Deciphering the Mechanism of Siwu Decoction Inhibiting Liver Metastasis by Integrating Network Pharmacology and In Vivo Experimental Validation.

BACKGROUND: Siwu Decoction (SWD) is a well-known classical TCM formula that has been shown to be effective as a basis for preventing and reducing liver metastases (LM). However, the active ingredients and potential molecular mechanisms remain unclear. OBJECTIVE: This study aimed to systematically analyze the active ingredients and potential molecular mechanisms of SWD on LM and validate mechanisms involved. MATERIALS AND METHODS: The active ingredients in SWD were extracted by UHPLC-MS/MS in a latest study. Protox II was retrieved to obtain toxicological parameters to detect safety. Swiss Target Prediction database was exploited to harvest SWD targets. Five databases, Gene Cards, DisGeNET, Drugbank, OMIM, and TTD, were employed to filter pathogenic targets of LM. STRING database was utilized to construct the protein-protein interaction network for therapeutic targets, followed by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. GEPIA database and the Human Protein Atlas were taken to observe the expression of core genes and proteins. ImmuCellAI algorithm was applied to analyze the immune microenvironment and survival relevant to core genes. Molecular docking was performed to verify the affinity of SWD effective ingredients to core targets. In vivo experiments were carried out to validate the anti-LM efficacy of SWD and verify the pivotal mechanisms of action. RESULTS: Eighteen main bioactive phytochemicals identified were all non-hepatotoxic. PPI network acquired 118 therapeutic targets, of which VEGFA, CASP3, STAT3, etc. were identified as core targets. KEGG analysis revealed that HIF-1 pathway and others were critical. After tandem targets and pathways, HIF-1/VEGF was regarded as the greatest potential pathway. VEGFA and HIF-1 were expressed differently in various stages of cancer and normal tissues. There was a negative regulation of immunoreactive cells by VEGFA, which was influential for prognosis. Molecular docking confirmed the tight binding to VEGFA. This study revealed the exact effect of SWD against LM, and identified significant inhibition the expression of HIF-1α, VEGF, and CD31 in the liver microenvironment. CONCLUSION: This study clarified the active ingredients of SWD, the therapeutic targets of LM and potential molecular mechanisms. SWD may protect against LM through suppressing HIF-1/VEGF pathway.

SUPT5H mutations associated with elevation of Hb A2 level: Identification of two novel variants and literature review.

ß-thalassemia is one of the most common monogenic disorders in areas of the tropics and subtropics, which represents a major familial and social burden to local people. The elevated Hb A2 level, generally specified as greater than 3.5 %, is commonly used as a high efficiency index for screening of ß-thalassemia carriers. However, mutations in other genes such as GATA1 and KLF1, could also result in increased Hb A2 level. In this study, we identified two novel variants in the SUPT5H gene: a frameshift mutation (SUPT5H: c.3032_3033delTG, p.M1011Mfs*9) and a nonsense mutation (SUPT5H: c.397C > T, p.Arg133*) in two Chinese individuals. Utilizing a combination of phenotype analysis, bioinformatics analysis, and functional analysis, we deduced that these two variants modified the SUPT5H protein's structure, thereby impacting its function and consequently leading to the heightened Hb A2 level phenotype found in the carriers. Furthermore, through a comprehensive literature review, a mutation spectrum was consolidated for SUPT5H, an investigation into the genotype-phenotype correlation was conducted, and factors known to influence Hb A2 levels were identified. Based on this in-depth understanding, clinicians are better equipped to carry out large scale screenings in regions with high prevalence of ß-thalassemia.

Review of pre-metastatic niches in lung metastasis: From cells to molecules, from mechanism to clinics.

Distant metastasis is responsible for high mortality in most cancer cases and the lung is one of the most common target organs, severely affecting the quality of daily life and overall survival of cancer patients. With relevant research breakthroughs accumulating, scientists have developed a deeper understanding of lung metastasis (LM) from the rudimentary "seed and soil" theory to a more vivid concept of the pre-metastatic niche (PMN). Thus, the mechanisms of PMN formation become considerably complicated, involving various types of cells, chemokines, cytokines, and proteins, providing potential biomarkers for improved LM diagnosis and treatment techniques. Here we summarized the latest findings (in 3 years) of lung PMN and systematically collated it from basic research to clinical application, which clearly exhibited the influences of the primary tumor, stromal, and bone marrow-derived cells (BMDCs) and associated molecules in the formation of lung PMN.

A novel gain-of-function PIP4K2A mutation elevates the expression of ß-globin and aggravates the severity of α-thalassemia.

