Leveraging a KRAS-based signature to predict the prognosis and drug sensitivity of colon cancer and identifying SPINK4 as a new biomarker.

KRAS is one of the leading mutations reported in colon cancer. However, there are few studies on the application of KRAS related signature in predicting prognosis and drug sensitivity of colon cancer patient. We identified KRAS related differentially expressed genes (DEGs) using The Cancer Genome Atlas (TCGA) database. A signature closely related to overall survival was recognized with Kaplan-Meier survival analysis and univariate cox regression analysis. Then we validated this signature with overall expression score (OE score) algorithm using both scRNA-seq and bulk RNA-seq data. Based on this signature, we performed LASSO cox regression to establish a prognostic model, and corresponding scores were calculated. Differences in genomic alteration, immune microenvironment, drug sensitivity between high- and low-KRD score groups were investigated. A KRAS related signature composed of 80 DEGs in colon cancer were recognized, among which 19 genes were selected to construct a prognostic model. This KRAS related signature was significantly correlated with worse prognosis. Furthermore, patients who scored lower in the prognostic model presented a higher likelihood of responding to chemotherapy, targeted therapy and immunotherapy. Furthermore, among the 19 selected genes in the model, SPINK4 was identified as an independent prognostic biomarker. Further validation in vitro indicated the knockdown of SPINK4 promoted the proliferation and migration of SW48 cells. In conclusion, a novel KRAS related signature was identified and validated based on clinical and genomic information from TCGA and GEO databases. The signature was proved to regulate genomic alteration, immune microenvironment and drug sensitivity in colon cancer, and thus might serve as a predictor for individual prognosis and treatment.

FOXD1 promotes chemotherapy resistance by enhancing cell stemness in colorectal cancer through ß­catenin nuclear localization.

Forkhead box D1 (FOXD1) serves a critical role in colorectal cancer (CRC). FOXD1 expression is an independent prognostic factor in patients with CRC; however, the molecular mechanism and signaling pathway of FOXD1 that regulates cell stemness and chemoresistance has not been fully characterized. The aim of the present study was to further validate the effect of FOXD1 on the proliferation and migration of CRC cells, and to delve into the possible potential of FOXD1 in the clinical treatment of CRC. The effect of FOXD1 on cell proliferation was assessed using Cell Counting Kit 8 (CCK­8) and colony formation assays. The effect of FOXD1 on cell migration was assessed by wound­healing and Transwell assays. The effect of FOXD1 on cell stemness was assessed by spheroid formation in vitro and limiting dilution assays in vivo. The expression of stemness associated proteins, leucine rich repeat containing G protein­coupled receptor 5 (LGR5), OCT4, Sox2 and Nanog, and epithelial­mesenchymal transition associated proteins, E­cadherin, N­cadherin and vimentin, were detected by western blotting. Proteins interrelationships were assessed by a co­immunoprecipitation assay. Oxaliplatin resistance was assessed using CCK­8 and apoptosis assays in vitro, and using a tumor xenograft model in vivo. By constructing FOXD1 overexpression and knockdown stably transfected strains of colon cancer cells, it was revealed that the overexpression of FOXD1 increased CRC cell stemness and chemoresistance. By contrast, knockdown of FOXD1 produced the opposite effects. These phenomena were caused by the direct interaction between FOXD1 and ß­catenin, thus promoting its nuclear translocation and the activation of downstream target genes, such as LGR5 and Sox2. Notably, inhibition of this pathway with a specific ß­catenin inhibitor (XAV­939) could impair the effects induced by the overexpression of FOXD1. In summary, these results indicated that FOXD1 may promote cell stemness and the chemoresistance of CRC by binding directly to ß­catenin and enhancing ß­catenin nuclear localization; therefore, it may be considered a potential clinical target.

IL-17A-mediated mitochondrial dysfunction induces pyroptosis in colorectal cancer cells and promotes CD8 + T-cell tumour infiltration.

