Adjacent Normal Colorectal Tissues Research Articles

MicroRNA-675-5p Overexpression Is an Independent Prognostic Molecular Biomarker of Short-Term Relapse and Poor Overall Survival in Colorectal Cancer.

Colorectal cancer (CRC) is the main cause of cancer-related deaths globally, highlighting the importance of accurate biomarkers for early detection and accurate prognosis. MicroRNAs (miRNAs) have emerged as effective cancer biomarkers. The aim of this study was to investigate the prognostic potential of miR-675-5p as a molecular prognostic biomarker in CRC. For this reason, a quantitative PCR assay was developed and applied to determine miR-675-5p expression in cDNAs from 218 primary CRC and 90 paired normal colorectal tissue samples. To assess the significance of miR-675-5p expression and its association with patient outcome, extensive biostatistical analysis was performed. miR-675-5p expression was found to be significantly downregulated in CRC tissue samples compared to that in adjacent normal colorectal tissues. Moreover, high miR-675-5p expression was associated with shorter disease-free (DFS) and overall survival (OS) in CRC patients, while it maintained its unfavorable prognostic value independently of other established prognostic factors. Furthermore, TNM stage stratification demonstrated that higher miR-675-5p levels were associated with shorter DFS and OS intervals, particularly in patients with CRC of TNM stage II or III. In conclusion, our findings suggest that miR-675-5p overexpression constitutes a promising molecular biomarker of unfavorable prognosis in CRC, independent of other established prognostic factors, including TNM staging.

Synaptophysin-like 2 expression correlates with lymph node metastasis and poor prognosis in colorectal cancer patients.

Colorectal cancer (CRC) is one of the most common and fatal cancers worldwide. Synaptophysin-like 2 (SYPL2) is a neuroendocrine-related protein highly expressed in skeletal muscle and the tongue. The involvement of SYPL2 in CRC, including its level of expression and function, has not been evaluated. To evaluate the correlations of SYPL2 expression with lymph node metastasis (LNM) and prognosis in patients with CRC. The levels of expression of SYPL2 in CRC and normal colorectal tissues were analyzed in multiple public and online databases. The associations between clinical variables and SYPL2 expression were evaluated statistically, and the associations between SYPL2 expression and prognosis in patients with CRC were analyzed using the Kaplan-Meier method and univariate/multivariate Cox regression analyses. SYPL2 expression was assessed in 20 paired CRC tissue and adjacent normal colorectal tissue samples obtained from Fuyang People's Hospital, and the associations between SYPL2 expression and the clinical characteristics of these patients were investigated. Correlations between the levels of expression of SYPL2 and key targeted genes were determined by Pearson's correlation analysis. The distribution of immune cells in these samples was calculated using the CIBERSORT algorithm. Gene set enrichment analysis (GSEA) was performed to evaluate the biofunction and pathways of SYPL2 in CRC. SYPL2 expression was significantly lower in CRC tissue samples than in normal colorectal tissue samples (P < 0.05). High SYPL2 levels in CRC tissues correlated significantly with LNM (P < 0.05) and a poorer patient prognosis, including significantly shorter overall survival (OS) [hazard ratio (HR) = 1.9, P < 0.05] and disease-free survival (HR = 1.6, P < 0.05). High SYPL2 expression was an independent risk factor for OS in both univariate (HR = 2.078, P = 0.014) and multivariate (HR = 1.754, P = 0.018) Cox regression analyses. In addition, SYPL2 expression correlated significantly with the expression of KDR (P < 0.0001, r = 0.47) and the BRAF V600E mutation (P < 0.05). Higher SYPL2 expression was associated with the enrichment of CD8 T-cells and M0 macrophages in the tumor microenvironment. GSEA revealed that SYPL2 was associated with the regulation of epithelial cell migration, vasculature development, pathways in cancer, and several vital tumor-related pathways. SYPL2 expression was lower in CRC tissue than in normal colorectal tissue. Higher SYPL2 expression in CRC was significantly associated with LNM and poorer survival.

Regulation of long non-coding RNAs XIST and ROR induced by homeodomain protein TGIF2LX in colorectal cancer.