Haemoglobin H (Hb H) disease (intermediate status of α-thalassemia) shows marked phenotypic variability from asymptomatic to severe anaemia. Apart from the combined ß-thalassemia allele ameliorating clinical severity, reports of genetic modifier genes affecting the phenotype of Hb H disease are scarce which bring inconvenience to precise diagnosis and genetic counselling of the patients. Here, we present a novel mutation (c.948C>A, p.S316R) in the PIP4K2A gene in a female Hb H disease patient who displayed moderate anaemia and a relatively high Hb H level. Haematological analysis in her family members revealed that individuals carrying this mutation have upregulated ß-globin expression, leading to a more imbalanced ß/α-globin ratio and more Hb H inclusion bodies in peripheral red blood cells. According to functional experiments, the mutant PIP4K2A protein exhibits enhanced protein stability, increased kinase activity and a stronger regulatory effect on downstream proteins, suggesting a gain-of-function mutation. Moreover, introduction of the S316R mutation into HUDEP-2 cells increased expression of ß-globin, further inhibiting erythroid differentiation and terminal enucleation. Thus, the S316R mutation is a novel genetic factor associated with ß-globin expression, and the PIP4K2A gene is a new potential modifier gene affecting the α-thalassemia phenotype.

Biomarkers and factors in small cell lung cancer patients treated with immune checkpoint inhibitors: A meta-analysis.

OBJECTIVE: The aim of this meta-analysis was to summarize the available results of immunotherapy predictors for small cell lung cancer (SCLC) and to provide evidence-based information for their potential predictive value of efficacy. METHODS: We searched PubMed, EMBASE, Web of Science, The Cochrane Library, and ClinicalTrials (from January 1, 1975 to November 1, 2021). The hazard ratios (HR) and its 95% confidence intervals (CIs) and tumor response rate of the included studies were extracted. RESULTS: Eleven studies were eventually included and the pooled results showed that programmed cell death ligand 1 (PD-L1) positive: objective response rate (ORR) (relative risk [RR] = 1.39, 95% CI [0.48, 4.03], p = 0.54), with high heterogeneity (p = 0.05, I2  = 56%); disease control rate [DCR] (RR = 1.31, 95% CI [0.04, 38.57], p = 0.88), with high heterogeneity (p = 0.04, I2  = 75%); overall survival (OS) (HR = 0.89, 95% CI [0.74, 1.07], p = 0.22); and progression-free survival (PFS) (HR = 0.83, 95% CI [0.59, 1.16], p = 0.27), with high heterogeneity (p = 0.005, I2  = 73.1%). TMB-High (TMB-H): OS (HR = 0.86, 95% CI [0.74, 1.00], p = 0.05); PFS (HR = 0.71, 95% CI [0.6, 0.85], p < 0.001). Lactate dehydrogenase (LDH) >upper limit of normal (ULN): OS (HR = 0.95, 95% CI [0.81, 1.11], p = 0.511). Asian patients: OS (HR = 0.87, 95% CI [0.72, 1.04], p = 0.135); White/Non-Asian patients: OS (HR = 0.83, 95% CI [0.76, 0.90], p < 0.001). Liver metastasis patients: OS (HR = 0.93, 95% CI [0.83, 1.05], p = 0.229); PFS (HR = 0.84, 95% CI [0.67, 1.06], p = 0.141). Central nervous system (CNS) metastasis patients: OS (HR = 0.91, 95% CI [0.71, 1.17], p = 0.474); PFS (HR = 1.03, 95% CI [0.66, 1.60], p = 0.903). CONCLUSION: The available research results do not support the recommendation of PD-L1 positive and TMB-H as predictors for the application of immune checkpoint inhibitors (ICIs) in SCLC patients. LDH, baseline liver metastasis and CNS metastasis may be used as markers/influencing factors for predicting the efficacy of ICIs in SCLC patients. Non-Asian SCLC patients had better efficacy with ICIs in our results.

Hairy gene homolog increases nasopharyngeal carcinoma cell stemness by upregulating Bmi-1.

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is overexpressed in various cancer types. We found that Bmi-1 mRNA levels were elevated in nasopharyngeal carcinoma (NPC) cell lines. In immunohistochemical analyses, high Bmi-1 levels were observed in not only 5 of 38 non-cancerous nasopharyngeal squamous epithelial biopsies, but also in 66 of 98 NPC specimens (67.3%). High Bmi-1 levels were detected more frequently in T3-T4, N2-N3 and stage III-IV NPC biopsies than in T1-T2, N0-N1 and stage I-II NPC samples, indicating that Bmi-1 is upregulated in advanced NPC. In 5-8F and SUNE1 NPC cells, stable depletion of Bmi-1 using lentiviral RNA interference greatly suppressed cell proliferation, induced G1-phase cell cycle arrest, reduced cell stemness and suppressed cell migration and invasion. Likewise, knocking down Bmi-1 inhibited NPC cell growth in nude mice. Both chromatin immunoprecipitation and Western blotting assays demonstrated that Hairy gene homolog (HRY) upregulated Bmi-1 by binding to its promoter, thereby increasing the stemness of NPC cells. Immunohistochemistry and quantitative real-time PCR analyses revealed that HRY expression correlated positively with Bmi-1 expression in a cohort of NPC biopsies. These findings suggested that HRY promotes NPC cell stemness by upregulating Bmi-1, and that silencing Bmi-1 can suppress NPC progression.

