[This retracts the article DOI: 10.7150/ijbs.7723.].

Gastroesophageal reflux disease (GERD) is a common clinical disease associated with upper gastrointestinal motility disorders. Recently, with improvements in living standards and changes in lifestyle and dietary habits, the incidence of GERD has been increasing yearly. However, the mechanism of GERD has not been fully elucidated due to its complex pathogenesis, and this had led to unsatisfactory therapeutic outcomes. Currently, the occurrence and development of GERD involve multiple factors. Its pathogenesis is mainly thought to be related to factors, such as lower esophageal sphincter pressure, transient lower esophageal sphincter relaxation, crural diaphragmatic dysfunction, hiatus hernia, and impaired esophageal clearance. Therefore, explaining the pathogenesis of GERD more clearly and systematically, exploring potential and effective therapeutic targets, and choosing the best treatment methods have gradually become the focus of scholars' attention. Herein, we reviewed current advancements in the dynamic mechanism of GERD to better counsel patients on possible treatment options.

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro, promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1. Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/β-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis. Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection.

Diabetic nephropathy (DN) has become the common and principal microvascular complication of diabetes that could lead to end-stage renal disease. It was reported endothelial-to-mesenchymal transition (EndMT) in glomeruli plays an important role in DN. Enolase1 (ENO1) and Lysine Methyltransferase 5A (KMT5A) were found to modulate epithelial-to-mesenchymal transition in some situations. In the present study, we speculated KMT5A regulates ENO1 transcript, thus participating in hyperglycemia-induced EndMT in glomeruli of DN. Our study represented vimentin, αSMA and ENO1 expression elevated, and CD31 expression decreased in glomeruli of DN participants and rats. In vitro, high glucose induced EndMT by increase of ENO1 levels. Moreover, high glucose downregulated KMT5A levels and increased regulatory factor X1 (RFX1) levels. KMT5A upregulation or si-RFX1 decreased high glucose-induced ENO1 expression and EndMT. RFX1 overexpression- or sh-KMT5A-induced EndMT was attenuated by si-ENO1. Further, the association between KMT5A and RFX1 was verified. Furthermore, histone H4 lysine20 methylation (the direct target of KMT5A) and RFX1 positioned on ENO1 promoter region. sh-KMT5A enhanced positive action of RFX1 on ENO1 promoter activity. KMT5A reduction and RFX1 upregulation were verified in glomeruli of DN patients and rats. KMT5A associated with RFX1 to modulate ENO1, thus involved in hyperglycemia-mediated EndMT in glomeruli of DN.

Systemic application of glucocorticoids is an essential anti-inflammatory and immune-modulating therapy for severe inflammatory or autoimmunity conditions. However, its long-term effects on articular cartilage of patients' health need to be further investigated. In this study, we studied the effects of dexamethasone (Dex) on the homeostasis of articular cartilage and the progress of destabilization of medial meniscus (DMM)-induced osteoarthritis (OA) in adult mice. Long-term administration of Dex aggravates the proteoglycan loss of articular cartilage and drastically accelerates cartilage degeneration under surgically induced OA conditions. In addition, Dex increases calcium content in calcified cartilage layer of mice and the samples from OA patients with a history of long-term Dex treatment. Moreover, long term usage of Dex results in decrease subchondral bone mass and bone density. Further studies showed that Dex leads to calcification of extracellular matrix of chondrocytes partially through activation of AKT, as well as promotes apoptosis of chondrocytes in calcified cartilage layer. Besides, Dex weakens the stress-response autophagy with the passage of time. Taken together, our data indicate that long-term application of Dex may predispose patients to OA and or even accelerate the OA disease progression development of OA patients.