Homeodomain protein transforming growth factor beta-induced factor 2 like, X-linked (TGIF2 LX) has been demonstrated to act as a transcription factor and regulate cancer cell proliferation. Long non-coding RNAs (lncRNAs) are well known as molecular regulators of colorectal cancer (CRC). Our aim was to evaluate the clinical and biological significance of TGIF2 LX and its effect on lncRNAs regulator of reprogramming (ROR) and X-inactive specific transcript (XIST) expression in CRC cells. Thirty-six CRC tissues and 22 adjacent normal colorectal tissues were subjected to RNA extraction and analysis of TGIF2 LX gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). The human SW1116 cell line was transfected with cDNA for the TGIF2 LX gene. Microscopic analysis, reverse transcriptase PCR, and western blotting were used for confirming at transcriptional and translational levels. Methyl thiazolyl tetrazolium and colony formation assays were applied for evaluating the in vitro cell viability and colony-forming ability, respectively. LncRNA expression analysis was carried out using qRT-PCR. The results showed that the expression levels of TGIF2 LX were significantly downregulated in CRC tissues compared to adjacent normal tissues (P = 0.032). Furthermore, the overexpression of TGIF2 LX could reduce the CRC cell line proliferation. The gene expression analysis revealed a significantly reduced level of lncRNA ROR and lncRNA XIST in TGIF2 LX-transfected SW1116 cells compared to nontransfected cells. Our findings provided evidence of molecular mechanisms by which TGIF2 LX may interact with lncRNAs ROR and XSIST to regulate CRC development by acting as a tumor suppressor. Thus, this protein may potentially be a promising option for CRC gene-based therapeutic strategies.

The novel circSLC6A6/miR-1265/C2CD4A axis promotes colorectal cancer growth by suppressing p53 signaling pathway

BackgroundColorectal cancer (CRC) is one of the most frequent malignancy and a leading cause of cancer-related deaths. Therefore, further researches are required to identify novel and more effective diagnoses and to identify molecular targets in treatment of CRC.MethodsC2CD4A expression in CRC tissues and cell lines was detected by qRT-PCR and western blot. The biological functions of C2CD4A were performed both in vitro and in vivo. Western blot, cDNA array, IP-MS, Co-immunoprecipitation assay, and Ubiquitination assay were used to analyze the interaction between C2CD4A and p53. Bioinformatics analysis, FISH, RNA sequencing, luciferase reporter assay, RNA immunoprecipitation, RNA pull-down and rescue experiments, were deployed to detect upstream regulation mechanism of C2CD4A.ResultsC2CD4A was elevated in CRC tissues compared with adjacent normal colorectal tissues. C2CD4A knockdown significantly promoted cell apoptosis and with inhibited proliferation in vitro, and tumorigenicity in vivo, whereas C2CD4A overexpression led to opposite effects. Moreover, circSLC6A6 was upregulated and shown to positively regulate C2CD4A expression via sponging miR-1265. Fundamentally, C2CD4A inhibited p53 signaling pathway through interacting with p53 and increasing its ubiquitination and degradation.ConclusionOur results identified that circSLC6A6/miR-1265/C2CD4A axis, which was involved in CRC via the p53 signaling pathway, may serve as a therapeutic target for CRC.

Identification of Cysteine Protease Inhibitor CST2 as a Potential Biomarker for Colorectal Cancer.

Additional biomarkers for the development and progression of colorectal cancer (CRC) remain to be identified. Hence, the current study aimed to identify potential diagnostic markers for CRC. Analyses of cysteine protease inhibitor [cystatins (CSTs)] expression in CRC samples and its correlation with cancer stage or survival in patients with CRC demonstrated that CRC tissues had greater CST1 and CST2 mRNA expression compared to noncancerous adjacent tissues, while higher CST2 mRNA expression in CRC tissues was correlated with advanced stages and disease-free survival in patients with CRC, encouraging further exploration on the role of CST2 in CRC. Through an online database search and tissue microarray (TMA), we confirmed that CRC samples had higher CST2 expression compared to noncancerous adjacent tissue or normal colorectal tissues at both the mRNA and protein levels. TMA also revealed that colorectal adenoma, CRC, and metastatic CRC tissues exhibited a significantly increased CST2 protein expression. Accordingly, survival analysis demonstrated that the increase in CST2 protein expression was correlated with shorter overall survival of patients with CRC. Moreover, our results found a significant upregulation of CST2 in multiple cancer tissues. Taken together, these findings suggest the potential role of CST2 as a diagnostic and prognostic biomarker for CRC.

