Experimental Cell Research

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

Fibroblast growth factor 2 (FGF2) is known to play a role in skeletal muscle development and growth. We examined two populations of myogenic precursor cells for their responses to FGF2 in vitro using both extraocular and limb skeletal muscle. Fluorescence-activated cell sorting (FACS) was used to isolate two different populations of myogenic precursor cells, the EECD34 cells [positive for CD34, and negative for Sca1, CD31, and CD45] and PAX7-positive cells, from tibialis anterior and extraocular muscles of mice. These cells were cultured and treated with either proliferation or differentiation media in the absence or the presence of FGF2, followed by assays to determine its effects on proliferation and differentiation. These cells were also assessed for expression of fibroblast growth factor receptor (FGFR) 1, FGFR2, and FGFR4. Both the EECD34 cells and the PAX7-positive cells responded to FGF2 with significantly increased proliferation. Both myogenic precursor cell populations showed increased differentiation in the presence of FGF2, but also showed decreased rates of fusion into multinucleated myotubes in this in vitro system relative to control cells. FGF2 has pleiotropic effects on skeletal muscles. Contrary to the literature, FGF2 did not inhibit differentiation, but did appear to decrease fusion into multinucleated myofibers in vitro. These results provide a potential mechanism for reduction in myofiber number and size in the extraocular muscles in individuals with Apert syndrome, where FGF receptor 2 mutations maintain the receptor in an activated state.

Mitochondria are the key generators of ATP in a cell. Visually, they are highly dynamic organelles that undergo cellular fission and fusion events in response to changing cellular energy requirements. Mitochondria are now emerging as regulators of mammalian cell motility. Here we show that mitochondria infiltrate the leading edge of NIH3T3 fibroblasts during migration. At the leading edge, we find that mitochondria move to and tether to Focal Adhesions (FA). FA regulate cell migration by coupling the cytoskeleton to the Extracellular Matrix through integrin receptors. Importantly, we find that inhibition of mitochondrial ATP generation concomitantly inhibits FA size. This suggests that mitochondrial energy production regulates migration through FA control.

Host genetics is known to influence cancer susceptibility. However, the specific candidate genes and molecular mechanisms that confer resistance remain poorly understood. Here, we demonstrate the power of haploinsufficiency screen to uncover host genetic regulators of cancer in Drosophila and identify the long noncoding RNA (lncRNA) hsr{omega}, a structural component of nuclear biomolecular condensate known as omega speckles, as a key host cancer susceptibility locus. Loss of hsr{omega} disrupts proteostasis and cell fitness, while its haploinsufficiency accelerates epithelial tumor progression driven by loss of the Lethal giant larvae (Lgl) tumor suppressor. Further validating the breadth of this screening strategy, we independently identified Drosophila STING (innate immunity) and Keap1 (oxidative stress defense) as genetic modifiers of cancer. Moreover, in humans, copy number variations (CNV) in these genes and Sat III (a functional human homolog of hsr{omega}) correlate with poor cancer prognosis, thereby revealing conserved stress pathways as potential host genetic susceptibility regulators.

Cellular architecture depends on keratin intermediate filament as a fundamental component, which offers essential mechanical support to fight environmental stresses. Our previous research demonstrated that keratin fusion variants increase tumor aggressiveness through enhanced cancer stemness in oral squamous cell carcinoma. The proper functioning of keratins plays an essential role in maintaining cell structure while determining cell fate. The present study demonstrates that keratin fusion variant drives genomic instability through cytokinesis defects, which results in the formation of polyaneuploid cancer cells (PACC). The cells expressing keratin fusion show elevated DNA damage repair gene expression, which serves as a key factor for mitotic slippage during cancer development. The PACCs generated by keratin fusion make cancer cells resistant to cisplatin treatment while simultaneously reducing {gamma}-H2AX induction and increasing survival rates. The Gene Set Enrichment Analysis results showed increased "regulation of actin cytoskeleton" activity in keratin fusion-expressing cells correlated with elevated actin filament networks and increased cell motility in these cells. In summary, the keratin fusion variant enhances cancer aggressiveness through three mechanisms: it creates genomic instability that leads to PACC formation and enables cancer cells to evade cGAS/STING-mediated death signals and modifies cytoskeleton structures, which results in drug resistance and metastasis.

