Integrating scRNA-seq and bulk RNA-seq to characterize infiltrating cells in the colorectal cancer tumor microenvironment and construct molecular risk models.

Colorectal cancer (CRC) is a malignancy that is both highly lethal and heterogeneous. Although the correlation between intra-tumoral genetic and functional heterogeneity and cancer clinical prognosis is well-established, the underlying mechanism in CRC remains inadequately understood. Utilizing scRNA-seq data from GEO database, we re-isolated distinct subsets of cells, constructed a CRC tumor-related cell differentiation trajectory, and conducted cell-cell communication analysis to investigate potential interactions across cell clusters. A prognostic model was built by integrating scRNA-seq results with TCGA bulk RNA-seq data through univariate, LASSO, and multivariate Cox regression analyses. Eleven distinct cell types were identified, with Epithelial cells, Fibroblasts, and Mast cells exhibiting significant differences between CRC and healthy controls. T cells were observed to engage in extensive interactions with other cell types. Utilizing the 741 signature genes, prognostic risk score model was constructed. Patients with high-risk scores exhibited a significant correlation with unfavorable survival outcomes, high-stage tumors, metastasis, and low responsiveness to chemotherapy. The model demonstrated a strong predictive performance across five validation cohorts. Our investigation involved an analysis of the cellular composition and interactions of infiltrates within the microenvironment, and we developed a prognostic model. This model provides valuable insights into the prognosis and therapeutic evaluation of CRC.

A random forest algorithm-based prediction model for moderate to severe acute postoperative pain after orthopedic surgery under general anesthesia.

BACKGROUND: Postoperative pain is one of the most common complications after surgery. In order to detect early and intervene in time for moderate to severe postoperative pain, it is necessary to identify risk factors and construct clinical prediction models. This study aimed to identify significant risk factors and establish a better-performing model to predict moderate to severe acute postoperative pain after orthopedic surgery under general anesthesia. METHODS: Patients who underwent orthopedic surgery under general anesthesia were divided into patients with moderate to severe pain group (group P) and patients without moderate to severe pain group (group N) based on VAS scores. The features selected by Lasso regression were processed by the random forest and multivariate logistic regression models to predict pain outcomes. The classification performance of the two models was evaluated through the testing set. The area under the curves (AUC), the accuracy of the classifiers, and the classification error rate for both classifiers were calculated, the better-performing model was used to predict moderate to severe acute postoperative pain after orthopedic surgery under general anesthesia. RESULTS: A total of 327 patients were enrolled in this study (228 in the training set and 99 in the testing set). The incidence of moderate to severe postoperative pain was 41.3%. The random forest model revealed a classification error rate of 25.2% and an AUC of 0.810 in the testing set. The multivariate logistic regression model revealed a classification error rate of 31.3% and an AUC of 0.764 in the testing set. The random forest model was chosen for predicting clinical outcomes in this study. The risk factors with the greatest and second contribution were immobilization and duration of surgery, respectively. CONCLUSIONS: The random forest model can be used to predict moderate to severe acute postoperative pain after orthopedic surgery under general anesthesia, which is of potential clinical application value.

[Construction of a Prognostic Model of Multiple Myeloma Based on Metabolism-Related Genes].

OBJECTIVE: To screen the prognostic biomarkers of metabolic genes in patients with multiple myeloma (MM), and construct a prognostic model of metabolic genes. METHODS: The histological database related to MM patients was searched. Data from MM patients and healthy controls with complete clinical information were selected for analysis.The second generation sequencing data and clinical information of bone marrow tissue of MM patients and healthy controls were collected from human protein atlas (HPA) and multiple myeloma research foundation (MMRF) databases. The gene set of metabolism-related pathways was extracted from Molecular Signatures Database (MSigDB) by Perl language. The biomarkers related to MM metabolism were screened by difference analysis, univariate Cox risk regression analysis and LASSO regression analysis, and the risk prognostic model and Nomogram were constructed. Risk curve and survival curve were used to verify the grouping effect of the model. Gene set enrichment analysis (GSEA) was used to study the difference of biological pathway enrichment between high risk group and low risk group. Multivariate Cox risk regression analysis was used to verify the independent prognostic ability of risk score. RESULTS: A total of 8 mRNAs which were significantly related to the survival and prognosis of MM patients were obtained (P<0.01). As molecular markers, MM patients could be divided into high-risk group and low-risk group. Survival curve and risk curve showed that the overall survival time of patients in the low-risk group was significantly better than that in the high risk group (P<0.001). GSEA results showed that signal pathways related to basic metabolism, cell differentiation and cell cycle were significantly enriched in the high-risk group, while ribosome and N polysaccharide biosynthesis signaling pathway were more enriched in the low-risk group. Multivariate Cox regression analysis showed that the risk score composed of the eight metabolism-related genes could be used as an independent risk factor for the prognosis of MM patients, and receiver operating characteristic curve (ROC) showed that the molecular signatures of metabolism-related genes had the best predictive effect. CONCLUSION: Metabolism-related pathways play an important role in the pathogenesis and prognosis of patients with MM. The clinical significance of the risk assessment model for patients with MM constructed based on eight metabolism-related core genes needs to be confirmed by further clinical studies.

