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Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancer diagnoses across the world. Despite recent appreciable improvements in treatment plans for patients with NSCLC, the prognosis for those with the cancer still remains poor. Recently, a growing number of studies have shown that N-myristoyltransferases (NMTs) may be critical in carcinogenesis, however, the functional and clinical significance of this pathway in NSCLC remains unclear and requires further research.Initially, we evaluated the expression levels of NMT1 or NMT2 in a clinical cohort comprising of 303 paired primary NSCLC tissues and matched normal mucosae by using ELISA. We subsequently performed a tissue microarray analysis (TMA) to confirm its expression pattern in an independent validation cohort (n = 78). Then, we used a publicly available KM plotter database (n = 1921) to evaluate the prognostic impact of NMT1 and NMT2 in NSCLC. Lastly, a series of in-vitro molecular/cellular and animal experiments were performed for mechanistic understanding of the role of N-myristoyltransferases in NSCLC.Our ELISA data revealed that the expression level of NMT1 and NMT2 was down-regulated in tumor tissues (n = 303, P 0.0001), which was confirmed in an independent validation cohort by TMA (n = 78, P = 0.014 for NMT1 and P 0.0001 for NMT2). On the other hand, patients with low expression of NMT1 or NMT2 had shorter overall survival (P = 0.013, HR = 0.85 for NMT1; P = 0.00059, HR = 0.8, for NMT2). Mechanistically, we revealed that the interaction and co-localization of NMT1 and NMT2 in NSCLC, and N-terminus of NMT1 and NMT2 was observed to be crucial for their interaction as well as for their catalytic activity. Moreover, we found that NMT1 can significantly promote the expression of NMT2 by enhancing its stability. We corroborated these findings by performing functional assays in which the knockout of NMT1 and NMT2 resulted in enhanced cell proliferation, migration and invasion as well as increased tumorxenograftgrowth. In addition, we identified miR-182 as a novel regulator of both NMT1 and NMT2. More specifically, the overexpression or inhibition of miR-182 modulated globe N-myristoylation level, contributed to phenotypic alterations in NSCLS cells.NMT1 and NMT2 can act as potential tumor suppressors in NSCLC, and the inhibition of miR-182 expression or therapeutic NMTs replenishment may be a promising treatment option for patients with NSCLC.

Colorectal cancer (CRC) is one of most common malignancies worldwide and its incidence is still growing. In spite of recent advances in targeted therapies, their clinical efficacy has been limited, non-curative and unaffordable. A growing body of literature indicates that CRC is a multi-modal disease, where a variety of factors within the tumor microenvironment play a significant role in its pathogenesis. For instance, imbalance in gut microbial profiles and impaired intestinal barrier function contribute to the overall intestinal inflammation and initiation of CRC. Moreover, persistent chronic inflammation favors a tumor microenvironment for the growth of cancer. In addition, autophagy or ‘self-eating’ is a surveillance mechanism involved in the degradation of cellular constituents that are generated under stressful conditions. Cancer stem cells (CSCs), on the other hand, engage in the onset of CRC and are able to endow cancer cells with chemo-resistance. Furthermore, the aberrant epigenetic alterations promote CRC. These evidences highlight the need for multi-targeted approaches that are not only safe and inexpensive but offer a more effective alternative to current generation of targeted drugs. Curcumin, derived from the plant Curcuma longa, represents one such option that has a long history of its use for a variety of chronic disease including cancer, in Indian ayurvedic and traditional Chinese medicine. Scientific evidence over the past few decades have overwhelmingly shown that curcumin exhibits a multitude of anti-cancer activities orchestrated through key signaling pathways associated with cancer. In this article, we will present a current update and perspective on this natural medicine – incorporating the basic cellular mechanisms it effects and the current state of clinical evidence, challenges and promise for its use as a cancer preventative and potential adjunct together with modern therapies for CRC patients.

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Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients.Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings.Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae (P = 0.0018), and its overexpression was associated with poor patient survival (P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes.Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918–28. ©2017 AACR.