Immune checkpoint blockade has revolutionized cancer therapy, but the therapeutic efficacy is limited. Clinical trials on blockade of newly identified immune checkpoints didn't show promising result, suggesting that it might be insufficient to understand the function of immune checkpoints in cancer merely in the context of immunity. Here, we found mutually exclusive expression patterns of the immune checkpoint VISTA (or VSIR) and the neural stemness factor SETDB1, an oncoprotein that promotes immunoevasion, in xenograft tumors, suggesting that cells with high VISTA expression represents a differentiated, and hence, less or non-malignant state in tumor. Non-neural differentiation factors HHEX, MYOD1 and PPARG promote, whereas oncoproteins KRAS (and the mutant KRAS(G12D)) and SOX2, both being embryonic neural factors, repress VISTA expression. This tendency can be inferred from the finding that neural stemness is the core property of cancer cell. Manipulated expression of VISTA in cancer cells generated no significant effect on cell tumorigenicity and differentiation state, but led to change in cell morphology and actin cytoskeleton. Mechanistically, VISTA regulates a key cytoskeleton regulator, WASF2, leading to the change in cell morphology, which might interfere with signal transduction of immune response. The results suggest that 1) high expression of a protein in tumor might represent a less or non-malignant state, targeting of which would leave malignant cells intact, and consequently, leading to weak or even no therapeutic efficacy, a key factor worth considering for target selection; 2) immune checkpoints might play other roles in cells that interfere with regulation of anti-tumor immunity.

Cancer stem cells (CSCs) are amongst the group of cells constituting tumors, being characterized by their strong self-renewal and survival properties. Cancer cells, CSCs included, are thought to rely mostly on glycolysis, even in the presence of oxygen, which confers them adaptative advantages. Adenine nucleotide translocator 2 (ANT2), responsible for the exchange of ADP and ATP in the mitochondrial inner membrane, has been correlated with a higher glycolytic metabolism and is known to be overexpressed in cancer cells. Using P19 embryonal carcinoma stem cells (P19SCs) as a CSCs model, we inhibit ANT2 translation by using siRNA. ANT2 protein levels were shown to be overexpressed in P19SC when compared to their differentiated counterparts. Furthermore, we showed here that the OXPHOS machinery and mitochondrial membrane potential are compromised after ANT2 depletion, exhibiting a metabolic adaptation towards a less oxidative phenotype. Interestingly, hexokinase II levels were downregulated, which was also accompanied by decreased cell growth, and decreased ability to form spheroids. Our findings underscore ANT2 as a key regulator of metabolic remodeling and cell survival of CSCs, suggesting its potential as a therapeutic target for controlling CSC-driven tumor progression. HighlightsO_LIANT2 silencing promotes cell growth arrest and metabolic remodeling in CSCs. C_LIO_LIANT2 depletion modulates HKII protein levels. C_LIO_LIANT2 induce anoikis resistance in P19SCs C_LI

To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate in this paper that cells migrating in 3D collagen use CSE to cover the developing protrusions. After retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. For the coordinated process of CSE storage and release, all cells should have specific mechanisms of regulation, and we hypothesize that microtubules (MT) play an important role in this mechanism. We show here that different effects of MT depolymerization on cell motility such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by the essential role of MT in CSE regulation and dynamics.

Phospholipase C{beta}1 is activated by Gq to generate calcium signals in response to hormones and neurotransmitters, and is found at high levels in mammalian neuronal tissue. Besides carrying out this key plasma membrane function, PLC{beta}1 has a cytosolic population that helps, in part, to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLC{beta}1 or reducing its cytosolic population by activating Gq to drive it to the plasma membrane, returns differentiated PC12 cells to an undifferentiated state. In this state, the cells return to a spherical morphology, resume proliferation and express the stem cell transcription factors nanog and Oct4. Similar changes are seen with C3PO down-regulation. This return to a stem-like state is accompanied by shifts in multiple miR populations, such as increased levels of rno-miR-21 and rno-miR-26a. Surprisingly, we find that de-differentiation can also be induced by extended stimulation of the Gq. In this case, the neurites completely retract over a 10-minute period, and while levels of nanog remain unchanged, the levels of some miRs begin to return to their undifferentiated values. In complementary studies, we followed the real time hydrolysis of a fluorescent-tagged miR in cells where PLC{beta}1 or C3PO were down-regulated. These samples showed substantial differences in miR processing in cells both the undifferentiated and differentiated states. Taken together, our studies suggest that PLC{beta}1, through its ability to regulate C3PO and endogenous miR populations, plays a key role in mediating PC12 cell differentiation.

Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not defined completely. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth and metastasis in mouse melanoma models. Finally, analysis in human melanoma samples showed that tumor cells with high levels of EMILIN-1 are reduced in metastatic lesions compared to primary tumors or nevi. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.

