Long non-coding RNAs (lncRNAs) function as key molecules in cancer progression. The lncRNA CYTOR plays oncogenic roles in multiple types of cancer, yet the detailed molecular mechanisms of those roles remain unknown. The aim of this study was to investigate the clinical significance, biological function and interacting partners of CYTOR in colorectal cancer (CRC). A systematic and comprehensive analysis of CYTOR expression was performed in 138 CRC samples and in the TCGA and GEO databases. Biological function was investigated through knockdown and overexpression of CYTOR in vitro and in vivo. In addition, its protein binding partner was identified and validated using ChIRP-MS and RNA immunoprecipitation assays. Their key interaction sites on CYTOR were verified by CRISPR/Cas9 and a series of mutant constructs. Furthermore, the downstream targets of CYTOR were confirmed via immunoblotting and luciferase reporter assays. CYTOR was significantly up-regulated in CRC samples and associated with poor prognosis, promoting proliferation and metastasis in vitro and in vivo. NCL and Sam68 could recognize their specific motifs and directly bind to EXON1 of CYTOR. Moreover, EXON1 was the key functional site mediating the interaction of CYTOR with NCL and Sam68. NCL and Sam68 functioned as oncogenes to promote CRC progression. Furthermore, we confirmed that the heterotrimeric complex of CYTOR, NCL and Sam68 activated the NF-κB pathway and EMT to contribute to CRC progression. CYTOR plays important roles in CRC progression by interacting with NCL and Sam68 and may serve as a prognostic biomarker and/or an effective target for CRC therapies.

Semaphorin 3D (SEMA3D) plays important roles in the genesis and progress of many cancers. However, the relationship between SEMA3D and colorectal cancer (CRC) remains unknown. The aim of this study was to investigate whether SEMA3D can be used as a predictive marker for the diagnosis, metastasis, and prognosis of CRC by assessing the expression of SEMA3D in the tissues and serum of CRC patients. Real-time quantitative polymerase chain reaction (qPCR) was used to measure the expression of SEMA3D mRNA in 100 CRC tissues and matched normal tissues. qPCR was also used to detect the expression of SEMA3D mRNA in the CRC cell line RKO. RKO cells were transfected with SEMA3D small-interring RNA (siRNA) to interfere with endogenous SEMA3D. The migratory ability of control and SEMA3D siRNA-transfected RKO cells was determined by transwell assays. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect the levels of SEMA3D in the serum of 80 CRC patients and 100 normal healthy controls. The expression of SEMA3D in 215 CRC tissues was assessed using immunohistochemistry (IHC). Then, statistical analyses were adopted to assess SEMA3D protein levels and clinical pathological characteristics. The mRNA expression of SEMA3D was significantly lower in CRC tissues than in paired normal tissues (t = 5.027, P < 0.0001). Compared with normal healthy controls, the serum levels of SEMA3D were decreased significantly in CRC patients (t = 3.656, P = 0.0003). The expression of SEMA3D protein was linked to lymph node metastasis, and low expression led to lymph node metastasis (χ 2 = 8.415, P = 0.004). The expression of SEMA3D in CRC tissues was a favorable prognostic factor. Patients with a higher expression of SEMA3D experienced longer survival (P = 0.002, log-rank [Mantel-Cox]; Kaplan-Meier). In addition, multivariate Cox’s proportional hazard model revealed that SEMA3D is an independent prognostic marker (hazard ratio [HR] 1.818, 95% CI 1.063–3.110, P = 0.029). Moreover, transwell assays showed that knocking down SEMA3D significantly increased RKO cell migration (t = 9.268, P = 0.0008). SEMA3D might function as a tumor suppressor during the formation and development of CRC. SEMA3D might become a predictive marker for the diagnosis, metastasis, and prognosis of CRC and provide a novel target for the prevention and treatment of CRC.

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Aim. To investigate whether USP18 can be used as a predictive marker for the diagnosis and development of colorectal cancer. Methods. The Gene Expression Omnibus (GEO) Dataset and the Cancer Genome Atlas (TCGA) database were used to select differential proteins for the ubiquitin-specific peptidases (USPs). The extensive target prediction and network analysis methods were used to assess the association with the USP18 interacting proteins, as well as the statistical correlation between USP18 and the clinical pathology parameters. The effects of USP18 on the proliferation of colorectal cancer were examined using CCK8. The effects of USP18 on the migration of colorectal cancer were examined using wound healing assays. Immunohisto-chemistry (IHC) was performed on the tissue microarray. Results. The results showed that the expression of USP18 was related to age (P=0.014). The positive rates of the USP18 protein in T1, T2, T3, and T4 were 0.00%, 22.92%, 78.38%, and 95.35%, respectively (P<0.00). The positive rates of the USP18 protein in I, II, III, and IV were 47.43%, 83.12%, 66.67%, and 100.00%, respectively (P<0.00). The Western blot assay showed that the expression of USP18 in colorectal cancer tissues was significantly higher than that in matched paracancerous tissues (P<0.05). The CCK8 experiments suggested that USP18 promoted the migration of CRC cells. Wound healing assays suggested that USP18 promoted the proliferation of CRC cells. Conclusion. This study showed that USP18 can promote the proliferation of colorectal cancer cells and might be a potential biomarker for the diagnosis of CRC.