UBA2 SUMOylates NQO1 and promotes the proliferation of hepatocellular carcinoma by modulating the MAPK pathway.

In our previous study, we found that small ubiquitin-related modifier (SUMO)-activating enzyme ubiquitin-associated-2 domain (UBA2) was upregulated in hepatocellular carcinoma (HCC) patients who were insensitive to chemoembolization. In this study, we aimed to investigate the role of UBA2 in HCC progression. Three cohorts were used to evaluate the efficacy of UBA2 as a prognostic factor for HCC. Our results indicated that UBA2 was associated with aggressive clinical behaviors and was a strong indicator of poor prognosis in HCC. In vitro experiments demonstrated that UBA2 accelerated cell growth, invasion, and migration. These results were further supported by in vivo experiments. RNA-sequencing analysis indicated NQO1 as a target of UBA2, with its levels altering following UBA2 manipulation. The results were verified by western blotting (WB) and quantitative PCR. The SUMOplot Analysis Program predicted lysine residue K240 as a modification target of UBA2, which was confirmed by immunoprecipitation (IP) assays. Subsequent mutation of NQO1 at K240 in HCC cell lines and functional assays revealed the significance of this modification. In addition, the oncogenic effect of UBA2 could be reversed by the SUMO inhibitor ML792 in vivo and in vitro. In conclusion, our study elucidated the regulatory mechanism of UBA2 in HCC and suggested that the SUMO inhibitor ML792 may be an effective combinatory treatment for patients with aberrant UBA2 expression.

Metabolic heterogeneity in clear cell renal cell carcinoma revealed by single-cell RNA sequencing and spatial transcriptomics.

BACKGROUND: Clear cell renal cell carcinoma is a prototypical tumor characterized by metabolic reprogramming, which extends beyond tumor cells to encompass diverse cell types within the tumor microenvironment. Nonetheless, current research on metabolic reprogramming in renal cell carcinoma mostly focuses on either tumor cells alone or conducts analyses of all cells within the tumor microenvironment as a mixture, thereby failing to precisely identify metabolic changes in different cell types within the tumor microenvironment. METHODS: Gathering 9 major single-cell RNA sequencing databases of clear cell renal cell carcinoma, encompassing 195 samples. Spatial transcriptomics data were selected to conduct metabolic activity analysis with spatial localization. Developing scMet program to convert RNA-seq data into scRNA-seq data for downstream analysis. RESULTS: Diverse cellular entities within the tumor microenvironment exhibit distinct infiltration preferences across varying histological grades and tissue origins. Higher-grade tumors manifest pronounced immunosuppressive traits. The identification of tumor cells in the RNA splicing state reveals an association between the enrichment of this particular cellular population and an unfavorable prognostic outcome. The energy metabolism of CD8+ T cells is pivotal not only for their cytotoxic effector functions but also as a marker of impending cellular exhaustion. Sphingolipid metabolism evinces a correlation with diverse macrophage-specific traits, particularly M2 polarization. The tumor epicenter is characterized by heightened metabolic activity, prominently marked by elevated tricarboxylic acid cycle and glycolysis while the pericapsular milieu showcases a conspicuous enrichment of attributes associated with vasculogenesis, inflammatory responses, and epithelial-mesenchymal transition. The scMet facilitates the transformation of RNA sequencing datasets sourced from TCGA into scRNA sequencing data, maintaining a substantial degree of correlation. CONCLUSIONS: The tumor microenvironment of clear cell renal cell carcinoma demonstrates significant metabolic heterogeneity across various cell types and spatial dimensions. scMet exhibits a notable capability to transform RNA sequencing data into scRNA sequencing data with a high degree of correlation.

Development and validation of a clinical-radiomics model for prediction of prostate cancer: a multicenter study.

