Suppression of IRF9 Promotes Osteoclast Differentiation by Decreased Ferroptosis via STAT3 Activation.

Osteoporosis is a chronic disease that endangers the health of the elderly. Inhibiting osteoclast hyperactivity is a key aspect of osteoporosis prevention and treatment. Several studies have shown that interferon regulatory factor 9 (IRF9) not only regulates innate and adaptive immune responses but also plays an important role in inflammation, antiviral response, and cell development. However, the exact role of IRF9 in osteoclasts has not been reported. To elucidate the role of IRF9 in osteoclast differentiation, we established the ovariectomized mouse model of postmenopausal osteoporosis and found that IRF9 expression was reduced in ovariectomized mice with overactive osteoclasts. Furthermore, knockdown of IRF9 expression enhanced osteoclast differentiation in vitro. Using RNA sequencing, we identified that the differentially expressed genes enriched by IRF9 knockdown were related to ferroptosis. We observed that IRF9 knockdown promoted osteoclast differentiation via decreased ferroptosis in vitro and further verified that IRF9 knockdown reduced ferroptosis by activating signal transducer and activator of transcription 3 (STAT3) to promote osteoclastogenesis. In conclusion, we identified an essential role of IRF9 in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

The High Risk Factors and Preventive Measures of Percutaneous Nephrolithotomy under the Guidance of B-Ultrasound in the Treatment of Postoperative Renal Calculi.

Objective: The aim of this study is to explore and analyze the high risk factors and preventive measures of percutaneous nephrolithotomy under the guidance of B-ultrasound in the treatment of postoperative renal calculi. Methods: The clinical data of 220 patients with renal calculi admitted to our hospital from 2018 to October 2021 were retrospectively analyzed. All patients were treated with percutaneous nephrolithotomy n = 36) and nonbleeding group (n = 184), comparing the personal data, disease-related data, surgical operation related data of the two groups of patients, single factor and logistic multifactor regression analysis to explore the influence of B-guided percutaneous. Nephrolithotomy is a high-risk factor for postoperative bleeding in patients with kidney stones, and preventive measures are based on high-risk factors. Results: There was no significant difference in the proportion of patients with different genders, whether they had renal surgery, whether they had hypertension, and those with postoperative hepatic insufficiency in the hemorrhagic group and the nonbleeding group (p > 0.05). There was no significant difference in age and body mass index between the bleeding group and the nonbleeding group (p > 0.05). The proportion of patients with diabetes in the bleeding group was higher than that in the nonbleeding group, and the difference between the groups was statistically significant (p < 0.05). Compared with the nonbleeding group, the bleeding group had a higher proportion of patients with calculus diameter ≥2 cm. The proportion of patients with staghorn calculi in the bleeding group was higher than that in the nonbleeding group. The difference between the groups was statistically significant (p < 0.05). There was no significant difference in the proportion of patients with hemorrhage, single or multiple renal stones, and ureteral stones in the hemorrhage group compared with the nonbleeding group (p > 0.05). Compared with the nonbleeding group, the proportion of patients with bleeding in the first stage was higher, and the proportion of patients with operation time >90 min was higher. The difference between the groups was statistically significant (p < 0.05). There was no significant difference in the proportion of patients in the bleeding group compared with the nonbleeding group (p > 0.05). Using Logic multifactorial regression analysis, independent risk factors for bleeding after percutaneous nephrolithotomy under ultrasound-guided bovery include diabetes mellitus, stone diameter, staghorn kidney stones, surgical timing, and staging surgery (p < 0.05). Conclusion: The independent high-risk factors affecting bleeding after percutaneous nephrolithotomy guided by B-ultrasound include diabetes, stone diameter, staghorn type kidney stones, operation time, and staged surgery. According to this, effective preventive measures can effectively reduce the operation and the occurrence of postbleeding.

Serum neuron-specific enolase: A promising biomarker of silicosis.

BACKGROUND: Silicosis is a type of chronic pulmonary fibrosis caused by long-term inhalation of silica dust particles. There has been no ideal biomarker for the diagnosis and differential diagnosis of silicosis until now. Studies have found that elevated neuron-specific enolase (NSE) concentration in the serum of silicosis patients is helpful for diagnosis and severity assessment of the disease. However, the number of cases in these studies was not enough to arouse attention. AIM: To investigate the clinical significance of serum NSE in the diagnosis and staging of silicosis. METHODS: From January 2017 to June 2019, 326 cases of silicosis confirmed in Quanzhou First Hospital Affiliated to Fujian Medical University were included in the silicosis group. A total of 328 healthy individuals or medical patients without silicosis were included in the control group. Serum NSE concentrations of all subjects were determined by electrochemical luminescence. RESULTS: There were no significant differences in sex, age, smoking index and complications between the silicosis and control groups. The mean serum NSE concentration was 26.57 ± 20.95 ng/mL in the silicosis group and 12.42 ± 2.68 ng/mL in the control group. The difference between the two groups was significant (U = 15187, P = 0.000). Among the 326 patients with silicosis, 103 had stage I silicosis, and the mean serum NSE concentration was 15.55 ± 6.23 ng/mL. The mean serum NSE concentration was 21.85 ± 12.05 ng/mL in 70 patients with stage II silicosis. The mean serum NSE concentration was 36.14 ± 25.72 ng/mL in 153 patients with stage III silicosis. Kruskal-Wallis H test suggested that the difference in serum NSE concentration in silicosis patients in the three groups was significant (H = 130.196, P = 0.000). Receiver operating characteristic curve analysis indicated that the area under the curve was 0.858 (95% confidence interval: 0.828-0.888; P = 0.000). When the NSE concentration was 15.82 ng/mL, the Jorden index was the largest, the sensitivity was 72%, and the specificity was 90%. CONCLUSION: Serum NSE concentration may be a promising biomarker for the diagnosis and assessment of severity of silicosis.

TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.

Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter.

Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effective method to visualize the trafficking of GLUT1 between the plasma membrane (PM) and endolysosomal system in live cells using an mCherry-EGFP-GLUT1 tandem fluorescence tracing system. We found that GLUT1 localized at the PM exhibited both red (mCherry) and green (EGFP) fluorescence (yellow when overlapping). However, a significant increase in red punctate fluorescence (mCherry is resistant to acidic pH), but not green fluorescence (EGFP is quenched by acidic pH), was observed upon glucose deprivation, indicating that the mCherry-EGFP-GLUT1 functional protein was trafficked to the acidic endolysosomal system. Besides, we were able to calculate the relative ratio of mCherry to EGFP by quantification of the translocation coefficient, which can be used as a readout for GLUT1 internalization and subsequent lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-ΔC4, were also constructed, which indirectly confirmed the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking. By using a series of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we were able to define a model of GLUT1 trafficking in live cells in which upon glucose deprivation, GLUT1 dissociates from the PM and experiences a pH gradient from 6.8-6.1 in the early endosomes to 6.0-4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.

Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair.

BACKGROUND: Chondrosarcoma is the second-most common type of bone tumor and has inherent resistance to conventional chemotherapy. Present study aimed to explore the therapeutic effect and specific mechanism(s) of combination BET family protein and HDAC inhibition in chondrosarcoma. METHODS: Two chondrosarcoma cells were treated with BET family protein inhibitor (JQ1) and histone deacetylase inhibitors (HDACIs) (vorinostat/SAHA or panobinostat/PANO) separately or in combination; then, the cell viability was determined by Cell Counting Kit-8 (CCK-8) assay, and the combination index (CI) was calculated by the Chou method; cell proliferation was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation and colony formation assay; cell apoptosis and reactive oxygen species (ROS) level were determined by flow cytometry; protein expressions of caspase-3, Bcl-XL, Bcl-2, γ-H2AX, and RAD51 were examined by Immunoblotting; DNA damage was determined by comet assay; RAD51 and γ-H2AX foci were observed by immunofluorescence. RESULTS: Combined treatment with JQ1 and SAHA or PANO synergistically suppressed the growth and colony formation ability of the chondrosarcoma cells. Combined BET and HDAC inhibition also significantly elevated the ROS level, followed by the activation of cleaved-caspase-3, and the downregulation of Bcl-2 and Bcl-XL. Mechanistically, combination treatment with JQ1 and SAHA caused numerous DNA double-strand breaks (DSBs), as evidenced by the comet assay. The increase in γ-H2AX expression and foci formation also consistently indicated the accumulation of DNA damage upon cotreatment with JQ1 and SAHA. Furthermore, RAD51, a key protein of homologous recombination (HR) DNA repair, was found to be profoundly suppressed. In contrast, ectopic expression of RAD51 partially rescued SW 1353 cell apoptosis by inhibiting the expression of cleaved-caspase-3. CONCLUSION: Taken together, our results disclose that BET and HDAC inhibition synergistically inhibit cell growth and induce cell apoptosis through a mechanism that involves the suppression of RAD51-related HR DNA repair in chondrosarcoma cells.

PRMT5-TRIM21 interaction regulates the senescence of osteosarcoma cells by targeting the TXNIP/p21 axis.

Osteosarcoma (OS) is the most common bone malignancy in adolescents and has poor clinical outcomes. Protein arginine methyltransferase 5 (PRMT5) has recently been shown to be aberrantly expressed in various cancers, yet its role in OS remains elusive. Here, we found that PRMT5 was overexpressed in OS and its overexpression predicted poor clinical outcomes. PRMT5 knockdown significantly triggered pronounced senescence in OS cells, as evidenced by the increase in senescence-associated ß-galactosidase (SA-ß-gal)-stained cells, induction of p21 expression, and upregulation of senescence-associated secretory phenotype (SASP) gene expression. In addition, we found that PRMT5 plays a key role in regulating DNA damaging agents-induced OS cell senescence, possibly, via affecting the repair of DNA damage. Furthermore, we found that TXNIP acts as a key factor mediating PRMT5 depletion-induced DNA damage and cellular senescence. Mechanistically, TRIM21, which interacts with PRMT5, was essential for the regulation of TXNIP/p21 expression. In summary, we propose a model in which PRMT5, by interaction with TRIM21, plays a key role in regulating the TXNIP/p21 axis during senescence in OS cells. The present findings suggest that PRMT5 overexpression in OS cells might confer resistance to chemotherapy and that targeting the PRMT5/TRIM21/TXNIP signaling may enhance the therapeutic efficacy in OS.