were treated with polystyrene MNPLs (PS-MNPLs, 10μm, 5μm and 80nm) at 60μg doses for 42days by intragastric administration. We evaluated the hematopoietic toxicity induced by MNPLs and potential mechanisms via combining 16S rRNA, metabolomics, and cytokine chips. The results demonstrated that PS-MNPLs induced hematopoietic toxicity, which was manifested by the disorder of bone marrow cell arrangement, the reduction in colony-forming, self-renewal and differentiation capacity, and the increased proportion of lymphocytes. PS-MNPLs also disrupted the homeostasis of the gut microbiota, metabolism, and inflammation, all of which were correlated with hematotoxicity, suggesting that abnormal gut microbiota-metabolite-cytokine axes might be the crucial pathways in MNPLs-induced hematopoietic injury. In conclusion, our study systematically demonstrated that multi-scale PS-MNPLs induced hematopoietic toxicity via the crosstalk of gut microbiota, metabolites, and cytokines and provided valuable insights into MNPLs toxicity, which was conducive to health risk assessment and informed policy decisions regarding PS-MNPLs.

Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

Immortalization-upregulated protein (IMUP) plays a vital role in cell proliferation and tumor progression. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we select IMUP as an alternative gene based on GeneChip analysis of clinical PDAC tissues and transcriptome data from The Cancer Genome Atlas. IMUP expression is upregulated in PDAC tumor tissues. Moreover, high IMUP expression correlates with poor prognosis, while IMUP depletion inhibits PDAC cell proliferation and colony formation capacity in vitro, and decreases xenograft tumor growth in vivo. IMUP downregulation leads to cell-cycle arrest in the S phase. IMUP knockdown increases the expression of four-and-a-half LIM domain protein 1 (FHL1), which regulates the phosphorylation of cell division cycle 25A (CDC25A) by cycle checkpoint kinase 1 (CHK1) and promotes cytoplasmic distribution of CDC25A by interaction with 14–3-3ξ. Furthermore, FHL1 knockdown restores the effects induced by IMUP depletion. Liquid chromatography tandem mass spectrometry and immunoprecipitation analysis further show that IMUP interacts directly with nucleophosmin (NPM1) and enhances its stability. DNA methylation sequencing shows that FHL1 promoter methylation decreases when IMUP is downregulated. Overexpression of NPM1 can increase the methylation level of FHL1, thereby decreasing its expression. Our study provides a novel perspective on IMUP/NPM1/FHL1-mediated cell-cycle arrest by regulating CDC25A phosphorylation in PDAC. These findings may provide a new therapeutic target for PDAC.Graphical abstract

Silencing of TP73-AS1 impairs prostate cancer cell proliferation and induces apoptosis via regulation of TP73.

Prostate cancer is a malignant disease that severely affects thehealth and comfort of themale population. The long non-coding RNA TP73-AS1 has been shown to be involved in the malignant transformation of various human cancers. However, whether TP73-AS1 contributes toprostate cancer progression has not been reported yet. Accordingly, here weaimed to report the role of TP73-AS1 in the development and progression of prostate cancer and determine its relationship with TP73. TP73-AS1-specific siRNA oligo duplexes were used to silence TP73-AS1 in DU-145 and PC-3 cells. Results indicated that TP73-AS1 was upregulated whereas TP73 was downregulated in prostate cancer cells compared to normal prostate cells and there was a negative correlation between them. Besides, loss of function experiments of TP73-AS1 in prostate cancer cells strongly induced cellular apoptosis, interfered with the cell cycle progression, and modulated related pro- and anti-apoptotic gene expression. Colony formation and migration capacities of TP73-AS1-silenced prostate cancer cells were also found to be dramatically reduced. Our findings provide novel evidence that suggests a chief regulatory role for the TP73-TP73-AS1 axis in prostate cancer development and progression, suggesting that the TP73/TP73-AS1 axis can be a promising diagnostic and therapeutic target for prostate cancer.