PURPOSE: To develop an early diagnosis model of prostate cancer based on clinical-radiomics to improve the accuracy of imaging diagnosis of prostate cancer. METHODS: The multicenter study enrolled a total of 449 patients with prostate cancer from December 2017 to January 2022. We retrospectively collected information from 342 patients who underwent prostate biopsy at Minhang Hospital. We extracted T2WI images through 3D-Slice, and used mask tools to mark the prostate area manually. The radiomics features were extracted by Python using the "Pyradiomics" module. Least Absolute Shrinkage and Selection Operator (LASSO) regression was used for data dimensionality reduction and feature selection, and the radiomics score was calculated according to the correlation coefficients. Multivariate logistic regression analysis was used to develop predictive models. We incorporated the radiomics score, PI-RADS, and clinical features, and this was presented as a nomogram. The model was validated using a cohort of 107 patients from the Xuhui Hospital. RESULTS: In total, 110 effective radiomics features were extracted. Finally, 9 features were significantly associated with the diagnosis of prostate cancer, from which we calculated the radiomics score. The predictors contained in the individualized prediction nomogram included age, fPSA/tPSA, PI-RADS, and radiomics score. The clinical-radiomics model showed good discrimination in the validation cohort (C-index = 0.88). CONCLUSION: This study presents a clinical-radiomics model that incorporates age, fPSA/PSA, PI-RADS, and radiomics score, which can be conveniently used to facilitate individualized prediction of prostate cancer before prostate biopsy.

Renal anatomical classification systems cannot predict the occurrence of vascular complications after partial nephrectomy.

OBJECTIVES: To determine the relationship between renal tumor complexity and vascular complications after partial nephrectomy using PADUA, RENAL, and ZS scores. METHODS: Between January 2007 and December 2018, a total of 1917 patients with available cross-sectional imaging were enrolled in the study. Logistic regressions were used to identify independent predictors of vascular complications. RESULTS: Of 1917 patients, 31 (1.6%) developed vascular complications, including 10 females and 21 males. The high-complexity category was significantly associated with a decreased risk of vascular complication in PADUA (OR = 0.256; 95%CI = 0.086-0.762; P = 0.014) and ZS score (OR = 0.279; 95%CI = 0.083-0.946; P = 0.040). Laparoscopic partial nephrectomy and robot-assisted laparoscopic partial nephrectomy were independent risk factors for vascular complications. Meanwhile, the incidence was significantly reduced in the recent 4 years in the high score tumor group alone in PADUA (0.2% [1/474] vs. 2.2% [3/139], P = 0.038) and ZS score (0.2% [1/469] vs. 2.7% [3/112], P = 0.024). In the first 8 years, laparoscopic partial nephrectomy and robot-assisted laparoscopic partial nephrectomy were the only two independent risk factors for vascular complications. In the recent 4 years, only the high-complexity category was significantly associated with a decreased risk of vascular complication in the PADUA score (OR = 0.110; 95%CI = 0.013-0.938; P = 0.044). CONCLUSION: The renal anatomic classification system cannot predict the occurrence of vascular complications after partial nephrectomy.

A novel mini-retractor for retroperitoneal laparoscopic partial nephrectomy.

BACKGROUND: Retroperitoneal partial nephrectomy (RLPN) is the premier treatment for localized renal tumors despite narrow operation space. Many efforts have been taken to facilitate the operation of RLPN, but the optimal resolution remains debatable. OBJECTIVE: To explore the feasibility of using Mini-lap to improve workspace and surgical vision in RLPN. DESIGN, SETTING, AND PARTICIPANTS: A multicenter retrospective review of 51 patients who underwent RLPN with Mini-lap from January 2018 to December 2020 was conducted. SURGICAL PROCEDURE: Standard RLPN under three poles was performed in all cases. We highlighted the usage of Mini-lap (Teleflex Minilap percutaneous Surgical System) as a novel retractor in RLPN. OUTCOME AND MEASUREMENTS AND STATICAL ANALYSIS: Demographics, preoperative, intraoperative, and postoperative outcomes were assessed. RESULTS AND LIMITATIONS: All 51 cases completed RLPN with three ports successfully and no conversion to open surgery. The mean diameter of tumors was (3.53 ± 1.05) cm, in which 62.7% (32/51) were located anteriorly. The operation time and warm ischemic time (WIT) were (86.7 ± 15.9) min and (25.6 ± 5) min respectively. Minor complications (Clavien grade 1-2) occurred in 6 cases. The limitations were small sample size, retrospective design, and absence of control. CONCLUSIONS: Mini-lap could be used as a mini-retractor in RLPN, sparing extra assistant ports, expanding workspace, and optimizing vision. PATIENT SUMMARY: With highlights of larger workspace and less instrument interference, mini-lap could be applied in retroperitoneal laparoscopic partial nephrectomy.

One day versus two days of hepatic arterial infusion with oxaliplatin and fluorouracil for patients with unresectable hepatocellular carcinoma.

