This study was to investigate the functional role of RNA methyltransferase METTL3, an enzyme catalyzes the formation of N6-methyladenosine (m6A) on the target mRNA, in the development of ...osteoarthritis (OA) and the underlying mechanism.
Cytokine IL-1β was used to stimulate the chondroprogenitor cell line ATDC5 cells to mimic the inflammatory condition in vitro. The level of METTL3 mRNA and m6A as well as inflammatory cytokines were detected by qRT-PCR. Cell activity was detected by CCK-8. The rate of apoptotic cell was measured by flow cytometry. Western blot was used to detect the levels of NF-κB signaling molecules and collagen in cells. Methylation inhibitor cycloleucine and methyl donor betaine were used to treat collagenase-induced OA mice.
In IL-1β-treated ATDC5 cells, the METTL3 mRNA levels and the percentage of m6A methylated mRNA of total mRNA were increased in a dose-dependent manner. Silencing of METTL3 by shRNA reduced the percentage of IL-1β-induced apoptosis, suppressed IL-1β-induced increased inflammatory cytokines levels and activation of NF-κB signaling in chondrocytes. Moreover, silencing of METTL3 promotes degradation of extracellular matrix (ECM) by reducing the expression of MMP-13 and Coll X, elevating the expression of Aggrecan and Coll II. In a OA mouse model induced by collagenase, injection of methylation inhibitor cycloleucine or methyl donor betaine does not affects METTL3 mRNA expression, but significantly inhibits or promotes the total level of m6A as well as inflammatory condition and ECM degradation, respectively.
METTL3 has a functional role in mediates osteoarthritis progression by regulating NF-κB signaling and ECM synthesis in chondrocytes that shed insight on developing preventive and curative strategies for OA by focusing on METTL3 and mRNA methylation.
•METTL3 levels and m6A methylated mRNA were increased in IL-1β-treated ATDC5 cells.•Silencing of METTL3 promotes degradation of extracellular matrix by reducing the expression of MMP-13 and Coll X.•Methylation inhibitor significantly inhibits the total level of m6A as well as inflammatory condition and ECM degradation.•METTL3 promotes osteoarthritis progression by regulating NF-κB signaling and ECM synthesis in chondrocytes.
Osteoarthritis (OA), the most common form of arthritis, is a very common joint disease that often affects middle-aged to elderly people. However, current treatment options for OA are predominantly ...palliative. Thus, understanding its pathological process and exploring its potential therapeutic approaches are of great importance. Rat chondrocytes were isolated and exposed to hydrogen peroxide (H
2
O
2
) to mimic OA. The effects of H
2
O
2
on ubiquitin-specific protease 7 (USP7) expression, reactive oxygen species (ROS) levels, proliferation, inflammatory cytokine release, and pyroptosis were measured. USP7 was knocked down (KD) or overexpressed to investigate the role of USP7 in OA. Co-immunoprecipitation (Co-IP) was used to study the interaction between USP7 and NAD(P)H oxidases (NOX)4 as well as NOX4 ubiquitination. NOX4 inhibitor was applied to study the involvement of NOX4 in USP7-mediated OA development. USP7 inhibitor was given to OA animals to further investigate the role of USP7 in OA
in vivo
. Moreover, H
2
O
2
treatment significantly increased USP7 expression, enhanced ROS levels, and inhibited proliferation in rat chondrocytes. The overexpression of USP7 enhanced pyroptosis, ROS production, interleukin (IL)-1β and IL-18 levels, and the expression level of NLRP3, GSDMD-N, active caspase-1, pro-caspase-1, matrix metalloproteinases (MMP) 1, and MMP13, which was abolished by ROS inhibition. The USP7 KD protected rat chondrocytes against H
2
O
2
-induced injury. Co-IP results showed that USP7 interacted with NOX4, and USP7 KD enhanced NOX4 ubiquitinylation. The inhibition of NOX4 blocked the pro-OA effect of USP7. Moreover, the USP7 inhibitor given to OA animals suppressed OA
in vivo
. USP7 inhibited NOX4 ubiquitination for degradation which leads to elevated ROS production. ROS subsequently activates NLPR3 inflammasome, leading to enhanced production of IL-1β and IL-18, GSDMD-N-dependent pyroptosis, and extracellular matrix remodeling. Thus, UPS7 contributes to the progression of OA via NOX4/ROS/NLPR3 axis.