Loss of AKR1B10 promotes colorectal cancer cells proliferation and migration via regulating FGF1-dependent pathway.

Colorectal cancer (CRC) is a common malignancy worldwide with poor prognosis and survival rates. The aldo-keto reductase family 1 member B10 (AKR1B10) plays an important role in metabolism, cell proliferation and mobility, and is downregulated in CRC. We hypothesized that AKR1B10 would promote CRC genesis via a noncanonical oncogenic pathway and is a novel therapeutic target. In this study, AKR1B10 expression levels in 135 pairs of CRC and para-tumor tissues were examined, and its oncogenic role was determined using in vitro and in vivo functional assays following genetic manipulation of CRC cells. AKR1B10 was downregulated in CRC tissues compared to the adjacent normal colorectal tissues, and associated with the clinicopathological status of the patients. AKR1B10 depletion promoted the proliferation and migration of CRC cells in vitro, while its ectopic expression had the opposite effect. AKR1B10 was also significantly correlated with FGF1 gene and protein levels. Knockdown of AKR1B10 promoted tumor growth in vivo, and increased the expression of FGF1. Finally, AKR1B10 inhibited FGF1, and suppressed the proliferation and migration ability of CRC cells in an FGF1-dependent manner. In conclusion, AKR1B10 acts as a tumor suppressor in CRC by inactivating FGF1, and is a novel target for combination therapy of CRC.

Long noncoding RNA KCNQ1OT1 promotes colorectal carcinogenesis by enhancing aerobic glycolysis via hexokinase-2.

In this study, we investigated the mechanistic role and prognostic significance of the long coding RNA (lncRNA) KCNQ1OT1 in colorectal cancer (CRC). KCNQ1OT1 levels were significantly higher in CRC tissues than adjacent normal colorectal tissues (n=79). High KCNQ1OT1 expression correlated with poorer prognosis in CRC patients. KCNQ1OT1-silenced CRC cells showed reduced proliferation, colony formation, extracellular acidification, and lactate and glucose secretion. This suggests KCNQ1OT1 promotes CRC cell proliferation by increasing aerobic glycolysis. RNA pull-down assays with biotinylated KCNQ1OT1 followed by mass spectrometry analysis showed that KCNQ1OT1 directly binds to hexokinase 2 (HK2). This was confirmed by RNA immunoprecipitation assays using anti-hexokinase 2 antibody. HK2 protein levels were reduced in KCNQ1OT1 knockdown CRC cells, but were restored by treatment with the proteasomal inhibitor MG132. KCNQ1OT1 knockdown CRC cells also showed higher ubiquitinated-HK2 levels, suggesting KCNQ1OT1 enhances aerobic glycolysis by stabilizing HK2. HK2 overexpression in KCNQ1OT1 knockdown CRC cells restored proliferation and aerobic glycolysis. KCNQ1OT1 levels correlated positively with HK2 expression and prognosis in CRC patients. These findings show that KCNQ1OT1 promotes colorectal carcinogenesis by increasing aerobic glycolysis through HK2.

High expression of CDCA7 predicts tumor progression and poor prognosis in human colorectal cancer.

Colorectal cancer (CRC) is one of the most fatal types of cancer worldwide. This study aimed to determine the predictive and prognostic values of cell division cycle associated protein 7 (CDCA7) in CRC. Firstly, the relationship between CDCA7 and CRC was assessed through bioinformatics analysis. Subsequently, CDCA7 expression levels were detected in various CRC cell lines, as well as 15 fresh human CRC tissues and their paired adjacent normal colorectal tissues using reverse transcription-quantitative PCR and western blotting. Additionally, immunohistochemical staining was used to determine the levels of CDCA7 in 104 CRC tissues and their paired adjacent normal colorectal tissues. The present study revealed that CDCA7 expression was upregulated in CRC tissues and cell lines. The positive expression rates of CDCA7 in normal and CRC tissues were 26.92 and 75.96%, respectively. The intensities of CDCA7 immunostaining were significantly associated with CRC invasion depth, lymph node metastasis, tumor-node-metastasis stage and distant metastasis. However, no significant differences in sex, age, tumor size and CRC differentiation were found between high and low CDCA7 expression groups. Furthermore, patients with low CDCA7 expression exhibited a greater overall survival rate of CRC compared to those with high CDCA7 expression. The findings of this study indicated that CDCA7 may serve a significant role in CRC prognosis and progression, and may be considered a novel biomarker for the prediction of patient survival after colectomy.