TRIM32 biallelic defects cause limb-girdle muscular dystrophy R8: identification of two novel mutations and investigation of genotype-phenotype correlation.

BACKGROUND: Limb-girdle muscular dystrophy R8 (LGMD R8) is a rare autosomal recessive muscle disease caused by TRIM32 gene biallelic defects. The genotype-phenotype correlation of this disease has been reported poorly. Here, we report a Chinese family with two female LGMD R8 patients. METHODS: We performed whole-genome sequencing (WGS) and Sanger sequencing on the proband. Meanwhile, the function of mutant TRIM32 protein was analyzed by bioinformatics and experimental analysis. In addition, a summary of the reported TRIM32 deletions and point mutations and an investigation of genotype-phenotype correlation were performed through a combined analysis of the two patients and other cases reported in previous literature. RESULTS: The two patients displayed typical symptoms of LGMD R8, which worsened during pregnancy. Genetic analysis by whole-genome sequencing (WGS) and Sanger sequencing showed that the patients were compound heterozygotes of a novel deletion (chr9.hg19:g.119431290_119474250del) and a novel missense mutation (TRIM32:c.1700A > G, p.H567R). The deletion encompassed 43 kb and resulted in the removal of the entire TRIM32 gene. The missense mutation altered the structure and further affected function by interfering with the self-association of the TRIM32 protein. Females with LGMD R8 showed less severe symptoms than males, and patients carrying two mutations in NHL repeats of the TRIM32 protein had earlier disease onset and more severe symptoms than other patients. CONCLUSIONS: This research extended the spectrum of TRIM32 mutations and firstly provided useful data on the genotype-phenotype correlation, which is valuable for the accurate diagnosis and genetic counseling of LGMD R8.

Network pharmacology and molecular docking analysis reveal insights into the molecular mechanism of shiliao decoction in the treatment of cancer-associated malnutrition.

Purpose: Shiliao Decoction (SLD) was developed for treatment and prevention of cancer-associated malnutrition (CAM) in China. In this study, we aim to discover SLD's active compounds and demonstrate the mechanisms of SLD that combat CAM through network pharmacology and molecular docking techniques. Methods: All components of SLD were retrieved from the pharmacology database of Traditional Chinese Medicine Systems Pharmacology (TCMSP). The GeneCards database and the Online Mendelian Inheritance in Man database (OMIM) were used to identify gene encoding target compounds, and Cytoscape was used to construct the drug compound-target network. The network of target protein-protein interactions (PPI) was constructed using the STRING database, while gene ontology (GO) functional terms and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways associated with potential targets were analyzed using a program in R language (version 4.2.0). Core genes linked with survival and the tumor microenvironment were analyzed using the Kaplan-Meier plotter and TIMER 2.0 databases, respectively. Protein expression and transcriptome expression levels of core gene were viewed using the Human Protein Atlas (HPA) and the Cancer Genome Atlas (TCGA). A component-target-pathway (C-T-P) network was created using Cytoscape, and Autodock Vina software was used to verify the molecular docking of SLD components and key targets. Results: The assembled compound-target network primarily contained 134 compounds and 147 targets of the SLD associated with JUN, TP53, MAPK3, MAPK1, MAPK14, STAT3, AKT1, HSP90AA1, FOS, and MYC, which were identified as core targets by the PPI network. KEGG pathway analysis revealed pathways involved in lipid and atherosclerosis, the PI3K/Akt signaling pathway, and immune-related pathways among others. JUN is expressed at different levels in normal and cancerous tissues, it is closely associated with the recruitment of different immune cells and has been shown to have a significant impact on prognosis. The C-T-P network suggests that the active component of SLD is capable of regulating target genes affecting these related pathways. Finally, the reliability of the core targets was evaluated using molecular docking technology. Conclusion: This study revealed insights into SLD's active components, potential targets, and possible molecular mechanisms, thereby demonstrating a potential method for examining the scientific basis and therapeutic mechanisms of TCM formulae.