BACKGROUND: Hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin and 5-fluorouracil was effective in unresectable hepatocellular carcinoma (HCC). The program of FOLFOX-HAIC in HCC was performed for 1 day (HAIC 1d) or 2 days (HAIC 2d). We hereby retrospectively compared the efficacy and safety between these two treatment regimens and explored the predictive power of thymidylate synthase (TYMS), an enzyme involved in the DNA synthesis process and metabolism of fluorouracil. METHODS: This study included patients with a primary diagnosis of unresectable HCC. These patients received HAIC for 1 day or 2 days. The overall survival (OS), progression-free survival (PFS), tumor response, and adverse events were compared. The propensity score matching (PSM) was used to reduce bias. Peripheral blood samples before the treatments were collected and used to measure the concentration of TYMS through enzyme-linked immunosorbent assay (ELISA). ELISA was performed according to the manufacturers' guidelines. RESULTS: We included 368 patients for this study: 248 in the HAIC 1d group and 120 in the HAIC 2d group. There was no significant difference of OS between the two groups (14.5 for HAIC 1d vs 15.3 months for HAIC 2d, p=0.46). Compared with the HAIC 1d group, the HAIC 2d group did not prolong the PFS (7.3 vs 7.5 months, p=0.91) or elevate the tumor response (42.5% vs 39.1%, p=0.53) per RECIST 1.1. In the PSM cohort, the efficacy between the two groups was similar. The total frequencies of grade 3-4 events were higher with the HAIC 2d group than with the HAIC 1d group, especially in the PSM cohort (p=0.043). Additionally, patients with TYMS low level might benefit longer OS from the HAIC 2d group (18.7 vs 13.6 months, p=0.014). CONCLUSIONS: There was not much of a difference in efficacy between the two groups, but the HAIC for 1 day might be safer, which needed further research. The level of TYMS might be the predictive biomarkers.

Comparison of HBV reactivation between patients with high HBV-DNA and low HBV-DNA loads undergoing PD-1 inhibitor and concurrent antiviral prophylaxis.

BACKGROUND: Programmed cell death protein-1 (PD-1) inhibitor is recommended to treat advanced hepatocellular carcinoma (HCC). However, the safety of PD-1 inhibitor in patients with high HBV-DNA load is unknown because of the potential risk of hepatitis B virus (HBV) reactivation. This study was to compare the HBV reactivation between patients with low HBV-DNA loads and high HBV-DNA loads undergoing antiviral prophylaxis and PD-1 inhibitor. METHODS: This was a retrospective study including consecutive hepatitis B surface antigen-positive HCC patients who received PD-1 inhibitor and concurrent antiviral prophylaxis for prevention of clinical hepatitis. Patients were divided into low HBV-DNA group (low group, ≤ 500 IU/ml) and high HBV-DNA group (high group, > 500 IU/ml) according to the baseline HBV-DNA level. The incidences of HBV reactivation, HBV-associated hepatitis, and PD-1 inhibitor disruption were compared between the two groups. RESULTS: Two hundred two eligible patients were included: 94 in the low group and 108 in the high group. Seven patients (5 in the low group and 2 in the high group) developed HBV reactivation, and all recovered from HBV reactivation and HBV-associated hepatitis. The incidence of HBV reactivation in the two groups was low (5.3% vs 1.9%, P = 0.34). There was also no difference in the incidence of HBV-associated hepatitis (P = 0.56), or PD-1 inhibitor disruption (P = 0.82). The multivariable analysis showed PD-1 inhibitor with hepatic arterial infusion chemotherapy was the only significant risk factor for HBV reactivation (P = 0.04) and hepatitis (P = 0.002). CONCLUSION: With concurrent antiviral prophylaxis, HBV-DNA load higher than 500 IU/ml should not be a contraindication for PD-1 inhibitor.

Construction of a single nucleotide variant score-related gene-based prognostic model in hepatocellular carcinoma: analysis of multi-independent databases and validation in vitro.

