Comprehensive bioinformatics analysis and cell line experiments revealed the important role of CDCA3 in sarcoma.

Background: Sarcoma mainly originate from bone and soft tissue and are highly aggressive malignant tumors. Cell division cycle-related protein 3 (CDCA3) is a protein involved in the regulation of the cell cycle, which is highly expressed in a variety of malignant tumors. However, its role in sarcoma remains unclear. This study aims to investigate the function and potential mechanism of CDCA3 in sarcoma and to elucidate its importance in sarcoma. Methods: We first studied the expression and prognosis of CDCA family members in sarcoma by Oncomine and the Gene Expression Profiling Interactive Analysis (GEPIA). The role of CDCA3 protein in sarcoma was further analyzed by the Cancer Genome Atlas Program (TCGA), the Cancer Cell Lineage Encyclopedia (CCLE), and Linke-dOmics. In addition, immunohistochemistry and Western blot were used to verify the expression of CDCA3 protein in clinical samples as well as sarcoma cell lines (U2OS, SAOS2, MG63, and HOS). Subsequently, in vitro experiments (cloning and scratching experiments) were performed using sh-NC as well as sh-CDCA3 group cells to reveal the biological functions of CDCA3. Results: We found that the CDCA family (CDCA3, CDCA4, and CDCA8) is highly expressed in sarcoma, and the expression level of CDCA3, CDCA4, and CDCA8 negatively correlates with the prognosis of sarcoma patients. CDCA3 mRNA was highly expressed in pan-cancer by CCLE and TCGA database analysis. KEGG analysis showed that CDCA3 was mainly enriched in the cell cycle signaling pathway (It promoted the transition of the cell cycle from the G0/G1 phase to the S phase). In the level of immune infiltration, CDCA3 was negatively correlated with pDC cells, CD8+T cells, and cytotoxic cells. Finally, patients with high CDCA3 expression in sarcoma were analyzed for resistance to NU7441 and others, while sensitive to Fulvestrant and Dihydrorotenone. Furthermore, we demonstrated high expression of CDCA3 protein in sarcoma tissues and cell lines by immunohistochemistry and Western blot experiments. Cloning, EDU, scratching, and migration experiments showed that the knockdown of CDCA3 inhibited the Proliferation and progression of sarcoma cells. Conclusion: These results suggest for the first time that knockdown of CDCA3 may inhibit sarcoma progression. CDCA3 may be an effective target for the treatment of sarcoma.

LAPTM4B counteracts ferroptosis via suppressing the ubiquitin-proteasome degradation of SLC7A11 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics.

miR-137-LAPTM4B regulates cytoskeleton organization and cancer metastasis via the RhoA-LIMK-Cofilin pathway in osteosarcoma.

Osteosarcoma (OS) is a rare malignant bone tumor but is one leading cause of cancer mortality in childhood and adolescence. Cancer metastasis accounts for the primary reason for treatment failure in OS patients. The dynamic organization of the cytoskeleton is fundamental for cell motility, migration, and cancer metastasis. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is an oncogene participating in various biological progress central to cancer biogenesis. However, the potential roles of LAPTM4B in OS and the related mechanisms remain unknown. Here, we established the elevated LAPTM4B expression in OS, and it is essential in regulating stress fiber organization through RhoA-LIMK-cofilin signaling pathway. In terms of mechanism, our data revealed that LAPTM4B promotes RhoA protein stability by suppressing the ubiquitin-mediated proteasome degradation pathway. Moreover, our data show that miR-137, rather than gene copy number and methylation status, contributes to the upregulation of LAPTM4B in OS. We report that miR-137 is capable of regulating stress fiber arrangement, OS cell migration, and metastasis via targeting LAPTM4B. Combining results from cells, patients' tissue samples, the animal model, and cancer databases, this study further suggests that the miR-137-LAPTM4B axis represents a clinically relevant pathway in OS progression and a viable target for novel therapeutics.

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N6 -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.

