LINC00571 drives tricarboxylic acid cycle metabolism in triple-negative breast cancer through HNRNPK/ILF2/IDH2 axis.

BACKGROUND: Triple-negative breast cancer is a complex breast malignancy subtype characterized by poor prognosis. The pursuit of effective therapeutic approaches for this subtype is considerably challenging. Notably, recent research has illuminated the key role of the tricarboxylic acid cycle in cancer metabolism and the complex landscape of tumor development. Concurrently, an emerging body of evidence underscores the noteworthy role that long non-coding RNAs play in the trajectory of breast cancer development. Despite this growing recognition, the exploration of whether long non-coding RNAs can influence breast cancer progression by modulating the tricarboxylic acid cycle has been limited. Moreover, the underlying mechanisms orchestrating these interactions have not been identified. METHODS: The expression levels of LINC00571 and IDH2 were determined through the analysis of the public TCGA dataset, transcriptome sequencing, qRT‒PCR, and Western blotting. The distribution of LINC00571 was assessed using RNA fluorescence in situ hybridization. Alterations in biological effects were evaluated using CCK-8, colony formation, EdU, cell cycle, and apoptosis assays and a tumor xenograft model. To elucidate the interaction between LINC00571, HNRNPK, and ILF2, RNA pull-down, mass spectrometry, coimmunoprecipitation, and RNA immunoprecipitation assays were performed. The impacts of LINC00571 and IDH2 on tricarboxylic acid cycle metabolites were investigated through measurements of the oxygen consumption rate and metabolite levels. RESULTS: This study revealed the complex interactions between a novel long non-coding RNA (LINC00571) and tricarboxylic acid cycle metabolism. We validated the tumor-promoting role of LINC00571. Mechanistically, LINC00571 facilitated the interaction between HNRNPK and ILF2, leading to reduced ubiquitination and degradation of ILF2, thereby stabilizing its expression. Furthermore, ILF2 acted as a transcription factor to enhance the expression of its downstream target gene IDH2. CONCLUSIONS: Our study revealed that the LINC00571/HNRNPK/ILF2/IDH2 axis promoted the progression of triple-negative breast cancer by regulating tricarboxylic acid cycle metabolites. This discovery provides a novel theoretical foundation and new potential targets for the clinical treatment of triple-negative breast cancer.

Identification and Preliminary Clinical Validation of Key Extracellular Proteins as the Potential Biomarkers in Hashimoto's Thyroiditis by Comprehensive Analysis.

Hashimoto's thyroiditis (HT) is an autoimmune disruption manifested by immune cell infiltration in thyroid tissue and the production of antibodies against thyroid-specific antigens, such as the thyroid peroxidase antibody (TPOAb) and thyroglobulin antibody (TGAb). TPOAb and TGAb are commonly used in clinical tests; however, handy indicators of the diagnosis and progression of HT are still scarce. Extracellular proteins are glycosylated and are likely to enter body fluids and become readily available and detectable biomarkers. Our research aimed to discover extracellular biomarkers and potential treatment targets associated with HT through integrated bioinformatics analysis and clinical sample validations. A total of 19 extracellular protein-differentially expressed genes (EP-DEGs) were screened by the GSE138198 dataset from the Gene Expression Omnibus (GEO) database and protein annotation databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the function and pathway of EP-DEGs. STRING, Cytoscape, MCODE, and Cytohubba were used to construct a protein-protein interaction (PPI) network and screen key EP-DEGs. Six key EP-DEGs (CCL5, GZMK, CXCL9, CXCL10, CXCL11, and CXCL13) were further validated in the GSE29315 dataset and the diagnostic curves were evaluated, which all showed high diagnostic accuracy (AUC > 0.95) for HT. Immune profiling revealed the correlation of the six key EP-DEGs and the pivotal immune cells in HT, such as CD8+ T cells, dendritic cells, and Th2 cells. Further, we also confirmed the key EP-DEGs in clinical thyroid samples. Our study may provide bioinformatics and clinical evidence for revealing the pathogenesis of HT and improving the potential diagnosis biomarkers and therapeutic strategies for HT.

