Expression In Colorectal Cancer Cells Research Articles

Heterogenous Nuclear Ribonucleoprotein H1 Promotes Colorectal Cancer Progression through the Stabilization of mRNA of Sphingosine-1-Phosphate Lyase 1.

The oncogenic properties of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) have been reported, although the tumor-promoting mechanism remains unclear. We herein report the mechanism underlying colorectal cancer cell progression mediated by hnRNP H1. The growth of colorectal cancer cells was suppressed by hnRNP H1 downregulation. A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed the anti-apoptotic effect of hnRNP H1 in colorectal cancer cells. An RNA immunoprecipitation assay revealed that hnRNP H1 bound to sphingosine-1-phosphate lyase 1 (SGPL1). Reverse transcription-polymerase chain reaction revealed the high expression of hnRNP H1 mRNA in colorectal cancer cells and Spearman’s rank correlation coefficient showed a strong positive correlation between hnRNP H1 mRNA and SGPL1 mRNA. An siRNA of hnRNP H1 decreased SGPL1 mRNA expression in colorectal cancer cells, but not in non-tumorous cells. These findings suggested that hnRNP H1 increased SGPL1 mRNA expression specifically in cancer cells through direct binding. Targeted knockdown of hnRNP H1 or SGPL1 with siRNAs upregulated p53 phosphorylation and p53-associated molecules, resulting in cell growth inhibition, while hnRNP H1 upregulated the mRNA of SGPL1 and inhibited p53 activation, thereby promoting tumor cell growth. This is a novel mechanism underlying colorectal cancer cell progression mediated by hnRNP H1–SGPL1 mRNA stabilization.

Scoulerine promotes cell viability reduction and apoptosis by activating ROS-dependent endoplasmic reticulum stress in colorectal cancer cells

Scoulerine, an isoquinoline alkaloid isolated from Corydalis plants, has been reported to possess potent anti-proliferative and pro-apoptotic function in cancer cells. However, the effects and underlying mechanisms of scoulerine on colorectal cancer (CRC) progression remain elusive. CCK-8 and LDH assays were used to evaluate cell viability. Apoptosis was assessed by flow cytometry analysis, caspase-3/7 activity assay, and Western blot analysis of Bax, Bcl-2 and cytochrome c (Cyt C) expression. Oxidative stress level was examined by measuring reactive oxygen species (ROS) and glutathione (GSH) contents and superoxide dismutase (SOD) activity. Endoplasmic reticulum (ER) stress activation was detected by Western blot analysis of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) expression. Results showed that scoulerine dose-dependently suppressed CRC cell viability. Scoulerine induced apoptosis and increased caspase-3/7 activity in CRC cells. Bax and cytosolic Cyt C expression was enhanced while Bcl-2 and mitochondrial Cyt C expression was reduced in scoulerine-treated CRC cells. Additionally, scoulerine induced oxidative damage in CRC cells by increasing ROS generation and reducing GSH content and SOD activity. Scoulerine activated ER stress, as evidenced by the increased GRP78 and CHOP expression in CRC cells. Interestingly, blocking ROS production by ROS scavenger N-acetyl-cysteine (NAC) attenuated scoulerine-induced ER stress. Inhibition of ER stress by 4-phenyl butyric acid (4-PBA) abolished scoulerine-induced ROS generation in CRC cells. Blockage of ROS and ER stress attenuated scoulerine-induced cell viability reduction and apoptosis in CRC cells. In conclusion, scoulerine promoted cell viability reduction and apoptosis by activating ROS-dependent ER stress in CRC cells.

Lidocaine Suppresses Cell Proliferation and Aerobic Glycolysis by Regulating circHOMER1/miR-138-5p/HEY1 Axis in Colorectal Cancer.