Bioinformatics analysis of SARS-CoV-2 infection-associated immune injury and therapeutic prediction for COVID-19.

Background: Severe acute respiratory syndrome coronavirus 2 is a highly contagious viral infection, without any available targeted therapies. The high mortality rate of COVID-19 is speculated to be related to immune damage. Methods: In this study, clinical bioinformatics analysis was conducted on transcriptome data of coronavirus infection. Results: Bioinformatics analysis revealed that the complex immune injury induced by coronavirus infection provoked dysfunction of numerous immune-related molecules and signaling pathways, including immune cells and toll-like receptor cascades. Production of numerous cytokines through the Th17 signaling pathway led to elevation in plasma levels of cytokines (including IL6, NF-κB, and TNF-α) followed by concurrent inflammatory storm, which mediates the autoimmune response. Several novel medications seemed to display therapeutic effects on immune damage associated with coronavirus infection. Conclusions: This study provided insights for further large-scale studies on the target therapy on reconciliation of immunological damage associated with COVID-19.

Qingyi decoction protects against myocardial injuries induced by severe acute pancreatitis.

BACKGROUND: We studied the protective effects of Qingyi decoction (QYD) (a Traditional Chinese Medicine) against severe acute pancreatitis (SAP)-induced myocardial infarction (MI). AIM: To study the function and mechanism of QYD in the treatment of myocardial injuries induced by SAP. METHODS: Ultrasonic cardiography, hematoxylin and eosin staining, immunohistochemistry, qRT-PCR, western blot, enzyme-linked immunosorbent assays, and apoptosis staining techniques were used to determine the effects of QYD following SAP-induced MI in Sprague-Dawley rats. RESULTS: Our SAP model showed severe myocardial histological abnormalities and marked differences in the symptoms, mortality rate, and ultrasonic cardiography outputs among the different groups compared to the control. The expression of serum cytokines [interleukin (IL)-1ß, IL-6, IL-8, IL-12, amyloid ß, and tumor necrosis factor-α] were significantly higher in the SAP versus QYD treated group (P < 0.05 for all). STIM1 and Orai1 expression in myocardial tissue extracts were significantly decreased post QYD gavage (P < 0.001). There was no significant histological difference between the 2-aminoethyl diphenylborinate inhibitor and QYD groups. The SAP group had a significantly higher apoptosis index score compared to the QYD group (P < 0.001). CONCLUSION: QYD conferred cardio-protection against SAP-induced MI by regulating myocardial-associated protein expression (STIM1 and Orai1).

Gamma-enolase predicts lung damage in severe acute pancreatitis-induced acute lung injury.

Severe acute pancreatitis (SAP) associated acute lung injury (ALI) accounts for about 70% mortality of SAP patients. However, there are no precise biomarkers for the disease currently. Herein, we evaluated the potential of gamma-enolase (ENO2), against its universal isoform alpha-enolase (ENO1), as a marker of SAP-ALI in a rat model. Firstly, 16 male Sprague-Dawley rats were randomly divided into two groups, Sham (n = 8) and SAP-ALI (n = 8), for pancreatitis induction. Ultra-structure examination by electron microscopy and HE staining were used for lung injury assessment. Lung tissue expressions of alpha-enolase and gamma-enolase were evaluated by qRT-PCR and immunohistochemistry. In a prospective validation experiment, 28 rats were used: sham (n = 8), SAP-ALI at 3 h (3 h, n = 10), and SAP-ALI at 24 h (24 h, n = 10). Lung tissue damage, tissue expression and circulating alpha-enolase and gamma-enolase levels were evaluated. Elevated serum levels of α-amylase and TNF-α were observed in SAP rats but not in sham-operated rats. Histological examination of pancreatic and lung tissues indicated marked damage in SAP rats. While alpha-enolase was universally expressed, gamma-enolase was expressed only in damaged lung tissues. Gamma-enolase was detected in lung tissues, BALF, and serum as early as 3 h post-surgery when physical pathological damage was not apparent. Unlike alpha-enolase, secreted and/or circulating gamma-enolase level progressively increased, especially in serum, as lung damage progressed. Thus, gamma-enolase may signal and correlate lung tissue damage well before obvious physical pathological tissue damage and might be a candidate diagnostic and/or prognostic marker.

