CircCDK14 ameliorates interleukin-1ß-induced chondrocyte damage by the miR-1183/KLF5 pathway in osteoarthritis.

BACKGROUND: The pathogenesis of osteoarthritis (OA), an endemic and debilitating disease, remains unclear. The study aimed to reveal the role of circular RNA cyclin dependent kinase 14 (circCDK14) in OA development and the underlying mechanism. METHODS: Human chondrocytes were stimulated by 10 ng/mL interleukin-1ß (IL-1ß) to mimic OA cell model. The RNA expression of circCDK14, microRNA-1183 (miR-1183) and kruppel like factor 5 (KLF5) was checked through quantitative real-time polymerase chain reaction. Western blot was employed to detect protein expression. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine and flow cytometry analysis, respectively. Starbase online database was performed to identify the interaction between miR-1183 and circCDK14 or KLF5. Exosomes were isolated by differential centrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. RESULTS: CircCDK14 and KLF5 expression were significantly decreased, while miR-1183 was increased in OA cartilage tissues and IL-1ß-treated chondrocytes in comparison with controls. CircCDK14 overexpression attenuated the inhibitory effect of IL-1ß treatment on cell proliferation and the promoting effects on cell apoptosis and extracellular matrix degradation. Additionally, miR-1183 was targeted by circCDK14, and miR-1183 mimics reversed circCDK14-mediated actions in IL-1ß-treated chondrocytes. The knockdown of KLF5, a target mRNA of miR-1183, also rescued the effects of miR-1183 inhibitors in IL-1ß-induced chondrocytes. Moreover, circCDK14 could induce KLF5 expression by interacting with miR-1183. Further, exosomal circCDK14 had a high diagnostic value in OA. CONCLUSION: CircCDK14 reintroduction assuaged IL-1ß-caused chondrocyte damage by the miR-1183/KLF5 pathway, providing a diagnostic biomarker for OA.

Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.

Positive feedback between retinoic acid and 2-phospho-L-ascorbic acid trisodium salt during somatic cell reprogramming.

Retinoic acid (RA) and 2-phospho-L-ascorbic acid trisodium salt (AscPNa) promote the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. In the current studies, the lower abilities of RA and AscPNa to promote reprogramming in the presence of each other suggested that they may share downstream pathways at least partially. The hypothesis was further supported by the RNA-seq analysis which demonstrated a high-level overlap between RA-activated and AscPNa activated genes during reprogramming. In addition, RA upregulated Glut1/3, facilitated the membrane transportation of dehydroascorbic acid, the oxidized form of L-ascorbic acid, and subsequently maintained intracellular L-ascorbic acid at higher level and for longer time. On the other hand, AscPNa facilitated the mesenchymal-epithelial transition during reprogramming, downregulated key mesenchymal transcriptional factors like Zeb1 and Twist1, subsequently suppressed the expression of Cyp26a1/b1 which mediates the metabolism of RA, and sustained the intracellular level of RA. Furthermore, the different abilities of RA and AscPNa to induce mesenchymal-epithelial transition, pluripotency, and neuronal differentiation explain their complex contribution to reprogramming when used individually or in combination. Therefore, the current studies identified a positive feedback between RA and AscPNa, or possibility between vitamin A and C, and further explored their contributions to reprogramming.

Epithelial-Mesenchymal Transition and Metabolic Switching in Cancer: Lessons From Somatic Cell Reprogramming.

Epithelial-mesenchymal transition (EMT) and its critical roles during cancer progression have long been recognized and extensively reviewed. Recent studies on the generation of induced pluripotent stem cells (iPSCs) have established the connections among EMT, energy metabolism, DNA methylation, and histone modification. Since energy metabolism, DNA methylation, and histone modification are important for cancer development and there are common characteristics between cancer cells and stem cells, it is reasonable to identify mechanisms that have been established during both reprogramming and cancer progression. In the current review, we start from a brief review on EMT and related processes during cancer progression, and then switch to the EMT during somatic cell reprogramming. We summarize the connection between EMT and metabolic switch during reprogramming, and further review the involvements of DNA methylation and cell proliferation. The connections between EMT and mesenchymal-epithelial transition (MET) and cellular aspects including DNA methylation, histone modification and energy metabolism may provide potential new targets for cancer diagnosis and treatment.

Naloxone regulates the differentiation of neural stem cells via a receptor-independent pathway.

The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosine dioxygenase ten-eleven translocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.

Morphine and Naloxone Facilitate Neural Stem Cells Proliferation via a TET1-Dependent and Receptor-Independent Pathway.

Normally, opioids function in a receptor-dependent manner. They bind to opioid receptors, activate or inhibit receptor activation, and subsequently modulate downstream signal transduction. However, the complex functions of opioids and the low expression of opioid receptors and their endogenous peptide agonists in neural stem cells (NSCs) suggest that some opioids may also modulate NSCs via a receptor-independent pathway. In the current study, two opioids, morphine and naloxone, are demonstrated to facilitate NSC proliferation via a receptor-independent and ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-dependent pathway. Morphine and naloxone penetrate cell membrane, bind to TET1 protein via three key residues (1,880-1,882), and subsequently result in facilitated proliferation of NSCs. In addition, the two opioids also inhibit the DNA demethylation ability of TET1. In summary, the current results connect opioids and DNA demethylation directly at least in NSCs and extend our understanding on both opioids and NSCs.