BACKGROUND: The accumulation of single nucleotide variants (SNVs) and the emergence of neoantigens can affect tumour proliferation and the immune microenvironment. However, the SNV-related immune microenvironment characteristics and key genes involved in hepatocellular carcinoma (HCC) are still unclear. We aimed to evaluate differences in the SNV-related immune microenvironment, construct a prognostic model and validate the key genes in vitro. METHODS: The categories of samples were defined by the expression of SNV score-related genes to evaluate the differences in mutational features, immune environment and prognosis. The survival model was constructed with survival-associated genes and verified in two independent test datasets. RCAN2, the key gene screened out for biofunction, was validated in vitro. RESULTS: IC2, among the three integrated clusters (IC1, IC2, IC3) classified by the 82 SNV score-related genes, was distinct from the rest in SNV score and immune cell infiltration, showing a better prognosis. Seven prognostic markers, HTRA3, GGT5, RCAN2, LGALS3, CXCL1, CLEC3B, and CTHRC1, were screened to construct a prognostic model. The survival model distinguished high-risk patients with poor prognoses in three independent datasets (log-rank P < 0.0001, 0.011, and 0.0068, respectively) with acceptable sensitivity and specificity. RCAN2 was inversely correlated with NK cell infiltration, and knockdown of RCAN2 promoted proliferation in HCC. CONCLUSIONS: This study revealed the characteristics of the HCC SNV-associated subgroup and screened seven latent markers for their accuracy of prognosis. Additionally, RCAN2 was preliminarily proven to influence proliferation in HCC and it had a close relationship with NK cell infiltration in vitro. With the capability to predict HCC outcomes, the model constructed with seven key differentially expressed genes offers new insights into individual therapy.

Denonvilliers' fascia acts as the fulcrum and hammock for continence after radical prostatectomy.

BACKGROUND: Radical prostatectomy (RP) is the primary treatment of localized prostate cancer. Immediate urinary incontinence post-RP was still common and depressing without specific reason. METHODS: A multicenter cohort of 154 consecutive patients from 2018 to 2020, who was diagnosed with localized prostate cancer underwent either modified mini-incision retropubic radical prostatectomy (Mmi-RRP) or laparoscopic radical prostatectomy (LRP) or robotic-assisted radical prostatectomy (RARP). Seventy-two patients with Denonvilliers' fascia (DF) spared were included in DFS (Denonvilliers' fascia sparing) group. Whereas eighty-two patients with DF completely or partially dissected were set as Group Control. The primary outcome was immediate continence (ImC). Continuous data and categorical data were analyzed with t-test and Chi-square test, respectively. Odds ratios (ORs) were calculated with logistic regression. RESULTS: Urinary continence of Group DFS was significantly better than that of Group Control at each time point within one year after operation. Incidence rate of continence in Group DFS and Group Control were 83.3% vs 13.4% (P < 0.01) for ImC, 90.3% vs 30.5% (P < 0.01) at 3 months, 91.7% vs 64.6% (P < 0.01) at 6 months, and 93.1% vs 80.5% (P = 0.02) at 1 year after operation, respectively. Positive surgical margin (PSM) showed no significant difference (20.8% vs 20.7%, P = 1.0). In multivariate analysis, DFS showed importance for ImC post RP (OR = 26.4, P < 0.01). CONCLUSIONS: Denonvilliers' fascia acted as the fulcrum and hammock for continence post RP. Preservation of DF contributed to better continence after RP without increase of PSM. Trail registration Our research was conducted retrospectively and approved by the ethical committees of Minhang Hospital, but not registered.

NAP1L1 is a prognostic biomarker and contribute to doxorubicin chemotherapy resistance in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most fatal cancers, and its molecular basis needs to be delineated to identify biomarkers for its potential treatment. The purpose of this study was to identify a novel gene, nucleosome assembly proteins 1-like 1 protein (NAP1L1), associated with aggressive phenotypes of HCC. METHODS: Immunohistochemical staining was used to detect NAP1L1 protein expression in HCC tissues. The prognostic value of NAP1L1 expression was determined using Kaplan-Meier analysis and the Cox proportional hazards model. CCK-8 and apoptosis assays were used to detect the chemosensitivity in vitro. Xenograft tumor models were used to evaluate tumor cell proliferation and chemosensitivity in vivo. RESULTS: NAP1L1 expression was significantly upregulated in tumor tissues as compared to adjacent non-tumor tissues. High NAP1L1 expression in HCC tissues was associated with aggressive clinicopathologic features, such as serum AFP levels, tumor size and tumor number. Patients with high NAP1L1 expression had poor overall survival in our cohort and in the extra-validation cohort analyzed by TCGA microarray dataset and was further identified as an independent prognostic factor in HCC patients treated with radical resection. Both in vitro and in vivo assays showed that NAP1L1 promoted HCC cell proliferation and contribute to chemotherapy resistance. Further analyses found that some certain stemness associated genes were decreased concurrently with NAP1L1 down-regulation in HCC cell lines. CONCLUSIONS: Our findings support that NAP1L1 is a prognostic biomarker and may contribute to chemotherapy resistance in human hepatocellular carcinoma.