Laron syndrome (LS) is an autosomal recessive genetic disease mainly caused by mutations in the human growth hormone receptor (GHR) gene. Previous studies have focused on Ghr mutant mice, but compared with LS patients, Ghr knockout (KO) mice exhibit differential lipid metabolism. To elucidate the relationship between GHR mutation and lipid metabolism, the role of GHR in lipid metabolism was examined in GHR KO pigs and hepatocytes transfected with siGHR. We observed high levels of free fatty acids and hepatic steatosis in GHR KO pigs, which recapitulates the abnormal lipid metabolism in LS patients. RNAseq analysis revealed that genes related to the fatty acid oxidation pathway were significantly altered in GHR KO pigs. AHR, a transcription factor related to lipid metabolism, was significantly downregulated in GHR KO pigs and siGHR-treated human hepatocytes. We found that AHR directly regulated fatty acid oxidation by directly binding to the promoters of ACOX1 and CPT1A and activating their expression. These data indicate that loss of GHR disturbs the ERK-AHR-ACOX1/CPT1A pathway and consequently leads to hepatic steatosis. Our results established AHR as a modulator of hepatic steatosis, thereby providing a therapeutic target for lipid metabolism disorder.

Abnormal lipid metabolism including synthesis, uptake, modification, degradation and transport has been considered a hallmark of malignant tumors and contributes to the supply of substances and energy for rapid cell growth. Meanwhile, abnormal lipid metabolism is also associated with lipid peroxidation, which plays an important role in a newly discovered type of regulated cell death termed ferroptosis. Long noncoding RNAs (lncRNAs) have been proven to be associated with the occurrence and progression of cancer. Growing evidence indicates that lncRNAs are key regulators of abnormal lipid metabolism and ferroptosis in cancer. In this review, we mainly summarized the mechanism by which lncRNAs regulate aberrant lipid metabolism in cancer, illustrated that lipid metabolism can also influence the expression of lncRNAs, and discussed the mechanism by which lncRNAs affect ferroptosis. A comprehensive understanding of the interactions between lncRNAs, lipid metabolism and ferroptosis could help us to develop novel strategies for precise cancer treatment in the future.

Bone remodeling is a dynamic process between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Disrupted bone remodeling is a key factor in postmenopausal osteoporosis, a metabolic disorder characterized by deteriorated bone microarchitecture and increased risk of fracture. Recent studies have shown that piwi-binding RNA (piRNA) is involved in the pathogenesis of certain diseases at the post-transcriptional level. Here, we analyzed piRNA-63049 (piR-63049), which may play an essential role in bone remodeling. The expression of piR-63049 significantly increased in both bone tissues and plasma of osteoporotic rats and postmenopausal osteoporotic patients. Overexpressing piR-63049 could inhibit the osteoblastogenesis of bone marrow stromal cells (BMSCs) while knocking down piR-63049 could promote the osteoblastogenesis of BMSCs through the Wnt2b/β-catenin signaling pathway. Moreover, knocking-down piR-63049 (piR-63049-antagonist) in vivo could attenuate the bone loss in ovariectomized rats by promoting bone formation. Taken together, the current study shows that piR-63049 inhibits bone formation through the Wnt2b/β-catenin signaling pathway. This novel piRNA may be a potential target to increase bone formation in bone loss disorders such as postmenopausal osteoporosis.

Background: Autophagy regulates many cell functions related to cancer, ranging from cell proliferation and angiogenesis to metabolism. Due to the close relationship between autophagy and tumors, we investigated the predictive value of autophagy-related genes. Methods: Data from patients with hepatocellular carcinoma were obtained from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases. A regression analysis of differentially expressed genes was performed. Based on a prognostic model, patients were divided into a high-risk or low-risk group. Kaplan-Meier survival analyses of patients were conducted. The immune landscapes, as determined using single-sample gene set enrichment analysis (ssGSEA), exhibited different patterns in the two groups. The prognostic model was verified using the ICGC database and clinical data from patients collected at Zhongnan Hospital. Based on the results of multivariate Cox regression analysis, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC) had the largest hazard ratio, and thus we studied the effect of ATIC on autophagy and tumor progression by performing in vitro and in vivo experiments. Results: Fifty-eight autophagy-related genes were differentially expressed (false discovery rate (FDR)<0.05, log2 fold change (logFC)>1); 23 genes were related to the prognosis of patients. A prognostic model based on 12 genes (ATG10, ATIC, BIRC5, CAPN10, FKBP1A, GAPDH, HDAC1, PRKCD, RHEB, SPNS1, SQSTM1 and TMEM74) was constructed. A significant difference in survival rate was observed between the high-risk group and low-risk group distinguished by the model (P<0.001). The model had good predictive power (area under the curve (AUC)>0.7). Risk-related genes were related to the terms type II IFN response, MHC class I (P<0.001) and HLA (P<0.05). ATIC was confirmed to inhibit autophagy and promote the proliferation, invasion and metastasis of liver cancer cells through the AKT/Forkhead box subgroup O3 (FOXO3) signaling pathway in vitro and in vivo. Conclusions: The prediction model effectively predicts the survival time of patients with liver cancer. The risk score reflects the immune cell features and immune status of patients. ATIC inhibits autophagy and promotes the progression of liver cancer through the AKT/FOXO3 signaling pathway.