BackgroundIncreasing evidence has uncovered the anticancer activity of lidocaine in many cancers. However, the role and the underlying molecular mechanism of lidocaine in colorectal cancer (CRC) remain poorly understood.Materials and MethodsCell viability and apoptosis were measured by cell counting kit-8 assay and flow cytometry. Western blot was used to detect the protein of p53, CyclinD1, Pro-caspase-3, Cleaved-caspase-3, Pro-caspase-9, Cleaved-caspase-9, and hes-related family bHLH transcription factor with YRPW motif 1 (HEY1). Glycolytic metabolism was calculated by measuring the glucose consumption, lactate production and adenosine triphosphate (ATP) contents. The expression of circRNA homer scaffold protein 1 (circHOMER1), microRNA (miR)-138-5p and HEY1 mRNA was detected by quantitative real-time polymerase chain reaction. The interaction between miR-138-5p and circHOMER1 or HEY1 was analyzed using the dual-luciferase reporter assay. In vivo experiments were performed using the murine xenograft model.ResultsLidocaine suppressed CRC cell viability and aerobic glycolysis but promoted cell apoptosis in vitro as well as hindered tumor growth in vivo. CircHOMER1 was elevated in CRC tissues and cells, while lidocaine decreased circHOMER1 expression in CRC cells. Additionally, circHOMER1 overexpression reversed the anti-tumor activity of lidocaine in CRC cells. miR-138-5p was confirmed to interact with circHOMER1 and HEY1 in CRC cells directly, and circHOMER1 regulated HEY1 expression through repressing miR-138-5p expression. Besides, rescue assay indicated the anti-tumor activity mediated by lidocaine could be regulated by circHOMER1/miR-138-5p/HEY1 axis.ConclusionLidocaine mediated CRC cell viability loss, apoptosis induction and aerobic glycolysis inhibition by regulating circHOMER1/miR-138-5p/HEY1 axis, providing a novel treatment option for lidocaine to prevent the progression of CRC.

Restoration of KMT2C/MLL3 in human colorectal cancer cells reinforces genome-wide H3K4me1 profiles and influences cell growth and gene expression

BackgroundThe histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant.ResultsGene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation.ConclusionsHere, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.

Long noncoding RNA ZFAS1 promoting small nucleolar RNA-mediated 2\u2032-O-methylation via NOP58 recruitment in colorectal cancer

BackgroundIncreasing evidence supports the role of small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs) as master gene regulators at the epigenetic modification level. However, the underlying mechanism of these functional ncRNAs in colorectal cancer (CRC) has not been well investigated.MethodsThe dysregulated expression profiling of lncRNAs-snoRNAs-mRNAs and their correlations and co-expression enrichment were assessed by GeneChip microarray analysis. The candidate lncRNAs, snoRNAs, and target genes were detected by in situ hybridization (ISH), RT-PCR, qPCR and immunofluorescence (IF) assays. The biological functions of these factors were investigated using in vitro and in vivo studies that included CCK8, trans-well, cell apoptosis, IF assay, western blot method, and the xenograft mice models. rRNA 2′-O-methylation (Me) activities were determined by the RTL-P assay and a novel double-stranded primer based on the single-stranded toehold (DPBST) assay. The underlying molecular mechanisms were explored by bioinformatics and RNA stability, RNA fluorescence ISH, RNA pull-down and translation inhibition assays.ResultsTo demonstrate the involvement of lncRNA and snoRNAs in 2′-O-Me modification during tumorigenesis, we uncovered a previously unreported mechanism linking the snoRNPs NOP58 regulated by ZFAS1 in control of SNORD12C, SNORD78 mediated rRNA 2′-O-Me activities in CRC initiation and development. Specifically, ZFAS1 exerts its oncogenic functions and significantly up-regulated accompanied by elevated NOP58, SNORD12C/78 expression in CRC cells and tissues. ZFAS1 knockdown suppressed CRC cell proliferation, migration, and increased cell apoptosis, and this inhibitory effect could be reversed by NOP58 overexpression in vitro and in vivo. Mechanistically, the NOP58 protein could be recognized by the specific motif (AAGA or CAGA) of ZFAS1. This event accelerates the assembly of SNORD12C/78 to allow for further guiding of 2′-O-Me at the corresponding Gm3878 and Gm4593 sites. Importantly, silencing SNORD12C or 78 reduced the rRNAs 2′-O-Me activities, which could be rescued by overexpression ZFAS1, and this subsequently inhibits the RNA stability and translation activity of their downstream targets (e.g., EIF4A3 and LAMC2).ConclusionThe novel ZFAS1-NOP58-SNORD12C/78-EIF4A3/LAMC2 signaling axis that functions in CRC tumorigenesis provides a better understanding regarding the role of lncRNA-snoRNP-mediated rRNAs 2′-O-Me activities for the prevention and treatment of CRC.