Rho-GTPase-activating proteins (Rho-GAPs) are essential upstream regulators of the Rho family of GTPases. Currently, it remains unclear if the phenotypic change caused by perturbations to a Rho-GAP is predictable from its structural sequence. Here we analyze the relationship between the morphological response of cells to the silencing of Rho-GAPs and their primary structure. For all possible pairs of 57 different Rho-GAPs expressed in MCF10A epithelial cells, the similarity in the Rho-GAP silencing-induced morphological change was quantified and compared to the similarity in the primary structure of the corresponding pairs. We found a distinct correlation between the morphological and structural similarities in a specific group of RhoA-targeting Rho-GAPs. Thus, the family-wide analysis revealed a common feature shared by the specific Rho-GAPs.

Nuclear deformability plays a critical role in cell migration. During this process, the remodeling of internal components of the nucleus has a direct impact on DNA damage and cell behavior; however, how persistent migration promotes nuclear changes leading to phenotypical and functional consequences remains poorly understood. Here, we described that the persistent migration through physical barriers was sufficient to promote permanent modifications in migratory-altered cells. We found that lamin B1 altered its localization, concomitant with morphological and transcriptional changes. Migratory-altered cells showed alterations in cellular functions such as DNA repair and cell migration. We applied biochemical and biophysical approaches to identify that confined conditions altered the biomechanical response of the nucleus. Mechanistically, we determined that actin dynamics controlled the redistribution of lamin, and the basal levels of DNA damage in migratory-altered cells. Our observations reveal a novel role for confined cell conditions in consistent nuclear and genomic alterations that might handle the genetic instability and cellular heterogeneity in aging diseases and cancer. HighlightsO_LIPersistent confined migration promotes permanent mophological changes. C_LIO_LILamin B1 is redistribution in the nucleus of migratory altered cells. C_LIO_LIMigratory-altered cells exhibit transcriptional and functional changes related to cell migration and survival. C_LIO_LIActin polymerization controls nuclear changes induced by cell migration. C_LI

BRD4 (Bromodomain containing protein 4) is a chromatin reader binds to acetylated lysine residues on histones interacting with RNA Pol II, p-TFeb. PDGF-BB was presented here, in soft-tissue tumor, as an oncogenic factor driving cell proliferation, and aberrant BRD4 knockdown significantly reduced tumor aggressiveness and unfavorable prognosis in soft-tissue tumors. To identify suppressive key drivers impeding demoid tumor growth, shRNA drop-out screen analysis identified signature of "transcription from RNA polymerase II promoter" including DDX, Stat3, SMARCA, ATM, SIRT1, cMyc that were recruited with BRD4 interation in activating {beta}-catenin, which is a major key driver mutated in soft-tissue tumor, and its depletion ceased soft-tissue tumor cell growth. Sepcifically, BRD4 mediated PDGF-BB signaling in GSK stimulation through transcriptional regulation from RNA polymerase II activity with PI3K as target, and thus not only canonical {beta} -catenin/TCF4 signaling, but also non-canonical {beta} -catenin conjunction complex response was activated by BRD4 in nucleus involved in promoting cell proliferation. Our study delineated a signaling axis that may allow soft-tissue tumor cells to escape apoptosis during colonization by activating PDGFBB-BRD4-GSK-{beta} -catenin and non-canonical-{beta}-catenin pathway through BRD4 in cancer cells. An efficient treatment for soft-tissue tumors could be accomplished by targeting PDGF and BRD4 survival pathways on soft-tissue tumor cells.