Causal inference between aggressive extrathyroidal extension and survival in papillary thyroid cancer: a propensity score matching and weighting analysis.

Background: Extrathyroidal extension is a major risk factor for poor prognosis in papillary thyroid cancer. However, the effect of different degrees of extrathyroidal extension on prognosis remains controversial. We performed a retrospective study to elucidate how the extent of extrathyroidal extension in papillary thyroid cancer affected the clinical prognosis of patients and its covariates. Methods: The study included 108,426 patients with papillary thyroid cancer. We categorized the extent of extension into none, capsule, strap muscles, and other organs. Three causal inference methods for retrospective studies, namely, inverse probability of treatment weighting, standardized mortality ratio weighting, and propensity score matching analysis, were used to minimize potential selection bias. Kaplan-Meier analysis and univariate Cox regression analyses were applied to analyze the precise effect of ETE on survival in papillary thyroid cancer patients. Results: In the Kaplan-Meier survival analysis, only extrathyroidal extension into or beyond the strap muscles was statistically significant for both overall survival (OS) and thyroid cancer-specific survival (TCSS). In univariate Cox regression analyses before and after matching or weighting based on causal inference, extrathyroidal extension into soft tissues or other organs is a high-risk factor for both overall survival and thyroid cancer-specific survival. Sensitivity analysis revealed that lower overall survival was observed in patients with older age (≥55) and larger tumor size (>2 cm) of papillary thyroid cancer with extrathyroidal extension into or beyond the strap muscles. Conclusions: Our study indicates that extrathyroidal extension into soft tissues or other organs is a high-risk factor in all papillary thyroid cancer. Even though invasion into the strap muscles did not seem to be a marker for poor prognosis, it still impaired the overall survival of patients with older age (≥55 years old) or larger tumor size (>2 cm). Further investigation is needed to confirm our results and to clarify further risk factors independent of extrathyroidal extension.

In vitro comparative study of multimodal imaging nano-assembled microspheres with two clinical drug-eluting beads loaded with doxorubicin.

DC Beads and CalliSpheres are commonly used microspheres in clinical transcatheter arterial chemoembolization, but these microspheres cannot be visualized by themselves. Therefore, in our previous study, we developed multimodal imaging nano-assembled microspheres (NAMs), which are visualized under CT/MR and the location of embolic microspheres can be determined during postoperative review, facilitating the evaluation of embolic areas and guiding subsequent treatment. Moreover, the NAMs can be carried with positively and negatively charged drugs, increasing the choice of drugs. Systematic comparative analysis of the pharmacokinetics of NAMs with commercially available DC Bead and CalliSpheres microspheres is important for evaluating the clinical application of NAMs. In our study, we compared the similarities and differences between NAMs and two drug-eluting beads (DEBs) in respect to drug loading capacity, drug release profiles, diameter variation and morphological characteristics. The results indicate that NAMs had good drug delivery and release characteristics as well as DC Bead and CalliSpheres in vitro experimental stage. Therefore, NAMs have a good application prospect in transcatheter arterial chemoembolization treatment of hepatocellular carcinoma.

[Prediction of immune-related genes associated with prognosis in patients with hepatocellular carcinoma by bioinformatics methods].