Development and prospective validation of a novel weighted quantitative scoring system aimed at predicting the pathological features of cystic renal masses.

OBJECTIVES: To develop and prospectively validate a novel weighted quantitative scoring system based on CT findings, namely, the renal cyst index (RCI), aimed at preoperatively predicting the pathological features of cystic renal masses (CRMs). METHODS: The RCI was based on four critical features of CRMs: the cyst wall, septal, nodule, and cyst contents. These parameters were scored with 1, 2, or 3 points. Weight coefficients for these parameters were determined by the multivariable logistic regression. The odds ratio (OR) and 95% confidence interval (95% CI) were used to summarise the results. The RCI was defined as the sum of these four weight coefficients. Malignancy risk prediction models were built based on the retrospective evaluation of 441 patients. We also compared the prediction ability of the RCI with the Bosniak classification in the 441 patients and applied these novel models to 152 masses resected in our institution to prospectively validate the efficiency of the RCI. RESULTS: The wall point (OR = 5.71 [95% CI = 1.734-18.808, p = 0.004, point = 2], OR = 12.665 [95% CI = 3.750-42.770, p < 0.001, point = 3]), septal point (OR = 3.325 [95% CI = 1.272-8.692, p = 0.014, point = 3]), nodule point (OR = 4.588 [95% CI = 1.429-14.729, p < 0.001, point = 2], OR = 17.032 [95% CI = 5.017-57.820, p = 0.010, point = 3]), content point (OR = 22.822 [95% CI = 1.041-495.995, p = 0.047, point = 2], OR = 2.723 [95% CI = 1.296-10.696, p = 0.015, point = 3]), and RCI (OR = 1.247 [95% CI = 1.197-1.299, p < 0.001]) were significantly associated with malignancy. Masses with an RCI < 6 were regarded as benign masses; masses with an RCI ≥ 10 were regarded as malignant masses. The malignancy risk of masses with an RCI > 6 but < 10 were determined by a nomogram. The prediction ability of the RCI was significantly superior to the Bosniak classification for Bosniak IIF and III masses (AUC: 0.912 vs. 0.753, p = 0.001). The RCI also accurately predicted the pathological features of 152 masses. CONCLUSION: The RCI is a reliable quantitative scoring system in predicting the malignancy risk of CRMs, and it outperformed the Bosniak classification system in some ways. KEY POINTS: • The renal cyst index (RCI) is a useful weighted quantitative classification system based on CT findings for diagnosing cystic renal masses. • The RCI outperforms the Bosniak classification system in some ways, especially for Bosniak IIF and III masses. • Masses with an RCI < 6 can be regarded as a simple cyst, while those with an RCI > 10 can be regarded as malignant masses.

Responses of bladder smooth muscle to the stretch go through extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor-κB (NF-κB) Pathway.

AIMS: The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch. METHODS: Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real-time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO. RESULTS: The bioinformatics analysis showed that chronic obstruction activated mitogen-activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor-κB (NF-κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF-κB in HBSMCs in a time-dependent manner. The mRNA expressions of α-smooth muscle actin and SM22 also increased and filamentous actin (F-actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF-κB pathway reversed mechanical stretch-induced changes of contractile phenotypic expression and F-action polymerization. CONCLUSIONS: Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F-actin. This process partially goes through ERK/p90RSK/NF-κB pathway.

Comparison of Stable and Unstable Ethiodized Oil Emulsions for Transarterial Chemoembolization of Hepatocellular Carcinoma: Results of a Single-Center Double-Blind Prospective Randomized Controlled Trial.