Sodium Butyrate Selectively Kills Cancer Cells and Inhibits Migration in Colorectal Cancer by Targeting Thioredoxin-1.

BackgroundSodium butyrate (NaB) is a short-chain fatty acid which is produced by bacterial fermentation of nondigestible dietary fiber and has been reported to exert anti-tumor effects in many tumors including colorectal cancer (CRC). However, the role of thioredoxin-1 (Trx-1) in NaB-induced anti-tumor effect has not been completely clarified.Materials and MethodsEffects of NaB on the growth of CRC cell lines HT29 and SW480 were detected by the Cell Counting Kit-8 (CCK-8) and colony formation assays. The apoptotic cells were determined by flow cytometry, and cell migration was assessed by a Transwell assay. Western blot analysis was used to test the Trx-1 and epithelial-to-mesenchymal transition (EMT)-related proteins level. Reactive oxygen species (ROS) level was determined and N-acetylcysteine (NAC) recovery experiment was performed in CRC cells. In addition, mice xenograft model was established to test the effect of NaB on CRC growth in vivo. Further, the effects of NaB on CRC cells with overexpression or knockdown were tested by the CCK-8 and Transwell assays.ResultsNaB treatment significantly inhibited cell growth and decreased Trx-1 protein expression in CRC cells but not in normal colon epithelial cells. NaB also induced apoptosis, inhibited colony formation, migration and EMT in CRC cells. Besides, NaB increased ROS level in CRC cells and NAC reversed NaB-induced inhibition of cell proliferation. Moreover, downregulation of Trx-1 significantly enhanced NaB-induced inhibitory effects on cell growth and migration, whereas overexpression of Trx-1 attenuated NaB-induced inhibitory effects on growth and migration in CRC cells.ConclusionThese findings indicate that the NaB-mediated anti-tumor effects on CRC cells are related to downregulation of Trx-1.

Circular RNA circ_0007142 Facilitates Colorectal Cancer Progression by Modulating CDC25A Expression via miR-122-5p.

BackgroundColorectal cancer (CRC) is a common malignant tumor in digestive system. Circular RNA (circRNA) circ_0007142 has been identified as an oncogene in CRC. However, the mechanism of circ_0007142 in CRC was rarely reported.Materials and MethodsThe levels of circ_0007142, dedicator of cytokinesis 1 (DOCK1), microRNA-122-5p (miR-122-5p), and cell division cycle 25A (CDC25A) in CRC tissues (n=31) and cells were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability and colony-forming ability were evaluated via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony-formation assay, respectively. The migrated and invaded abilities were monitored by Transwell assay. The dual-luciferase reporter assay was performed to validate the interactions between miR-122-5p and circ_0007142 or CDC25A. The protein level of CDC25A was detected via Western blot assay. The biological role of circ_0007142 was examined by xenograft tumor model in vivo.ResultsThe levels of circ_0007142 and CDC25A were enhanced and the level of miR-122-5p was declined in CRC tissues and cells, while the level of DOCK1 had no fluctuation. Circ_0007142 sponged miR-122-5p and CDC25A was a target of miR-122-5p. Circ_0007142 knockdown impeded cell proliferation, colony formation, migration, and invasion in CRC cells by regulating miR-122-5p. Besides, miR-122-5p inhibitor promoted cell proliferation, colony formation, migration, and invasion in CRC cells by modulating CDC25A. Circ_0007142 regulated CDC25A expression in CRC cells by sponging miR-122-5p. Moreover, circ_0007142 knockdown blocked CRC tumor growth in vivo.ConclusionCirc_0007142 modulated CDC25A expression to promote CRC progression by sponging miR-122-5p.

