Circadian Rhythm Disruption in Hepatocellular Carcinoma Investigated by Integrated Analysis of Bulk and Single-Cell RNA Sequencing Data.

Circadian rhythms are essential regulators of a multitude of physiological and behavioral processes, such as the metabolism and function of the liver. Circadian rhythms are crucial to liver homeostasis, as the liver is a key metabolic organ accountable for the systemic equilibrium of the body. Circadian rhythm disruption alone is sufficient to cause liver cancer through the maintenance of hepatic metabolic disorder. Although there is evidence linking CRD to hepatocarcinogenesis, the precise cellular and molecular mechanisms that underlie the circadian crosstalk that leads to hepatocellular carcinoma remain unknown. The expression of CRD-related genes in HCC was investigated in this study via bulk RNA transcriptomic analysis and single-cell sequencing. Dysregulated CRD-related genes are predominantly found in hepatocytes and fibroblasts, according to the findings. By using a combination of single-cell RNA sequencing and bulk RNA sequencing analyses, the dysregulated CRD-related genes ADAMTS13, BIRC5, IGFBP3, MARCO, MT2A, NNMT, and PGLYRP2 were identified. The survival analysis using the Kaplan-Meier method revealed a significant correlation between the expression levels of BIRC5 and IGFBP3 and the survival of patients diagnosed with HCC.

MicroRNA-29a Manifests Multifaceted Features to Intensify Radiosensitivity, Escalate Apoptosis, and Revoke Cell Migration for Palliating Radioresistance-Enhanced Cervical Cancer Progression.

Radioresistance remains a major clinical challenge in cervical cancer therapy and results in tumor relapse and metastasis. Nevertheless, the detailed mechanisms are still largely enigmatic. This study was conducted to elucidate the prospective impacts of microRNA-29a (miR-29a) on the modulation of radioresistance-associated cervical cancer progression. Herein, we established two pairs of parental wild-type (WT) and radioresistant (RR) cervical cancer cells (CaSki and C33A), and we found that constant suppressed miR-29a, but not miR-29b/c, was exhibited in RR-clones that underwent a dose of 6-Gy radiation treatment. Remarkably, radioresistant clones displayed low radiosensitivity, and the reduced apoptosis rate resulted in augmented surviving fractions, measured by the clonogenic survival curve assay and the Annexin V/Propidium Iodide apoptosis assay, respectively. Overexpression of miR-29a effectively intensified the radiosensitivity and triggered the cell apoptosis in RR-clones. In contrast, suppressed miR-29a modestly abridged the radiosensitivity and abolished the cell apoptosis in WT-clones. Hence, ectopically introduced miR-29a into RR-clones notably attenuated the wound-healing rate and cell migration, whereas reduced miR-29a aggravated cell mobilities of WT-clones estimated via the in vitro wound-healing assay and time-lapse recording assay. Notably, we further established the in vivo short-term lung locomotion metastasis model in BALB/c nude mice, and we found that increased lung localization was shown after tail-vein injection of RR-CaSki cells compared to those of WT-CaSki cells. Amplified miR-29a significantly eliminated the radioresistance-enhanced lung locomotion. Our data provide evidence suggesting that miR-29a is a promising microRNA signature in radioresistance of cervical cancer cells and displays multifaceted innovative roles involved in anti-radioresistance, escalated apoptosis, and anti-cell migration/metastasis. Amalgamation of a nucleoid-based strategy (miR-29a) together with conventional radiotherapy may be an innovative and eminent strategy to intensify the radiosensitivity and further protect against the subsequent radioresistance and the potential metastasis in cervical cancer treatment.

TIAM2S as a novel regulator for serotonin level enhances brain plasticity and locomotion behavior.

