Smad2/3/4 complex could undergo liquid liquid phase separation and induce apoptosis through TAT in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-ß signaling pathway may be associated with tumor progression. However, the relationship between TGF-ß signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-ß signaling pathway on HCC progression as well as the potential regulatory mechanism involved. METHODS: We conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni-NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo. RESULTS: We found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is -1032 to -1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis. CONCLUSION: This study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-ß signaling pathway and HCC, which contributes to discover the mechanism of HCC development.

Identification of cuproptosis hub genes contributing to the immune microenvironment in ulcerative colitis using bioinformatic analysis and experimental verification.

Instruction: Ulcerative colitis (UC) can cause a variety of immune-mediated intestinal dysfunctions and is a significant model of inflammatory bowel disease (IBD). Colorectal cancer (CRC) mostly occurs in patients with ulcerative colitis. Cuproptosis is a type of procedural death that is associated with different types of diseases to various degrees. Methods: We used a combination of bioinformatic prediction and experimental verification to study the correlation between copper poisoning and UC. We used the Gene Expression Omnibus database to obtain disease gene expression data and then identified relevant genes involved in various expression levels in normal and UC samples. The Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed to cluster the genes that are highly responsible and find the central interaction in gene crosstalk. Notably, DLD, DLAT, and PDHA1 were present in high-scoring PPI networks. In addition, hub gene expression information in UC tissues was integrated to estimate the relationship between UC copper poisoning and the immune environment. Results: In our study, the expression of DLD, DLAT, and PDHA1 in UC tissues was lower than that in normal tissues. The key genes associated with cuproptosis have therapeutic effects on immune infiltration. We verified the expression of DLD, DLAT, and PDHA1 using real-time quantitative polymerase chain reaction in mouse models of UC induced by DSS. Discussion: Notably, this study clearly indicates that bioinformatic analysis performed to verify the experimental methods provides evidence that cuproptosis is associated with UC. This finding suggests that immune cell infiltration in UC patients is associated with cuproptosis. The key genes associated with cuproptosis can be helpful for discovering the molecular mechanism of UC, thus facilitating the improvement of UC treatment and preventing the associated CRC.

Influence of different region of interest sizes on CT-based radiomics model for microvascular invasion prediction in hepatocellular carcinoma. / æ„Ÿå ´è¶£åŒºèŒƒå›´å¯¹CT影像组学模型预测肝细胞癌微血管侵犯的影响.

OBJECTIVES: Microvascular invasion (MVI) is an important predictor of postoperative recurrence or poor outcomes of hepatocellular carcinoma (HCC). Radiomics is able to predict MVI in HCC preoperatively. This study aims to investigate the influence of different region of interest (ROI) sizes on CT-based radiomics model for MVI prediction in HCC. METHODS: Patients with HCC with or without MVI confirmed by pathology and those who underwent preoperative plain or enhanced abdominal CT scans in the Third Xiangya Hospital of Central South University from January 2010 to December 2020 were retrospectively and consecutively included. According to the ratio of 7 to 3, the patients were randomly assigned into a training set and a validation set. Clinical data were collected from medical records, and radiomics features were extracted from the arterial phase (AP) and portal venous phase (PVP) of preoperatively acquired CT in all patients. Six different ROI sizes were employed. The original ROI (OROI) was manually delineated along the visible borders of the tumor layer-by-layer. The OROI was expanded out by 1-5 mm. The OROI was combined with 5 different peritumoral regions to generate the other 5 ROIs, named Plus1-Plus5. Feature extraction, dimension reduction, and model development were conducted in 6 different ROIs separately. Supporter vector machine (SVM) was used for model construction. Model performance was assessed via receiver operating characteristic (ROC) curve. RESULTS: A total of 172 HCC patients were included, in which 83 (48.3%) were MVI positive, and 89 (51.7%) were MVI negative. Three hundred and ninety-six features based on AP or PVP images were extracted from each ROI. After feature selection and dimension reduction, 4, 5, 15, 11, 6, and 3 features of OROI, Plus1, Plus2, Plus 3, Plus4, and Plus5 were selected for model construction, respectively. In the training set, the sensitivity, specificity, and area under the curve (AUC) of OROI were 0.759, 0.806, and 0.855, respectively. The AUC values of Plus2 (0.979) and Plus3 (0.954) were higher than that of OROI. The AUC values of Plus1 (0.802), Plus4 (0.792), and Plus5 (0.774) were not significantly different from those of OROI. In the validation set, the sensitivity, specificity, and AUC value of OROI were 0.640, 0.630, and 0.664, respectively. The AUC value of Plus3 was 0.903, which was higher than that of OROI. The AUC values of Plus1 (0.679), Plus2 (0.536), Plus4 (0.708), and Plus5 (0.757) were not significantly different from that of OROI (P>0.05). CONCLUSIONS: The size of ROI significantly inflluences on the performance of CT-based radiomics model for MVI prediction in HCC. Including appropriate area around the tumor into ROI could improve the predictive performance of the model, and 3 mm might be appropriate distance.