FLI-1 mediates tumor suppressor function via Klotho signaling in regulating CRC.

Colorectal cancer (CRC) is an aggressive malignancy with a high incidence and mortality rate. Although a targeting therapy has been developed, the 5-year survival rate is still very low in CRC patients with distant metastasis. Thus, the identification of new targets is still significant for improving CRC treatment. Klotho is a tumor suppressor, and its expression is aberrant in CRC. In this study, the roles of theFLI-1 gene in regulating Klotho gene expression and Klotho-associated signaling, as well as the effects of FLI-1 on colony formation, invasion, and apoptosis were investigated in CRC cell lines. The methylation of the FLI-1 gene was analyzed using a commercial methylation kit. Results showed that FLI-1 messenger RNA and protein expression were downregulated in six CRC cell lines when compared with the normal colon mucosal epithelial cell line, which negatively correlated with the level of DNA methylation. Silencing of FLI-1 gene expression decreased Klotho protein expression and phosphorylation of β-catenin protein at Thr41 /Ser45 , but increased Wnt3a and β-catenin protein expression and IGF-1R phosphorylation in HT29 cells. In contrast to silencing FLI-1, overexpressing FLI-1 significantly increased Klotho protein expression and phosphorylation of β-catenin protein at Thr41 /Ser45 , but decreased Wnt3a and β-catenin protein expression and IGF-1R phosphorylation in Caco-2 cells. Silencing of FLI-1 gene expression significantly increased colony formation and invasion, but decreased apoptosis in HT29 cells. In contrast, overexpressing theFLI-1 gene significantly decreased colony formation and invasion, but increased apoptosis in Caco-2 cells. These findings suggest that FLI-1 functions as a tumor suppressor in CRC cells and positively regulates Klotho signaling. Hypermethylation may be one of the causes of the loss of FLI-1 gene expression in CRC cells.

The Agpat4/LPA axis in colorectal cancer cells regulates antitumor responses via p38/p65 signaling in macrophages

Lipid metabolic reprogramming plays an essential role in regulating the progression of colorectal cancer (CRC). However, the effect of lysophosphatidic acid (LPA) metabolism on CRC development is incompletely characterized. Here, we compared the mRNA levels of human CRC tissues to those of paracarcinoma tissues and focused on the notably enriched LPA metabolic pathways. We identified and verified that 1-acylglycerol-3-phosphate O-acyltransferase 4 (Agpat4) was aberrantly expressed in CRC tissues and predicted poor survival in CRC patients. Manipulating Agpat4 expression in CRC cells did not affect the growth or migration of CRC cells in vitro, whereas Agpat4 silencing suppressed CRC cell growth in subcutaneous and peritoneal xenograft models. Mechanistically, Agpat4 silencing-induced LPA release from CRC cells and polarized macrophages to an M1-like phenotype through LPA receptors 1 and 3. This M1 activation, characterized by elevated p38/p65 signaling and increased proinflammatory cytokines, promoted the infiltration and activation of CD4+ and CD8+ T cells in the tumor microenvironment. Modulation of the Agpat4/LPA/p38/p65 axis regulated macrophage polarization, T-cell activity and CRC progression. Notably, combined therapy with LPA and regular chemotherapy drugs synergistically suppressed CRC development. Taken together, our results showed that the Agpat4/LPA axis in CRC cells regulated p38/p65 signaling-dependent macrophage polarization, T-cell activation, and CRC progression. The Agpat4/LPA/p38/p65 axis might represent a potential target for therapy in the clinic.

BCL‑3 promotes cyclooxygenase‑2/prostaglandinE2 signalling in colorectal cancer.