Metabolic switch and epithelial-mesenchymal transition cooperate to regulate pluripotency.

Both metabolic switch from oxidative phosphorylation to glycolysis (OGS) and epithelial-mesenchymal transition (EMT) promote cellular reprogramming at early stages. However, their connections have not been elucidated. Here, when a chemically defined medium was used to induce early EMT during mouse reprogramming, a facilitated OGS was also observed at the same time. Additional investigations suggested that the two events formed a positive feedback loop via transcriptional activation, cooperated to upregulate epigenetic factors such as Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5, and accelerated pluripotency induction at the early stage. However, at late stages, by over-inducing glycolysis and preventing the necessary mesenchymal-epithelial transition, the two events trapped the cells at a new pluripotency state between naïve and primed states and inhibited further reprogramming toward the naïve state. In addition, the pluripotent stem cells at the new state have high similarity to epiblasts from E4.5 and E5.5 embryos, and have distinct characteristics from the previously reported epiblast-like or formative states. Therefore, the time-dependent cooperation between OGS and EMT in regulating pluripotency should extend our understanding of related fields.

Regulated expression of PTPRJ by COX-2/PGE2 axis in endothelial cells.

BACKGROUND: This study was designed to examine a novel role of COX-2/PGE2 signaling as a regulator of PTPRJ expression in endothelial cells. METHODS: A bioinformatics analysis of a whole genome array was carried out to search for regulators of PTPRJ expression in endothelial cells. PTPRJ expression was also measured in endothelial cells derived from a balloon injury-induced neointimal hyperplasia model in male New Zealand Rabbits. Changes in PTPRJ expression in HUVEC cells was examined by RT-PCR and western blotting after transfection of COX-2 plasmids or treatment with varying concentrations of a COX-2 inhibitor. RESULTS: A significant correlation was identified between COX-2 and PTPRJ in GSE39264 (Pearson correlation coefficient  =  -0.87; n = 22; P < 0.01, two-tailed). PTPRJ expression was reduced during the progression of neointimal hyperplasia after balloon injury, which correlated with an increase in COX-2 expression. In HUVECs, after transfection with 1 µg/ml, 0.5 µg/ml, or 0.25 µg/ml COX-2 plasmids, PTPRJ protein expression was reduced to 0.60- (± 0.08), 0.75- (± 0.09), and 0.88- (± 0.04) fold, respectively, while mRNA expression was reduced to 0.15- (± 0.03), 0.26- (± 0.05), and 0.47- (± 0.09) fold, respectively. After treatment of HUVECs with 10 µmol/L or 20 µmol/L celecoxib, the reduction in PTPRJ expression induced by COX-2 over-expression was not only rescued but in fact increased by 2.05-fold (± 0.28) and 3.34-fold (± 0.37), respectively, compared with control. CONCLUSIONS: Our results suggest that COX-2/PGE2 signaling may function as a negative regulator of PTPRJ expression in endothelial cells both in vivo and vitro.

Endoscopic management of early-stage chronic pancreatitis based on M-ANNHEIM classification system: a prospective study.

OBJECTIVE: The aim of this study was to evaluate the M-ANNHEIM classification system to categorize patients with chronic pancreatitis (CP). METHODS: All symptomatic patients recruited from the gastroenterology outpatient clinic of Changhai Hospital (n = 89) were routinely evaluated by magnetic resonance cholangiopancreatography and contrast-enhanced computed tomography. M-ANNHEIM clinical staging was used to categorize patients. The primary outcome measure was pain during the 2-year follow-up period, expressed as mean Izbicki pain scores obtained before and after endotherapy. RESULTS: There was a significant improvement in mean (SD) Izbicki pain scores obtained at 24 months among patients receiving endoscopic therapy at stage 1a compared with those at stage 1b (4.9 [3.0] vs 14.5 [6.9], P = 0.012). Furthermore, significantly more patients receiving endoscopic therapy at stage 1a achieved complete + partial pain relief after 2-year follow-up than those at stage 1b (95.2% vs 78.0%, P = 0.021). There was no exocrine or endocrine insufficiency, but a significantly greater number of patients treated at stage 1a had post-endoscopic retrograde cholangiopancreatography pancreatitis compared with those at stage 1b (10.5% vs 2.7%, P = 0.025). CONCLUSIONS: We demonstrated that a sophisticated M-ANNHEIM classification system for CP will improve diagnosis by allowing for more timely intervention. Furthermore, prompt treatment of CP may achieve improved pain relief and patient outcomes.

Comprehensive screening for PRSS1, SPINK1, CFTR, CTRC and CLDN2 gene mutations in Chinese paediatric patients with idiopathic chronic pancreatitis: a cohort study.