Overexpression of LBX2 associated with tumor progression and poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common carcinomas with high morbidity and mortality worldwide. However, the underlying molecular mechanisms of CRC are unclear. The aim of the present study was to establish the role that overexpression of LBX2 serves in CRC and to investigate the associated biological pathways. The difference in the expression levels of LBX2 between CRC tissues and adjacent normal colorectal tissues was assessed using the Oncomine database and Tumor Immune Estimation Resource. The expression levels of LBX2 and its prognostic significance in CRC were analyzed using a t-test and χ2 test using data from The Cancer Genome Atlas database. The Kaplan-Meier method and Cox regression analysis were used to estimate the prognostic value of LBX2 in CRC. Furthermore, the potential mechanisms of LBX2 dysregulation and its underlying molecular mechanisms in CRC were investigated using Gene Set Enrichment Analysis (GSEA). LBX2 expression levels were significantly upregulated in CRC samples compared with corresponding normal colorectal tissues (P<0.05). LBX2 upregulation was correlated with advanced tumor stage (III or IV), vascular invasion, lymphatic invasion and the male sex (all P<0.05). Kaplan-Meier analyses showed that high expression levels of LBX2 were associated with a less favorable overall survival (OS) and disease-free survival (DFS) in CRC (all P<0.05). Multivariate analyses further confirmed that LBX2 upregulation was an independent indicator of less favorable OS and DFS (all P<0.05). In addition, LBX2 DNA hypomethylation and microRNA (miR)-378a-3p downregulation correlated with LBX2 upregulation in CRC (all P<0.05). The downregulation of miR-378a-3p in CRC was also significantly associated with less favorable OS and DFS, as demonstrated using Kaplan-Meier analyses (all P<0.05). Moreover, GSEA indicated that ‘VEGF signaling’, ‘Cell adhesion molecules CAMs’, ‘Toll-like receptor signaling’ and ‘Natural killer cell-mediated cytotoxicity’ signaling pathways were enriched in the high LBX2 expressing cohort (all P<0.05). Thus, overexpression of LBX2 may be associated with the development of CRC and may serve as a novel prognostic marker and therapeutic target in CRC. The mechanisms of LBX2 upregulation in CRC are possibly associated with LBX2 DNA hypomethylation and miR-378a-3p downregulation. The potential mechanisms of LBX2 upregulation in CRC might be regulated via the ‘Cell adhesion molecules CAMs’, ‘Toll-like receptor signaling’ and ‘Natural killer cell-mediated cytotoxicity’ signaling pathways.

AZGP1 suppresses the process of colorectal cancer after upregulating FASN expression via mTOR signal pathway.

Colorectal cancer (CRC) is the most common malignant gastrointestinal tumor. Obesity has been confirmed to be closely related to the occurrence of CRC, but the specific mechanism is not clear. This study mainly explored the roles of obesity-related genes, fatty acid synthase (FASN) and zinc-alpha-2-glycoprotein (AZGP1), in CRC. 30 cases of CRC tissues and adjacent normal colorectal tissues were obtained to quantify the levels of FASN and AZGP1 using qRT-PCR and Western blotting. Overexpression-AZGP1, overexpression-FASN and FASN shRNA were transfected into SW480 cells. CCK-8, wound healing and Transwell assays were used to evaluate the roles of FASN and AZGP1 on cell proliferation, migration as well as invasion. Western blot was performed to investigate the expression of MMP-2, MMP-9 and mTOR signaling-related proteins. AZGP1 expression was decreased in CRC tissues, which was negatively correlated with FASN expression. Overexpression-AZGP1 showed a significant inhibitory effect on cell proliferation, invasion and migration via inhibiting MMP-2 and MMP-9 expressions. Furthermore, up-regulation of AZGP1 suppressed the expression of mTOR pathway downstream proteins 4EBP and eIF4E through inhibiting FASN expression. Reintroduction of overexpression-FASN could partially reverse and inhibition of FASN further decrease the anti-tumor effect of AZGP1. AZGP1 suppresses CRC cellular activities by regulating FASN via mTOR pathway, suggesting that AZGP1 and FASN may be the targets for CRC therapy.