Objective To establish a prognosis model using immune-related genes in hepatocellular carcinoma (HCC) patients, which could provide a theoretical foundation for HCC immunotherapy. Methods Immune-related genes were identified by differential expression analysis, and risk prognosis prediction models were established using univariate, multivariate Cox and Least absolute shrinkage and selection operator (LASSO) regression analysis. The predictive value of the prognostic model was evaluated using the concordance index (C-index), receiver operating characteristic curve (ROC curve), and calibration curve and decision curve. In addition, risk score was used to stratify patients to assess prognostic differences in patients at different risk levels. Results We identified 1403 immune-related genes, mainly involved in biological processes such as immune response, adaptive immune response, and immunoglobulin production, as well as pathways such as cytokine interactions, chemokine signaling pathways and allograft rejection. Univariate Cox analysis found that 53 immune-related genes were associated with prognosis, and eight prognostic immune-related genes cytochrome P450 1A2(CYP1A2), ficolin 3(FCN3), hepatoma derived growth factor-like 1(HDGFL1), lipocalin 2(LCN2), mitochondrially encoded cytochrome C oxidase II pseudogene 12(MTCO2P12), peptidyl arginine deiminase 3(PADI3) and regulator of G protein signaling 16(RGS16) were further screened by LASSO and multivariate Cox regression analysis. Subsequently, a prognostic nomogram based on risk score was established. The ROC curve, calibration curve and decision curve confirmed that the model has good discrimination, accuracy and clinical value. Furthermore, stratified analysis showed that patients with higher risk scores had poorer prognosis. Conclusion We establish a prognostic nomogram model using eight immune-related genes, which can reliably predict the prognosis of HCC patients.

LncRNA SEMA3B-AS1 inhibits breast cancer progression by targeting miR-3940/KLLN axis.

Long noncoding RNAs (lncRNAs) play crucial regulatory roles in the progression of various cancers. However, the functional roles of lncRNAs in breast cancer remain unclear. In this study, we investigated the functional role of a novel long noncoding RNA SEMA3B-AS1 (lncRNA SEAS1) in breast cancer progression and the underlying mechanisms. SEAS1 was downregulated in the triple-negative breast cancer (TNBC) tissues compared with the para-carcinoma tissues, which was associated with poor prognosis of TNBC patients. We demonstrated that SEAS1 knockdown significantly increased the proliferation, migration, and invasion of TNBC cell lines, whereas SEAS1 overexpression reversed these effects. Bioinformatics analysis demonstrated that microRNA (miR)-3940-3p was a potential target of SEAS1. Mechanistically, RNA immunoprecipitation (RIP) and luciferase reporter assays confirmed that lncRNA SEMA3B-AS1 acted as sponge for miR-3940-3p, preventing the degradation of its target gene KLLN, which acts as a tumor-inhibiter in TNBC. Moreover, RNA pulldown, mass spectrometry, ChIP, and luciferase reporter assays confirmed that SMAD3 directly interacted with the promoter of SEAS1 and suppressed its transcription, thereby promoting TNBC progression. The clinical samples of TNBC confirmed SEAS1 was correlated inversely with lymphatic and distant metastasis. In conclusion, our findings reveal a novel pathway for TNBC progression via SMAD3/lncRNA SEAS1/miR-3940-3p/KLLN axis, and suggest that SEAS1 may serve as a potential biomarker and therapeutic target for TNBC.

TSH-TSHR axis promotes tumor immune evasion.

BACKGROUND: Hormones are identified as key biological variables in tumor immunity. However, previous researches mainly focused on the immune effect of steroid hormones, while the roles that thyroid-stimulating hormone (TSH) played in the antitumor response were far from clear. METHODS: The source of TSH was determined using single-cell transcriptomic, histologic, quantitative PCR, and ELISA analysis. The influence of TSH on tumor proliferation, invasion, and immune evasion was evaluated in multiple cell lines of thyroid cancer, glioma, and breast cancer. Then transcriptomic sequencing and cellular experiments were used to identify signaling pathways. TSH receptor (TSHR) inhibitor was injected into homograft mouse tumor models with or without anti-programmed cell death protein-1 antibody. RESULTS: Monocyte-derived dendritic cells (moDCs) highly expressed TSHα and TSHß2 and were the primary source of TSH in the tumor microenvironment. TSH released by moDCs promoted proliferation and invasion of tumors with high TSHR expressions, such as thyroid cancers and glioma. TSH also induced tumor programmed death-ligand 1 (PD-L1) expression through the TSHR-AC-PKA-JNK-c-JUN pathway. TSHR inhibitors reversed tumor immune evasion by inhibiting PD-L1 expression in tumor and myeloid cells and enhancing Teff activation. CONCLUSIONS: TSH-TSHR axis promotes tumor evasion in thyroid cancers and glioma. TSH suppression therapy is an effective therapeutic strategy for combination in immune checkpoint blockades.

