A novel prognostic N7-methylguanosine-related long non-coding RNA signature in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is regulated by methylation modifications and long noncoding RNAs (lncRNAs). However, knowledge of N7-methylguanosine (m7G)-related lncRNAs that predict ccRCC prognosis remains insufficient. A prognostic multi-lncRNA signature was created using LASSO regression to examine the differential expression of m7G-related lncRNAs in ccRCC. Furthermore, we performed Kaplan-Meier analysis and area under the curve (AUC) analysis for diagnosis. In all, a model based on five lncRNAs was developed. Principal component analysis (PCA) indicated that the risk model precisely separated the patients into different groups. The IC50 value for drug sensitivity divided patients into two risk groups. High-risk group of patients was more susceptible to A.443654, A.770041, ABT.888, AMG.706, and AZ628. Moreover, a lower tumor mutation burden combined with low-risk scores was associated with a better prognosis of ccRCC. Quantitative real-time polymerase chain reaction (qRT-PCR) exhibited that the expression levels of LINC01507, AC093278.2 were very high in all five ccRCC cell lines, AC084876.1 was upregulated in all ccRCC cell lines except 786-O, and the levels of AL118508.1 and DUXAP8 were upregulated in the Caki-1 cell line. This risk model may be promising for the clinical prediction of prognosis and immunotherapeutic responses in patients with ccRCC.

Machine learning-based construction of a ferroptosis and necroptosis associated lncRNA signature for predicting prognosis and immunotherapy response in hepatocellular cancer.

Introduction: Liver hepatocellular carcinoma (LIHC), one of the most common malignancies worldwide, occurs with high incidence and mortality. Ferroptosis and necroptosis are critically associated with LIHC prognosis. Some long non-coding RNAs (lncRNAs) have been found to induce ferroptosis and necroptosis in hepatocellular carcinoma cells. Methods: Cox regression analysis was used to construct a risk model for LIHC based on differentially expressed ferroptosis and necroptosis related lncRNAs (F-NLRs), and their expression in SMMC7721, HepG2 and WRL68 cells was detected by qPCR. Results: Five F-NLRs were associated with LIHC prognosis, including KDM4A-AS1, ZFPM2-AS1, AC099850.3, MKLN1-AS, and BACE1-AS. Kaplan-Meier survival analysis indicated that patients with LIHC in the high-risk group were associated with poor prognosis. The combined F-NLR signature model demonstrated a prognostic AUC value of 0.789 and was more accurate than standard clinical variables for predicting LIHC prognosis. T cell functions and immunotherapy responses differed significantly between patients in the low- and high-risk groups. Additionally, immune checkpoints and m6A-related genes were differentially expressed between patients in the two risk groups. Furthermore, proteins encoded by the five F-NLRs were overexpressed in four liver cancer cell lines compared to that in human liver cell line WRL68. Pan-cancer examination revealed that expression levels of the five F-NLRs differed between most common tumor types and normal tissues. Conclusion: F-NLRs identified in this study provide a predictive signature representing ferroptosis and necroptosis in LIHC, which correlated well with patient prognosis, clinicopathological characteristics, and immunotherapy responses. The study findings help to elucidate the mechanisms of F-NLRs in LIHC and provide further guidance for the selection and development of immunotherapeutic agents for LIHC.

Identification and Validation of Cuproptosis-Related LncRNA Signatures in the Prognosis and Immunotherapy of Clear Cell Renal Cell Carcinoma Using Machine Learning.