Homo Sapiens Circular RNA 0079993 (hsa_circ_0079993) of the POLR2J4 Gene Acts as an Oncogene in Colorectal Cancer Through the microRNA-203a-3p.1 and CREB1 Axis.

BackgroundWorldwide, dietary changes have resulted in an increased incidence of colorectal cancer (CRC). Circular RNAs (circRNAs) are involved in tumorigenesis of several human tumors, but their role in CRC remains unknown. This study aimed to investigate the expression and effects of Homo sapiens (hsa)_circ_0079993 of POLR2J4 and its impact on CRC.Material/MethodsPaired CRC tissue and adjacent normal colorectal tissue samples (N=41), and HCT116 and SW620 human CRC cells were studied. The expression of circ_0079993 and its parental gene, POLR2J4, were examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Two small-interfering RNAs (siRNAs) against circ_0079993 were used to silence circ_0079993 expression in HCT116 and SW620 CRC cells. Cell proliferation was evaluated using the cell counting kit-8 (CCK-8) assay, colony formation, and in vivo tumor growth assays. The target miRNAs of circ_0079993 was predicted using TargetScan, and the interaction between circ_0079993 and its target miRNAs were verified by the dual-luciferase reporter (DLR) assay.ResultsIn CRC tissue POLR2J4 expression was reduced, and circ_0079993 expression was increased compared with normal tissue. Knockdown of circ_0079993 significantly inhibited the proliferation of CRC cells in vitro. Also, circ_0079993 was predicted to sponge multiple miRNAs, miR-203a-3p.1 was verified as a target of circ_0079993, and circ_0079993 indirectly regulated mRNA expression of the CREB1 gene by sponging miR-203a-3p.1 in CRC cells. The use of anti-miR-203a-3p.1 reversed the inhibitory effects of circ_0079993 knockdown on CRC cell proliferation.ConclusionsThe findings supported that hsa_circ_0079993 acts as an oncogene in CRC through the miRNA-203a-3p.1/CREB1 axis.

KRT18 is correlated with the malignant status and acts as an oncogene in colorectal cancer.

Keratin 18 (KRT18) has been suggested to be overexpressed in most types of human tumor, but the expression pattern of KRT18 in colorectal cancer (CRC) remained unknown. In our research, KRT18 protein expression was markedly increased in CRC cancer tissues and cell lines compared with adjacent normal colorectal tissues and normal colonic epithelial cell line, respectively. Meanwhile, we observed high KRT18 expression was associated with advanced clinical stage, deep tumor invasion, lymph node metastasis, distant metastasis, poor differentiation and unfavorable prognosis in CRC patients. Multivariate Cox regression analysis showed high expression of KRT18 was an unfavorable independent predictor for overall survival in CRC patients. The in vitro studies indicated down-regulation of KRT18 expression depressed CRC cell viability, migration and invasion. In conclusion, KRT18 serves as an oncogenic role in CRC progression and may be a therapeutic target for promoting CRC patients’ prognosis.

Plasma Inter-Alpha-Trypsin Inhibitor Heavy Chains H3 and H4 Serve as Novel Diagnostic Biomarkers in Human Colorectal Cancer.