YTHDF1 promotes breast cancer progression by facilitating FOXM1 translation in an m6A-dependent manner.

BACKGROUND: N6-methyladenosine (m6A) is the most common post-transcriptional modification at the RNA level. However, the exact molecular mechanisms of m6A epigenetic regulation in breast cancer remain largely unknown and need to be fully elucidated. METHODS:  The integrating bioinformatics analyses were used to screen clinical relevance and dysregulated m6A "reader" protein YTHDF1 in breast cancer from TCGA databases, which was further validated in a cohort of clinical specimens. Furthermore, functional experiments such as the CCK-8 assay, EdU assay, wound healing assay, transwell invasion assay and cell cycle assay were used to determine the biological role of YTHDF1 in breast cancer. RIP, m6A-IP, and CLIP assays were used to find the target of YTHDF1 and further verification by RT-qPCR, western blot, polysome profiling assay. The protein-protein interaction between YTHDF1 and FOXM1 was detected via co-immunoprecipitation. RESULTS: Our study showed that YTHDF1 was overexpressed in breast cancer cells and clinical tissues specimens. At the same time, the high expression level of YTHDF1 was positively correlated with tumor size, lymph node invasion, and distant metastasis in breast cancer patients. YTHDF1 depletion repressed the proliferation, invasion and epithelial-mesenchymal transformation (EMT) and induced G0/G1 phase cell cycle arrest of breast cancer cells in vitro and in vivo. We also demonstrated that FOXM1 is a target of YTHDF1. Through recognizing and binding to the m6A-modified mRNA of FOXM1, YTHDF1 accelerated the translation process of FOXM1 and promoted breast cancer metastasis. Whereas overexpression of FOXM1 in breast cancer cells partially counteracted the tumor suppressed effects caused by YTHDF1 silence, which further verified the regulatory relationship between YTHDF1 and FOXM1. CONCLUSION: Our study reveals a novel YTHDF1/FOXM1 regulatory pathway that contributes to metastasis and progression of breast cancer, suggesting that YTHDF1 might be applied as a potential biomarker and therapeutic target. That also advances our understanding of the tumorigenesis for breast cancer from m6A epigenetic regulation.

A Metabolic-associated Nomogram Predicts Recurrence Survival of Thyroid Cancer.

OBJECTIVE: Various studies have suggested that metabolic genes play a significant role in papillary thyroid cancer (PTC). The current study aimed to identify a metabolic signature related biomarker to predict the prognosis of patients with PTC. METHODS: We conducted a comprehensive analysis on the data obtained from the Cancer Genome Atlas (TCGA) database. The correlation between survival result and metabolic genes was evaluated based on the univariate Cox analyses, least absolute shrinkage and selection operator (LASSO) and multivariate Cox analyses. The performance of a 7-gene signature was assessed according to Kaplan-Meier and receiver operating characteristic (ROC) analysis. Multivariate Cox regression analysis was adopted to unearth clinical factors related to the recurrence free survival (RFS) of patients with PTC. Finally, a prognostic nomogram was developed based on risk score, cancer status and cancer width to improve the prediction for RFS of PTC patients. RESULTS: Seven metabolic genes were used to establish the prognostic model. The ROC curve and C-index exhibited high value in training, testing and the whole TCGA datasets. The established nomogram, incorporating the 7-metabolic gene signature and clinical factors, was able to predict the RFS with high effectiveness. The 7-metabolic gene signature-based nomogram had a good performance to predict the RFS of patients with PTC. CONCLUSION: Our study identified a 7-metabolic gene signature and established a prognostic nomogram, which were useful in predicting the RFS of PTC.