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

miRNA-223 has been previously reported to play an essential role in hepatic cholesterol homeostasis. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.

Given the heterogeneity of solid tumors, single-target CAR-T cell therapy often leads to recurrence, especially in ovarian cancer (OV). Here, we constructed a Tandem-CAR targeting two antigens with secretory activity (IL-12) to improve the effects of CAR-T cell therapy. Twenty coexpressed upregulated genes were identified from the GEO database, and we found FOLR1 (folate receptor 1) and MSLN (mesothelin) were specifically and highly expressed in cancer tissues and only 11.25% of samples were negative for both antigens. We observed an increased proliferation rate for these three CAR-T cells, and Tandem CAR-T cells could efficiently lyse antigen-positive OV cells in vitro and secrete higher levels of cytokines than single-target CAR-T cells. More importantly, in vivo experiments indicated that Tandem CAR-T cells markedly decreased tumor volume, exhibited enhanced antitumor activity, and prolonged mouse survival. Furthermore, the infiltration and persistence of T cells in the Tandem-CAR group were higher than those in the MSLN-CAR and Control-T groups but comparable to those in the FOLR1-CAR group. Collectively, this study demonstrated that Tandem CAR-T cells secreting IL-12 could enhance immunotherapeutic effects by reducing tumor antigen escape and increasing T cell functionality, which could be a promising therapeutic strategy for OV and other solid tumors.

Cepharanthine (CEP), a natural compound extracted from Stephania cepharantha Hayata, has been found to have the potential to treat a variety of tumors in recent years. This study aims to evaluate the anti-hepatocellular carcinoma (HCC) effect of CEP and determine its in-depth mechanism. In this study, Hep3B and HCCLM3 cells were selected to evaluate the antitumor effects of CEP in vitro, whereas tumor xenograft in nude mice was performed to make in vivo anti-tumor assessment. RNA-sequence (RNA-seq) was used to identify possible molecular targets and pathways. Further, gas chromatography mass spectrometry (GC-MS) was performed to assess the differential metabolites involved in mediating the effect of CEP on the HCC cell line. Our results showed that CEP treatment resulted in the dose-dependent inhibition of cell viability, migration, and proliferation and could also induce apoptosis in HCC cells. RNA-seq following CEP treatment identified 168 differentially expressed genes (DEGs), which were highly enriched in metabolism-associated pathways. In addition, CEP down-regulated many metabolites through the amino acid metabolism pathway. In vivo experiment showed that CEP significantly suppressed tumor growth. Our results indicate that CEP has significant antitumor effects and has the potential to be a candidate drug for HCC treatment.

Atherosclerotic vascular calcification contributes to increased risk of death in patients with cardiovascular diseases. Assessing the type and severity of inflammation is crucial in the treatment of numerous cardiovascular conditions. IL-1β, a potent proinflammatory cytokine, plays diverse roles in the pathogenesis of atherosclerotic vascular calcification. Several large-scale, population cohort trials have shown that the incidence of cardiovascular events is clinically reduced by the administration of anti-IL-1β therapy. Anti-IL-1β therapy might reduce the incidence of cardiovascular events by affecting atherosclerotic vascular calcification, but the mechanism underlying this effect remains unclear. In this review, we summarize current knowledge on the role of IL-1β in atherosclerotic vascular calcification, and describe the latest results reported in clinical trials evaluating anti-IL-1β therapies for the treatment of cardiovascular diseases. This review will aid in improving current understanding of the pathophysiological roles of IL-1β and mechanisms underlying its activity.

