Classification of molecular subtypes for colorectal cancer and development of a prognostic model based on necroptosis-related genes.

Background: Necroptosis could regulate immunity in cancers, and stratification of colorectal cancer (CRC) subtypes based on key genes related to necroptosis might be a novel strategy for CRC treatment. Method: The RNA-sequencing data of CRC and other 31 types of cancers were obtained from The Cancer Genome Atlas (TCGA) database. Consensus clustering was performed based on protein-coding genes (PCGs) related to necroptosis score calculated by single sample gene set enrichment analysis (ssGSEA). Module genes showing a significant positive correlation with the necroptosis score were identified by weighted correlation network analysis (WGCNA) and further used to develop a risk stratification model applying least absolute shrinkage and selection operator (LASSO) and Cox regression analysis. The risks score for each sample in CRC cohorts, immunotherapy cohorts and pan-cancer study cohorts was calculated. Result: Two subgroups (C1 cluster and C2 cluster) of CRC were identified based on the necroptosis score. Compared with C1 cluster, the survival possibility of C2 cluster was greatly reduced, the levels of necroptosis score, immune cell infiltration, immune score and expression of immune checkpoint molecules were significantly increased and immunotherapy response was less active. Low-risk patients defined by the risk model had a significant survival advantage than high-risk counterparts in both CRC and the other 31 cancer types. Furthermore, the risk model was also more efficient than the Tumor Immune Dysfunction and Exclusion (TIDE) tool in predicting OS and immunotherapy response for the samples in the immunotherapy cohort. Conclusion: CRC patients were classified by necroptosis score-related PCGs, and a risk model was designed to evaluate the immunotherapy and prognosis of patients with CRC. The current molecular subtype and prognostic model could help stratify patients with different risks and predict their prognosis and immunotherapy sensitivity.

Single-cell data revealed CD14-type and FCGR3A-type macrophages and relevant prognostic factors for predicting immunotherapy and prognosis in stomach adenocarcinoma.

Background: Stomach adenocarcinoma (STAD) exhibits profound tumor heterogeneity and represents a great therapeutic challenge. Single-cell sequencing technology is a powerful tool to identify characteristic cell types. Methods: Single-cell sequencing data (scRNA-seq) GSE167297 and bulk RNA-seq data from TCGA, GTEx, GSE26901 and GSE15459 database were included in this study. By downscaling and annotating the cellular data in scRNA-seq, critical cell types in tumor progression were identified by AUCell score. Relevant gene modules were then identified by weighted gene co-expression network analysis (WGCNA). A prognostic scoring system was constructed by identifying prognostic factors in STAD by Least absolute shrinkage and selection operator (LASSO) COX model. The prognosis and model performance in the RiskScore groups were measured by Kaplan-Meier (K-M) curves and Receiver operating characteristic (ROC) curves. Nomogram was drawn based on RiskScore and prognosis-related clinical factors. In addition, we evaluated patient's feedback on immunotherapy in the RiskScore groups by TIMER, ESTIMATE and TIDE analysis. Finally, the expression levels of prognostic factors were verified in gastric cancer cell lines (MKN7 and MKN28) and human normal gastric mucosal epithelial cells (GES-1), and the effects of prognostic factors on the viability of gastric cancer cells were examined by the CCK8 assay and cell cycle. Results: scRNA-seq analysis revealed that 11 cell types were identified, and macrophages exhibited relatively higher AUCell scores and specifically expressed CD14 and FCGR3A. High macrophage scores worsened the prognosis of STAD patients. We intersected the specifically expressed genes in macrophages subgroups (670) and macrophage module genes (2,360) obtained from WGCNA analysis. Among 86 common genes, seven prognostic factors (RGS2, GNAI2, ANXA5, MARCKS, CD36, NRP1 and PDE4A) were identified and composed a RiskScore model. Patients in low Risk group showed a better survival advantage. Nomogram also provided a favorable prediction for survival at 1, 3 and 5 years in STAD patients. Besides, we found positive feedback to immunotherapy in patients with low RiskScore. The expression tendency of the seven prognostic factors in MKN7 and MKN28 was consistent with that in the RNA-seq data in addition to comparison of protein expression levels in the public HPA (The Human Protein Atlas) database. Further functional exploration disclosed that MARCKS was an important prognostic factor in regulating cell viability in STAD. Conclusion: This study preliminary uncovered a single cell atlas for STAD patients, and Macrophages relevant gene signature and nomogram displayed favorable immunotherapy and prognostic prediction ability. Collectively, our work provides a new insight into the molecular mechanisms and therapeutic approach for LUAD patients.