(1) Objective: We aimed to mine cuproptosis-related LncRNAs with prognostic value and construct a corresponding prognostic model using machine learning. External validation of the model was performed in the ICGC database and in multiple renal cancer cell lines via qPCR. (2) Methods: TCGA and ICGC cohorts related to renal clear cell carcinoma were included. GO and KEGG analyses were conducted to determine the biological significance of differentially expressed cuproptosis-related LncRNAs (CRLRs). Machine learning (LASSO), Kaplan-Meier, and Cox analyses were conducted to determine the prognostic genes. The tumor microenvironment and tumor mutation load were further studied. TIDE and IC50 were used to evaluate the response to immunotherapy, a risk model of LncRNAs related to the cuproptosis genes was established, and the ability of this model was verified in an external independent ICGC cohort. LncRNAs were identified in normal HK-2 cells and verified in four renal cell lines via qPCR. (3) Results: We obtained 280 CRLRs and identified 66 LncRNAs included in the TCGA-KIRC cohort. Then, three hub LncRNAs (AC026401.3, FOXD2-AS1, and LASTR), which were over-expressed in the four ccRCC cell lines compared with the human renal cortex proximal tubule epithelial cell line HK-2, were identified. In the ICGC database, the expression of FOXD2-AS1 and LASTR was consistent with the qPCR and TCGA-KIRC. The results also indicated that patients with low-risk ccRCC-stratified by tumor-node metastasis stage, sex, and tumor grade-had significantly better overall survival than those with high-risk ccRCC. The predictive algorithm showed that, according to the three CRLR models, the low-risk group was more sensitive to nine target drugs (A.443654, A.770041, ABT.888, AG.014699, AMG.706, ATRA, AP.24534, axitinib, and AZ628), based on the estimated half-maximal inhibitory concentrations. In contrast, the high-risk group was more sensitive to ABT.263 and AKT inhibitors VIII and AS601245. Using the CRLR models, the correlation between the tumor immune microenvironment and cancer immunotherapy response revealed that high-risk patients are more likely to respond to immunotherapy than low-risk patients. In terms of immune marker levels, there were significant differences between the high- and low-risk groups. A high TMB score in the high-risk CRLR group was associated with worse survival, which could be a prognostic factor for KIRC. (4) Conclusions: This study elucidates the core cuproptosis-related LncRNAs, FOXD2-AS1, AC026401.3, and LASTR, in terms of potential predictive value, immunotherapeutic strategy, and outcome of ccRCC.

A Novel Prognostic Ferroptosis-Related Long Noncoding RNA Signature in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common primary malignancy of renal cancer in adults. Ferroptosis is critically associated with the prognosis of ccRCC. However, knowledge of long noncoding RNA- (lncRNA-) related ferroptosis that affects the prognosis of ccRCC is still insufficient. Using the LASSO regression, we created a risk model based on differentially expressed ferroptosis-related lncRNAs (FRLRS) in ccRCC. The analysis of Kaplan-Meier for survival, area under the curve (AUC) for diagnosis, nomogram for predicting overall survival, and gene expression for immune checkpoints were performed based on the screened independent prognostic factors. Nine lncRNAs were found to be associated with ccRCC prognosis. Furthermore, the prognostic AUC of the FRLRS signature was 0.78, demonstrating its usefulness in predicting ccRCC prognosis. The lncRNA risk model outperformed the standard clinical variables in predicting ccRCC prognosis. Finally, The Cancer Genome Atlas revealed that T cell functions, such as cytolytic activity, human leukocyte antigen activity, inflammation regulation, and type II interferon response coordination, are significantly different between two different risk levels of ccRCC. Immune checkpoints were also expressed differently in programmed cell death 1 receptor, inducible T cell costimulator, cytotoxic T-lymphocyte antigen-4, and leukocyte-associated immunoglobulin-like receptor 1. The nine FRLRS signature models may affect the prognosis of ccRCC.

Diagnosis of Lung Cancer by ATR-FTIR Spectroscopy and Chemometrics.

Lung cancer is the leading cause of cancer-related death in the world. Early diagnosis has great significance for the survival of patients with lung cancer. In this paper, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy combined with chemometrics was used to study the serum samples from patients with lung cancer and healthy people. The results of spectral band area comparison showed that the concentrations of protein, lipid and nucleic acids molecules in serum of patients with lung cancer were increased compared with those in healthy people. The original spectra were preprocessed to improve the accuracy of principal component regression (PCR) and partial least squares-discriminant analysis (PLS-DA) models. PLS-DA results for first derivative spectral data in nucleic acids (1250-1000cm-1) band showed 80% sensitivity, 91.89% specificity and 87.10% accuracy with high R c 2 of 0.8949 and R v 2 of 0.8153, low RMSEC of 0.3136 and RMSEV of 0.4180. It is shown that ATR-FTIR spectroscopy combined with chemometrics might be developed as a simple method for clinical screening and diagnosis of lung cancer.

Nomogram for the Prediction of Intrahospital Mortality Risk of Patients with ST-Segment Elevation Myocardial Infarction Complicated with Hyperuricemia: A Multicenter Retrospective Study.