Rationale: Focal segmental glomerulosclerosis (FSGS) is characterized by the dysfunction of "post-mitotic" podocytes. The reentry of podocytes in the cell cycle will ultimately result in cell death. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of anaphase-promoting complex (APC)/cyclosome, precisely controls the metaphase to anaphase transition and ordered cell cycle progression. However, the role of MAD2B in FSGS podocyte injury remains unknown. Methods: To explore MAD2B function in podocyte cell cycle reentry, we used conditional mutant mice lacking MAD2B selectively in podocytes in ADR-induced FSGS murine model. Additionally, KU-55933, a specific inhibitor of ataxia-telangiectasia mutated (ATM) was utilized in vivo and in vitro to explore the role of ATM in regulating MAD2B. Results: The expression of MAD2B in podocytes was dramatically increased in patients with FSGS and ADR-treated mice along with podocyte cell cycle reentry. Podocyte-specific knockout of MAD2B effectively attenuated proteinuria, podocyte injury, and prevented the aberrant cell cycle reentry. By bioinformatics analysis we revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase abolished MAD2B-driven cell cycle reentry and alleviated podocyte impairment in FSGS murine model. In vitro studies by site-directed mutagenesis and immunoprecipitation we revealed ATM phosphorylated MAD2B and consequently hampered the ubiquitination of MAD2B in a phosphorylation-dependent manner. Conclusions: ATM kinase-MAD2B axis importantly contributes to the cell cycle reentry of podocytes, which is a novel pathogenic mechanism of FSGS, and may shed light on the development of its therapeutic approaches.

Extracellular vesicles derived from trophoblasts (T-EVs) play an important role in pregnancy, but the mechanism is not entirely clear. In this study, we found that HLA-E, which is mostly confined to the cytoplasm of trophoblast cells, was secreted by T-EVs. The level of HLA-E in T-EVs from unexplained recurrent spontaneous abortion (URSA) patients was lower than that in normal pregnancy (NP) and RSA patients who had an abnormal embryo karyotype (AK-RSA). T-EVs promoted secretion of IFN-γ and VEGFα by decidual NK (dNK) cells from URSA patients via HLA-E, VEGFα was necessary for angiogenesis and trophoblast growth, and IFN-γ inhibited Th17 induction. Glycolysis and oxidative phosphorylation (OxPhos) were involved in this process. Glycolysis but not OxPhos of dNK cells facilitated by T-EVs was dependent on mTORC1 activation. Inhibition of T-EV production in vivo increased the susceptibility of mice to embryo absorption, which was reversed by transferring exogenous T-EVs. T-EVs promoted secretion of IFN-γ and VEGFα by dNK cells to maintain pregnancy via Qa-1 in abortion-prone mouse models. This study reveals a new mechanism of pregnancy maintenance mediated by HLA-E via T-EVs.

While a number of therapeutic advances have been made in recent years, the overall survival of patients with head and neck squamous cell cancer (HNSCC) remains poor. MicroRNAs (miRNAs) are key drivers of oncogenic progression, with miR-34a-5p downregulation having been observed in many different tumor types. Here, we assessed the link between miR-34a-5p and HNSCC progression and the mechanistic basis for this relationship. Levels of miR-34a-5p in HNSCC tumors and cell lines were assessed via qPCR, after which we explored the functional importance of this miRNA in this oncogenic setting. Through luciferase reporter assays, the ability of miR-34a-5p to regulate flotillin-2 (FLOT-2) was further clarified. Overall, these analyses revealed that HNSCC tumors and cells exhibited marked miR-34a-5p downregulation that was linked to the progression of this tumor type. At a functional level, miR-34a-5p constrained the proliferation, migratory/invasive activity, and epithelial-mesenchymal transition induction in HNSCC cells. At the mechanistic level, miR-34a-5p was found to suppress FLOT-2 expression and to activate the MEK/ERK1/2 pathway. Overall, these results suggest that miR-34a-5p can function as a tumor suppressor miRNA in HNSCC owing to its ability to target FLOT-2, highlighting the promise of targeting this regulatory axis to treat HNSCC.