TIAM2S, the short form of human T-cell lymphoma invasion and metastasis 2, can have oncogenic effects when aberrantly expressed in the liver or lungs. However, it is also abundant in healthy, non-neoplastic brain tissue, in which its primary function is still unknown. Here, we examined the neurobiological and behavioral significance of human TIAM2S using the human brain protein panels, a human NT2/D1-derived neuronal cell line model (NT2/N), and transgenic mice that overexpress human TIAM2S (TIAM2S-TG). Our data reveal that TIAM2S exists primarily in neurons of the restricted brain areas around the limbic system and in well-differentiated NT2/N cells. Functional studies revealed that TIAM2S has no guanine nucleotide exchange factor (GEF) activity and is mainly located in the nucleus. Furthermore, whole-transcriptome and enrichment analysis with total RNA sequencing revealed that TIAM2S-knockdown (TIAM2S-KD) was strongly associated with the cellular processes of the brain structural development and differentiation, serotonin-related signaling, and the diseases markers representing neurobehavioral developmental disorders. Moreover, TIAM2S-KD cells display decreased neurite outgrowth and reduced serotonin levels. Moreover, TIAM2S overexpressing TG mice show increased number and length of serotonergic fibers at early postnatal stage, results in higher serotonin levels at both the serum and brain regions, and higher neuroplasticity and hyperlocomotion in latter adulthood. Taken together, our results illustrate the non-oncogenic functions of human TIAM2S and demonstrate that TIAM2S is a novel regulator of serotonin level, brain neuroplasticity, and locomotion behavior.

MicroRNA-128 Confers Anti-Endothelial Adhesion and Anti-Migration Properties to Counteract Highly Metastatic Cervical Cancer Cells' Migration in a Parallel-Plate Flow Chamber.

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells' adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells' adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells' adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.

Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells.

BACKGROUND: VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1. METHODS: Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were co-expressed in cells using plasmid transfection. RESULTS: A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm. CONCLUSION: Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm.

Human Umbilical Cord Mesenchymal Stem Cells Extricate Bupivacaine-Impaired Skeletal Muscle Function via Mitigating Neutrophil-Mediated Acute Inflammation and Protecting against Fibrosis.

Skeletal muscle injury presents a challenging traumatological dilemma, and current therapeutic options remain mediocre. This study was designed to delineate if engraftment of mesenchymal stem cells derived from umbilical cord Wharton's jelly (uMSCs) could aid in skeletal muscle healing and persuasive molecular mechanisms. We established a skeletal muscle injury model by injection of myotoxin bupivacaine (BPVC) into quadriceps muscles of C57BL/6 mice. Post BPVC injection, neutrophils, the first host defensive line, rapidly invaded injured muscle and induced acute inflammation. Engrafted uMSCs effectively abolished neutrophil infiltration and activation, and diminished neutrophil chemotaxis, including Complement component 5a (C5a), Keratinocyte chemoattractant (KC), Macrophage inflammatory protein (MIP)-2, LPS-induced CXC chemokine (LIX), Fractalkine, Leukotriene B4 (LTB4), and Interferon-γ, as determined using a Quantibody Mouse Cytokine Array assay. Subsequently, uMSCs noticeably prevented BPVC-accelerated collagen deposition and fibrosis, measured by Masson's trichrome staining. Remarkably, uMSCs attenuated BPVC-induced Transforming growth factor (TGF)-ß1 expression, a master regulator of fibrosis. Engrafted uMSCs attenuated TGF-ß1 transmitting through interrupting the canonical Sma- And Mad-Related Protein (Smad)2/3 dependent pathway and noncanonical Smad-independent Transforming growth factor beta-activated kinase (TAK)-1/p38 mitogen-activated protein kinases signaling. The uMSCs abrogated TGF-ß1-induced fibrosis by reducing extracellular matrix components including fibronectin-1, collagen (COL) 1A1, and COL10A1. Most importantly, uMSCs modestly extricated BPVC-impaired gait functions, determined using CatWalk™ XT gait analysis. This work provides several innovative insights into and molecular bases for employing uMSCs to execute therapeutic potential through the elimination of neutrophil-mediated acute inflammation toward protecting against fibrosis, thereby rescuing functional impairments post injury.

MicroRNA-29a Exhibited Pro-Angiogenic and Anti-Fibrotic Features to Intensify Human Umbilical Cord Mesenchymal Stem Cells-Renovated Perfusion Recovery and Preventing against Fibrosis from Skeletal Muscle Ischemic Injury.