miR-1249-3p accelerates the malignancy phenotype of hepatocellular carcinoma by directly targeting HNRNPK.

BACKGROUND: microRNAs (miRNAs) have been implicated to play crucial roles in carcinogenesis. miR-1249-3p was reported to be abnormally expressed in multiple human cancers. However, its biological role and the associated underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unknown. METHODS: miR-1249-3p expression level in HCC cell lines and normal cell line was measured by quantitative real-time PCR. Role of miR-1249-3p on HCC cell proliferation, colony formation, and invasion was examined by cell counting kit-8 assay, colony formation assay, and transwell invasion assay, respectively. Luciferase activity reporter assay and western blot were performed to validate whether heterogeneous nuclear ribonucleoprotein K (HNRNPK) was a direct target of miR-1249-3p. Effect of miR-1249-3p on overall survival of HCC patients was analyzed at KM Plotter website. RESULTS: We found miR-1249-3p expression level was increased, while HNRNPK expression level was decreased in HCC cell lines compared with normal cell line. Knockdown miR-1249-3p expression inhibits HCC cell proliferation, colony formation, and cell invasion through regulating HNRNPK in vitro. We also showed high miR-1249-3p expression was a predictor for poor overall survival of HCC patients. CONCLUSIONS: These findings about miR-1249-3p/HNRNPK pair provide a novel therapeutic method for HCC patients.

Long noncoding RNA GAS5 inhibits angiogenesis and metastasis of colorectal cancer through the Wnt/ß-catenin signaling pathway.

OBJECTIVE: Angiogenesis plays a key role in the tumorigenesis and progression of colorectal cancer (CRC). In this study, we investigated the effect of long noncoding RNA (lncRNA) GAS5 on the angiogenesis, invasion, and metastasis of CRC, and the involvement of the Wnt/ß-catenin signaling pathway. METHODS: CRC tissues and adjacent normal tissues were collected from 52 patients with CRC. GAS5 expression was determined in vivo and in vitro by real-time quantitative polymerase chain reaction (RT-qPCR). Then RT-qPCR and Western blot were used to identify expression of key genes of Wnt/ß-catenin signaling pathway. CRC cells with lowest GAS5 expression were selected and subjected to si-GAS5, oe-GAS5, or XAV939 to validate the effect of GAS5 and Wnt/ß-catenin signaling pathway on CRC cell activities. The activation of Wnt/ß-catenin signaling pathway was determined in response to GAS5. Subcutaneous tumor growth and microvascular density were observed in nude mice, in which in vivo metastasis was observed following tail vein injection of CRC cells. RESULTS: Initially, poor expression of GAS5 was observed in CRC tissues and cells. Upregulated GAS5 repressed CRC cell invasion and migration in vitro, as well as subcutaneous tumor growth, angiogenesis, and liver metastases in vivo. Furthermore, the Wnt/ß-catenin signaling pathway was determined to be activated in CRC tissues and cells, while its activation was inhibited by GAS5. The Wnt/ß-catenin signaling pathway promoted the CRC cell invasion and migration in vitro, subcutaneous tumor growth, angiogenesis and, liver metastases in vivo. CONCLUSION: Taken together, the results of the study conclude that lncRNA GAS5 inhibited the activation of the Wnt/ß-catenin signaling pathway, thereby suppressing the angiogenesis, invasion, and metastasis of CRC.