Background: Insulin therapy is the major treatment of glycaemic control in type I diabetes mellitus (DM) and advanced type II DM patients who fail to respond to oral hypoglycemic agents. Nonetheless, insulin therapy is deemed unsuccessful in controlling the incidence of diabetic retinopathy (DR) and is likely a risk factor. Berberine, an isoquinoline alkaloid, has caught great attention towards its anti-diabetic mechanisms. This study aims to investigate the effect of berberine in decelerating DR progression in insulin-treated DM. Methods: To better understand the therapeutic potential of berberine in the presence of insulin, we elaborated the action of mechanism whether berberine inhibited retinal expression of HIF-1α and VEGF through regulating AKT/mTOR pathway. Suppression of insulin-induced neovasculature of retina endothelial cells by berberine was also studied. Lastly, the in vivo efficacy and safety of berberine as adjuvant therapy for the treatment of DR were systemically investigated in experimental type I and type II DM mice with insulin treatment. Results: Among various types of retinal cells, the activity of HIF-1α and VEGF in retinal endothelial cells could be particularly and exclusively stimulated by insulin intervention, which could be inhibited by berberine treatment in a dose- and time-dependent manner. Berberine suppressed Akt/mTOR activity in these cells, and restoration of Akt/mTOR signalling attenuated berberine's inhibition on HIF-1α and VEGF expression. Berberine suppressed the progression of DR in experimental type I and type II diabetic mice receiving insulin therapy. Conclusion: Berberine improves insulin-induced diabetic retinopathy in type I and II diabetes through inhibiting insulin-induced activation of retinal endotheliocytes via Akt/mTOR/ HIF-1α/VEGF pathway.

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3'UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects. Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish. Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway. Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.

Circular RNAs (circRNAs) is a novel class of non-coding RNAs resulting from the non-canonical splicing of linear pre-mRNAs. However, the role of circRNAs in gastric cancer (GC) remains indistinct. This study aims to explore their potential modulation in GC and its prognostic value. We first screen for circRNA expression patterns in GC through GC and adjacent noncancerous tissues by microarray. Based on the bioinformatics analysis of the microarray data, we screened out a novel circRNA, circ-PTPDC1. Then we demonstrated that circ-PTPDC1 was up-regulated in GC cells, tissues, and serum. Its overexpression was positively correlated with age, invasion depth, advanced clinical stages, and worse survival in patients with GC. We further revealed that circ-PTPDC1 promotes the proliferation, migration, and invasion of GC cell lines via sponging miR-139-3p by regulating ELK1. Importantly, we identified that circ-PTPDC1 promotes tumor upgrowth and metabasis in vivo. Additionally, we established its prognostic prediction model based on the follow-up data of the patients. Our study revealed a novel regulatory mechanism and a comprehensive landscape of circ-PTPDC1 in GC, suggesting that circ-PTPDC1 has the potential to be a biomarker for early detection and prognostic prediction of GC.

Rationale: MicroRNAs (miRNAs) are endogenous ~22nt RNAs that play critical regulatory roles in various biological and pathological processes, including various cancers. Their function in renal cancer has not been fully elucidated. It has been reported that miR-196a can act as oncogenes or as tumor suppressors depending on their target genes. However, the molecular target for miR-196a and the underlying mechanism in miR-196a promoted cell migration and invasion in renal cancer is still not clear. Methods: The expression, survival and correlation between miR-196a and BRAM1 were investigated using TCGA analysis and validated by RT-PCR and western blot. To visualize the effect of Bram1 on tumor metastasis in vivo, NOD-SCID gamma (NSG) mice were intravenously injected with RCC4 cells (106 cells/mouse) or RCC4 overexpressing Bram1. In addition, cell proliferation assays, migration and invasion assays were performed to examine the role of miR-196a in renal cells in vitro. Furthermore, immunoprecipitation was done to explore the binding targets of Bram1. Results: TCGA gene expression data from renal clear cell carcinoma patients showed a lower level of Bram1 expression in patients' specimens compared to adjacent normal tissues. Moreover, Kaplan‑Meier survival data clearly show that high expression of Bram1correlates to poor prognosis in renal carcinoma patients. Our mouse metastasis model confirmed that Bram1 overexpression resulted in an inhibition in tumor metastasis. Target-prediction analysis and dual-luciferase reporter assay demonstrated that Bram1 is a direct target of miR-196a in renal cells. Further, our in vitro functional assays revealed that miR-196a promotes renal cell proliferation, migration, and invasion. Rescue of Bram1 expression reversed miR-196a-induced cell migration. MiR-196a promotes renal cancer cell migration by directly targeting Bram1 and inhibits Smad1/5/8 phosphorylation and MAPK pathways through BMPR1A and EGFR. Conclusions: Our findings thus provide a new mechanism on the oncogenic role of miR-196a and the tumor-suppressive role of Bram1 in renal cancer cells. Dysregulated miR-196a and Bram1 represent potential prognostic biomarkers and may have therapeutic applications in renal cancer.