This study was conducted to elucidate whether microRNA-29a (miR-29a) and/or together with transplantation of mesenchymal stem cells isolated from umbilical cord Wharton's jelly (uMSCs) could aid in skeletal muscle healing and putative molecular mechanisms. We established a skeletal muscle ischemic injury model by injection of a myotoxin bupivacaine (BPVC) into gastrocnemius muscle of C57BL/6 mice. Throughout the angiogenic and fibrotic phases of muscle healing, miR-29a was considerably downregulated in BPVC-injured gastrocnemius muscle. Overexpressed miR-29a efficaciously promoted human umbilical vein endothelial cells proliferation and capillary-like tube formation in vitro, crucial steps for neoangiogenesis, whereas knockdown of miR-29a notably suppressed those endothelial functions. Remarkably, overexpressed miR-29a profitably elicited limbic flow perfusion and estimated by Laser Dopple. MicroRNA-29a motivated perfusion recovery through abolishing the tissue inhibitor of metalloproteinase (TIMP)-2, led great numbers of pro-angiogenic matrix metalloproteinases (MMPs) to be liberated from bondage of TIMP, thus reinforced vascular development. Furthermore, engrafted uMSCs also illustrated comparable effect to restore the flow perfusion and augmented vascular endothelial growth factors-A, -B, and -C expression. Notably, the combination of miR29a and the uMSCs treatments revealed the utmost renovation of limbic flow perfusion. Amplified miR-29a also adequately diminished the collagen deposition and suppressed broad-wide miR-29a targeted extracellular matrix components expression. Consistently, miR-29a administration intensified the relevance of uMSCs to abridge BPVC-aggravated fibrosis. Our data support that miR-29a is a promising pro-angiogenic and anti-fibrotic microRNA which delivers numerous advantages to endorse angiogenesis, perfusion recovery, and protect against fibrosis post injury. Amalgamation of nucleic acid-based strategy (miR-29a) together with the stem cell-based strategy (uMSCs) may be an innovative and eminent strategy to accelerate the healing process post skeletal muscle injury.

Low P16INK4A Expression Associated with High Expression of Cancer Stem Cell Markers Predicts Poor Prognosis in Cervical Cancer after Radiotherapy.

Previous studies have suggested that cancer stem cells (CSCs) resisted radiotherapy and chemotherapy. P16INK4A is a biomarker for cervical carcinogenesis and reduces proliferation of stem cells. We aimed to investigate the expression and clinical significance of cyclin-dependent kinase inhibitor 2A (P16INK4A), sex determining region Y-box 2 (SOX2), and Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) in cervical cancer treated with radiotherapy and cervical cell line models. The expressions of P16INK4A, SOX2, and ALDH1A1 were performed by immunohistochemical staining of tumor samples from 139 cervical cancer patients with International Federation of Gynecology and Obstetrics stages Ib to IV. The staining showed high expression in 100, 107, and 13 patients with P16INK4A (>80%), SOX2 (≥10%), and ALDH1A1 (50%), respectively. The high-P16INK4A group had a higher five-year overall survival (OS) rate and disease-free survival (DFS) than the low-P16INK4A group (OS: 62.0% and 35.2%, p = 0.016; DFS: 60.0% and 31.2%, p = 0.002). The low-P16INK4A/high-SOX2 and low-P16INK4A/high-ALDH1A1 groups had a worse five-year OS and DFS rate than the high-P16INK4A/low-SOX2 and high-P16INK4A/low-ALDH1A1 groups, respectively. Depletion of P16INK4A promoted chemoresistance and radioresistance of cervical cancer cells increased the expression of SOX2 and ALDH1A1 and exhibited higher self-renewal ability. These results suggest that lower P16INK4A expression associated with higher CSC markers predicts poor prognostic outcomes and is a promising target in patients with cervical cancer.

MicroRNA-29a promotion of nephrin acetylation ameliorates hyperglycemia-induced podocyte dysfunction.