The biological roles of epithelial-mesenchymal transition (EMT) in the pathogenesis of radiation-induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6 -methyladenosine (m6 A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6 A modification participates in ionizing radiation (IR)-induced EMT and RILI remain unclear. Here, significantly increased m6 A levels upon IR-induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase-like 3 (METTL3) expression and downregulated α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3-mediated m6 A modification suppresses IR-induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3-mediated mRNA m6 A modification in a YTH-domain family 2 (YTHDF2)-dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR-responsive METTL3 is involved in IR-induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2-dependent FOXO1 m6 A modification, which may be a novel mechanism involved in the occurrence and development of RILI.

[Simulation of the responses of spring wheat yield to the rates and depths of nitrogen application in dryland based on APSIM model]. / 基于APSIM模型的旱地春小麦产量对施氮量和施氮深度的响应模拟.

Nitrogen limitation is an important factor for the improvement of crop water production potential in rain-fed areas of the Loess Plateau. The reasonable deep application of nitrogen fertilizer is a promising method to increase yield of rain-fed crop. Based on APSIM model, this study simulated spring wheat yield under different nitrogen application rates and depths, by using meteorological observation data from 1990 to 2020 in the semiarid areas of central Gansu Province, aiming to provide theoretical reference for optimizing wheat fertilization strategy. The results showed that the determination coefficient of simulated spring wheat yield, biomass and soil water content in 0-200 cm soil profile was greater than 0.80, the normalized root mean square error was less than 0.2, and the model validity index was greater than 0.5. These results indicated that the model had good fitting and adaptability in the test area. Across all the levels within the experimental design, increasing nitrogen application rates could significantly increase the yield of spring wheat in different precipitation years, and increasing nitrogen application depth could significantly increase spring wheat yield in wet and normal years, but had no effect in dry years. The rate and depth of nitrogen application had significant interaction effects on spring wheat yield in wet and normal years, but not in dry years. According to the binary quadratic regression fitting equation, when the potential maximum yield reached 2749 kg·hm-2 in wet year, nitrogen application depth was 22.7 cm, and nitrogen application rate was 245 kg·hm-2. When the maximum potential yield reached 2596 kg·hm-2 in normal year, nitrogen application depth was 20.6 cm, and nitrogen application rate was 235 kg·hm-2. Integrating the effects of nitrogen application rate and depth on yield, biomass and agronomic efficiency of nitrogen fertilizer, and farmer's fertilizer application habits, the recommended nitrogen application depth was 20-23 cm, and nitrogen application amount was 120-150 kg·hm-2, which could further improve water productivity and nitrogen use efficiency of spring wheat in arid areas of central Gansu Province.

Eriodictyol, a plant flavonoid, attenuates LPS-induced acute lung injury through its antioxidative and anti-inflammatory activity.

Acute lung injury (ALI) is characterized by excessive inflammatory responses and oxidative injury in the lung tissue. It has been suggested that anti-inflammatory or antioxidative agents could have therapeutic effects in ALI, and eriodictyol has been reported to exhibit antioxidative and anti-inflammatory activity in vitro. The aim of the present study was to investigate the effect of eriodictyol on lipopolysaccharide (LPS)-induced ALI in a mouse model. The mice were divided into four groups: Phosphate-buffered saline-treated healthy control, LPS-induced ALI, vehicle-treated ALI (LPS + vehicle) and eriodictyol-treated ALI (LPS + eriodictyol). Eriodictyol (30 mg/kg) was administered orally once, 2 days before the induction of ALI. The data showed that eriodictyol pretreatment attenuated LPS-induced ALI through its antioxidative and anti-inflammatory activity. Furthermore, the eriodictyol pretreatment activated the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway in the ALI mouse model, which attenuated the oxidative injury and inhibited the inflammatory cytokine expression in macrophages. In combination, the results of the present study demonstrated that eriodictyol could alleviate the LPS-induced lung injury in mice by regulating the Nrf2 pathway and inhibiting the expression of inflammatory cytokines in macrophages, suggesting that eriodictyol could be used as a potential drug for the treatment of LPS-induced lung injury.

[Efficacy and safety of delayed thrombolytic therapy for acute massive pulmonary throboembolism].