PURPOSE: This study aimed to establish an accurate and easy predictive model for ST-segment elevation myocardial infarction (STEMI) patients with hyperuricemia, using readily available features to estimate intrahospital mortality risk. PATIENTS AND METHODS: This was a multicenter retrospective study involving the development of risk prediction models for intrahospital mortality among all STEMI patients with hyperuricemia from Zunyi Medical University Chest Pain Center's specialized alliance between January 1, 2016 and June 30, 2020. The primary outcome was intrahospital mortality. A total of 48 candidate variables were considered from demographic and clinical data. The least absolute shrinkage and selection operator (LASSO) was used to develop a nomogram. Concordance index values, decision curve analysis, the area under the curve (AUC), and clinical impact curves were examined. In this study, 489 patients with STEMI were included in the training dataset and an additional 209 patients from the 44 chest pain centers were included in the test cohort. B-type natriuretic peptides, α-hydroxybutyrate dehydrogenase (α-HBDH), cystatin C, out-of-hospital cardiac arrest (OHCA), shock index, and neutrophil-to-lymphocyte ratio were associated with intrahospital mortality and included in the nomogram. RESULTS: The model showed good discrimination power, and the AUC generated to predict survival in the training set was 0.875 (95% confidence interval, 0.825-0.925). In the validation set, the AUC of survival predictions was 0.87 (95% confidence interval, 0.792-0.947). Calibration plots and decision curve analysis showed good model performance in both datasets. A web-based calculator (https://bzxzmu.shinyapps.io/STEMI-with-Hyperuricemia-intrahospital-mortality/) was established based on the nomogram model, which was used to measure the levels of OHCA, neutrophil-to-lymphocyte ratio, shock index, α-HBDH, cystatin C, and B-type natriuretic peptides. CONCLUSION: For practical applications, this model may prove clinically useful for personalized therapy management in patients with STEMI with hyperuricemia.

Hypoxia-reoxygenation induces macrophage polarization and causes the release of exosomal miR-29a to mediate cardiomyocyte pyroptosis.

To investigate the mechanism by which hypoxia-reoxygenation (HR) mediates macrophage polarization to the M1 phenotype and then mediates cardiomyocyte (CM) pyroptosis through exosome release. Mouse bone marrow macrophages and CMs were cultured in vitro under hypoxia for 12 h and reoxygenation for 6 h to establish an HR cell model. qPCR was used to detect the M1 or M2 macrophage markers IL-1ß, TNF-α, MR, and Arg, and a macrophage and CM coculture system was then established. Macrophages were transfected with an exosome-CD63-red fluorescent protein (RFP) lentivirus, allowing secretion of exosomes expressing RFP, and GW4869 was used to inhibit exosome release by macrophages. qPCR detected miR-29 expression in macrophage-derived exosomes, and macrophages were transfected with miR-29a inhibitors to obtain exosomes with low miR-29a expression (siR-exos). Pyroptosis indicators were detected by Western blot and ELISA. Importantly, LPS induced bone marrow macrophage polarization to the M1 type as a positive control to further verify that these exosomes (LPS-exos) regulated CM pyroptosis by delivering miR29a. Dual luciferase reporter and Western blot assays were adopted to analyze the miR-29a and MCL-1 target relationship. In addition, MCL-1 overexpression was used as a rescue experiment to determine whether miR-29a regulates pyroptosis in CM by targeting MCL-1. Macrophages expressed the M1 macrophage markers IL-1ß and TNF-α after HR exposure. After CM coculture, RFP expression was significantly higher in the HR group than in the normal (Nor) group but significantly reduced in the GW4869 group. Immunofluorescence showed that caspase-1 mRNA and protein expression in the HR group was significantly higher than that in the Nor group (P < 0.05). Caspase-1 expression was significantly decreased in the GW4869 group compared with the HR group (P < 0.05). Western blotting showed that the pyrolysis-related NLRP3 and ASC protein expression levels were significantly upregulated in the HR group compared with the control (Ctr) and Nor groups (P < 0.05). However, GW4869 effectively inhibited pyroptosis-related protein expression (P < 0.05). In addition, ELISA showed that the expression of the inflammation indicators IL-1ß and IL-18 was significantly increased in the HR group compared to the Ctr group (P < 0.05) but decreased in the GW4869 group (P < 0.05). qPCR showed that miR-29a was upregulated in the HR group compared to the Nor group. Moreover, HR-induced exosomes (HR-exos) from macrophages exacerbated HR-induced CM pyroptosis, while inhibition of miR-29a in exosomes partially offset CM pyroptosis induction. LPS-exos promoted pyroptosis-related protein expression, as the IL-1ß and IL-18 concentrations were increased in the LPS-exos group. However, pyroptosis-related proteins were observably decreased, and IL-1ß and IL-18 were also significantly decreased after miR-29a inhibition when compared with that in the HR-exos and LPS-exos groups. Mcl-1 overexpression reversed miR-29a-mediated CM pyroptosis in an HR environment. HR treatment induced macrophage polarization towards the M1 phenotype, which mediated CM pyroptosis through exosomal miR-29a transfer by targeting MCL-1.