Purpose: Colorectal cancer ranks as the third most frequent cancer type, and its incidence continues to rise gradually worldwide, highlighting the need to identify previously unrecognized molecular events that propel development of this malignancy. Recent evidence suggests that dysregulated expression of FOX family of transcription factors may be critical in various genetic disorders as well as cancer; however, the functional and clinical significance of this pathway in colorectal cancer remains unclear.Experimental Design and Results: Herein, we performed a systematic and comprehensive discovery step by evaluating the expression of FOX family members, and identified that FOXM1 and FOXQ1 are frequently overexpressed in colorectal cancer. We subsequently confirmed these findings in two large testing cohorts (n = 550) and an independent clinical validation cohort (n = 134), in which high expression of FOXM1 and FOXQ1 emerged as an independent prognostic factor in colorectal cancer patients. We corroborated these findings by performing functional assays in which knockdown of FOXM1 and FOXQ1 resulted in inhibited cell proliferation and suppressed migration and invasion in colorectal cancer cells. Furthermore, using bioinformatic approaches, we identified miR-342 as a novel regulator of both FOXM1 and FOXQ1. Overexpression or inhibition of miR-342 modulated the expression of both genes and contributed to phenotypic alterations in colorectal cancer cells, which was subsequently validated in a xenograft animal model.Conclusions: Collectively, we have firstly identified FOXM1 and FOXQ1 as promising prognostic biomarkers in colorectal cancer patients, and provided novel evidence that therapeutic targeting of these genes or miR-342 may be a potential treatment approach in colorectal cancer patients. Clin Cancer Res; 22(19); 4947–57. ©2016 AACR.

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Supplementary methods and figure legends

Supplementary Figure 1: The siRNA knockdown efficiency of FOXM1 and FOXQ1 in CRC cells; Supplementary Figure-2: Down-regulation of FOXM1 and FOXQ1 inhibited migration in CRC cell lines; Supplementary Figure-3:Down-regulation of FOXM1 and FOXQ1 inhibited invasion in CRC cell lines; Supplementary Figure-4: MiR-342 directly targets 3'-UTR of FOXM1 and FOXQ1 in HT-29 CRC cells; Supplementary Figure-5: MiR-342 overexpression inhibited cell migration and invasion in SW480 CRC cells; Supplementary Figure 6: Inhibition of miR-342 promotes cell migration and invasion in SW480 CRC cells; Supplementary Figure 7: MiR-342 inhibited cell migration and invasion in HT-29 CRC cells; Supplementary Figure-8: MiR-342 overexpression resulted in down-regulation of FOXM1 and FOXQ1 expression in xenograft tissues.

The aim of this study was to build up a novel chiral mesoporous silica called PEIs@TA-CMS through a facile biomimetic strategy and to explore its potential to serve as a drug carrier for improving the delivery efficiency of poorly water-soluble drug. PEIs@TA-CMS was synthesized by using a chiral crystalline complex associated of tartaric acid and polyethyleneimine (PEIs) as templates, scaffolds and catalysts. The structural features including morphology, size, pore structure and texture properties were systematacially studied. The results showed that PEIs@TA-CMS was monodispersed spherical nanoparticles in a uniformed diameter of 120–130 nm with well-developed pore structure (SBET: 1009.94 m2/g, pore size

Long noncoding RNAs (lncRNAs) are abnormally expressed in many malignant tumors and involved in regulating the malignant phenotypes of cancer cells. However, the role of LINC00665 in colorectal cancer (CRC) and its regulatory mechanism remain unclear. In this study, real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of LINC00665, miR-9-5p and activating transcription factor 1 (ATF1) mRNA in CRC tissues. The expression of ATF1 in CRC tissues was also detected by immunohistochemistry and Western blot. CCK-8 and colony formation assays were employed to detect cell proliferation. Cell cycle and apoptosis were detected by flow cytometry analysis. Scratch healing assay and Transwell test were exploited to detect cell migration and invasion. The targeting relationships between LINC00665 and miR-9-5p, and miR-9-5p and ATF1 were validated by dual luciferase reporter assay. We found that LINC00665 was significantly overexpressed in CRC tissues, and it was also negatively correlated with the expression of miR-9-5p and positively associated with the expression of ATF1. Besides, LINC00665 promoted the proliferation, migration and invasion of CRC cells, and inhibited cell apoptosis by sponging miR-9-5p. ATF1 was proved to be the downstream target of miR-9-5p and was indirectly regulated by LINC00665. Collectively, it is concluded that LINC00665 contributes to the progression of CRC by regulating miR-9-5p/ATF1 axis.