To investigate the low-temperature adaptability of different provenances of Ziziphus jujuba var. spinosa, we used 21 clones from seven provenances as experimental materials and observed the changes ...in physiological and biochemical indicators and the characteristics of anatomical structures under low-temperature stress. A comprehensive evaluation of their cold resistance was conducted using the membership function method. As the temperature decreased, the relative electrical conductivity (REC) of clone 89 became stable and had the lowest LT50 value (−44.04 °C). The cold-resistant Z. jujuba var. spinosa had a higher bound water/free water (BW/FW) ratio and antioxidant enzyme activity and accumulated large quantities of osmotic regulatory substances. Higher xylem, phloem, and xylem–cortex ratios and greater conduit density enhanced the cold resistance of Z. jujuba var. spinosa. The membership function values of clones 89, 90, 91, 604, and 612 were greater than 0.6, indicating that they could be evaluated as resources with the potential for low-temperature resistance. The cold resistance rankings for the different provenances were as follows: Kazuo, Liaoning > Jiaxian, Shaanxi > Fuxing, Heibei > Changqing, Shandong > Neiqiu, Heibei > Yanchuan, Shaanxi > Xiaxian, Shanxi. These results provide a scientific basis for the rapid and accurate identification of cold resistance in Z. jujuba var. spinosa resources and the breeding and cultivation of new cold-resistant varieties of this subspecies.
The basic helix-loop-helix (bHLH) family is one of the most well-known transcription factor families in plants, and it regulates growth, development, and abiotic stress responses. However, systematic ...analyses of the bHLH gene family in
Prunus sibirica
have not been reported to date. In this study, 104
PsbHLHs
were identified and classified into 23 subfamilies that were unevenly distributed on eight chromosomes. Nineteen pairs of segmental replication genes and ten pairs of tandem replication genes were identified, and all duplicated gene pairs were under purifying selection.
PsbHLHs
of the same subfamily usually share similar motif compositions and exon-intron structures.
PsbHLHs
contain multiple stress-responsive elements.
PsbHLHs
exhibit functional diversity by interacting and coordinating with other members. Twenty
PsbHLHs
showed varying degrees of expression. Eleven genes up-regulated and nine genes down-regulated in −4°C. The majority of
PsbHLHs
were highly expressed in the roots and pistils. Transient transfection experiments demonstrated that transgenic plants with overexpressed
PsbHLH42
have better cold tolerance. In conclusion, the results of this study have significant implications for future research on the involvement of bHLH genes in the development and stress responses of
Prunus sibirica
.
Wear particle-induced periprosthetic osteolysis (PPO) is a common long-term complication of total joint arthroplasty, and represents the major cause of aseptic loosening and subsequent implant ...failure. Previous studies have identified the central role of osteoclast-mediated bone resorption in the pathogenesis of PPO. Thus, therapeutic approaches of inhibiting osteoclast formation and activity are considered to be of great potential to prevent and treat this osteolytic disease. Hedgehog (Hh) signaling has been shown to play an important role in promoting osteoblast differentiation and bone formation. While Hh signaling is also implicated in regulating osteoclastogenesis, whether it can directly inhibit osteoclast differentiation and bone resorption remains controversial. Moreover, its potential therapeutic effects on PPO have never been assessed. In this study, we explored the cell-autonomous role of Hh signaling in regulating osteoclastogenesis and its therapeutic potential in preventing wear particle-induced osteolysis.
Hh signaling was activated in macrophages by genetically ablating
in these cells using
or by treating them with purmorphamine (PM), a pharmacological activator of Smoothened (Smo).
cell-autonomous effects of Hh pathway activation on RANKL-induced osteoclast differentiation and activity were evaluated by TRAP staining, phalloidin staining, qPCR analyses, and bone resorption assays.
evaluation of its therapeutic efficacy against PPO was performed in a murine calvarial model of titanium particle-induced osteolysis by μCT and histological analyses. Mechanistic details were explored in RANKL-treated macrophages through Western blot analyses.
We found that
deletion or PM treatment potently activated Hh signaling in macrophages, and strongly inhibited RANKL-induced TRAP
osteoclast production, F-actin ring formation, osteoclast-specific gene expression, and osteoclast activity
. Furthermore, we found that
deletion or PM administration significantly attenuated titanium particle-induced osteoclast formation and bone loss
. Our mechanistic study revealed that activation of Hh signaling suppressed RANKL-induced activation of JNK pathway and downregulated protein levels of two key osteoclastic transcriptional factors, c-Fos and its downstream target NFATc1.
Both genetic and pharmacological activation of Hh signaling can cell-autonomously inhibit RANKL-induced osteoclast differentiation and activity
and protect against titanium particle-induced osteolysis
. Mechanistically, Hh signaling hinders osteoclastogenesis partly through suppressing the JNK/c-Fos-NFATc1 cascade. Thus, Hh signaling may serve as a promising therapeutic target for the prevention and treatment of PPO and other osteolytic diseases.
Histology-based analyses are important tools to dissect cellular and molecular mechanisms of skeletal homeostasis, diseases, and regeneration. The success of these efforts is highly dependent on ...rapidly obtaining high-quality sections of mineralized skeletal tissues suitable for various analyses. However, the current techniques for preparing such sections are still far from satisfactory. This study aimed to develop a new approach for preparing high-quality undecalcified bone sections applicable to various histological analyses.
Two important modifications were made to the conventional Cryojane Tape-Transfer System, including utilization of an optimized adhesive to prepare adhesive glass slides for improving the transfer efficiency, and a cheap conventional benchtop UV transilluminator for UV curing. Cryosections of undecalcified rodent bones were prepared using this modified tape transfer approach, and their tissue morphology and structural integrity were visually examined. A variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as 5-Ethynyl-2’-deoxyuridine (EdU) and TUNEL assays, were performed on these sections.