Mastitis causes great psychological and physical pain among women. Our previous studies found that niacin has anti-inflammatory effect, and the realization of this function depends on GPR109A. However, there are no previous reports about the anti-inflammatory function of GPR109A in mastitis. In our study, we observed the effect of niacin on the WT and GPR109A-/- mice mastitis model. The results showed that administration of niacin to WT mice reduced the damage, proinflammatory mediators and protected the integrity of the blood milk barrier in mammary gland. While in GPR109A-/- mice, there was no effect on the above indexes. In mammary epithelial cells, GPR109A was able to promote autophagy and Nrf2 nuclear import through AMPK. In LPS-induced mammary epithelial cells, niacin inhibited the LPS-induced inflammatory response and downregulation of tight junction proteins, and these effects were eliminated by knocking down GPR109A, blocking autophagy or inhibiting Nrf2 nuclear import. These results indicate that in mastitis, GPR109A promotes autophagy and Nrf2 nuclear import through AMPK, thereby inhibiting inflammatory damage to the mammary gland and repairing the blood milk barrier. Our results suggested that GPR109A may be a potential target for the treatment of mastitis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. CD38 was initially identified as a lymphocyte surface antigen and then has been found to exist in a variety of cell types. Our previous studies showed that CD38-/- mice were resistant to high-fat diet (HFD)-induced obesity. However, the role and mechanism of CD38 in HFD-induced NAFLD is still unclear. Here, we reported that CD38-/- mice significantly alleviated HFD-induced hepatic steatosis. HFD or oleic acid (OA) remarkably increased the mRNA and protein expressions of CD38 in mouse hepatic tissues and primary hepatocytes or hepatic cell lines in vitro and in vivo, suggesting that CD38 might play a role in HFD-induced hepatic steatosis. We observed that CD38 deficiency markedly decreased HFD- or OA-induced the lipid accumulation and oxidative stress in CD38-/- livers or primary hepatocytes, respectively. In contrast, overexpression of CD38 in Hep1-6 cells aggravated OA-induced lipid accumulation and oxidative stress. Furthermore, CD38 deficiency markedly inhibited HFD- or OA-induced the expressions of NOX4, and increased the expression of PPARα, CPT1, ACOX1 and SOD2 in liver tissue and hepatocytes from CD38-/- mice, indicating that CD38 deficiency-mediated the enhancement of fatty acid oxidation and the inhibition of oxidative stress contributed to protecting NAFLD. More importantly, Ex527 (Sirt1 inhibitor) and 3-TYP (Sirt3 inhibitor) significantly enhanced OA-induced lipid accumulation and oxidative stress in CD38-/- primary hepatocytes, suggesting that the anti-lipid accumulation of CD38 deficiency might be dependent on NAD/Sirtuins-mediated enhancement of FAA β-oxidation and suppression of oxidative stress in hepatocytes. In conclusion, we demonstrated that CD38 deficiency protected mice from HFD-induced NAFLD by reducing lipid accumulation and suppressing oxidative stress via activating NAD/Sirtuins signaling pathways.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.