First discovered as an oncogene in leukaemia, recent reports highlight an emerging role for the proto‑oncogene BCL‑3 in solid tumours. Importantly, BCL‑3 expression is upregulated in >30% of colorectal cancer cases and is reported to be associated with a poor prognosis. However, the mechanism by which BCL‑3 regulates tumorigenesis in the large intestine is yet to be fully elucidated. In the present study, it was shown for the first time that knocking down BCL‑3 expression suppressed cyclooxygenase‑2 (COX‑2)/prostaglandinE2 (PGE2) signalling in colorectal cancer cells, a pathway known to drive several of the hallmarks of cancer. RNAi‑mediated suppression of BCL‑3 expression decreased COX‑2 expression in colorectal cancer cells both at the mRNA and protein level. This reduction in COX‑2 expression resulted in a significant and functional reduction (30‑50%) in the quantity of pro‑tumorigenic PGE2 produced by the cancer cells, as shown by enzyme linked immunoassays and medium exchange experiments. In addition, inhibition of BCL‑3 expression also significantly suppressed cytokine‑induced (TNF‑α or IL‑1β) COX‑2 expression. Taken together, the results of the present study identified a novel role for BCL‑3 in colorectal cancer and suggested that expression of BCL‑3 may be a key determinant in the COX‑2‑meditated response to inflammatory cytokines in colorectal tumour cells. These results suggest that targeting BCL‑3 to suppress PGE2 synthesis may represent an alternative or complementary approach to using non‑steroidal anti‑inflammatory drugs [(NSAIDs), which inhibit cyclooxygenase activity and suppress the conversion of arachidonic acid to prostaglandin], for prevention and/or recurrence in PGE2‑driven tumorigenesis.

LncRNA FTX Contributes to the Progression of Colorectal Cancer Through Regulating miR-192-5p/EIF5A2 Axis.

BackgroundLong non-coding RAN five prime to Xist (LncRNA FTX) has been revealed to be a cancer-related lncRNA and implicated in the progression of colorectal cancer (CRC). Besides, miR-192-5p (miR-192) or eukaryotic initiation factor 5A2 (EIF5A2) also was identified to link with the tumorigenesis of CRC. Here, we further explored the function of FTX and the regulatory relationship among FTX, miR-192 and EIF5A2 in CRC progression.MethodsLevels of FTX, miR-192-5p and EIF5A2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot, respectively. Cell proliferation, apoptosis, migration and invasion were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry or transwell assay, respectively. The interaction between miR-192-5p and FTX or EIF5A2 was confirmed by dual-luciferase reporter and pull-down assay. Murine xenograft model was established using LoVo cells transfected with sh-FTX.ResultsFTX was up-regulated in CRC tissues and cell lines, knockdown of FTX inhibited CRC cell proliferation, migration and invasion in vitro as well as suppressed CRC tumor growth in vivo. FTX was confirmed to directly bind to miR-192-5p and negatively regulated miR-192-5p expression in CRC cells. Besides that overexpressed FTX positively modulated EIF5A2, a direct target of miR-192-5p, via miR-192-5p in CRC cells. Importantly, the inhibitory activities on CRC progression mediated by FTX deletion were reversed miR-192-5p down-regulation or EIF5A2 up-regulation.ConclusionLncRNA FTX functioned as an oncogene to contribute to CRC progression by regulating miR-192-5p/EIF5A2 axis, providing a novel insight into the pathogenesis of CRC and a promising therapeutic target for CRC treatment.

DNA-methylated gene markers for colorectal cancer in TCGA database.

Colorectal cancer (CRC) is characterized by the accumulation of genetic and epigenetic alterations in neoplastic processes. DNA methylation, as an important epigenetic process, contributes to the development of CRC. In the present study, the epigenetic landscape of genes in CRC was characterized by analyzing the dataset from The Cancer Genome Atlas database and 177 DNA-methylated genes were screened based on the criterion of the Pearson correlation (R) between expression and methylation levels being >0.4. Pathway enrichment analysis revealed prominent pathways, including transcription and metabolism, further implying their significant role in tumorigenesis. Among the methylated genes, only zinc finger protein (ZNF)726 with aberrant expression was determined to affect overall survival (OS) as well as disease-free survival of patients with CRC. In addition, ZNF726 was identified as an independent prognostic risk factor for OS in patients with CRC. The methylation-based regulation of ZNF726 expression in CRC cells was further assessed using the Cancer Cell Line Encyclopedia database. Finally, the CpG island methylation of the ZNF726 promoter was evaluated to further elucidate its role in the development of CRC. In conclusion, the epigenetic landscape of genes in terms of promoter methylation in CRC was characterized, revealing that aberrant expression of ZNF726 may be an independent prognostic risk factor for OS in patients with CRC.

Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for colorectal cancer

Berberine (BBR), a natural isoquinoline alkaloid, has been shown to be a promising therapeutic agent for colorectal cancer (CRC), but the molecular mechanism remains unclear. Here, we used mass spectrometry-based label-free proteomics to explore the potential targets of BBR in CRC cells. Comprehensive proteomic profiles demonstrated that of 8051 identified proteins, 503 and 277 differentially expressed proteins (DEPs) were screened out of CACO2 and LOVO cells, respectively. 83 DEPs were overlapped and most of these were down-regulated. A pathway enrichment analysis pinpointed mitochondrial translation, respiratory electron transport and the citric acid (TCA) cycle as biological effectors. The data of proteomics was subsequently confirmed by citrate synthase (CS), Tu translation elongation factor (TUFM), pentatricopeptide repeat domain 3 (PTCD3) and mitochondrial ribosomal protein L48 (MRPL 48) protein measurement. CS protein expression in CRC cells and tissues was higher than it was in normal specimens. Additionally, forcible downregulation of CS led to remarkable cell proliferation inhibition. Taken together, we concluded that the anticancer effects of BBR are attributable to mitochondrial protein synthesis, TCA and respiratory electron transport inhibition and that CS might be a useful therapeutic target in CRC treatment.

MiR-654-5p Targets HAX-1 to Regulate the Malignancy Behaviors of Colorectal Cancer Cells.

Introduction. The biological roles of microRNA-654-5p (miR-654-5p) in cancers have been previously reported. However, its role in colorectal cancer (CRC) remains largely unknown. The purpose of this work was to investigate the roles and associated mechanisms in CRC. Methods Quantitative Real-Time PCR (qRT-PCR) was utilized to explore the expression pattern of miR-654-5p in CRC cells. Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and transwell invasion assay were conducted to investigate the effects of miR-654-5p on CRC cell proliferation, migration, and invasion, respectively. Moreover, the mechanisms behind miR-654-5p regulates CRC progression were investigated. Results Compared with normal cell line, miR-654-5p expression level was significantly suppressed in CRC cells. After overexpression of miR-654-5p, the malignancy behaviors of CRC cells including cell proliferation, migration, and invasion were remarkably decreased. Subsequently, we found hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) was a putative target for miR-654-5p. Rescue experiments showed overexpression of HAX-1 could partially reversed the effects of miR-654-4p on CRC cell events. Conclusion miR-654-5p was reduced expression in CRC cells and could regulate CRC progression via targeting HAX-1.

Capicua suppresses colorectal cancer progression via repression of ETV4 expression

BackgroundAlthough major driver gene mutations have been identified, the complex molecular heterogeneity of colorectal cancer (CRC) remains unclear. Capicua (CIC) functions as a tumor suppressor in various types of cancers; however, its role in CRC progression has not been examined.MethodsDatabases for gene expression profile in CRC patient samples were used to evaluate the association of the levels of CIC and Polyoma enhancer activator 3 (PEA3) group genes (ETS translocation variant 1 (ETV1), ETV4, and ETV5), the best-characterized CIC targets in terms of CIC functions, with clinicopathological features of CRC. CIC and ETV4 protein levels were also examined in CRC patient tissue samples. Gain- and loss-of function experiments in cell lines and mouse xenograft models were performed to investigate regulatory functions of CIC and ETV4 in CRC cell growth and invasion. qRT-PCR and western blot analyses were performed to verify the CIC regulation of ETV4 expression in CRC cells. Rescue experiments were conducted using siRNA against ETV4 and CIC-deficient CRC cell lines.ResultsCIC expression was decreased in the tissue samples of CRC patients. Cell invasion, migration, and proliferation were enhanced in CIC-deficient CRC cells and suppressed in CIC-overexpressing cells. Among PEA3 group genes, ETV4 levels were most dramatically upregulated and inversely correlated with the CIC levels in CRC patient samples. Furthermore, derepression of ETV4 was more prominent in CIC-deficient CRC cells, when compared with that observed for ETV1 and ETV5. The enhanced cell proliferative and invasive capabilities in CIC-deficient CRC cells were completely recovered by knockdown of ETV4.ConclusionCollectively, the CIC-ETV4 axis is not only a key module that controls CRC progression but also a novel therapeutic and/or diagnostic target for CRC.