Podocyte dysfunction is a detrimental feature in diabetic nephropathy, with loss of nephrin integrity contributing to diabetic podocytopathy. MicroRNAs (miRs) reportedly modulate the hyperglycemia-induced perturbation of renal tissue homeostasis. This study investigated whether regulation of histone deacetylase (HDAC) actions and nephrin acetylation by miR-29 contributes to podocyte homeostasis and renal function in diabetic kidneys. Hyperglycemia accelerated podocyte injury and reduced nephrin, acetylated nephrin, and miR-29a levels in primary renal glomeruli from streptozotocin-induced diabetic mice. Diabetic miR-29a transgenic mice had better nephrin levels, podocyte viability, and renal function and less glomerular fibrosis and inflammation reaction compared with diabetic wild-type mice. Overexpression of miR-29a attenuated the promotion of HDAC4 signaling, nephrin ubiquitination, and urinary nephrin excretion associated with diabetes and restored nephrin acetylation. Knockdown of miR-29a by antisense oligonucleotides promoted HDAC4 action, nephrin loss, podocyte apoptosis, and proteinuria in nondiabetic mice. In vitro, interruption of HDAC4 signaling alleviated the high glucose-induced apoptosis and inhibition of nephrin acetylation in podocyte cultures. Furthermore, HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), the enrichment of H3K9Ac in miR-29a proximal promoter, and miR-29a transcription in high glucose-stressed podocytes. In conclusion, hyperglycemia impairs miR-29a signaling to intensify HDAC4 actions that contribute to podocyte protein deacetylation and degradation as well as renal dysfunction. HDAC4, via epigenetic H3K9 hypoacetylation, reduces miR-29a transcription. The renoprotective effects of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis highlights the importance of post-translational acetylation reactions in podocyte microenvironments. Increasing miR-29a action may protect against diabetic podocytopathy.

MicroRNA-29a protects against glucocorticoid-induced bone loss and fragility in rats by orchestrating bone acquisition and resorption.

OBJECTIVE: Excessive glucocorticoid treatment increases the incidence of osteopenia and osteonecrosis. MicroRNAs (miRNAs) reportedly target messenger RNA expression and regulate osteoblastogenesis and skeletal development. We undertook this study to investigate whether miR-29a regulates glucocorticoid-mediated bone loss. METHODS: Rats were given methylprednisolone, lentivirus-mediated miR-29a precursor, or lentivirus-mediated miR-29a inhibitor. Dual x-ray absorptiometry, micro-computed tomography, material testing, and enzyme-linked immunosorbent assay were performed to quantify bone mass, microarchitecture, peak load, and serum Dkk-1 levels. Differential miRNA expression profiles were detected using polymerase chain reaction arrays. The abundance of signaling molecules was assessed using immunoblotting. RESULTS: Glucocorticoid treatment induced loss of bone mineral density and trabecular microstructure in association with reduced miR-29a expression. Treatment with miR-29a precursor attenuated the adverse effects of glucocorticoid on bone mass, trabecular bone volume fraction, and biomechanical load-bearing capacity of bone tissue. Gain of miR-29a function alleviated the detrimental effects of glucocorticoid treatment on mineral acquisition and ex vivo osteoblast differentiation, and also reduced osteoclast surface, ex vivo osteoclast differentiation, and RANKL expression in bone microenvironments. Knockdown of miR-29a accelerated osteoclast resorption, cortical bone porosity, bone fragility, and loss of ex vivo osteogenic differentiation capacity. MicroRNA-29a regulated the abundance of Wnt signaling components (Wnt-3a, glycogen synthase kinase 3ß, and ß-catenin), the Wnt inhibitor Dkk-1, Akt, and phosphorylated ERK, and the expression of the osteogenic factors RUNX-2 and insulin-like growth factor 1 in bone tissue. CONCLUSION: MicroRNA-29a signaling protected against glucocorticoid-induced disturbance of Wnt and Dkk-1 actions and improved osteoblast differentiation and mineral acquisition. Promotion of miR-29a signaling is an alternative strategy for alleviating glucocorticoid-induced bone deterioration.

Radiation-induced increase in cell migration and metastatic potential of cervical cancer cells operates via the K-Ras pathway.