Adenovirus-mediated overexpression of sST2 attenuates cardiac injury in the rat with severe acute pancreatitis.

AIMS: Severe acute pancreatitis (SAP) is a serious disease associated with systematic inflammation and multiple organs dysfunction. Soluble ST2 (sST2), a member of the Toll interleukin (IL)-1 receptor (TIR) superfamily, has been demonstrated to exert immune-regulatory and anti-inflammatory properties in several inflammation-related diseases. In this study, we investigated whether transfer of sST2 gene by adenovirus vector could attenuate sodium taurocholate-induced SAP and associated cardiac injury. MAIN METHODS: A rat model of SAP was induced by retrograde injection of 5% sodium taurocholate (1 ml/kg) into the biliopancreatic duct. Rats in the treatment groups were intravenously injected with adenovirus expressing sST2 (Ad-sST2, 1 × 109 particles/rat) or green fluorescent protein (Ad-GFP) via the tail vein 48 h before SAP induction. Histological changes in the pancreatic and heart tissues, and parameters for evaluating SAP and associated cardiac injury were determined at 24 h after SAP. KEY FINDINGS: Sodium taurocholate induced obvious pathological changes in pancreas and elevated serum levels of amylase and lipase. Furthermore, SAP animals exhibited significant cardiac impairment, evidenced by decreased cardiac function, increased myocardial apoptosis and cardiac-related enzymes including creatine kinase isoenzyme, lactate dehydrogenase, and Troponin T. Administration of Ad-sST2 markedly improved the structure of pancreas and heart tissues, and reversed the alterations in serum amylase, lipase and cardiac-related enzymes. In addition, Ad-sST2 treatment downregulated pro-inflammatory cytokines production, demonstrating the anti-inflammatory property of sST2. SIGNIFICANCE: Our results suggest that administration of Ad-sST2 significantly attenuated the severity of SAP and associated cardiac damage, and the cardioprotective effect is associated with its anti-inflammatory action.

GP73 promotes invasion and metastasis of bladder cancer by regulating the epithelial-mesenchymal transition through the TGF-ß1/Smad2 signalling pathway.

This study investigated the effects of Golgi membrane protein 73 (GP73) on the epithelial-mesenchymal transition (EMT) and on bladder cancer cell invasion and metastasis through the TGF-ß1/Smad2 signalling pathway. Paired bladder cancer and adjacent tissue samples (102) and normal bladder tissue samples (106) were obtained. Bladder cancer cell lines (T24, 5637, RT4, 253J and J82) were selected and assigned to blank, negative control (NC), TGF-ß, thrombospondin-1 (TSP-1), TGF-ß1+ TSP-1, GP73-siRNA-1, GP73-siRNA-2, GP73-siRNA-1+ TSP-1, GP73-siRNA-1+ pcDNA-GP73, WT1-siRNA and WT1-siRNA + GP73-siRNA-1 groups. Expressions of GP73, TGF-ß1, Smad2, p-Smad2, E-cadherin and vimentin were detected using RT-qPCR and Western blotting. Cell proliferation, migration and invasion were determined using MTT assay, scratch testing and Transwell assay, respectively. Compared with the blank and NC groups, levels of GP73, TGF-ß1, Smad2, p-Smad2, N-cadherin and vimentin decreased, and levels of WT1 and E-cadherin increased in the GP73-siRNA-1 and GP73-siRNA-2 groups, while the opposite results were observed in the WT1 siRNA, TGF-ß, TSP-1 and TGF-ß + TSP-1 groups. Cell proliferation, migration and invasion notably decreased in the GP73-siRNA-1 and GP73-siRNA-2 groups in comparison with the blank and NC groups, while in the WT1 siRNA, TGF-ß, TSP-1 and TGF-ß + TSP-1 groups, cell migration, invasion and proliferation showed the reduction after the EMT. These results suggest that GP73 promotes bladder cancer invasion and metastasis by inducing the EMT through down-regulating WT1 levels and activating the TGF-ß1/Smad2 signalling pathway.

Effects of MicroRNA-19b on the Proliferation, Apoptosis, and Migration of Wilms' Tumor Cells Via the PTEN/PI3K/AKT Signaling Pathway.

