Single-cell combined bioinformatics analysis: construction of immune cluster and risk prognostic model in kidney renal clear cells based on CD8+ T cell-associated genes.

BACKGROUND: Kidney cancer is an immunogenic solid tumor, characterized by high tumor burden and infiltration of CD8+ T cells. Although immunotherapy targeting the PD1/CTLA-4 axis has demonstrated excellent clinical efficacy, clinical outcomes in most patients are poor. METHODS: We used the RNA sequencing data from the GEO database for KIRC GSE121636 and normal kidney tissue GSE131685, and performed single-cell analysis for cluster identification, pathway enrichment, and CD8+ T cell-associated gene identification. Subsequently, the significance of different CD8+ T-cell associated gene subtypes was elucidated by consensus clustering, pathway analysis, mutated gene analysis, and KIRC immune microenvironment analysis in the TCGA-KIRC disease cohort. Single gene analysis identified LAG3 as the most critical CD8+ T-cell-associated gene and its function was verified by cell phenotype and immunohistochemistry in KIRC. RESULTS: In the present study, CD8+ T-cell associated genes in KIRC were screened, including GZMK, CD27, CCL4L2, FXYD2, LAG3, RGS1, CST7, DUSP4, CD8A, and TRBV20-1 and an immunological risk prognostic model was constructed (risk score = - 0.291858656434841*GZMK - 0.192758342489394*FXYD2 + 0.625023643446193*LAG3 + 0.161324477181591*RGS1 - 0.380169045328895*DUSP4 - 0.107221347575037*TRBV20-1). LAG3 was identified and proved as the most critical CD8+ T cell-associated gene in KIRC. CONCLUSION: We proposed and constructed an immunological risk prognostic model for CD8+ T cell-associated genes and identified LAG3 as a pivotal gene for KIRC progression and CD8+ T-cell infiltration. The model comprehensively explained the immune microenvironment and provided novel immune-related therapeutic targets and biomarkers in KIRC.

Single-cell disulfidptosis regulator patterns guide intercellular communication of tumor microenvironment that contribute to kidney renal clear cell carcinoma progression and immunotherapy.

Background: Disulfidptosis, an emerging type of programmed cell death, plays a pivotal role in various cancer types, notably impacting the progression of kidney renal clear cell carcinoma (KIRC) through the tumor microenvironment (TME). However, the specific involvement of disulfidptosis within the TME remains elusive. Methods: Analyzing 41,784 single cells obtained from seven samples of KIRC through single-cell RNA sequencing (scRNA-seq), this study employed nonnegative matrix factorization (NMF) to assess 24 disulfidptosis regulators. Pseudotime analysis, intercellular communication mapping, determination of transcription factor activities (TFs), and metabolic profiling of the TME subgroup in KIRC were conducted using Monocle, CellChat, SCENIC, and scMetabolism. Additionally, public cohorts were utilized to predict prognosis and immune responses within the TME subgroup of KIRC. Results: Through NMF clustering and differential expression marker genes, fibroblasts, macrophages, monocytes, T cells, and B cells were categorized into four to six distinct subgroups. Furthermore, this investigation revealed the correlation between disulfidptosis regulatory factors and the biological traits, as well as the pseudotime trajectories of TME subgroups. Notably, disulfidptosis-mediated TME subgroups (DSTN+CD4T-C1 and FLNA+CD4T-C2) demonstrated significant prognostic value and immune responses in patients with KIRC. Multiple immunohistochemistry (mIHC) assays identified marker expression within both cell clusters. Moreover, CellChat analysis unveiled diverse and extensive interactions between disulfidptosis-mediated TME subgroups and tumor epithelial cells, highlighting the TNFSF12-TNFRSF12A ligand-receptor pair as mediators between DSTN+CD4T-C1, FLNA+CD4T-C2, and epithelial cells. Conclusion: Our study sheds light on the role of disulfidptosis-mediated intercellular communication in regulating the biological characteristics of the TME. These findings offer valuable insights for patients with KIRC, potentially guiding personalized immunotherapy approaches.