Liver hepatocytes possess remarkable regenerative capabilities, yet severe damage may compromise this process. Liver progenitor ("oval") cells exhibit the potential to differentiate into both hepatocytes and cholangiocytes, making them promising candidates for cell therapy. However, their mechanisms in liver regeneration are not clear. Here, on rat liver oval stem-like epithelial cells (WB-F344) a wound healing assay was performed. The scratched near-confluent monolayers (70% area removed) underwent the G1-arrest, bi-nucleation at 10-12 hours post-wounding, starting movement of epithelial to mesenchymal transition (EMT) cell portion into the wounded areas. Nanog nuclear upregulation, fragmentation, and transition as granules into cytoplasm and around, along with p16Ink4a nuclear intrusion from the cytoplasm, loss of epithelial markers, and YAP1/Hippo activation were seen near the wound edge. The replicative stress and proliferation boost followed, documented at 24 hours. Proliferation concluded at 40-48 hours, accomplished by reconstitution of epithelial tissue, the disappearance of Nanog granulation and p16Ink4a return to the cytoplasm, releasing excess. This investigation reveals novel regulatory facets in liver regeneration by oval cells. It accentuates the stemness-senescence bistable switch regulated by reciprocal nucleo-cytoplasmic transitions of opposite regulators, coordinated with Hippo-pathway switch, replicative stress, and boost, along with ploidy, EMT-MET and paracrine secretome circuits - enabling successfully resolving the massive injury. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/586724v2_fig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@7dc56dorg.highwire.dtl.DTLVardef@12cabacorg.highwire.dtl.DTLVardef@1bacfc2org.highwire.dtl.DTLVardef@19dfbae_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig 1.C_FLOATNO Graphical abstract. Bistable nuclear-cytoplasmic switch between stemness and senescence regulators in the wound healing process by oval liver cells: (1-2) Priming phase: (1) at the wound edge, (2) in the wound; (3) Proliferative phase, wound closure. Nanog - green; p16INK4A - red, EMT cell - with blue nucleus. C_FIG

Germ cell 1 spermatogonial (GC-1spg) cells are multipotent progenitor cells. We previously confirmed that bone morphogenetic protein (BMP) 9 is among the most osteogenic BMPs. However, whether GC-1spg cells are driven toward osteogenic differentiation under proper stimuli is uncertain. Additionally, the molecular mechanism of BMP9 remains unclear. In the present study, we aimed to determine whether BMP9 can induce osteogenic differentiation of GC-1spg cells. Recombinant adenoviruses were generated by the AdEasy system to regulate the BMP9 expression in GC-1spg cells. We identified osteogenic markers by real-time PCR and staining techniques in vitro. Ectopic ossification assays and histological analysis were also performed to verify the in vivo activity of BMP9. Finally, potential signaling pathways of BMP9 were assessed by transcriptome sequencing and KEGG enrichment analysis. Using recombinant adenoviruses, we demonstrate that BMP9 upregulates osteogenic markers including Runx2, osteocalcin, osteopontin, and Sox9. BMP9 also activates alkaline phosphatase activity and calcium deposition in GC-1spg cells. In vivo results show that BMP9 overexpression in GC-1spg cells promotes ectopic bone formation and chondrogenesis. In addition, RNA-sequencing and KEGG pathway analysis demonstrate that several signaling pathways are involved in BMP9-mediated osteogenesis. GC-1spg cells not only maintain spermatogenesis but also retain the ability to form bone tissue. Therefore, BMP9 activity in GC-1spg cells may help identify signaling pathways implicated in bone formation and could be of use in regenerative medicine.

A cavernous hemangioma, well-known as vascular malformation, is present at birth, grows proportionately with the child, and does not undergo regression. Although a cavernous hemangioma has well-defined histopathological characteristics, its origin and formation remain unknown. In the present study, we characterized the cellular heterogeneity of cavernous hemangioma using single-cell RNA sequencing (scRNA-seq). The main contribution of this study is the discovery of mesenchymal stem cells (MSCs) that cause tumour formation in cavernous hemangioma and we propose that these MSCs may be abnormally differentiated or incompletely differentiated from epiblast stem cells. Other new findings include the responsive ACKR1 positive endothelial cell (ACKR1+EC) and BTNL9 positive endothelial cell (BTNL9+EC) and the BTNL9-caused checkpoint blockade enhanced by the CXCL12-CXCR4 signalling. The activated CD8+T and NK cells may highly express CCL5 for their infiltration in cavernous hemangiomas, independent on the tumor cell-derived CCL5-IFNG-CXCL9 pathway. The highly co-expression of CXCR4 and GZMB suggested that plasmacytoid dendritic cells (pDCs) function for anti-tumour as CD8+T cells in cavernous hemangiomas. The oxidised low-density lipoprotein (oxLDL) in the TME of cavernous hemangiomas may play an important role as a signalling molecular in the immune responses. Notably, we propose that oxLDL induces the oxLDL-OLR1-NLRP3 pathway by over-expression of OLR1 in M1-like macrophages, whereas oxLDL induces the oxLDL-SRs-C1q (SRs are genes encoding scavenger receptors of oxLDL except OLR1) pathway by over-expression of other scavenger receptors in M2-like macrophages. The present study revealed the origin of cavernous hemangiomas and discovered marker genes, cell types and molecular mechanisms associated with the origin, formation, progression, diagnosis or therapy of cavernous hemangiomas. The information from the present study makes important contributions to the understanding of cavernous hemangioma formation and progression and facilitates the development of gold standard for molecular diagnosis and effective drugs for treatment.