PURPOSE: To compare the stability of stable and unstable water-in-oil emulsions and the efficacy and safety of these emulsions in a single-center, prospective double-blind trial of transarterial chemoembolization for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: A total of 812 patients with inoperable HCC were randomized (stable emulsion, n = 402; unstable emulsion, n = 410). The 2 emulsions were prepared by using the same protocol except that different solvents were used for chemotherapy agents, including epirubicin, lobaplatin, and mitomycin C. The solvent in the stable emulsion arm was contrast medium and distilled water, and the solvent in the unstable emulsion arm was distilled water. The primary endpoint was overall survival (OS), and secondary endpoints were time to progression (TTP), tumor response, adverse events (AEs), and plasma epirubicin concentrations. RESULTS: In vitro, stable emulsions did not occur until 1 day, and unstable emulsions, with a lower peak plasma concentration (P = .001) in vivo, exhibited rapid separation of the oil and aqueous phases after 10 minutes. Median OS times in the stable and unstable emulsion arms were 17.7 and 19.2 months, respectively (P = .81). No differences were found in TTP, tumor response, and AEs except for myelosuppression (anemia, 3.5% vs 7.6%; thrombocytopenia, 11.5% vs 17.7%), which was significantly more severe and frequent in the unstable emulsion arm (P = .013). CONCLUSIONS: Chemoembolization is equally effective with the use of stable and unstable emulsions, but the use of a stable emulsion has the advantage of less myelosuppression and a favorable pharmacokinetic profile.

An optimized transurethral catheterization cystometry in mice and comparison with classic suprapubic catheterization cystometry.

AIMS: The aim of this study is to establish an optimized, minimally invasive transurethral catheterization cystometry (TUCC) and a novel urethral pressure profile (UPP) measurement for mice. METHODS: The optimized TUCC and the UPP measurement were first established. This optimized TUCC was then performed in 16 anesthetized female mice and compared with the suprapubic catheterization cystometry (SCC) in parallel after suprapubic catheters implantation (SCI; on zero, third, and seventh day, respectively). Finally, the optimized TUCC and novel UPP measurement were applied to investigate in another eight mice of partial bladder outlet obstruction (pBOO) model. The urodynamic parameters including micturition pressure (MP), basal pressure (BP), threshold pressure (TP), bladder capacity (BC), micturition volume (MV), residual urine (RV), bladder compliance (COM), maximum urethral pressure (MUP), bladder pressure curve and UPP were recorded. Statistical cross-comparisons of parameters for two kinds of cystometries and pBOO model were performed. RESULTS: Compared with the optimized TUCC before SCI, the MV, RV, BC, and MP decreased significantly on the seventh day after SCI (270.4-132.5 µL, 46.13-20.09 µL, 316.4-152.5 µL, 30.01-24.34 cmH2 0, respectively). After SCI, the BP, MP, TP, MV, RV, BC, and COM showed no significant difference between the TUCC and SCC at the same time point. The MUP increased significantly after pBOO operation (19.1-46.6 cmH2 0, P < 0.05). CONCLUSIONS: The minimally invasive TUCC along with UPP measurement could be widely applied to study the bladder function of mice as a feasible, repeatable, and accessible method.

The Prognostic Hub Gene POLE2 Promotes BLCA Cell Growth via the PI3K/AKT Signaling Pathway.

BACKGROUND: BLCA is a common urothelial malignancy characterized by a high recurrence rate. Despite its prevalence, the molecular mechanisms underlying its development remain unclear. AIMS: This study aimed to explore new prognostic biomarkers and investigate the underlying mechanism of bladder cancer (BLCA). OBJECTIVE: The objective of this study is to identify key prognostic biomarkers for BLCA and to elucidate their roles in the disease. METHODS: We first collected the overlapping DEGs from GSE42089 and TCGA-BLCA samples for the subsequent weighted gene co-expression network analysis (WGCNA) to find a key module. Then, key module genes were analyzed by the MCODE algorithm, prognostic risk model, expression and immunohistochemical staining to identify the prognostic hub gene. Finally, the hub gene was subjected to clinical feature analysis, as well as cellular function assays. RESULTS: In WGCNA on 1037 overlapping genes, the blue module was the key module. After a series of bioinformatics analyses, POLE2 was identified as a prognostic hub gene in BLCA from potential genes (TROAP, POLE2, ANLN, and E2F8). POLE2 level was increased in BLCA and related to different clinical features of BLCA patients. Cellular assays showed that si-POLE2 inhibited BLCA proliferation, and si-POLE2+ 740Y-P in BLCA cells up-regulated the PI3K and AKT protein levels. CONCLUSION: In conclusion, POLE2 was identified to be a promising prognostic biomarker as an oncogene in BLCA. It was also found that POLE2 exerts a promoting function by the PI3K/AKT signaling pathway in BLCA.