BACKGROUND: Interleukin-17A (IL-17A), a proinflammatory cytokine primarily secreted by Th17 cells, γδT cells and natural killer T (NKT) cells, performs essential roles in the microenvironment of certain inflammation-related tumours by regulating cancer growth and tumour elimination proved in previous literature. In this study, the mechanism of IL-17A that induces mitochondrial dysfunction promoted pyroptosis has been explored in colorectal cancer cells. METHOD: The records of 78 patients diagnosed with CRC were reviewed via the public database to evaluate clinicopathological parameters and prognosis associations of IL-17A expression. The colorectal cancer cells were treated with IL-17A, and the morphological characteristics of those cells were indicated by scanning electron microscope and transmission electron microscope. After IL-17A treatment, mitochondrial dysfunction was tested by mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). The expression of pyroptosis associated proteins including cleaved caspase-4, cleaved gasdermin-D (GSDMD), IL-1ß, receptor activator of nuclear NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck like protein containing a card (ASC), and factor-kappa B was measured through western blotting. RESULTS: Positive IL-17A protein expression was observed in CRC compared to the non-tumour tissue. IL-17A expression indicates a better differentiation, earlier stage, and better overall survival in CRC. IL-17A treatment could induce mitochondrial dysfunction and stimulate intracellular reactive oxygen species (ROS) production. Furthermore, IL-17A could promote pyroptosis of colorectal cancer cells and significantly increase the secretion of inflammatory factors. Nevertheless, the pyroptosis induced by IL-17A could be inhibited through the pre-treatment with Mito-TEMPO (a mitochondria-targeted superoxide dismutase mimetic with superoxide and alkyl radical scavenging properties) or Z-LEVD-FMK (caspase-4 inhibitor, fluoromethylketone). Additionally, after being treated with IL-17A, an increasing number of CD8 + T cells showed in mouse-derived allograft colon cancer models. CONCLUSION: IL-17A, as a cytokine mainly secreted by γδT cells in the colorectal tumour immune microenvironment, can regulate the tumour microenvironment in multiple ways. IL-17A could induce mitochondrial dysfunction and pyroptosis through the ROS/NLRP3/caspase-4/GSDMD pathway, and promote intracellular ROS accumulation. In addition, IL-17A can promote the secretion of inflammatory factors such as IL-1ß、IL-18 and immune antigens, and recruit CD8 + T cells to infiltrate tumours.

[Study on mechanism of Rehmanniae Radix Praeparata for treatment of osteoarthritis based on network pharmacology and molecular docking].

The mechanism of Rehmanniae Radix Praeparata against osteoarthritis was investigated based on network pharmacology, molecular docking, and in vitro experiments in the present study. Osteoclast models were established via receptor activator of nuclear factor-κB ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) inducing RAW264.7 cells. Further, the influence of Rehmanniae Radix Praeparata on the activity of tartrate-resistant acid phosphatase(TRAP) was evaluated and the efficacy of Rehmanniae Radix Praeparata in the treatment of osteoarthritis was verified. The active components of Rehmanniae Radix Praeparata were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and literature, and the potential targets of the components were collected from SwissTargetPrediction. Osteoarthritis disease targets were searched in Online Mendelian Inheritance in Man(OMIM), Therapeutic Target Database(TTD), GeneCards, and DisGeNET. The intersection targets of Rehmanniae Radix Praeparata and osteoarthritis were obtained by Venny platform. The protein-protein interaction(PPI) network was constructed by Cytoscape 3.8.2, and key targets were obtained based on topology algorithm. The Database for Annotation, Visualization and Integrated Discovery(DAVID) was used to perform Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Finally, the mRNA expression of the key targets was determined by RT-qPCR and the binding activity between the components and key targets was validated by molecular docking. The results showed that Rehmanniae Radix Prae-parata inhibited the TRAP activity, thus inhibiting bone resorption by osteoclasts and treating osteoarthritis. By network pharmacology, 14 active components of Rehmanniae Radix Praeparata and 126 intersection targets were obtained. The network pharmacology enrichment results revealed 432 biological processes and 139 signaling pathways. Key targets such as proto-oncogene tyrosine-protein kinase Src(SRC), signal transducer and activator of transcription 3(STAT3) and transcription factor p65(RELA) were obtained according to the degree in topological analysis. SRC was highly expressed in osteoclasts, which accelerated the development of osteoarthritis. Therefore, SRC was selected for subsequent verification, and Rehmanniae Radix Praeparata decreased the gene expression level of SRC. The molecular docking showed that acteoside, isoacteoside, raffinose had good bonding activity with SRC, suggesting that they might be the critical components in treating osteoarthritis. In conclusion, Rehmanniae Radix Praeparata can inhibit bone resorption by osteoclasts and balance the metabolism of articular cartilage and subchondral bone via acting on SRC, thus playing a therapeutic role in osteoarthritis. In addition, Rehmanniae Radix Praeparata may exert overall efficacy on osteoarthritis through other targets such as STAT3 and RELA, and other related pathways such as PI3 K-AKT and IL-17 signaling pathways.

