Construction of an immune predictive model and identification of TRIP6 as a prognostic marker and therapeutic target of CRC by integration of single-cell and bulk RNA-seq data.

BACKGROUND: Investigations elucidating the complex immunological mechanisms involved in colorectal cancer (CRC) and accurately predicting patient outcomes via bulk RNA-Seq analysis have been notably limited. This study aimed to identify the immune status of CRC patients, construct a prognostic model, and identify prognostic signatures via bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq). METHODS: The scRNA-seq data of CRC were downloaded from Gene Expression Omnibus (GEO). The UCSC Xena database was used to obtain bulk RNA-seq data. Differentially expressed gene (DEG), functional enrichment, and random forest analyses were conducted in order to identify core genes associated with colorectal cancer (CRC) that were relevant to prognosis. A molecular immune prediction model was developed using logistic regression after screening features using the least absolute shrinkage and selection operator (LASSO). The differences in immune cell infiltration, mutation, chemotherapeutic drug sensitivity, cellular senescence, and communication between patients who were at high and low risk of CRC according to the predictive model were investigated. The prognostic genes that were closely associated with CRC were identified by random survival forest (RSF) analysis. The expression levels and clinical significance of the hub genes were analyzed in vitro. The LoVo cell line was employed to ascertain the biological role of thyroid hormone receptor-interacting protein 6 (TRIP6). RESULTS: A total of seven main cell subtypes were identified by scRNA-seq analysis. A molecular immune predictive model was constructed based on the risk scores. The risk score was significantly associated with OS, stage, mutation burden, immune cell infiltration, response to immunotherapy, key pathways, and cell-cell communication. The functions of the six hub genes were determined and further utilized to establish a regulatory network. Our findings unequivocally confirmed that TRIP6 upregulation was verified in the CRC samples. After knocking down TRIP6, cell proliferation, migration, and invasion of LoVo cells were inhibited, and apoptosis was promoted. CONCLUSIONS: The molecular predictive model reliably distinguished the immune status of CRC patients. We further revealed that TRIP6 may act as an oncogene in CRC, making it a promising candidate for targeted therapy and as a prognostic marker for CRC.

m6A­mediated LINC02038 inhibits colorectal cancer progression via regulation of the FAM172A/PI3K/AKT pathway via competitive binding with miR­552­5p.

Long noncoding RNAs (lncRNAs) are a type of regulatory molecule with potential roles in the development of several different malignancies. However, the underlying mechanisms of lncRNAs in colorectal cancer (CRC) are incompletely understood. The present study investigated the molecular mechanism of LINC02038 in CRC. LINC02038 expression was decreased in CRC tissues compared to the para­cancerous tissues and LINC02038 overexpression markedly reduced the proliferation, vitality, migration and invasive ability and greatly accelerated apoptosis of colorectal cancer cells. Bioinformatics examination indicated that LINC02038 may have targeted microRNA (miR)­552­5p. RNA immunoprecipitation and luciferase reporter assays showed that LINC02038 served as a sponge for miR­552­5p, hindering target gene FAM172A of miR­552­5p degradation. Moreover, methylated RNA immunoprecipitation (MeRIP)­qualitative PCR assays revealed that YTHDF2 could identify and regulate the METTL3­mediated LINC02038 N6­methyladenosine (m6A) modification and increase its degradation, thereby promoting CRC progression via the PI3K/AKT pathway. Based on the CRC clinical specimens, it was shown that LINC02038 was negatively associated with lymphatic metastasis and distant metastasis. These results revealed that m6A/LINC02038/miR­552­5p/FAM172A may be a novel anti­tumor axis and LINC02038 may serve as a biomarker and treatment option for colorectal cancer.

M6A Promotes Colorectal Cancer Progression via Regulating the miR-27a-3p/BTG2 Pathway.

Long noncoding (lnc) RNAs regulate cancer progression. However, the importance of lncRNAs and how they are regulated in colorectal cancer (CRC) are unclear. We aim to evaluate the function of lncRNA ADAMTS9-AS2 in CRC and its fundamental mechanism. Levels of ADAMTS9-AS2, miR-27a-3p, and B-cell translocation gene 2 (BTG2) were measured by qPCR. Cell viability was analyzed by CCK-8 and colony formation. Migration and invasion were tested by transwell assay. The interactions among ADAMTS9-AS2, miR-27a-3p, BTG2, and YTHDF2 were analyzed by luciferase test, immunoblotting, RNA pull-down, or RNA immunoprecipitation (RIP). An animal model was adopted to assess ADAMTS9-AS2's function. Overexpressing ADAMTS9-AS2 inhibited cell migration, invasion, colony formation capacity, and proliferation in vitro. The direct targeting of miR-27a-3p by ADAMTS9-AS2 abrogated the latter's effect in CRC cells. BTG2 was identified a target of miR-27a-3p, and silencing BTG2 weakened miR-27a-3p's effect. Knocking down ADAMTS9-AS2 abolished sh-YTHDF2's inhibitory effect on cell proliferation and invasion. Finally, overexpressing ADAMTS9-AS2 restrained xenograft growth. M6A reader YTHDF2-mediated degradation of ADAMTS9-AS2 promotes colon carcinogenesis via miR-27a-3p/BTG2 axis.

Regional methylome profiling reveals dynamic epigenetic heterogeneity and convergent hypomethylation of stem cell quiescence-associated genes in breast cancer following neoadjuvant chemotherapy.

BACKGROUND: Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ~ 30% of patients with breast cancer. However, aberrant DNA methylation alterations are frequent events during breast cancer progression and acquisition of chemoresistance. We aimed to characterize the inter- and intra-tumor methylation heterogeneity (MH) in breast cancer following NAC. METHODS: DNA methylation profiles of spatially separated regions of breast tumors before and after NAC treatment were investigated using high-density methylation microarray. Methylation levels of genes of interest were further examined using multiplexed MethyLight droplet digital PCR (ddPCR). RESULTS: We have discovered different levels of intra-tumor MH in breast cancer patients. Moreover, NAC dramatically altered the methylation profiles and such changes were highly heterogeneous between the patients. Despite the high inter-patient heterogeneity, we identified that stem cell quiescence-associated genes ALDH1L1, HOPX, WNT5A and SOX9 were convergently hypomethylated across all the samples after NAC treatment. Furthermore, by using MethyLight ddPCR, we verified that the methylation levels of these 4 genes were significantly lower in breast tumor samples after NAC than those before NAC. CONCLUSIONS: Our study has revealed that NAC dramatically alters epigenetic heterogeneity in breast cancer and induces convergent hypomethylation of stem cell quiescence-associated genes, ALDH1L1, HOPX, WNT5A and SOX9, which can potentially be developed as therapeutic targets or biomarkers for chemoresistance.

A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy.

Recent progress in neural stem cell- (NSC-) based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT) to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR) that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is "switched off" by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is "switched on" by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.