Mitochondria transplantation protects traumatic brain injury via promoting neuronal survival and astrocytic BDNF.

Traumatic brain injury (TBI) is one of the leading causes of disability and paralysis around the world. Secondary injury, characterized by progressive neuronal loss and astrogliosis, plays important roles in the post-TBI cognitive impairment and mood disorder. Unfortunately, there still lacks effective treatments, particularly surgery interferences for it. Recent findings of intercellular mitochondria transfer implies a potential therapeutic value of mitochondria transplantation for TBI, which has not been tested yet. In the present study, we demonstrated a quick dysfunction of mitochondria, up-regulation of Tom20 in the injured cortex and subsequent cognitive and mood impairment. Our data demonstrated that mitochondria derived from allogeneic liver or autogeneic muscle stimulated similar microglial activation in brain parenchyma. In vitro experiments showed that exogenous mitochondria could be easily internalized by neurons, astrocytes, and microglia, except for oligodendrocytes. Mitochondria transplantation effectively rescued neuronal apoptosis, restored the expression of Tom20 and the phosphorylation of JNK. Further analysis revealed that mitochondria transplantation in injured cortex induced a significant up-regulation of BDNF in reactive astrocytes, improved animals' spatial memory and alleviated anxiety. In together, our data indicate that mitochondria transplantation may has the potential of clinical translation for TBI treatment, in combination with surgery.

XBP1s Acts as a Tumor Suppressor to Inhibit the EMT Process and Metastasis of Papillary Thyroid Cancer.

PURPOSE: Papillary thyroid cancer (PTC) patients could obtain poor prognosis if they have lymph node metastasis, identification of informative and robust biomarkers for predicting cervical lymph node metastasis is critical for improving clinical decision-making and patient prognosis. MATERIALS AND METHODS: In this study, we analyzed the expression of X box binding protein 1 spliced-form (XBP1s) in 41 PTC tissue samples and extended our findings using public databases, then we investigated how XBP1s' contributed to PTC progression in vitro. RESULTS: We found that XBP1s' expression was lower in PTC patients with cervical lymph node metastasis than non-metastasis patients by immunohistochemical analysis. With publicly accessible dataset, we showed that the XBP1 transcription was significantly decreased in thyroid cancer (TC) tissues with lymph node metastasis as compared to that without lymph node metastasis. Moreover, we also found that XBP1 expression was significantly correlated with patients' gender, T classification, lymph node metastasis and PTC stages, and low XBP1 expression was associated with poor diseases free survival (DFS). In vitro, XBP1s overexpression could inhibit the invasion, migration, and wound healing capacity of PTC cells. Mechanistically, overexpression of XBP1s could enhance the expression of classical epithelial-mesenchymal transition (EMT) markers such as ZO-1 and E-cadherins, and downregulated N-cadherin in BCPAP cells. CONCLUSION: These findings suggest that XBP1s is a prognostic maker for thyroid carcinoma patients, and sustaining XBP1s expression might be a new strategy to control PTC progression.

MiR-3121-3p promotes tumor invasion and metastasis by suppressing Rap1GAP in papillary thyroid cancer in vitro.