OBJECTIVE: To observe the efficacy and safety of delayed thrombolytic therapy on acute massive pulmonary thromboembolism (PTE) and discuss the influence factors. METHODS: From 2009 to 2013, the clinical data of patients with acute massive pulmonary thromboembolism were analyzed retrospectively. Patients with over 14-day duration and treated with thrombolytic therapy (delayed thrombolytic group) were compared with those within 14-day duration and treated with thrombolytic therapy (normal thrombolytic group) in the same period. General conditions before treatment, case history, efficacy and the incidence of bleeding after one-week treatment were collected. The influence factors of delayed thrombolytic therapy were analyzed. RESULTS: Sixty two cases were collected and divided into the normal thrombolytic group with 32 cases and the delayed thrombolytic group with 30 cases. Compared with the normal thrombolytic group, the delayed thrombolytic group had a longer duration [(24.8 ± 0.9) vs.(7.2 ± 0.6)d, P<0.001], an aggravation time of (5.3 ± 0.8) d, and higher systolic pulmonary arterial pressure (SPAP) [(69 ± 4)vs. (55 ± 4)mmHg, 1 mmHg= 0.133 kPa, P= 0.016]. Ages, genders, D-Dimmer, CT subpulmonic obstruction index (CTI), brain natriuretic peptide (BNP), cardiactroponinI (TnI), PaCO2values and PaO2values had no statistical difference between two groups. After one-week treatment, the efficacy and the incidence of bleeding was 78% and 25% respectively in normal thrombolytic group, while they were 77% and 30% respectively in delayed thrombolytic group, and there was no significant difference between two groups (P>0.05). The single factor analysis showed that the delayed thrombolytic group had more patients with hypertension, were older and had a lower PaO2(P<0.05 or 0.01). Multivariate logistic regression analysis did not find the predictors of delayed thrombolytic therapy (P>0.05). CONCLUSIONS: For acute massive PTE patients with duration over 14 days, increased D-D and new exacerbation of symptoms,delayed thrombolytic therapy had the same efficacy with the normal thrombolytic therapy. The factors for predicting efficacy need further research.

[Construction and influence of human nuclear factor-κB p65 shRNA lentiviral 
vector on malignant biological behavior of lung cancer cells].

BACKGROUND AND OBJECTIVE: Nuclear factor-κB is an important transcription factor and is closely associated with a variety of malignant tumors. The biological behavior of lung tumor cells can be reversed by inhibiting the expression of NF-κBp65 directly or indirectly. Nuclear factor-κBp65 gene shRNA recombinant plasmids were constructed and then infected with A549 cells. New stable cell lines were selected, and the ability of migration and adhesion was identified. METHODS: Both scramble control sequence and interference sequence (shRNA) of human nuclear factor-κBp65 were designed and synthesized to build recombinant plasmids, with BamH I site at the 5' end and Xho I and EcoR I sites at the 3' end. A549 cells were infected, and stable transfection strains were selected by puromycin. Western blot and qRT-PCR methods were applied to assess the interference efficient of NF-κBp65 and the protein expression level of IκBα. Transwell and MTT assays were carried out to analyze the ability of migration and adhesion of A549 cells separately. RESULTS: Recombinant plasmids were successfully built, and A549/NF-κB p65 scramble and A549/NF-κB p65 shRNA stable transfection strains were also successfully screened. Both mRNA and protein expression levels of NF-κBp65 showed that A549/NF-κBp65 shRNA cells decreased compared with A549/NF-κB p65 scramble cells and A549 cells, whereas the protein level of IκBα significantly increased. Both migration and adhesion abilities were also reduced. CONCLUSIONS: In this study, both mRNA and protein expression levels of NF-κBp65 were effectively suppressed by RNA interference technique. NF-κBp65 inhibition can significantly reduce the migration and adhesion ability of A549 cells.

Preparation and biological evaluation of (99m) TcN-labeled pteroyl-lys derivative as a potential folate receptor imaging agent.