[High-salt exposure induces macrophage polarization to promote proliferation and phenotypic transformation of co-cultured renal fibroblasts].

OBJECTIVE: To investigate high-salt exposure-induced polarization of mononuclear macrophages and the changes in proliferation and phenotypic transformation of renal fibroblasts in a co-culture system. METHODS: Cultured mononuclear macrophages were exposed to high salt (161 mmol/L Na +) for 2 h and the surface markers of M0, M1 and M2-type macrophages were detected with RT-qPCR. The culture medium of the macrophages in normal and high-salt groups was collected for detection of the mRNA and protein levels of IL-6 and TGF-ß1 using RT-qPCR and ELISA. A co-culture system of high salt-exposed macrophages and renal fibroblasts (NRK-49F) was established using a Transwell chamber, and the changes in proliferation and migration of NRK-49F cells were examined using EdU assay and Transwell assay, respectively. Western blotting was performed to detect the expressions of collagen I, collagen III and collagen α-SMA in NRK-49F cells. RESULTS: The high salt-exposed macrophages showed significantly increased mRNA levels of M2-type macrophage surface markers mannose receptor and arginase (P < 0.05). The results of EdU and Transwell assays showed that NRK-49F cells co-cultured with high salt-exposed macrophages exhibited significantly increased proliferation and migration ability (P < 0.05). Co-culture with high salt-exposed macrophages resulted in significantly enhanced protein expressions of collagen I, collagen III and α-SMA in NRK-49F cells (P < 0.05) and significantly increased levels of IL-6 and TGF-ß1 in the culture medium (P < 0.05). CONCLUSIONS: High-salt exposure induces polarization of mononuclear macrophages into M2-type macrophages and promotes secretion of IL-6 and TGF-ß1 by the macrophages to induce the proliferation and phenotypic transformation of NRK-49F cells.

[Role of TGF-ß1/ILK/FSP1 signaling pathway in cyclosporin A-induced epithelialmesenchymal transition in cultured renal tubular epithelial cells].

OBJECTIVE: To explore the role of transforming growth factor-ß1/integrin-linked kinase/fibroblast-specific protein 1 (TGF- ß1/ILK/FSP1) signaling pathway in cyclosporine A (CsA)-induced renal tubular epithelial cell transdifferentiation. METHODS: Rat renal tubular epithelial NRK-52E cells were induced with 1 mg/L CsA, treated with TGF-ß1 inhibitor (SB431542, 10 µmol/L), or transfected with the ILK-RNAi lentiviral expression vector (ILKshRNA) or a negative control vector before CsA induction. The expressions of TGF-ß1, ILK and FSP-1 mRNAs and proteins in the cells were detected using real-time PCR and Western blotting. The positive cells for α-SMA expression were detected by immunohistochemistry. RESULTS: Compared with the blank control cells, the cells treated with CsA showed significantly increased levels of TGF-ß1, ILK and FSP-1 mRNAs and proteins (P < 0.05). The expressions of TGF-ß1, ILK and FSP-1 were significantly lower in TGF-ß1 inhibitor group than in CsA group (P < 0.05). The levels of ILK and FSP-1 were significantly decreased after shRNA-mediated ILK silencing (P < 0.05). The number of positive cells for α-SMA was significantly lower in cells treated with SB431542 and in cells with ILK silencing than in the cells treated with CsA alone (P < 0.05). CONCLUSIONS: The activation of TGF-ß1/ILK/FSP-1 signaling pathway is an important mechanism for CsA-induced transdifferentiation in rat renal tubular epithelial cells. ILK participates in CsA-induced epithelialmesenchymal transition of renal tubular epithelial cells.