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.

LncRNA OTUD6B-AS1 overexpression promoted GPX4-mediated ferroptosis to suppress radioresistance in colorectal cancer

Background Radiotherapy is widely employed in colorectal cancer (CRC) treatment but is often compromised by developed radioresistance. This study explored the mechanism of long non-coding RNA ovarian tumor domain containing 6B-antisense RNA1 (lncRNA OTUD6B-AS1) in CRC radioresistance through tripartite motif 16 (TRIM16). Methods CRC and non-cancerous tissues were collected and radioresistant CRC cells were established, with real-time quantitative polymerase chain reaction to determine gene expression in tissues and cells. Radioresistance was evaluated by cell counting kit-8 assay and immunofluorescence (γ-H2AX) and ferroptosis was tested by Western blot assay (ACSL4/GPX4) and assay kits (Fe2+/ROS/MDA/GSH). The association between ferroptosis and lncRNA OTUD6B-AS1-inhibited radioresistance was testified using ferroptosis inhibitor. The subcellular localization of lncRNA OTUD6B-AS1 was tested by the nuclear/cytoplasmic fractionation assay, with RNA immunoprecipitation assay to validate gene interactions. Rescue experiments were conducted to analyze the role of TRIM16 in CRC radioresistance. Results LncRNA OTUD6B-AS1 and TRIM16 were poorly expressed (P < 0.01) in CRC tissues and cells and further decreased (P < 0.01) in radioresistant CRC cells. OTUD6B-AS1 overexpression decreased cell survival (P < 0.01), increased γ-H2AX levels (P < 0.01), and elevated ferroptosis and oxidative stress (P < 0.01) after X-ray radiation. Ferroptosis inhibitor attenuated radioresistance (P < 0.01) caused by lncRNA OTUD6B-AS1 overexpression. LncRNA OTUD6B-AS1 stabilized TRIM16 mRNA via binding to HuR. TRIM16 knockdown reduced ferroptosis and increased radioresistance (P < 0.05). Conclusion OTUD6B-AS1 overexpression stabilized TRIM16 via binding to HuR and increased GPX4-mediated ferroptosis, thus attenuating CRC radioresistance. Our study provided a new rationale for the treatment of CRC (AU)

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.

LncRNA OTUD6B-AS1 overexpression promoted GPX4-mediated ferroptosis to suppress radioresistance in colorectal cancer.

BACKGROUND: Radiotherapy is widely employed in colorectal cancer (CRC) treatment but is often compromised by developed radioresistance. This study explored the mechanism of long non-coding RNA ovarian tumor domain containing 6B-antisense RNA1 (lncRNA OTUD6B-AS1) in CRC radioresistance through tripartite motif 16 (TRIM16). METHODS: CRC and non-cancerous tissues were collected and radioresistant CRC cells were established, with real-time quantitative polymerase chain reaction to determine gene expression in tissues and cells. Radioresistance was evaluated by cell counting kit-8 assay and immunofluorescence (γ-H2AX) and ferroptosis was tested by Western blot assay (ACSL4/GPX4) and assay kits (Fe2+/ROS/MDA/GSH). The association between ferroptosis and lncRNA OTUD6B-AS1-inhibited radioresistance was testified using ferroptosis inhibitor. The subcellular localization of lncRNA OTUD6B-AS1 was tested by the nuclear/cytoplasmic fractionation assay, with RNA immunoprecipitation assay to validate gene interactions. Rescue experiments were conducted to analyze the role of TRIM16 in CRC radioresistance. RESULTS: LncRNA OTUD6B-AS1 and TRIM16 were poorly expressed (P < 0.01) in CRC tissues and cells and further decreased (P < 0.01) in radioresistant CRC cells. OTUD6B-AS1 overexpression decreased cell survival (P < 0.01), increased γ-H2AX levels (P < 0.01), and elevated ferroptosis and oxidative stress (P < 0.01) after X-ray radiation. Ferroptosis inhibitor attenuated radioresistance (P < 0.01) caused by lncRNA OTUD6B-AS1 overexpression. LncRNA OTUD6B-AS1 stabilized TRIM16 mRNA via binding to HuR. TRIM16 knockdown reduced ferroptosis and increased radioresistance (P < 0.05). CONCLUSION: OTUD6B-AS1 overexpression stabilized TRIM16 via binding to HuR and increased GPX4-mediated ferroptosis, thus attenuating CRC radioresistance. Our study provided a new rationale for the treatment of CRC.

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.

miR-217 suppresses proliferation, migration, and invasion promoting apoptosis via targeting MTDH in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has frequent incidence and the third highest mortality rate among cancers in the world. This study aimed to clarify the roles of miR-217 and metadherin (MTDH) in HCC. First, we identified that miR-217 expression was downregulated and MTDH expression was upregulated in the HCC tissues. Functional studies revealed that miR-217 negatively regulated MTDH expression via binding to the 3'-untranslated region of MTDH mRNA in the HCC cells. In our further studies, the miR-217 overexpression resulted in downregulation of MTDH expression in HCC cells. The miR-217 overexpression in HCC cells suppressed proliferation, migration, and invasion inducing apoptosis. Taken together, our study provides the initial evidence that the increase of MTDH expression is associated with the decrease of miR-217 expression in HCC. This study also suggests that miR-217 inhibits malignant progression of HCC in vitro and may be used for miRNA-based therapy, possibly via directly targeting MTDH.