Objective Inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) are heavy chains of protein members belonging to the ITI family, which was associated with inflammation and carcinogenesis. However, the diagnostic value of ITIH3 and ITIH4 in human colorectal cancer (CRC) remains unknown. Methods In total, 101 CRC patients and 156 healthy controls were enrolled. The concentrations of ITIH3 and ITIH4 proteins in plasma samples of participants were assessed using enzyme-linked immunosorbent assay. ITIH3 and ITIH4 expressions in human CRC tissues were additionally assessed via immunohistochemical staining (IHC). Receiver operating characteristic (ROC) was applied to estimate the diagnostic power of the two proteins, and the net reclassification improvement (NRI) was adopted to evaluate the incremental predictive ability of ITIH3/ITIH4 when added to the tissue inhibitor of metalloproteinase-1 (TIMP-1). Results The plasma concentration of ITIH3 in CRC patients (median: 4.370 μg/mL; range: 2.152–8.170 μg/mL) was significantly lower than that in healthy subjects (median: 4.715 μg/mL; range: 2.665–10.257 μg/mL; p < 0.001), while the ITIH4 plasma level in subjects with CRC (median: 0.211 μg/mL; range: 0.099–0.592 μg/mL) was markedly increased relative to that in the control group (median: 0.134 μg/mL; range: 0.094–0.460 μg/mL, p < 0.001). Consistently, IHC score assessment showed a dramatic reduction in ITIH3 expression and, conversely, upregulation of ITIH4 in colorectal carcinoma specimens relative to adjacent normal colorectal tissues (p < 0.001 in both cases). The area under the curve (AUC) of the ROC for ITIH4 (AUC = 0.801, 95% CI: 0.745–0.857) was higher than that for ITIH3 (AUC = 0.638, 95% CI: 0.571–0.704, both p values < 0.001). The AUC of the ROC for combined ITIH3 and ITIH4 was even higher than that for carcinoembryonic antigen. NRI results showed that combining ITIH3 and ITIH4 with TIMP-1 significantly improved diagnostic accuracy (NRI = 17.12%, p = 0.002) for CRC patients compared to TIMP-1 alone. Conclusions Circulating ITIH3 and ITIH4 levels are associated with carcinogenesis in CRC, supporting their potential diagnostic utility as surrogate biomarkers for colorectal cancer detection.

The Long Noncoding RNA, LINC01555, Promotes Invasion and Metastasis of Colorectal Cancer by Activating the Neuropeptide, Neuromedin U

BackgroundThis study aimed to investigate the role of the long noncoding RNA (lncRNA), LINC01555, on the migration and invasion of colorectal cancer (CRC) cells, its expression in CRC tissue, and its interaction with the neuropeptide, neuromedin U (NmU).Material/MethodsLINC01555 expression in SW620 and HCT116 CRC cells, and NCM460 normal colorectal cells, and 48 resection specimens containing CRC and adjacent normal tissue, was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cox regression analysis was used to assess the relationship between LINC01555 expression and patient survival. The effects of LINC01555 expression on CRC cell proliferation, migration, and invasion were assessed using the cell counting kit-8 (CCK-8) assay, the colony formation assay, and the transwell assay. Functional studies determined the interaction between LINC01555 and NmU in the development of CRC.ResultsThe Cancer Genome Atlas (TCGA) dataset showed that LINC01555 was highly expressed in CRC tissue when compared with adjacent normal colorectal tissue. LINC01555 expression was positively correlated with tumor stage, but negatively correlated with disease-free survival (DFS) and overall survival (OS) and was an independent risk factor for CRC. The receiver operating characteristic (ROC) curve analysis showed the diagnostic specificity of LINC01555 in CRC. Knockdown of LINC01555 inhibited cell proliferation, migration, and invasion of CRC cells. Functional studies showed that knockdown of NmU reduced cell migration and invasion of CRC cells that overexpressed LINC01555.ConclusionsIncreased expression of LINC01555 was found in CRC tissues and promoted the invasion of CRC cells by upregulating the expression of NmU.

High Expression of LTBP2 Contributes to Poor Prognosis in Colorectal Cancer Patients and Correlates with the Mesenchymal Colorectal Cancer Subtype.