Retrospective research of neoadjuvant therapy on tumor-downstaging, post-operative complications, and prognosis in locally advanced rectal cancer.

BACKGROUND: Neoadjuvant therapy (NAT) is becoming increasingly important in locally advanced rectal cancer. Hence, such research has become a problem. AIM: To evaluate the downstaging effect of NAT, its impact on postoperative complications and its prognosis with different medical regimens. METHODS: Seventy-seven cases from Shanghai Ruijin Hospital affiliated with Shanghai Jiaotong University School of Medicine were retrospectively collected and divided into the neoadjuvant radiochemotherapy (NRCT) group and the neoadjuvant chemotherapy (NCT) group. The differences between the two groups in tumor regression, postoperative complications, rectal function, disease-free survival, and overall survival were compared using the χ 2 test and Kaplan-Meier analysis. RESULTS: Baseline data showed no statistical differences between the two groups, whereas the NRCT group had a higher rate of T4 (30/55 vs 5/22, P < 0.05) than the NCT groups. Twelve cases were evaluated as complete responders, and 15 cases were evaluated as tumor regression grade 0. Except for the reduction rate of T stage (NRCT 37/55 vs NCT 9/22, P < 0.05), there was no difference in effectiveness between the two groups. Preoperative radiation was not a risk factor for poor reaction or anastomotic leakage. No significant difference in postoperative complications and disease-free survival between the two groups was observed, although the NRCT group might have better long-term overall survival. CONCLUSION: NAT can cause tumor downstaging preoperatively or even complete remission of the primary tumor. Radiochemotherapy could lead to better T downstaging and promising overall survival without more complications.

[Study on components efficacy of Salviae Miltiorrhizae Radix et Rhizoma based on systematic traditional Chinese medicine].

This paper aimed to establish efficacy systems of tanshinones and salvianolic acids, two representative substances in Salviae Miltiorrhizae Radix et Rhizoma by using literature mining and biological network construction, based on systematic traditional Chinese medicine theory. The systematic study on the efficacy of traditional Chinese medicine was carried out from the basic unit, the structure and relationship between the basic units, the boundary of the research object and the function of the system, so as to explain the overall efficacy of the two kinds of components at the molecular level. Firstly, we collected the elements of the efficacy systems of these two kinds of components by literature mining, and defined their boundaries based on biological processes. After that, the structure of the efficacy systems was clarified according to the relationship in the KEGG database. Finally, the function of the efficacy systems was analyzed from the level of pharmacology, pharmacodynamics, and efficacy, revealing the scientific connotation of traditional Chinese medicine efficacy system. The results showed that there were 201 targets(elements), 12 target sets(boundary), and 12 pathway networks(structure) in salvianolic acids' efficacy system. Meanwhile, there were 189 targets(elements), 11 target sets(boundary), and 11 pathway networks(structure) in tanshinones' efficacy system. The results suggested that the functions of salvia-nolic acids' and tanshinones' efficacy systems were different in pharmacology and pharmacodynamics from aspects of elements, boundary, relationship and structure, but they were same in functional level as both of them could promote blood circulation, remove blood stasis, clear away heart-fire, relieve restlessness, and soothe the nerves. Based on systematic traditional Chinese medicine, we constructed the efficacy system of two representative components in Salviae Miltiorrhizae Radix et Rhizoma in this paper, elucidated the overall efficacy and builded the bridge between reductionism and holism in traditional Chinese medicine.

Designing principles of an ecological water storage basin on coastal saline: a case study.

The degradation of water source environment becomes serious problems accompanying with rapid urbanization in China. Ecological engineering provides ecologically sound and cost-effective solution to solving this problem. As a case study, a 15 hm2 ecological water storage basin for a water plant was designed and constructed on the TEDA area in Tianjin City. Located on saline, the construction of this project has to face serious difficulties, such as high salinity, scarce seed banks of macrophytes, and strong winds. Freshwater replacement, soil amendation and macrophytes planting at the basinshore, wooden water breaker and plastic membrane installation and other measures were conducted for the assistance of plant community establishment. The result showed that the chloride concentration in the basin water decreased from 11600 mg/L to less than 100 mg/L, and the chloride content in the basin sediment decreased from 2.1 % to 0.35% after freshwater soaking. The introduced macrophytes of 8 species all survived and 11 other macrophytes species were occurred in the basin. A new ecosystem was created with increased biological diversity in the original saline, and the water quality was improved. This ecological water storage basin also provided a pleasing landscape for local people.