LMNTD2-AS1 regulates immune cell infiltration and promotes prostate cancer progression by targeting FUS to regulate NRF2 signal pathway.

Numerous studies have demonstrated that long non-coding RNAs (lncRNAs) play crucial roles in tumor progression. This study aimed to identify lncRNAs associated with overall survival (OS) and progression-free interval (PFI) in prostate cancer (PCa) patients and to elucidate the driving mechanisms and functions of these lncRNAs. We utilized the TCGA database to screen for lncRNAs linked with OS and PFI. KM survival analysis, ROC curve analysis, and Cox survival analysis were employed to assess the prognostic significance of lncRNAs in PCa patients. We conducted a loss-of-function assay to explore the role of lncRNAs in PCa. Correlation analysis was performed to study the relationship between lncRNAs and immune cell infiltration. Lasso regression analysis was performed to screen proteins which might interact with lncRNAs, while rescue experiments verified the integrity of the signaling pathway. LMNTD2-AS1 was found to be the only lncRNA in PCa patients associated with both OS and PFI with significantly elevated levels in PCa. Elevated LMNTD2-AS1 expression was significantly linked to advanced stage, grade, primary treatment outcomes, residual tumors, and Gleason scores in PCa patients. Moreover, multivariate Cox regression analysis revealed that high LMNTD2-AS1 expression independently predicted PFI in PCa patients. The AUC of LMNTD2-AS1 for predicting 3-year OS and 5-year OS in PCa patients was 0.877 and 0.807, respectively, while for 3-year PFI and 5-year PFI it was 0.751 and 0.727, respectively. Overexpression of LMNTD2-AS1 correlated with infiltration of neutrophils, macrophages, pDC, NK CD56bright cells, and other immune cells. Furthermore, FUS and NRF2 are both potential binding proteins and related signaling pathways downstream of LMNTD2-AS1. Functional experiments demonstrated that LMNTD2-AS1 knockdown significantly inhibited migration, invasion, and proliferation of PCa cells while overexpression of FUS was found to rescue the functional inhibition caused by LMNTD2-AS1 knockdown. LMNTD2-AS1 functions as an oncogene in PCa, influencing patient prognosis and the immune microenvironment; it may regulate immune cell infiltration and promote PCa progression by interacting with the NRF2 signaling pathway via FUS binding.

Biological functions of RNA modification patterns that define tumor microenvironment and survival outcomes in testicular germ cell tumors.

BACKGROUND: Accumulating evidence has indicated that aberrant RNA modifications are associated with malignant progression and the immune microenvironment in various tumors. However, the function of RNA modification regulators in testicular germ cell tumors (TGCTs) remains to be discovered. This study aimed to investigate the biological functions of RNA modification regulators in testicular germ cell tumors and identify their potential clinical predictive value. METHODS: Expression level of 75 RNA modification regulators was acquired to generate differential expression patterns. RNA modification regulatory genes were applied to construct a progression-free survival (PFS) risk model. Meanwhile, three RNA modification clusters were identified using consensus clustering. Subsequently, the infiltration characteristics of cells in the microenvironment as well as the antitumor drug candidates have been further analyzed. Finally, to further validate our results, we examined the expression and biological behavior of seven selected RNA modification regulators both in TGCT cell lines and clinical tissues. RESULTS: We collected the differentially expressed regulators of RNA modification. RNA modification risk signature was developed to stratify the prognosis of TGCT patients. Furthermore, we found significant differences in immune microenvironment between subgroups. Ultimately, seven selected RNA modification regulators were further verified. CONCLUSIONS: We generated and validated a risk signature related to RNA modification which could accurately predict the relapse risk in TGCT patients. This risk signature was correlated with immune cells infiltration among tumor microenvironments. Furthermore, we screened antitumor drug candidates and evaluated the sensitivity and efficacy of class chemotherapeutic drugs, which could provide reference for clinical drug use.