Rationale: Nonalcoholic steatohepatitis (NASH), as one of the key stages in the development of nonalcoholic fatty liver disease (NAFLD), can directly progress to HCC, but the underlying mechanism is not fully understood. Methods: Differentially expressed genes (DEGs) in each stage of disease development were studied through a GEO dataset deriving from a Stelic Animal Model (STAM), which can simulate the evolution of NAFLD/NASH to HCC in humans. GSVA analysis was performed to analyze the differentially expressed oncogenic signatures in each stage. A human NAFLD-related dataset from GEO database was utilized for gene expression verification and further validated in the protein level in STAM mice. Small molecule inhibitors were applied to STAM mice for investigating whether inhibition of the LPL/FABP4/CPT1 axis could prevent the occurrence of NASH-related HCC in vivo. Microsphere formation and clonal formation assays in vitro were applied to study if inhibition of the LPL/FABP4/CPT1 axis can reduce the viability of liver cancer stem cells (LCSCs). Results: We found that upregulation of the LPL/FABP4/CPT1 molecular axis, as a fatty acid metabolic reprogramming process, occurred specifically during the NASH phase. GSVA analysis showed widespread activation of a large number of oncogenic signals, which may contribute to malignant transformation during NASH. Furthermore, inhibition of the LPL/FABP4/CPT1 axis could effectively delay the tumor growth in STAM mice. Cell assays revealed inhibitors targeting this axis can significantly reduce the sphere-forming, proliferation, and clonality of LCSCs. Conclusion: These results suggest that activation of the LPL/FABP4/CPT1 axis is essential for LCSCs maintenance, which acts synergistically with a variety of up-regulated oncogenic signals that drive the hepatocyte-LCSCs transdifferentiation during NASH to HCC progression. Thus, targeting the LPL/FABP4/CPT1 axis may provide a potential direction for NASH-related HCC prevention.

Lipid metabolites are emerging as pivotal regulators of protein function and cell signaling. The availability of intracellular fatty acid is tightly regulated by glycolipid metabolism and may affect human body through many biological mechanisms. Recent studies have demonstrated palmitate, either from exogenous fatty acid uptake or de novo fatty acid synthesis, may serve as the substrate for protein palmitoylation and regulate protein function via palmitoylation. Palmitoylation, the most-studied protein lipidation, encompasses the reversible covalent attachment of palmitate moieties to protein cysteine residues. It controls various cellular physiological processes and alters protein stability, conformation, localization, membrane association and interaction with other effectors. Dysregulation of palmitoylation has been implicated in a plethora of diseases, such as metabolic syndrome, cancers, neurological disorders and infections. Accordingly, it could be one of the molecular mechanisms underlying the impact of palmitate metabolite on cellular homeostasis and human diseases. Herein, we explore the relationship between lipid metabolites and the regulation of protein function through palmitoylation. We review the current progress made on the putative role of palmitate in altering the palmitoylation of key proteins and thus contributing to the pathogenesis of various diseases, among which we focus on metabolic disorders, cancers, inflammation and infections, neurodegenerative diseases. We also highlight the opportunities and new therapeutics to target palmitoylation in disease development.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.

Background: Inflammation and apoptosis play a crucial role in the progression of nonalcoholic steatohepatitis (NASH). Suppressor of cytokine signaling 2 (SOCS2) is one of classic negative regulators of cytokine signaling, which has recently been described as anti-inflammatory mediators. However, the role of SOCS2 in macrophages during NASH progression and the relationship among SOCS2, inflammation, apoptosis and NASH is largely unknown. Herein, we aimed to study the function of SOCS2 in NASH progression. Methods: We detected SOCS2 expression in macrophages in human subjects without steatosis, with simple steatosis and with NASH to confirm the relationship between SOCS2 and NASH. Free fatty acids was used to establish stress environment in RAW 264.7 cell lines stably overexpressing or knockdown SOCS2. In vitro and vivo assays also performed to study the molecular function of SOCS2 in NASH progression. Findings: Our human samples illustrated that SOCS2 was decreased in macrophages during NASH progression and was negatively correlated to NASH level. Meanwhile, In vitro assays showed SOCS2 overexpression in macrophages suppressed inflammation and apoptosis via inhibiting NF-κB signaling pathway, while SOCS2 knock-down in macrophages caused an increased activation of NF-κB, which could be blocked by ammonium 1-pyrrolidinedithiocarbamate (PDTC). In addition, SOCS2 in macrophages also suppressed inflammation via limiting the activation of inflammasomes. Consistent with these, our BMT model also confirmed the SOCS2 function in macrophages during NASH. Interpretation: Our data strongly indicate that SOCS2 plays a role in inhibiting inflammation and apoptosis via NF-κB and inflammasome signaling pathway in macrophages during NASH. Further studies are required to explore the potential preventive and therapeutic strategies of SOCS2 for this common liver disease.