MZF1 promotes tumour progression and resistance to anti-PD-L1 antibody treatment in hepatocellular carcinoma.

Background & Aims: The mechanism underlying resistance to immunotherapy involves engagement of immune checkpoint pathways. The transcriptional and epigenetic processes of checkpoint molecules, however, have not been well investigated. We thus studied whether the transcription factor myeloid zinc finger 1 (MZF1) may promote resistance to immunotherapy in hepatocellular carcinoma (HCC). Methods: Single-cell RNA-sequencing was performed to study the correlation between MZF1 and tumour microenvironment features in six patients with HCC. Combined immunohistochemistry and multi-immunofluorescence analyses were performed for verification. Ectopic expression of MZF1 was used in both orthotopic and genetically engineered hydrodynamic mouse HCC models for in vivo experiments. Proteome analysis, including protein degradation assays, ubiquitination assays, and co-immunoprecipitation assays, revealed the function of MZF1 in immune checkpoint pathways. Results: Single-cell RNA-sequencing suggested an immunosuppressive environment and a strong correlation with the immune checkpoint programmed death ligand 1 (PD-L1) in MZF1-overexpressing tumours. Analyses of 163 HCC samples demonstrated that MZF1 expression in HCC cells is associated with decreased T-cell infiltration. In vivo experiments showed that ectopic MZF1 expression in HCC cells impairs T-cell recruitment, resulting in resistance to immune checkpoint blockade. Mechanistically, MZF1 accelerated PD-L1 ubiquitination by binding to the cyclin-dependent kinase 4 (CDK4) activation site, while a direct bond between CDK4 and MZF1 led to increased MZF1 expression. Conclusions: MZF1 promotes PD-L1 ubiquitination via CDK4 and possibly MZF1. Inhibition of CDK4 can therefore restore PD-L1 expression and may be a potential strategy for combination with anti-PD-L1 antibodies. Impact and implications: Resistance to immune checkpoint blockade with anti-programmed death ligand 1 (PD-L1) antibody therapy is attributed to oncogenic alterations of tumour cells, however, effective countermeasures are yet to be established. Here, we report that the transcription factor myeloid zinc finger 1 (MZF1) can bind to the cyclin-dependent kinase 4 (CDK4) activation site and accelerate PD-L1 ubiquitination. A CDK4 inhibitor therefore enhances anti-PD-L1 antibody efficacy by blocking MZF1 signalling. This indicates a potential benefit of combining CDK4 inhibitors and anti-PD-L1 antibodies for the treatment of advanced HCC.

Clinical and biomarker analyses of hepatic arterial infusion chemotherapy plus lenvatinib and PD-1 inhibitor for patients with advanced intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with a dismal prognosis and few effective therapeutic approaches. This study aimed to investigate the efficacy, safety, and predictive biomarkers of hepatic arterial infusion chemotherapy (FOLFOX-HAIC) in combination with lenvatinib and PD-1 inhibitor for patients with advanced iCCA. Methods: Locally advanced or metastatic iCCA patients receiving the triple combination therapy of lenvatinib, PD-1 inhibitor, and FOLFOX-HAIC were included in this retrospective study. Primary endpoint was the progression-free survival, evaluated using the RECIST criterion. The secondary endpoints included overall survival, objective response rate, and safety. Whole exome and RNA sequencing of tumor biopsy tissues were performed for biomarker exploration. Results: Between May, 2019 and December 2022, a total of 46 patients were included in this study. The primary endpoint showed a median progression-free survival of 9.40 months (95% CI: 5.28-13.52), with a 6-month progression-free survival rate of 76.1%. The median overall survival was 16.77 months (95% CI, 14.20-19.33), with an objective response rate of 47.8% and disease control rate of 91.3% per RECIST. In addition, 4.3% and 8.7% of patients achieved complete response of all lesions and intrahepatic target lesions per mRECIST, respectively. The most common treatment-related adverse events were neutropenia, thrombocytopenia, elevated aspartate aminotransferase and alanine aminotransferase level. Furthermore, integrated analysis of genetic, transcriptomic, and immunohistochemistry data revealed that pre-existing immunity (high expression level of immune-related signatures and intra-tumoral CD8+ T cell density) in baseline tumor tissues was associated with superior clinical benefits. However, the evaluation of tumor mutation burden did not show potential predictive value in this triple combination. Conclusion: FOLFOX-HAIC in combination with lenvatinib and PD-1 inhibitor demonstrated a promising antitumor activity with manageable safety profiles in patients with advanced iCCA. Moreover, our study also revealed new perspectives on potential biomarkers for clinical efficacy.