A regulatory circuit of lncRNA NLGN1-AS1 and Wnt signalling controls clear cell renal cell carcinoma phenotypes through FZD4-modulated pathways.

BACKGROUND: Recent evidence has indicated that long non-coding RNAs (lncRNAs) were emerged as key molecules in clear cell renal cell carcinoma (ccRCC). TCGA database showed that the expression level of lncRNA NLGN1-AS1 was up-regulated in ccRCC; However, whether NLGN1-AS1 implicated in the malignant progression of ccRCC remained unclear. METHODS: Based on TCGA database, candidate lncRNAs were selected and quantitative real-time PCR (qRT-PCR) was utilized to verify the expression levels of candidate lncRNAs in human ccRCC tissues. Loss-of-function experiments were performed to examine the biological functions of NLGN1-AS1 both in vitro and in vivo. According to bioinformatics analysis, fluorescence reporter assays and rescue experiments, the underlying mechanisms of NLGN1-AS1 in ccRCC cell lines were so clearly understood. RESULTS: As a novel lncRNA, NLGN1-AS1 was up-regulated in ccRCC cell lines and associated with poor prognosis of and ccRCC patients, which was correlated with the progression of ccRCC. Functionally, the down-regulation of NLGN1-AS1 significantly decreased the proliferation of ccRCC cells both in vitro and in vivo. Bioinformatics analysis and luciferase report assays identified that miR-136-5p was a direct target of NLGN1-AS1. We also determined that FZD4 were inhibitory targets of miR-136-5p, and that Wnt/ß-catenin signaling was inhibited by both NLGN1-AS1 knockdown and miR-136-5p over-expression. In addition, we found that the suppression of proliferation and the inhibition of Wnt/ß-catenin pathway induced by NLGN1-AS1 knockdown would require the over-expression of FZD4. CONCLUSIONS: Our findings suggested that lncRNA NLGN1-AS1 could promote the progression of ccRCC by targeting miR-136-5p/FZD4 and Wnt/ß-catenin pathway, and might serve as a novel potential therapeutic target to inhibit the progression of ccRCC.

PSA density in the diagnosis of prostate cancer in the Chinese population: results from the Chinese Prostate Cancer Consortium.

We performed this study to investigate the diagnostic performance of prostate-specific antigen density (PSAD) in a multicenter cohort of the Chinese Prostate Cancer Consortium. Outpatients with prostate-specific antigen (PSA) levels ≥4.0 ng ml-1 regardless of digital rectal examination (DRE) results or PSA levels <4.0 ng ml-1 and abnormal DRE results were included from 18 large referral hospitals in China. The diagnostic performance of PSAD and the sensitivity and specificity for the diagnosis of prostate cancer (PCa) and high-grade prostate cancer (HGPCa) at different cutoff values were evaluated. A total of 5220 patients were included in the study, and 2014 (38.6%) of them were diagnosed with PCa. In patients with PSA levels ranging from 4.0 to 10.0 ng ml-1, PSAD was associated with PCa and HGPCa in both univariate (odds ratio [OR] = 45.15, P < 0.0001 and OR = 25.38, P < 0.0001, respectively) and multivariate analyses (OR = 52.55, P < 0.0001 and OR = 26.05, P < 0.0001, respectively). The areas under the receiver operating characteristic curves (AUCs) of PSAD in predicting PCa and HGPCa were 0.627 and 0.630, respectively. With the PSAD cutoff of 0.10 ng ml-2, we obtained a sensitivity of 88.7% for PCa, and nearly all (89.9%) HGPCa cases could be detected and biopsies could be avoided in 20.2% of the patients (359/1776 cases). Among these patients who avoided biopsies, only 30 cases had HGPCa. We recommend 0.10 ng ml-2 as the proper cutoff value of PSAD, which will obtain a sensitivity of nearly 90% for both PCa and HGPCa. The results of this study should be validated in prospective, population-based multicenter studies.