Colorectal cancer (CRC) is a complex and heterogeneous disease with four consensus molecular subtypes (CMS1-4). LTBP2 is a member of the fibrillin/LTBP super family and plays a critical role in tumorigenesis by activating TGF-β in the CMS4 CRC subtype. So far, the expression and prognostic significance of LTBP2 in CRC remains obscure. In this study, we aimed to analyze the mRNA and protein expression levels of LTBP2 in CRC tissues and then estimate their values as a potential prognostic biomarker. We detected the mRNA expression of LTBP2 in 28 cases of fresh CRC tissues and 4 CRC cell lines and the protein expression of LTBP2 in 483 samples of CRC tissues, matched tumor-adjacent tissues, and benign colorectal diseases. LTBP2 protein expression was then correlated to patients' clinical features and overall survival. Both LTBP2 mRNA and protein expression levels in CRC tissues were remarkably superior to those in adjacent normal colorectal tissues (P = 0.0071 and P < 0.001, respectively), according to TCGA dataset of CRC. High LTBP2 protein expression was correlated with TNM stage (P < 0.001), T stage (P < 0.001), N stage (P < 0.001), and M stage (P < 0.001). High LTBP2 protein expression was related to poor overall survival in CRC patients and was an independent prognostic factor for CRC. LTBP2 mRNA expression was especially higher in the CMS4 subtype (P < 0.001), which was confirmed in CRC cell lines. Our data suggested that LTBP2 may act as an oncogene in the development of colorectal cancer and have important significance in predicting CRC prognosis. LTBP2 could be a novel biomarker and potential therapeutic target for mesenchymal colorectal cancer and can improve the outcome of high-risk CRC.

Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer.

We reported that insulin receptor substrate 4 (IRS-4) levels increased in tissue from colorectal cancer (CRC) patients and promoted retinoblastoma-cyclin-dependent kinase activation. The aim of the present study was to evaluate the effect of IRS-4 on IGF-1 receptor pathway and its impact on procaspase 3 and PARP expression in RKO and HepG2 cancer cell lines. The results obtained in vitro were compared with those obtained from biopsies of patients with CRC (n = 18), tubulovillous adenomas (TA) (n = 2) and in matched adjacent normal colorectal (MANC) tissue (n = 20). IRS-4 overexpression in cultured cells induced the overactivation of IGF-1/BRK/AKT/GSK-3/β-catenin/cyclin D1 pathways, which led to increased expression of procaspase 3 and PARP protein levels. Studies carried out on CRC and TA tissues revealed the overactivation of the IGF-1 receptor signalling pathway, as well as the overexpression of procaspase 3 and PARP in tumoural tissue with respect to MANC tissue. The upregulation of IRS-4 in tumoural samples correlated significantly with the increase in pIGF-1 receptor (Tyr 1165/1166) (r = 0.84; p < 0.0001), procaspase 3 (r = 0. 77; p < 0. 0005) and PARP (r = 0. 89; p < 0. 0005). Similarly, we observed an increase in the proteolysis of procaspase 3 in tumoural tissue with respect to MANC tissue, which correlated significantly with the degradation of PARP (r = 0.86; p < 0.0001), p53 (r = 0.84; p < 0.0001), and GSK-3 (r = 0.78; p < 0.0001). The stratification of patient samples using the TNM system revealed that procaspase 3 and caspase 3 increased gradually with T values, which suggests their involvement in the size and local invasion of primary tumours. Taken together, our findings suggest that IRS-4 overexpression promotes the activation of the IGF-1 receptor pathway, which leads to the increase in procaspase 3 levels in CRC.

Insulin receptor substrate-4 is overexpressed in colorectal cancer and promotes retinoblastoma-cyclin-dependent kinase activation.

Insulin receptor substrate 4 (IRS-4) is an adaptor protein for which new evidence suggests plays a role in tumour promotion. We described nuclear IRS-4 in RKO colon cancer cell lines in biopsies of patients with colorectal cancer (CRC) (n=20) and in matched adjacent normal colorectal (MANC) tissue (n=20). Treatment with physiological doses of IGF-1 promoted nuclear influx of IRS-4 from cellular cytosol in RKO cells. When exogenous IRS-4 was overexpressed in RKO cells, there was an increase in cyclin D1, cyclin E, E2F1, pRB Ser 809/811 and pRB Ser 705 levels compared with the empty vector-transfected cells. Some of these changes returned to control values after wortmannin treatment. Subcellular fractionation showed an overexpression of IRS-4 in the cytoplasm, membrane, and nuclei of tumour samples, whereas the levels of the protein were barely detectable in the three compartments of normal samples. Immunohistochemical studies showed positive nuclear IRS-4 staining in over 74% of the tumour cells. IRS-4 was strongly overexpressed in tumoural tissues from CRC patients compared to MANC tissues. The up-regulation of IRS-4 in CRC samples correlated significantly with the increase of several G1 checkpoint proteins including cyclin D1 (r=0.6662), Rb (r=0.7779), pRb Serine 809/811 (r=0.6864), pRb serine 705 (r=0.6261) and E2F1 (r=0.8702). Taken together, our findings suggest that IRS-4 promotes retinoblastoma-cyclin-dependent kinase activation and it may serve as a pharmacological target since its expression is very low in normal tissue, including colonic epithelium.