OBJECTIVE: Genetic alterations may contribute to chronic pancreatitis (CP) in Chinese young patients. This study was designed to investigate mutations of cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor or serine protease inhibitor Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR), chymotrypsin C (CTRC) and CLDN2 genes and the copy number variations (CNVs) of PRSS1 and asses associations with the development of idiopathic CP (ICP) in Chinese children. DESIGN: Retrospective. SETTING: A single center. PARTICIPANTS: 75 ICP Chinese children (40 boys and 35 girls). PRIMARY AND SECONDARY OUTCOME MEASURES: Mutations of PRSS1, SPINK1, CFTR, CTRC and CLDN2 genes and CNVs. RESULTS: 7 patients had heterozygous mutations in PRSS1, that is, N29I (n=1), R122H or R122C (n=6). The CNVs of PRSS1 in five patients had abnormal copies (1 copy (n=4), five copies (n=1)). 43 patients had IVS3+2T>C (rs148954387) (10 homozygous and 33 heterozygous) in SPINK1. None of the PRSS1 mutation patients carried a SPINK1 mutation. Frequency of PRSS1 and SPINK1 mutations was 9.3% and 57.3%, respectively, with an overall frequency of 66.6% (50/75). In addition, one patient had a novel deletion of CFTR (GCTTCCTA from c.500 to c.508 leading to the shortened polypeptide molecule via a stop codon). Another patient had a novel missense in CLDN2 exon 2 (c.592A>C mutation). Clinically, patients with SPINK1 mutations had a higher rate of pancreatic duct stones, pancreatic pseudocyst and pancreatic calcification than those without SPINK1 mutations (p<0.05). CONCLUSIONS: SPINK1 mutations were more commonly associated with Chinese children with ICP. SPINK1 IVS3+2T>C mutation may play an important role in the pathogenesis of Chinese paediatric ICP. However, further study is needed to confirm and to investigate the role of these genes in the development of Chinese ICP.

[Correlation analysis of tissue factor promotor polymorphism -1208I/D and venous thromboembolism].

The study was aimed to investigate the correlation of tissue factor promotor polymorphism -1208I/D with the venous thromboembolism in patients. Tissue factor promotor polymorphism -1208 was detected by polymerase chain reaction (PCR) and DNA sequencing in 96 cases of DVT, 14 cases of PE and 59 nonthrombosis normal individuals. The results showed that the allele containing a 18-bp nucleotides insertion at -1208. 67.8% of normal individuals exhibited D/D, 25.4% were heterozygous I/D, and 6.8% were homozygous for I/I. DVT group and PE group exhibited a similar distributions (62.5%D/D, 29.8% I/D, 8.3% I/I and 57.1% D/D, 35.7% I/D, 7.1% I/I). The allele frequencies of D and the allele frequencies of I in the normal control, DVT and PE groups were 80.5%, 77.1%, 75.0% and 19.5%, 22.9%, 25.0% respectively. There was no significant difference in the TF-12081/D genotype frequency between the groups of patients and normal individuals. In conclusion, there is no correlation of the tissue factor promotor polymorphism -1208I/D in the patients with venous thromboembolism. The gene of promoter -1208I/D may not be a major susceptible gene of VTE in Chinese Han. Further investigations would be necessary to define accurately tissue factor gene polymorphisms.

[Determination of heavy metals for RoHS compliance by ICP-OES spectrometry coupled with microwave extraction system].

The harm of heavy metals contained in electronic and electrical equipment (EEE) on environment is of high concern by human. Aiming to handle the great challenge of RoHS compliance, the determinations of trace or ultratrace chromium (Cr), cadmium (Cd), mercury (Hg) and lead (Pb) by inductively coupled plasma optical emission spectrometry (ICP-OES) was performed in the present paper, wherein, microwave extraction technology was used to prepare the sample solutions. In addition, the precision, recovery, repeatability and interference issues of this method were also discussed. The results exhibited that using the microwave extraction system to prepare samples is more quick, lossless, contamination-free in comparison with the conventional extraction methods such as dry ashing, wet-oven extraction etc. By analyzing the recoveries of these four heavy metals over different working time and wavelengths, the good recovery range between 85% and 115% showed that there was only tiny loss or contamination during the process of microwave extraction, sample introduction and ICP detection. Repeatability experiments proved that ICP plasma had a good stability during the working time and the matrix effect was small. Interference was a problem troublesome for atomic absorption spectrometry (AAS), however, the techniques of standard additions or inter-element correction (IEC) method can effectively eliminated the interferences of Ni, As, Fe etc. with the Cd determination. By employing the multi-wavelengths and two correction point methods, the issues of background curve sloping shift and spectra overlap were successfully overcome. Besides, for the determinations of trace heavy metal elements, the relative standard deviation (RSD) was less than 3% and the detection limits were less than 1 microg x L(-10 (3sigma, n = 5) for samples, standard solutions, and standard additions, which proved that ICP-OES has a good precision and high reliability. This provided a reliable technique support for electronic and electrical (EE) industries to comply with RoHS directive.