Hypoxia Induces Growth Differentiation Factor 15 to Promote the Metastasis of Colorectal Cancer via PERK-eIF2α Signaling

Hypoxia plays an essential role in orchestrating Epithelial-mesenchymal transition and promoting metastasis of colorectal cancer. However, the underlying mechanisms are still not well elucidated. Here, we present that hypoxic exposure causes endoplasmic reticulum stress and activates the unfolded protein response pathways, which drives GDF15 expression in colorectal cancer cells. Mechanistically, upregulated CHOP led by activated PERK-eIF2α signaling promotes GDF15 transcription via directly binding to its promoter. Further study implicates that hypoxia-induced GDF15 is required for the EMT and invasion of colorectal cancer cells; enforced expression of GDF15 promotes the mitochondrial oxidation of fatty acids in colorectal cancer cells. Moreover, the abrogation of GDF15 results in smaller xenograft tumors in size and impaired metastasis. GDF15 is expressed much more in tumor tissues of CRC patients and displays positive correlations with CHOP and HIF1α in mRNA levels. Our study demonstrates a novel molecular mechanism underlying hypoxia-promoted metastasis of CRC and provides PERK signaling-regulated GDF15 as a new and promising therapeutic target for clinical treatment and drug discovery.

The long non-coding RNA HCG18 promotes the growth and invasion of colorectal cancer cells through sponging miR-1271 and upregulating MTDH/Wnt/β-catenin.

Long non-coding RNAs (lncRNAs) have recently emerged as key regulators of the occurrence and progression of various human cancers, including colorectal cancer. However, the regulatory mechanism of lncRNAs in the tumorigenesis of colorectal cancer remains poorly understood. In this study, we aimed to elucidate the potential role of lncRNA HCG18 in colorectal cancer. Herein, we found that HCG18 expression was significantly upregulated in colorectal cancer tissues and cell lines. Knockdown of HCG18 significantly inhibited the growth and invasion of colorectal cancer cells, while its overexpression had the opposite effect. Moreover, HCG18 was identified as a sponge of miR-1271. Our results showed that knockdown of HCG18 markedly upregulated miR-1271 expression in colorectal cancer cells. Notably, HCG18 expression was inversely correlated with miR-1271 expression in colorectal cancer specimens. Further investigation revealed that HCG18 contributed to the enhancement of MTDH/Wnt/β-catenin signalling in colorectal cancer cells. The antitumour effect of HCG18 inhibition was significantly reversed by miR-1271 inhibition or MTDH overexpression. Overall, the results of our study demonstrate that HCG18 exerts a potential oncogenic function in colorectal cancer by enhancing MTDH/Wnt/β-catenin signalling via sponging of miR-1271, highlighting the importance of HCG18/miR-1271/ MTDH/Wnt/β-catenin signalling in the progression of colorectal cancer.