Downregulation of miR-874-3p promotes chemotherapeutic resistance in colorectal cancer via inactivation of the Hippo signaling pathway.

Overcoming resistance to chemotherapy is an arduous challenge in the treatment of colorectal cancer (CRC), particularly since the underlying molecular mechanisms remain obscure. In the present study, we reported that miR-874-3p was markedly downregulated in CRC tissues compared with that in adjacent normal colorectal epithelial tissues. Upregulation of miR-874-3p attenuated the chemoresistance of CRC cells to 5-fluorouracil (5-FU) in vitro and in vivo. Conversely, inhibition of miR-874-3p yielded an opposite effect. Furthermore, our results demonstrated that miR-874-3p directly inhibited the expression of transcriptional co-activators YAP and TAZ of the Hippo signaling pathway, resulting in the inactivation of the TEAD transcription. Thus, our findings clarify a novel mechanism by which miR-874-3p restores chemotherapeutic sensitivity of CRC to 5-FU, indicating that offering miR-874-3p mimics in combination with 5-FU may serve as a new therapeutic strategy to circumvent the chemoresistance in CRC.

Long noncoding RNA ZEB1-AS1 expression predicts progression and poor prognosis of colorectal cancer.

ZEB1-AS1 acts as an oncogene in hepatocellular carcinoma, accelerating tumor growth and promoting metastasis. However, its roles in colorectal cancer (CRC) remain unclear. In this study, we determined the expression of ZEB1-AS1 in CRC tissues by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, we investigated the relationship between various clinicopathological features of CRC patients and ZEB1-AS1 expression, and evaluated the diagnostic and prognostic value of ZEB1-AS1 in CRC. We found that ZEB1-AS1 expression was significantly higher in CRC tissues than in adjacent normal colorectal tissues. Moreover, its expression was significantly correlated with tumor size, differentiation degree, TNM grade, metastasis, depth of invasion and Dukes' classification, but not with sex, age, location and organization. In addition, at the optimal cutoff value of 2.340, the values of diagnostic sensitivity and specificity amounted to 63.0% and 90.7%, respectively, with an area under the curve (AUC) of 0.846 (95% CI, 0.797-0.895). Finally, CRC patients of the high ZEB1-AS1 expression group had a poorer prognosis and a significantly lower survival rate than those of the low expression group, and Cox regression analysis indicated that ZEB1-AS1 expression and metastasis were independent predictors of poor prognosis. Our data suggest that ZEB1-AS1 has no obvious early diagnostic value, but it may be utilized as a new prognostic biomarker for CRC.

DNA methylation of CMTM3, SSTR2, and MDFI genes in colorectal cancer

Colorectal cancer (CRC) is increasingly common worldwide, including in China. Therefore, there is an increasing need to detect CRC at an early stage and to discover and evaluate diagnostic and prognostic biomarkers. DNA methylation of genes in CRC is a potential epigenetic biomarker for the early detection of CRC. This study was performed to analyze the methylation frequency of six candidate genes, CMTM3, SSTR2, MDFI, NDRG4, TGFB2, and BCL2L11, in fresh-frozen CRC tissues and adjacent normal colorectal tissues, from 42 patients with CRC. DNA isolation, bisulphite modification, and pyrosequencing were performed. The sensitivity, specificity, and the area under the receiver operator characteristic (ROC) curve (AUC) were evaluated to determine whether these genes showed any associations with tumor grade, stage, or diagnostic features. Among the tested genes, three genes, CMTM3, SSTR2, and MDFI were significantly methylated in CRC tissues when compared with adjacent normal colorectal tissues. The ROC analysis showed that a multigene model, including CMTM3, SSTR2, and MDFI, had a sensitivity of 81% and a specificity of 91% with an AUC value of 0.92. The findings of this study have shown that DNA methylation of the genes, CMTM3, SSTR2, and MDFI should be studied further with a view to determining their potential role as biomarkers for CRC.