Radiotherapy is a well established treatment for cervical cancer, the second most common cancer in women worldwide. However, metastasis often circumvents the efficacy of radiotherapy. This study was conducted to elucidate the molecular mechanism of radioresistance-associated metastatic potential of cervical cancer cells. We established three radioresistant cervical cancer cell lines by exposure of cells to a sublethal dose of radiation and screened for lines that exhibited an increased migration phenotype for at least 6 months before undertaking mechanistic studies. Radiation-associated metastatic potential was evaluated using a wound-healing assay, time-lapse recording, and cell locomotion into the lungs of BALB/c nude mice. The radioresistant C33A and CaSki cell lines, but not the radioresistant HeLa cell line, exhibited significantly increased cell migration and wound healing than did wild-type cells. Furthermore, K-Ras played a prometastatic role via the activation of c-Raf/p38, whereas interference of those mediators via either RNA interference-mediated knockdown or the use of chemical inhibitors substantially reversed the radioresistance-associated increase in cell migration. Clinical examination further showed the relative up-regulation of the K-Ras/c-Raf/p38 pathway in locally recurring tumors and distant metastases compared with in the primary cervical tumor. These findings demonstrate that a sublethal dose of radiation can enhance the metastatic potential of human cervical cancer cells via K-Ras/c-Raf/p38 signaling, highlighting the potential development of specific inhibitors for reducing metastatic potential during radiotherapy.

Suppression of annexin A2 by prostaglandin E2 impairs phagocytic ability of peritoneal macrophages in women with endometriosis.

STUDY QUESTION: Is annexin A2 involved in the reduced phagocytic ability of macrophages in endometriosis? SUMMARY ANSWER: Data from women with endometriosis and a murine model of the disease show that expression of annexin A2 in peritoneal macrophages is inhibited by prostaglandin E2 (PGE2) and this impairs the phagocytic ability of macrophages. WHAT IS ALREADY KNOWN: Endometriosis is a chronic inflammatory disease that recruits many immune cells, especially macrophages, to the peritoneal cavity. The phagocytic ability of peritoneal macrophages isolated from women with endometriosis is reduced. STUDY DESIGN, SIZE, DURATION: A laboratory study. Thirty-five patients (20 with and 15 without endometriosis) of reproductive age with normal menstrual cycles were recruited. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peritoneal macrophages isolated from women with or without endometriosis were cultured and treated with vehicle, PGE2 and different EP receptor agonists, and the expression of annexin A2 was quantified by RT-PCR and western blotting. Annexin A2 was knocked down (by small interfering RNA) in normal macrophages or overexpressed (by treatment with recombinant protein) in endometriotic macrophages and their phagocytic ability was measured by flow cytometry. Peritoneal macrophages were isolated from a mouse model of endometriosis and treated with PGE2 or cyclo-oxygenase (COX) inhibitors, and annexin A2 mRNA was quantified. MAIN RESULTS AND THE ROLE OF CHANCE: Levels of annexin A2 were markedly reduced in peritoneal macrophages from women with endometriosis versus controls (mRNA: P < 0.01). The level of annexin A2 mRNA in the macrophages was reduced by PGE2 (P < 0.01/P < 0.05 in women without/with endometriosis versus control) via the EP2/EP4 receptor-dependent signaling pathway. Treatment with PGE2 or knockdown of annexin A2 inhibited the phagocytic ability of macrophages (P < 0.05 versus control), while treatment with annexin A2 recombinant protein enhanced phagocytosis. Autologous transplantation animal studies further confirmed that levels of annexin A2 in peritoneal macrophages were markedly reduced in mice treated with PGE2 (P < 0.01 versus control). In contrast, treatment with COX inhibitors to inhibit PGE2 production enhanced annexin A2 expression in peritoneal macrophages (P < 0.05 versus control). LIMITATIONS, REASONS FOR CAUTION: We have provided no direct demonstration that phagocytic activity is indeed decreased in peritoneal cells from patients with endometriosis or that their endometriotic fluid contains increased amounts of PGE2 when compared with control subjects. WIDER IMPLICATIONS OF THE FINDINGS: Inhibiting PGE2 signaling, in order to restore or enhance the phagocytic capability of macrophages, may represent a new direction of thinking in developing novel strategies against endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from National Science Council of Taiwan, Republic of China (NSC97-2314-B-006-020-MY3) to M.-H.W. and (NSC98-2320-B-006-026-MY3) to S.-J.T., and grants from the Chang Gung Memorial Hospital, Taiwan, Republic of China (CMRPG891432 and CMRPG8A0531) to P.-C.C. None of the authors have any conflicts of interest.