TMB Signature-Related RCAN2 Promotes Apoptosis by Upregulating EHF/DR5 Pathway in Hepatocellular Carcinoma.

BACKGROUND: The tumour mutation burden (TMB) is a valuable indicator of the accumulation of somatic mutations, and is thought to be associated with the biological behaviour and prognosis of tumours. However, the related genetic mechanism for these association is still unclear. The aim of the present study was to identify the key gene(s) associated with TMB in hepatocellular carcinoma (HCC) and to investigate its biological functions, downstream transcription factors, and mechanism of action. METHODS: Patients in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database were classified according to TMB signature-related genes. Key genes related to the TMB signature and tumour prognosis were identified. Immunohistochemistry and Quantitative Real-Time Polymerase Chain Reaction (qPCR) were then used to assess gene expression in clinical HCC tissues and HCC cells. Cells with altered gene expression were evaluated for the effect on cell proliferation and apoptosis, both in vitro and in vivo. Three independent databases and cell sequencing data were used to identify the mechanisms involved and the downstream transcription factors. The mechanism was also studied by altering the expression of downstream transcription factors in vitro. RESULT: The integrated cluster (IC) 2 group, characterized by 99 TMB signature-related genes, showed a significant different TMB score compared to the IC1 group (p < 0.001), as well as more favourable tumour prognosis (p = 0.031). We identified five key prognostic genes that were differentially expressed between IC2 and IC1 and were associated with overall survival. The expression of one of these key prognostic genes, RCAN2, was negatively correlated with TMB in 18 out of 33 tumour types examined. A high level of RCAN2 was correlated with better overall survival in HCC (p = 0.0009). Overexpression of RCAN2 enhanced apoptosis in vitro and in vivo, whereas knockdown of RCAN2 attenuated apoptosis. The mechanism by which RCAN2 promotes apoptosis may involve upregulation of the expression of ETS homologous factor (EHF) and of death receptor 5 (DR5). CONCLUSIONS: Downregulation of RCAN2 expression was found to correlate with elevated TMB in multiple cancer types. RCAN2 was also found to be a biomarker of HCC prognosis, and to promote the apoptosis of HCC cells through the EHF/DR5 pathway. These findings provide a new perspective on systemic treatment for advanced HCC with a high TMB.

Predictive Factors for Acute Kidney Injury and Long-Term Renal Function Loss After Partial Nephrectomy: A Prospective Single-Center Study.

OBJECTIVE: To find factors related to postoperative acute kidney injury and long-term significant renal function (RF) loss after partial nephrectomy (PN) in Chinese population. METHODS: The main outcome was significant RF loss during the last follow-up, which was defined as >25% decrease in estimated glomerular filtration rate. RESULTS: A total of 416 patients were included with median age as 57 (interquartile ranges,IQR 49.8-65.0) year with body mass index as 24.2 (IQR 22.0-26.5) kg/m2 and preoperative estimated glomerular filtration rate as 90.5 (IQR 79.8-101) mL/min. Summarily, 259 (62.3%) patients were male, 54 (13%) had diabetes, 180 (43.3%) hypertension and 80 (19.2%) hyperuricemia. Median (IQR) tumor diameter was 3.1 (2.4-4.1) cm. All patients underwent PN, in which 135 (32.5%) by open PN approach, 109 (26.2%) by laparoscopic PN and 172 (41.3%) by robot assisted PN. RF was followed up for 16.88 (10.15-36.37) months, where 58 (13.9%) patients suffered significant RF loss. Multivariable analysis showed age (P = .0039), body mass index (P = .0049), diabetes (P = .0351), operative time > 110 minutes (P = .0034), diameter classification by Diameter-Axial-Polar score (diameter 2.4 cm-4.4 cm, P = .0225: diameter > 4.4 cm, P = .0207), postoperative acute kidney injury (P < .001) to be predictors of RF loss with area under the curve as 0.850. CONCLUSION: We prospectively found predictive factors of short and long-term significant RF loss in all operative methods and constructed a clinical nomogram for long-term Chinese patients RF loss.