14-3-3 protein family binds and regulate hundred of serine/threonine phosphorylated proteins. Considered as redundant, ubiquitous and constantly expressed this protein family was treated as an accessory for many signaling systems. Here we studied the reversible inhibition by acetylation of its essential N-{varepsilon}-lysine 49/51 residue during the osteogenic differentiation of human adipose-derived stem cells (ASC). We found that during the differentiation of ASC the levels of 14-3-3 acK49/51 increase showing that inhibition of 14-3-3 is necessary for this process. Among the 7 paralogs of this family, the inhibition by this posttranslational modification occurs mostly on the paralog {beta} located specifically in the nucleus where 14-3-3 was described to binds to H3 histone and many transcription factors. Short hairpin RNA silencing of 14-3-3{beta} gene but not 14-3-3{gamma} increases significantly the osteogenic potential of the cells. These results show that specifically 14-3-3{beta} is a negative regulator of osteogenesis and its inhibition by acetylation on lysine 51 is the cellular mechanism to regulate it.

Fish cells are largely affected by their culture media. Fibroblast-like cells obtained from the fish fins can differentiate to various kinds of cells such as skeletal muscle-like, neurofilaments and adipocytes. Our results suggest that the fins which are usually discarded as food wastes may practically applied to the clean meat technology.

Alaph-1 antitrypsin overexpressing mesenchymal stromal/stem cells (AAT-MSCs) showed improved innate properties with a faster proliferation rate when studied for their protective effects in mouse models of diseases. Here, we investigated the potential mechanism(s) by which AAT gene insertion increases MSC proliferation. Human bone marrow-derived primary or immortalized MSCs (iMSCs) or AAT-MSCs (iAAT-MSCs) were used in the study. Cell proliferation was measured by cell counting and cell cycle analysis. Possible pathways involved in the pro-proliferation effect of AAT were investigated by measuring mRNA and protein expression of key cell cycle genes. Interval cell counting showed increased proliferation in AAT-MSCs or iAAT-MSCs compared to their corresponding MSC controls. Cell cycle analysis revealed more cells progressing into the S and G2/M phases in iAAT-MSCs, with a notable increase in the cell cycle protein, Cyclin D1. Moreover, treatment with Cyclin D1 inhibitors showed that the increase in proliferation is due to Cyclin D1 and that the AAT protein is upstream and a positive regulator of Cyclin D1. Furthermore, AATs effect on Cyclin D1 is independent of the Wnt signaling pathway as there were no differences in the expression of regulatory proteins, including GSK3{beta} and {beta}-Catenin in iMSC and iAAT-MSCs. In summary, our results indicate that AAT gene insertion in an immortalized MSC cell line increases cell proliferation and growth by increasing Cyclin D1 expression and consequently causing cells to progress through the cell cycle at a significantly faster rate.

BackgroundAlthough mesenchymal stem cells (MSCs) are most commonly used in cell therapy and stem cell research, the mechanism and the locations of their self-renewal are still unknown. MethodMouse blood was collected, and examined under microscopy. The results were compared with the data of human umbilical cord blood (hUCB) collected 10 years ago. ResultsWe found that the procedure of self-renewal for the mesenchymal stem cells in mouse blood and hUCB needs at least 5 steps. First, tube-shaped stem cell niches release long segmented materials composed of sand-like particles and semitransparent granules. Second, the sand-like particles and semitransparent granules separate from the segmented materials. Third, each of the individual semitransparent granules releases groups of fusiform-shaped structures that do not stain to H&E. The sizes of the fusiform-shaped structures range from 1 to 100 m in length in mouse blood, but can be 200 m in hUCB. Fourth, the large-sized fusiform structures can directly transform into lineage-restricted cellular structures; the medium-sized fusiform structures fuse or engulf each other to form cellular structures. The cellular structures further acquire membranes from the adjacent nucleated cells. Fifth, the nucleolus appears in the new cellular structures before the nucleus. During all the procedures, the adjacent nucleated mesenchymal cells are must needed. Thus, these newly formed cellular structures will further differentiate into nucleated mesenchymal stem cells. ConclusionOur findings again provide new evidence that, in physiological conditions, mesenchymal stem cell self-renewal needs several steps to complete, which, however, does not occur by mitotic division. The tube-shaped structures are the niches of the stem cells.