Deciphering the Divergent Gene Expression Landscapes of m6A/m5C/m1A Methylation Regulators in Hepatocellular Carcinoma Through Single-Cell and Bulk RNA Transcriptomic Analysis.

Introduction: RNA modifications mediated by the m6A, m1A, and m5C regulatory genes are crucial for the progression of malignancy. This study aimed to explore the expression of regulator genes for m6A/m5C/m1A methylation at the single-cell level and to validate their expression in cancerous and adjacent para-cancerous liver tissues of adult patients with HCC who underwent tumor resection. Methods: The bulk sequencing from The Cancer Genome Atlas (TCGA) database and the single-cell RNA sequencing (scRNA-seq) data obtained from the Gene Expression Omnibus (GEO) database were used to identify the dysregulated m6A/m5C/m1A genes for hepatocellular carcinoma (HCC). A real-time polymerase chain reaction (real-time PCR) was used to measure the expression of dysregulated m6A/m5C/m1A genes in collected human HCC tissues and compared with adjacent para-cancerous liver tissues. Immune cell infiltration with these significantly expressed methylation-related genes was evaluated using Timer2.0. Results: A discrepancy in m6A/m5C/m1A gene expression was observed between bulk sequencing and scRNA-seq. The clustered heatmap of the scRNA-seq-identified dysregulated m6A/m5C/m1A genes in TCGA cohort revealed heterogeneous expression of these methylation regulators within the cancer, whereas their expression in the adjacent liver tissues was more homogeneous. The real-time PCR validated the significant overexpression of DNMT1, NSUN5, TRMT6, IGF2BP1, and IGFBP3, which were identified using scRNA-seq, and IGFBP2, which was identified using bulk sequencing. These dysregulated methylation genes are mainly correlated with the infiltration of natural killer cells. Discussion: This study suggests that cellular diversity inside tumors contributes to the discrepancy in the expression of methylation regulator genes between traditional bulk sequencing and scRNA-seq. This study identified five regulatory genes that will be the focus of further studies regarding the function of m6A/m5C/m1A in HCC.

Inhibition of CD36-dependent phagocytosis by prostaglandin E2 contributes to the development of endometriosis.

Dysfunction in macrophage-mediated phagocytosis of aberrant cells that undergo retrograde transport to the peritoneal cavity is considered an important factor in the development of endometriosis. However, the mechanisms responsible for the loss of function of macrophages remain largely unknown. Herein, we report that prostaglandin (PG) E(2), via the EP2 receptor-dependent signaling pathway, inhibits the expression of CD36 in peritoneal macrophages, resulting in reduced phagocytic ability. PGE(2)-mediated inhibition of macrophage phagocytic capability was restored by ectopic expression of CD36. Treatment with PGE(2) inhibited CD36-dependent phagocytosis of peritoneal macrophages and increased the number and size of endometriotic lesions in mice. In contrast, blockade of PGE(2) production by cyclooxygenase inhibitors enhanced the phagocytic ability of peritoneal macrophages and reduced endometriotic lesion formation. Taken together, our findings reveal a potential mechanism of immune dysfunction during endometriosis development and may contribute to the design of an effective prevention/treatment regimen.

Downregulation of CD36 results in reduced phagocytic ability of peritoneal macrophages of women with endometriosis.

Endometriosis, defined as the growth of endometrial tissues outside of the uterine cavity, is a severe and complex disease affecting more than 10% of women. The aetiology of endometriosis is unclear but immune dysfunction might be an important factor for its development. The natural function of the immune system is to detect and destroy aberrant or abnormal cells. Failure of the immune system to eradicate these aberrant cells often results in disease pathogenesis. We report here that the phagocytic ability of macrophages is reduced in peritoneal macrophages isolated from women with endometriosis. In-depth investigation revealed that the level of CD36, a class B scavenger receptor, in peritoneal macrophages derived from women with endometriosis was lower than that in normal macrophages. Blockage of CD36 function by neutralized antibody or knocking down CD36 using siRNA impaired the phagocytic ability of normal macrophages. In contrast, forced expression of CD36 in macrophages isolated from women with endometriosis restored phagocytic ability. Taken together, we identified that the scavenger receptor CD36 is reduced in the peritoneal macrophages of women with endometriosis, which leads to a decrease of the phagocytic ability of macrophages. These findings revealed a potential mechanism of immune dysfunction during endometriosis development.