In order to develop a novel (99m) Tc-labeled folate receptor (FR) imaging agent, a dithiocarbamate derivative, pteroyl-lys-DTC, was synthesized and radiolabeled with (99m) Tc through the [(99m) TcN](2+) intermediate. The radiochemical purity of the corresponding (99m) Tc-complex, (99m) TcN-pteroyl-lys-DTC, was over 95% as measured by reversed-phase HPLC. The (99m) TcN complex was stable under physiological conditions. (99m) TcN-pteroyl-lys-DTC exhibited specific FR binding in FR-positive KB cells in vitro. The biodistribution in tumor-bearing mice showed that the (99m) TcN-labeled radiotracer had good uptake (3.56 ± 0.09%ID/g at 2 h postinjection) in FR-positive KB tumors, as well as in the kidneys (30.34 ± 3.53%ID/g at 2 h postinjection). After coinjection with excess folic acid, the uptake in tumor and kidneys was significantly blocked. The results indicated that (99m) TcN-pteroyl-lys-DTC was able to target the FR-positive tumor cells and tissues specifically both in vitro and in vivo.

Synthesis and in vitro/in vivo evaluation of 99mTc-labeled folate conjugates for folate receptor imaging.

INTRODUCTION: Folate receptor (FR) is a potential molecular target for radionuclide imaging since it is overexpressed in many human epithelial tumor cells. In this study, a novel folate conjugate was synthesized and labeled with (99m)Tc using different coligands. In vitro and in vivo evaluations of these complexes have been done to explore the effect of coligands on the stable, affinity and pharmacokinetic properties. METHODS: A novel folate conjugate, HYNIC-NHHN-FA, was synthesized and characterized. This conjugate was radiolabeled with (99m)Tc using tricine, tricine/diphenylphosphinobenzene-3-sulfonic acid sodium (TPPMS) and tricine/trisodium triphenylphosphine-3,3',3''-trisulfonate (TPPTS) as coligands, respectively. The complexes were purified by high-pressure liquid chromatography (HPLC). In vitro and in vivo evaluations were performed with FR-positive KB cells, normal Kunming mice and athymic nude mice bearing KB tumors. RESULTS: Labeling with (99m)Tc using different coligands resulted in three complexes, (99m)Tc (HYNIC-NHHN-FA)(tricine), 5, (99m)Tc (HYNIC-NHHN-FA)(tricine/TPPMS), 6 and (99m)Tc (HYNIC-NHHN-FA)(tricine/TPPTS), 7. Complex 5 showed at least two isomers and was unstable after being purified by HPLC. Complexes 6 and 7 displayed high stability and similar affinity to FR in vitro. Biodistribution results in athymic nude mice bearing KB tumor showed that complex 7 had a high uptake in FR-positive tumor (9.79±1.66%ID/g at 4 h postinjection), and the results of blockade studies confirmed the specific accumulation of the radiotracer in vivo. However, complex 6 showed a low tumor uptake due to its fast excretion via the gastrointestinal tract. CONCLUSION: The modification of the coligands can significantly alter the pharmacokinetic properties of the corresponding (99m)Tc-HYNIC complexes. (99m)Tc (HYNIC-NHHN-FA)(tricine/TPPTS), 7 could be a promising radiotracer for FR imaging.

The synthesis of pteroyl-lys conjugates and its application as Technetium-99m labeled radiotracer for folate receptor-positive tumor targeting.

Aiming to develop a new (99m)Tc-labeled folate derivative for FR-positive tumor imaging, a simpler method has been established to synthesize the folate-drug conjugates with free α-carboxyl group. In this study, the conjugate pteroyl-lys-HYNIC was synthesized and labeled with (99m)Tc using tricine and TPPTS as co-ligands. The radiochemical purity of the final complex (99m)Tc(HYNIC-lys-pteroyl)(tricine/TPPTS), 5 was high (>98%), and it remained stable in saline and plasma over 6h after preparation. The biologic evaluation results showed that the (99m)Tc labeled pteroyl-lys conjugate was able to specifically target the FR-positive tumor cells and tissues both in vitro and in vivo, highlighting its potential as an effective folate receptor targeted agent for tumor imaging.