[Magnetic resonance-ultrasound fusion guided transperineal prostate biopsy for the diagnosis of prostate cancer].

OBJECTIVE: To investigate the application value of magnetic resonance-ultrasound fusion (MR-USF) guided transperineal prostate biopsy (TPPB) in the diagnosis of prostate cancer. METHODS: Relevant data were collected retrospectively from 77 patients undergoing MR-USF guided TPPB (n = 22) or 12-core systematic prostate biopsy (SPB) (n = 55) in Binhai People's Hospital from May to July 2019 and statistically analyzed using the software Graphad Prism 7.0 and SPSS 22.0. RESULTS: The patients were aged 51－91 (70.5 ± 9.7) years, with a mean PSA level of (35.1 ± 115.4) µg/L (1.02－959 µg/L) and an average prostate volume of (48.81 ± 38.4) cm3 (7.54－307.61 cm3). There were no statistically significant differences in age, PSA, and BMI between the two groups of patients (P > 0.05). Prostate cancer was confirmed in 31 of the cases, with a positive rate of 40.26% (31/77), significantly higher in the TPPB (45.45% ï¼»10/22ï¼½ than in the SPB group (38.18% ï¼»21/55ï¼½, P < 0.01). Based on the PI-RADS scores, the Gleason grade was also higher in the former than in the latter group. CONCLUSIONS: MR-USF guided TPPB can improve the biopsy performance. Whether it should be used is based on the patient's PSA level and PI-RADS scores.

Involvement of aberrantly activated HOTAIR/EZH2/miR-193a feedback loop in progression of prostate cancer.

BACKGROUND: Though androgen deprivation therapy is the standard treatment for prostate cancer (PCa), most patients would inevitably progress to castration-resistant prostate cancer (CRPC) which is the main cause of PCa death. Therefore, the identification of novel molecular mechanism regulating cancer progression and achievement of new insight into target therapy would be necessary for improving the benefits of PCa patients. This study aims to study the function and regulatory mechanism of HOTAIR/EZH2/miR-193a feedback loop in PCa progression. METHODS: MSKCC and TCGA datasets were used to identify miR-193a expression profile in PCa. Cell Counting Kit-8 (CCK-8) assays, colony formation, invasion, migration, flow cytometry, a xenograft model and Gene Set Enrichment Analysis were used to detect and analyze the biological function of miR-193a. Then, we assessed the role of HOTAIR and EZH2 in regulation of miR-193a expression by using plasmid, lentivirus and small interfering RNA (siRNA). Luciferase reporter assays and chromatin immunoprecipitation assays were performed to detect the transcriptional activation of miR-193a by EZH2 and HOTAIR. Further, qRT-PCR and luciferase reporter assays were conducted to examine the regulatory role of miR-193a controlling the HOTAIR expression in PCa. Finally, the correlation between HOTAIR, EZH2 and miR-193a expression were analyzed using In situ hybridization and immunohistochemistry. RESULTS: We found that miR-193a was significantly downregulated in metastatic PCa through mining MSKCC and TCGA datasets. In vitro studies revealed that miR-193a inhibited PCa cell growth, suppressed migration and invasion, and promoted apoptosis; in vivo results demonstrated that overexpression of miR-193a mediated by lentivirus dramatically reduced PCa xenograft tumor growth. Importantly, we found EZH2 coupled with HOTAIR to repress miR-193a expression through trimethylation of H3K27 at miR-193a promoter in PC3 and DU145 cells. Interestingly, further evidence illustrated that miR-193a directly targets HOTAIR showing as significantly reduced HOTAIR level in miR-193a overexpressed cells and tissues. The expression level of miR-193a was inversely associated with that of HOTAIR and EZH2 in PCa. CONCLUSION: This study firstly demonstrated that miR-193a acted as tumor suppressor in CRPC and the autoregulatory feedback loop of HOTAIR/EZH2/miR-193a served an important mechanism in PCa development. Targeting this aberrantly activated feedback loop may provide a potential therapeutic strategy.