Wilms' tumor (WT) is a most common renal cancer that occurs among children, and microRNA-19b (miR-19b) usually participates in various human cancers. Importantly, the PTEN/PI3K/Akt signaling pathway plays a key role in cell apoptosis, growth and proliferation. Thus, our present study aims to investigate the effect of miR-19b on the PTEN/PI3K/Akt signaling pathway during WT cell proliferation, migration, and apoptosis. WT tissues and adjacent normal tissues from WT patients were collected. qRT-PCR was applied to detect miR-19b expression in both the WT tissues and the adjacent normal tissues, immunohistochemistry was applied to detect the protein expressions of PTEN, P13K, and p-Akt, SK-NEP-1 cells were divided into the blank, negative control (NC), miR-19b mimics and miR-19b inhibitors groups. MTT assay, propidium iodide (PI) staining, Annexin-V/PI double-staining, Transwell assay and Western blotting were performed to examine cell proliferation, cycle, apoptosis, migration, and invasion, and the protein expressions of PTEN, P13K, Akt, and p-Akt. Increased miR-19b expression, positive expression rates of P13K and Akt, decreased PTEN expression rate, a negative correlation between PTEN expression and tumor lymph node metastasis, and a positive correlation between the expression of P13K and Akt and the clinical stages were observed in the WT tissues. The miR-19b inhibitors group exhibited decreased cell proliferation, cell cycle progression, migration and invasion, and protein expressions of PI3K and p-Akt but increased PTEN protein expression compared with the blank and NC groups. Thus, inhibition of miR-19b suppresses the progression of WT by modulating the PTEN/PI3K/AKT signaling pathway. J. Cell. Biochem. 118: 3424-3434, 2017. © 2017 Wiley Periodicals, Inc.

High selectivity of PI3Kß inhibitors in SETD2-mutated renal clear cell carcinoma.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is characterized with frequent mutations of SETD2 gene and our purpose was to explore targeted therapy for this entity. METHODS: By bioinformatic investigation of two major databases, the Genomics of Drug Sensitivity in Cancer (GDSC) database and The Cancer Genome Atlas (TCGA) database, we identified the selective PI3Kß inhibitors TGX221 and AZD6482 as selective inhibitors for ccRCC with SETD2 mutations, with AZD6482 additionally targeting PIK3CA and CDK6 mutations. RESULTS: Further investigation on AZD6482 profile revealed that mutations in RB1, KRAS, NRAS and APC contributed in drug resistance. Changes in both AZD6482-sensitive and -resistant gene sets showed limited impact on prognosis. Western blotting showed AZD6482 did not induce changes in a panel of major downstream effectors of AKT, but substantially increased PMS2 level. AZD6482 also selectively inhibited migration, invasiveness, and colony formation of ccRCC cells with SETD2 mutations. Integrative network analysis revealed complex interactions between these genes except SETD2. CONCLUSION: AZD6482 is a novel inhibitor with high selectivity for ccRCC SETD2 mutations. Increased activity of PI3K/AKT/PMS2 could play a role in SETD2 mutated ccRCC.

Therapeutic effect of Qingyi decoction in severe acute pancreatitis-induced intestinal barrier injury.

AIM: To investigate the effect of Qingyi decoction on the expression of secreted phospholipase A2 (sPLA2) in intestinal barrier injury. METHODS: Fifty healthy Sprague-Dawley rats were randomly divided into control, severe acute pancreatitis (SAP), Qingyi decoction-treated (QYT), dexamethasone-treated (DEX), and verapamil-treated (VER) groups. The SAP model was induced by retrograde infusion of 1.5% sodium deoxycholate into the biliopancreatic duct of the rats. All rats were sacrificed 24 h post-SAP induction. Arterial blood, intestine, and pancreas from each rat were harvested for investigations. The levels of serum amylase (AMY) and diamine oxidase (DAO) were determined using biochemical methods, and serum tumor necrosis factor (TNF)-α level was measured by an enzyme linked immunosorbent assay. Pathologic changes in the harvested tissues were investigated by microscopic examination of hematoxylin and eosin-stained tissue sections. The expressions of sPLA2 at mRNA and protein levels were detected by reverse transcriptase PCR and Western blot, respectively. A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used to investigate apoptosis of epithelial cells in the intestinal tissues. RESULTS: Compared to the control group, the expression of sPLA2 at both the mRNA and protein levels increased significantly in the SAP group (0.36 ± 0.13 vs 0.90 ± 0.38, and 0.16 ± 0.05 vs 0.64 ± 0.05, respectively; Ps < 0.01). The levels of AMY, TNF-α and DAO in serum were also significantly increased (917 ± 62 U/L vs 6870 ± 810 U/L, 59.7 ± 14.3 ng/L vs 180.5 ± 20.1 ng/L, and 10.37 ± 2.44 U/L vs 37.89 ± 5.86 U/L, respectively; Ps < 0.01). The apoptosis index of intestinal epithelial cells also differed significantly between the SAP and control rats (0.05 ± 0.02 vs 0.26 ± 0.06; P < 0.01). The serum levels of DAO and TNF-α, and the intestinal apoptosis index significantly correlated with sPLA2 expression in the intestine (r = 0.895, 0.893 and 0.926, respectively; Ps < 0.05). The levels of sPLA2, AMY, TNF-α, and DAO in the QYT, VER, and DEX groups were all decreased compared with the SAP group, but not the control group. Qingyi decoction intervention, however, gave the most therapeutic effect against intestinal barrier damage, although the onset of its therapeutic effect was slower. CONCLUSION: Qingyi decoction ameliorates acute pancreatitis-induced intestinal barrier injury by inhibiting the overexpression of intestinal sPLA2. This mechanism may be similar to that of verapamil.