We developed a modified version of tape transfer approach that can prepare cryosections of undecalcified rodent adult bones within 4 days at a low cost. Bone sections prepared by this approach exhibited good tissue morphology and structural integrity. Moreover, these sections were applicable to a variety of histological analyses, including calcein labeling, Von kossa staining, immunofluorescence, and enzymatic activity staining as well as EdU and TUNEL assays.
The tape transfer approach we developed provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections valuable for bone research.
Our research provides a rapid, affordable, and easy learning method for preparing high-quality undecalcified bone sections that can be potentially used for accurate diagnosis of various bone disorders and evaluation of the efficacy of different therapies in the treatment of these diseases.
Human osteosarcoma is the most common primary cancer of the bone. Multiple mechanisms underlying cell growth, apoptosis, bone development, and drug resistance are important in the development of ...osteosarcoma therapy, which remains to be fully studied.
We collected thirty-paired tumor tissues and the adjacent normal ones from osteosarcoma patients. Two osteosarcoma cell lines (SAOS2, U2OS) were used for in vitro experiments. RT-qPCR and Western blot were used for gene expression detection. We applied starBase to predict the potential binding sites. Then, the luciferase reporter assay was used to confirm the potential direct interaction. Besides, we applied CCK-8, EdU assay, and flow cytometric assays to detect cell growth and apoptosis rate. We used wound healing and transwell assays to determine cell migration and invasion abilities. Additionally, we constructed a cisplatin-resistant osteosarcoma cell line to study the potential impact of the regulatory axis on drug resistance.
Small nucleolar RNA host gene 16 (SNHG16) and autophagy-related 4B (ATG4B) were significantly upregulated in osteosarcoma tissues than the normal ones, and the higher expression level of SNHG16 predicted a poor prognosis in osteosarcoma patients. By contrast, the expression level of miR-16 was markedly lower in tumor tissues and was negatively correlated with SNHG16 (p < 0.001). SNHG16 was shown to promote cell growth, migration, and invasion, while miR-16 reversed this impact. Meanwhile, overexpression of ATG4B significantly promoted the development of osteosarcoma cells attenuated by SNHG16 knockdown or miR-16 mimics. Specifically, overexpression of ATG4B promoted cisplatin-induced autophagy and inhibited cell apoptosis rate, which enhanced the cisplatin resistance in osteosarcoma cell lines.
Overall, our findings showed the importance of the regulatory axis of SNHG16/miR-16/ATG4B underlying osteosarcoma progression and chemoresistance to cisplatin. This research would benefit the therapy development in the treatment of osteosarcoma.
•SNHG16 enhanced osteosarcoma cell activity and was associated with poor prognosis in osteosarcoma patients.•MiR-16 could significantly suppress the function of SNHG16 by direct interaction.•ATG4B directly interacted with miR-16 to rescue the cell activity attenuated by miR-16 mimics or SNHG16 knockdown.•Overexpression of ATG4B could induce cell autophagy to cisplatin resistance in osteosarcoma.
Artesunate (ART) is a semi-synthetic derivative of artemisinin and used preferentially in treatment of malaria in China. ART has strong anti-inflammatory, anti-tumor, and anti-angiogenic properties. ...Although the pharmacological effect of ART in osteoarthritis (OA) is unknown, evidence suggests that ART might regulate osteoclastogenesis through NF-κB signaling. In this study we explored the therapeutic effect of ART in an anterior cruciate ligament transection (ACLT)-induced OA model. We found that ART effectively relieved ACLT-induced osteoarthritis, as demonstrated by the improvement expression of pathological genes. We further demonstrated that ART markedly reduced OA-associated osteoclastogenesis and angiogenesis. In addition, ART also regulated inflammatory response and inhibited the activation of Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling in ACLT rats. Taken together our study has identified a novel function of ART and provided a molecular basis for ART potential applications in the treatment of OA and other joint disorders.
In this paper, we apply machine-learning techniques to construct detecting models of stock market manipulation. By combining manually collected China Securities Regulatory Commission punishment cases ...from 2014 to 2016 with financial information of listed companies, we construct a training set and a test set to compare the detecting ability of support vector machine (SVM) and logistic model. Considering imbalanced data, we further incorporate Borderline Synthetic Minority Oversampling Technique (Borderline SMOTE) to oversample minority class and then find that Borderline SMOTE–SVM performs better than SVM and benchmark model in detecting manipulation. To enhance detecting performance of the models, we innovatively introduce market sentiment indicators which are extracted from analyst rating reports, financial news, and Guba comments into our indicators set. The results indicate that the new indicators generate significant marginal increment to the model accuracy.
•Support vector machine (SVM) can commendably identify the stock market manipulation in China.•Borderline Synthetic Minority Oversampling Technique can enhance performance of the detecting models.•Market sentiment indicators which are extracted from analyst rating reports, financial news, and Guba comments enhance model accuracy.
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