BACKGROUND: Rap1GAP is a tumor suppressor and is downregulated in human malignancies including papillary thyroid cancer (PTC). The mechanism of its suppression in PTC remains unclear. METHODS: Bioinformatic analyses were carried out to evaluate clinical significance and to predict upstream miRNA bindings of Rap1GAP. Three PTC cell lines, TPC-1, B-CPAP, and K1, were employed for functional verification and further experiments. We used dual-luciferase reporter gene assay to confirm the miRNA binding prediction, Western blotting and quantitative polymerase chain reaction (qPCR) to explore miRNA and Rap1GAP regulation, Transwell and wound healing assays to compare cell migration and invasion after protein knockout or overexpression, and Cell Counting Kit-8 (CCK-8) assay to evaluate cell proliferation. RESULTS: Rap1GAP expression was suppressed in thyroid cancer compared to adjacent normal tissues and was a potential diagnostic marker of PTC. Rap1GAP suppression was correlated to younger age, advanced T stage, N stage, extrathyroidal extension, BRAF-like tumors, and higher risk of recurrence. Combined analysis of bioinformatic prediction and dual-luciferase assay revealed binding between miR-3121-3p with 3'UTR of Rap1GAP promoter. MiR-3121-3p promoted cell migration, invasion, and proliferation via inhibiting Rap1GAP and thus upregulating MAPK pathway. Overexpression and knockdown of Rap1GAP could counteract the influence on cell migration and invasion carried out by miR-3121-3p mimic and inhibitor, respectively. Rap1GAP partially impaired the effect of miR-3121-3p in cell growth in the CCK-8 assay. CONCLUSIONS: Rap1GAP expression is suppressed in PTC and is a potential diagnostic marker. Its upstream regulator, miR-3121-3p, affects tumor metastasis and proliferation via regulating Rap1GAP expression. MAPK signaling pathway may be involved in this effect.

CXCR4 Antagonist AMD3100 Reverses the Resistance to Tamoxifen in Breast Cancer via Inhibiting AKT Phosphorylation.

Endocrine therapy is a systemic therapy and has become the main treatment strategy for patients with estrogen receptor (ER)-positive breast cancer. However, tamoxifen resistance has become an insurmountable clinical challenge, and the underlying mechanisms are still poorly understood. In this study, we explored the roles of CXC chemokine receptor type 4 (CXCR4) in tamoxifen-treated breast cancer and tamoxifen resistance. Based on the Gene Expression Omnibus (GEO) database, high expression of CXCR4 was found to be associated with worse overall survival (hazard ratio [HR] = 4.646, p < 0.001) and cancer-specific survival (HR = 4.480, p < 0.001) in tamoxifen-treated breast cancer. CXCR4 was also positively correlated with the level of AKT phosphorylation and the resistance to tamoxifen in breast cancer. AMD3100 is a CXCR4 antagonist and was found to decrease phosphorylated (p)-AKT levels of tamoxifen-resistant cells. The reversal effect of AMD3100 on tamoxifen resistance was also confirmed in vitro and in vivo. Taken together, our study demonstrated that CXCR4 could be a potential prognostic biomarker for tamoxifen-treated breast cancer, and the combination of AMD3100 with tamoxifen could be a more efficacious therapeutic strategy for the treatment of tamoxifen resistance.

Combined MEK inhibition and tumor-associated macrophages depletion suppresses tumor growth in a triple-negative breast cancer mouse model.

Tumor-associated macrophages (TAMs) are closely related to poor prognosis in triple-negative breast cancer (TNBC). Thus, gaining insight into how TAMs support cancer progression could contribute to effective therapies. We utilized the 4 T1 murine TNBC cell line and murine bone marrow-derived macrophages to assess TAM-mediated pro-proliferative effects in vivo and in vitro. Further, Transcriptional analysis was performed to identify pathways activated in TAM-stimulated 4 T1 cells. We also explored the therapeutic efficacy of combining a mitogen-activated protein kinase kinase (MEK) inhibitor with TAM-targeted therapy using a TNBC mouse model. We found that the presence of TAMs was significantly associated with proliferating cancer cells in a TNBC mouse model. Moreover, RNA sequencing analysis showed that TAMs could enhance mitogen-activated protein kinase (MAPK) pathway activation in 4 T1 cells compared to that in control cells. Further, the depletion of TAMs by clodronate liposomes significantly reduced MAPK pathway activation in vivo. In addition, the blockade of MAPK signaling by a MEK inhibitor repressed TAM-mediated cancer cell proliferation. Most importantly, MEK inhibition combined with macrophage depletion significantly suppressed tumor growth and increased T lymphocyte infiltration in a TNBC model. Our study suggests the possibility that TAM-induced MAPK pathway activation promotes cancer cell proliferation. Thus, MEK inhibition combined with macrophage depletion might represent an effective treatment for TNBC.