Effects and mechanisms of alveolar type II epithelial cell apoptosis in severe pancreatitis-induced acute lung injury.

This study aimed to examine the role of alveolar type II epithelial cell (AEC II) apoptosis in severe pancreatitis-induced acute lung injury (ALI) and the intervening role of Qingyi decoction (QYT). An SAP model was established in male Sprague-Dawley rats. Immunohistochemical analysis was conducted to observe the pathological changes in the pancreas and lung tissue. AEC II apoptosis was detected by flow cytometry and the free Ca2+ concentration in AECs II was determined by laser scanning confocal microscopy. A radioimmunoassay was performed to determine serum TNF-α content. Quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis were performed to detect the mRNA and protein expression levels of Bax and caspase-8 in the lung tissue. Hematoxylin and eosin staining of lung tissue sections in the severe acute pancreatitis (SAP) group showed pathological changes from control tissue, consistent with acute lung injury (ALI). Flow cytometry showed that the level of AEC II apoptosis in the SAP group was significantly increased compared with that in the control group (P<0.01). Laser scanning confocal microscopy indicated that the free Ca2+ concentration in the AECs II of the SAP group was also significantly increased compared with that in the control (P<0.01). Radioimmunoassay demonstrated that the TNF-α levels were significantly increased in the SAP group compared with those in the control group (P<0.01), and qPCR results showed that the levels of Bax and caspase-8 apoptotic gene expression in the AECs II of the SAP group were significantly elevated (P<0.01). The aforementioned indicators were significantly lower following drug treatment compared with the levels observed in the SAP model group. These results suggest that AEC II apoptosis is involved in the ALI procedure associated with SAP. The mitochondrial pathway and death receptor pathway may have key regulatory roles in AEC II apoptosis. The use of QYT may significantly reduce the extent of lung injury.

Expression and significance of E-cadherin, N-cadherin, transforming growth factor-ß1 and Twist in prostate cancer.

OBJECTIVE: To study the expression of E-cadherin, N-cadherin, TGF-ß1 and Twist protein and investigate its significance in the occurrence and development of prostate cancer. METHODS: The expression of E-cadherin, N-cadherin, TGF-ß1 and Twist protein in 59 prostate cancer tissues and 21 adjacent tissues were detected by immunohistochemical SABC staining, and the correlation with clinicopathological features was analyzed. RESULTS: Positive rates of E-cadherin, N-cadherin, TGF-ß1 and Twist were 32.2%, 54.2%, 71.2% and 74.6%, respectively, in prostate cancer tissues and 85.7%, 9.52%, 19.0% and 9.52%, respectively, in cancer-adjacent tissues, with significant differences between the two groups (P<0.05). The reduced expression of E-cadherin was related to the differentiation of prostate cancer tissues and PSA level, but was not associated with clinical stage, lymph node metastasis, bony metastasis and age. The increased expression of N-cadherin, TGF-ß1 and Twist was related to the differentiation of prostate cancer tissues, clinical stage, lymph node metastasis, bony metastasis, but not to age. The difference in positive expression of N-cadherin and TGF-ß1 was significant between PSA≤20 µg/L group and PSA>20µg/L group, but the positive expression of Twist was not significant between groups. The expression of E-cadherin was highly negatively correlated with that of N-cadherin and also highly negatively correlated with that of Twist. The expression of TGF-ß1 was correlated with those of E-cadherin, N-cadherin and Twist. CONCLUSIONS: The reduced expression of E-cadherin, abnormal expression of N-cadherin, transformation form E-cadherin to N-cadherin and the increased expression of TGF-ß1 and Twist play an important role in the occurrence and development of prostate cancer.