The long noncoding RNA TUG1 is required for TGF-\u03b2/TWIST1/EMT-mediated metastasis in colorectal cancer cells

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, and metastasis is the major cause of CRC-related mortality. Transforming growth factor-beta (TGF-β) has a central role not only in the regulation of the normal colon but also in the development and metastasis of CRC. However, TGF-β is not considered an ideal therapeutic target because it shows both pro-tumorigenic and anti-tumorigenic activity, depending on the tumor stage. Therefore, it is important to find a downstream signaling component of TGF-β that can be targeted to impair CRC metastasis. Here, we show that TGF-β promotes CRC migration and upregulates the expression of long-noncoding RNA Taurine Upregulated Gene 1 (TUG1). TUG1 knockdown inhibited migration, invasion, and epithelial–mesenchymal transition (EMT) of CRC cells in vitro, and reduced CRC lung metastasis in vivo. TGF-β induced metastasis, and TUG1 knockdown inhibited these effects. In addition, TGF-β could not reverse the anti-metastasis effects of TUG1 knockdown. These data demonstrate that TUG1 is a downstream molecular of TGF-β. Moreover, TWIST1 expression was increased with TGF-β treatment, and TUG1 knockdown decreased TWIST1 expression in CRC cells. TWIST1 knockdown inhibited invasion and EMT in CRC cells; these effects were not changed by simultaneous TUG1 knockdown, indicating that TWIST1 is a downstream mediator of TUG1. Moreover, TUG1 was significantly overexpressed in CRC patients. In conclusion, TGF-β promotes metastasis of CRC via a TUG1/TWIST1/EMT signaling pathway. TUG1 may be a promising drug target to inhibit TGF-β pathway activation in the treatment of CRC.

MiR-744 mediates the Oxaliplatin chemoresistance in colorectal cancer through inhibiting BIN1.

Colorectal cancer (CRC) is one of the most common malignant cancers worldwide. However, lacking of novel and sensitive chemotherapy revealed the major challenge to improve the survival of CRC patients. The aim of this study was to explore the effect and mechanism of miR-744 on the oxaliplatin chemoresistance in CRC. Firstly, the levels of miR-744 were elevated significantly in CRC tissues from patients with oxaliplatin administration before surgery and in oxaliplatin-resistant HCT116 cells. Then, the oxaliplatin chemoresistance was enhanced by miR-744 overexpression, while was attenuated by miR-744 inhibition in HCT116 and T84 cells. Additionally, the level of BIN1 protein was found to be regulated negatively by miR-744, and BIN1 overexpression blocked the oxaliplatin chemoresistance induced by miR-744. Furthermore, BIN1 was proved to be a direct target of miR-744 by luciferase reporter assay. Taken together, these findings demonstrated that miR-744 might positively mediate the oxaliplatin chemoresistance through suppressing BIN1 expression in CRC cells, thus suggested a rationale target for the developing more effective strategies to reverse oxaliplatin resistance in CRC treatment.

TNNT1, negatively regulated by miR-873, promotes the progression of colorectal cancer.

BackgroundTroponin T1 (TNNT1) is a subunit of troponin that has been linked to neuromuscular disorder. Recently, it was reported that TNNT1 facilitates the proliferation of breast cancer cells. Interestingly, Cancer Genome Atlas data indicate that its overexpression is associated with an unfavorable prognosis of colorectal cancer (CRC) patients. The present study aimed to explore the expression, function and mechanism of dysregulation of TNNT1 in CRC.MethodsImmunohistochemical staining and a real‐time polymerase chain reaction were used to compare the expression level of TNNT1 in CRC tissues and adjacent tissues. Western blotting was used to detect the expression of TNNT1 in cell lines. Kaplan–Meier analysis and a chi‐squared test were applied to evaluate the potential of TNNT1 to function as a cancer biomarker. RNA interference was used to inhibit TNNT1 expression in CRC cells, followed by detection of cell proliferation, apoptosis, migration and invasion. A luciferase reporter gene assay was used to determine the regulatory relationship between miR‐873 and TNNT1.ResultsIn the present study, we found that TNNT1 was significantly up‐regulated in CRC samples and cell lines. The up‐regulation of TNNT1 was also associated with several clinicopathologic features, and its high expression was correlated with an unfavorable prognosis of the patients. Knockdown of TNNT1 markedly arrested proliferation, migration and invasion, whereas it also promoted apoptosis. TNNT1 was identified as a target gene of miR‐873, and there was a negative correlation among CRC samples.ConclusionsIn conclusion, we have demonstrated that TNNT1, regulated by miR‐873, is an oncogene of CRC associated with patient prognosis.