TIAM2S Mediates Serotonin Homeostasis and Provokes a Pro-Inflammatory Immune Microenvironment Permissive for Colorectal Tumorigenesis.

The short isoform of human TIAM2 has been shown to promote proliferation and invasion in various cancer cells. However, the roles of TIAM2S in immune cells in relation to tumor development have not been investigated. To characterize the effects of TIAM2S, we generated TIAM2S-overexpressing mouse lines and found that aged TIAM2S-transgenic (TIAM2S-TG) developed significantly higher occurrence of lymphocytic infiltration and tumorigenesis in various organs, including colon. In addition, TIAM2S-TG is more sensitized to AOM-induced colon tumor development, suggesting a priming effect toward tumorigenesis. In the light of our recent findings that TIAM2S functions as a novel regulator of cellular serotonin level, we found that serotonin, in addition to Cox2, is a unique inflammation marker presented in the colonic lesion sites in the aged TG animals. Furthermore, our results demonstrated that ectopic TIAM2S altered immunity via the expansion of T lymphocytes; this was especially pronounced in CD8+ T cells in combination with CXCL13/BCA-1 pro-inflammatory chemokine in the serum of TIAM2S-TG mice. Consequently, T lymphocytes and B cells were recruited to the lesion sites and stimulated IL-23/IL17A expression to form the tertiary lymphoid organs. Collectively, our research suggests that TIAM2S provokes a pro-inflammatory immune microenvironment permissive to colorectal tumorigenesis through the serotonin-induced immunomodulatory effects.

Prostaglandin E2 induces fibroblast growth factor 9 via EP3-dependent protein kinase Cdelta and Elk-1 signaling.

Fibroblast growth factor 9 (FGF-9) is a potent mitogen that controls the proper development of many tissues and organs. In contrast, aberrant expression of FGF-9 also results in the evolution of many human diseases, such as cancers and endometriosis. Despite its vital function being reported, the cellular and molecular mechanisms responsible for the regulation of FGF-9 expression are mostly unknown. We report here that prostaglandin E2 (PGE2) induces expression of FGF-9, which promotes endometriotic stromal cell proliferation, through the EP3 receptor-activated protein kinase Cdelta (PKCdelta) signaling pathway. Activation of PKCdelta leads to phosphorylation of ERK1/2, and the transcription factor Elk-1 thereby promotes transcription of FGF-9. Two Elk-1 cis-binding sites located at nucleotides -1324 to -1329 and -1046 to -1051 of the human FGF-9 promoter are identified as crucial for mediating PGE2 actions. Collectively, we demonstrate, for the first time, that PGE2 can directly induce FGF-9 expression via a novel signaling pathway involving EP3, PKCdelta, and a member of the ETS domain-containing transcription factor superfamily in primary human endometriotic stromal cells. Our findings may also provide a molecular framework for considering roles for PGE2 in FGF-9-related embryonic development and/or human diseases.

Mineral trioxide aggregate affects cell viability and induces apoptosis of stem cells from human exfoliated deciduous teeth.

BACKGROUND: Mineral trioxide aggregate (MTA) is widely used for pulp-capping procedures in permanent teeth and as a gold standard material in endodontics. The aim of the study was to investigate the effect of MTA on cell viability and apoptosis when MTA is directly in contact with Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs). METHODS: MTA was mixed and coated in the bottom of a 24-well plate. SHEDs collected and cultured from normal exfoliated human deciduous teeth (passages 3-4) were seeded on square cover glasses. The glasses with seeded SHEDs were incubated in the plates with or without MTA coating. They were divided into four groups: MTA direct contact, direct control, MTA indirect contact, and indirect control. After 1, 2 and 3 days of culturing, cell morphology was observed and cell viability was assessed by the WST-1 cell cytotoxicity assay. TUNEL assay, immunofluorescent labeling and western blot analysis were used to study the effects of MTA on SHEDs apoptosis. RESULTS: MTA impaired cell viability of SHEDs in 1, 2 and 3 days, and the effect of direct contact was more severe. Cell apoptosis with positive Annexin V and TUNEL staining was noted when there was direct contact with MTA. Western blot analysis revealed that Bcl-2 and Bcl-xL decreased after SHEDs were in contact with MTA. CONCLUSIONS: This study shows that direct contact with 1 week post-set MTA significantly decreases the viability of SHEDs and induced cell apoptosis. The results suggest that there is a possible cytotoxic effect of pulp tissue when there is direct contact with MTA. Different responses would be expected due to the strong alkaline characteristics of fresh mixed MTA.

Endometriosis: disease pathophysiology and the role of prostaglandins.

Endometriosis is considered to be a polygenic disease with a complex, multifactorial aetiology that affects about 10% of women in the reproductive age. Women with endometriosis have symptoms that include chronic pelvic pain, dysmenorrhoea and dyspareunia, significantly reducing their quality of life. Endometriosis is also the primary cause of infertility in women, with the prevalence rate ranging from 20% to 50%. The high prevalence and severe outcomes of this disease have made it a major public health concern in modern society. Currently, the mechanism(s) responsible for the initiation and promotion of this disease remains obscure. In this review, we focus on the expression, regulation and action of prostaglandins in the cellular and molecular mechanisms that contribute to the development and/or maintenance of endometriosis.

Histone demethylase UTX counteracts glucocorticoid deregulation of osteogenesis by modulating histone-dependent and -independent pathways.

Excess glucocorticoid administration impairs osteogenic activities, which raises the risk of osteoporotic disorders. Epigenetic methylation of DNA and histone regulates the lineage commitment of progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 6a (UTX) with regard to the glucocorticoid impediment of osteogenic differentiation. Osteogenic progenitor cells responded to supraphysiological glucocorticoid by elevating CpG dinucleotide methylation proximal to transcription start sites within Runx2 and osterix promoters and Wnt inhibitor Dickkopf-1 (Dkk1) expression concomitant with low UTX expression. 5'-Aza-deoxycystidine demethylation of Runx2 and osterix promoters abolished the glucocorticoid inhibition of mineralized matrix accumulation. Gain of UTX function attenuated the glucocorticoid-induced loss of osteogenic differentiation, whereas UTX silencing escalated adipogenic gene expression and adipocyte formation. UTX sustained osteogenic gene transcription through maintaining its occupancy to Runx2 and osterix promoters. It also mitigated the trimethylation of histone 3 at lysine 27 (H3K27me3), which reduced H3K27me3 enrichment to Dkk1 promoter and thereby lowered Dkk1 transcription. Modulation of ß-catenin and Dkk1 actions restored UTX signaling in glucocorticoid-stressed cells. In vivo, UTX inhibition by exogenous methylprednisolone and GSK-J4 administration, an effect that disturbed H3K27me3, ß-catenin, Dkk1, Runx2, and osterix levels, exacerbated trabecular microarchitecture loss and marrow adiposity. Taken together, glucocorticoid reduction of UTX function hindered osteogenic differentiation. Epigenetic hypomethylation of osteogenic transcription factor promoters and H3K27 contributed to the UXT alleviation of Dkk1 transcription and osteogenesis in glucocorticoid-stressed osteogenic progenitor cells. Control of UTX action has an epigenetic perspective of curtailing glucocorticoid impairment of osteogenic differentiation and bone mass. KEY MESSAGES: UTX attenuates glucocorticoid deregulation of osteogenesis and adipogenesis. UTX reduces Runx2 promoter methylation and H3K27me3 enrichment in the Dkk1 promoter. ß-catenin and Dkk1 modulate the glucocorticoid inhibition of UTX signaling. UTX inhibition exacerbates bone mass, trabecular microstructure and fatty marrow. UTX signaling is indispensable in fending off glucocorticoid-impaired osteogenesis.