Background: Lung cancer is one of most common malignancies worldwide. We have previously identified retinoic acid-induced gene G (Rig-G) as a tumor suppressor in not only acute promyelocytic leukemia, but as well in other solid tumors. However, the clinical significance of Rig-G and the underlying mechanism(s) for its biological function in lung cancer remain largely unexplored. Methods: We first compared the expression of Rig-G between lung cancer (n=138) and normal tissues (n=23), from public-available datasets and our patient cohort. We further analyzed the correlation of Rig-G expression with key clinico-pathological features and survival outcomes in a multi-site clinical cohort of 300 lung cancer patients. Functional studies for Rig-G were performed in cell lines, and an animal model to support clinical findings. Findings: We found that Rig-G was frequently downregulated in lung cancer tissues and celllines, and correlated with poor prognosis in lung cancer patients. Overexpression of Rig-G led to significantly reduced cell growth and suppressed migrationin A549 and NCI-H1944 cells, accompanied by reduced epithelial-mesenchymal transition. Likewise, restoration of Rig-G in Lewis lung carcinoma cells permitted development of fewer cancer metastases vs. controls in an animal model. Gene expression profiling results identified p53 pathway as a key downstream target of Rig-G, and p53 inhibition by pifithrin-α caused abrogation of tumor-suppressive effects of Rig-G in lung cancer. Interpretation: We for the first time have identified Rig-G as a novel and important tumor suppressor, which may serve as a potential therapeutic target for restoring p53 expression in lung cancer patients. Funding Statement: This work was supported by the National Cancer Institute, NIH (CA72851, CA184792, CA202797 and CA187956), the National Natural Science Foundation of China (81272603, 81472179 and 81873975), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Clinical Research and Cultivation Project of Shanghai Tongji Hospital grant [ITJ(ZD)1803]. This work was also supported by the National Natural Science Foundation of China (81672826 and 81874179), the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai (2017YQ044), the Shanghai Pujiang Talent Plan (18PJD047), the Natural Science Foundation of Shanghai (19ZR1448800), the Medical Guidance Science and Technology Support Project of Shanghai (9411964800) and the National Natural Science Foundation Training Program of Tongji Hospital (GJPY1804). Declaration of Interests: None of the authors have any potential conflicts to disclose. Ethics Approval Statement: A written informed consent was obtained from all patients and the study was approved by ethics committee of Shanghai Tongji Hospital. All animal experiments were approved by the Tongji Hospital of Tongji University Ethics Committee on the Use and Care of Animals.

A novel cascade signal amplification based on tyramide signal amplification (TSA) and surface-initiated enzymatic polymerization (SIEP) was first reported for the sensitive and template-free detection of colorectal cancer (CRC) exosomes. This assay exhibited 20.9-fold signal amplification with a low detection limit of 12.8 particles per μL. Furthermore, accurate and reproducible results were obtained for detecting exosomes in serum samples, suggesting its potential application in exosomes analysis and clinical diagnostics.

Emerging evidence suggests that hypermethylation of HOXD10 plays an important role in human cancers. However, the biological and clinical impacts of HOXD10 overmethylation and its downstream targets in colorectal cancer remain unknown. We evaluated the methylation level of HOXD10 in paired cancer and normal tissues (n = 42) by using pyrosequencing, followed by validation of the methylation status of HOXD10 from The Cancer Genome Atlas (TCGA) datasets with 302 cancer tissues and 38 normal tissues. The biological function of HOXD10 was characterized in cell lines. We further evaluated the effects of HOXD10 and its targets on chemoresistance in our established resistant cell lines and clinical cohort (n = 66). HOXD10 was found frequently methylated in colorectal cancer, and its hypermethylation correlates with its low expression level, advanced disease, and lymph node metastasis. Functionally, HOXD10 acts as a tumor suppressor gene, in which HOXD10-expressing cells showed suppressed cell proliferation, colony formation ability, and migration and invasion capacity. Mechanistically, DNMT1, DNMT3B, and MeCP2 were recruited in the HOXD10 promoter, and demethylation by 5-Aza-2′-deoxycytidine (5-Aza-CdR) treatment or MeCP2 knockdown can sufficiently induce HOXD10 expression. HOXD10 regulates the expressions of miR-7 and IGFBP3 in a promoter-dependent manner. Restoration of the expression of HOXD10 in 5-fluorouracil (5-FU)-resistant cells significantly upregulates the expressions of miR-7 and IGFBP3 and enhances chemosensitivity to 5-FU. In conclusion, we provide novel evidence that HOXD10 is frequently methylated, silenced, and contributes to the development of colorectal cancers. Restoration of HOXD10 activates the expressions of miR-7 and IGFBP3 and results in an inhibited phenotype biologically, suggesting its potential therapeutic relevance in colorectal cancer (CRC).

Graphical abstract Currently, the determination of DNA methylation is still a challenge due to the limited efficiency of enrichment, bisulfite modification, and detection. In this study, a dual-modality loop-mediated isothermal amplification integrated with magnetic bead isolation is proposed for the determination of methylated Septin9 gene in colorectal cancer. Magnetic beads modified with anti-methyl cytosine antibody were prepared for fast enrichment of methylated DNA through specific immunoaffinity (30 min). One-pot real-time fluorescence and colorimetric loop-mediated isothermal amplification were simultaneously developed for detecting methylated Septin9 gene (60 min). The real-time fluorescence generating by SYTO-9 dye (excitation: 470 nm and emission: 525 nm) and pH indicator (neutral red) was used for quantitative and visualized detection of methylated DNA. This method was demonstrated to detect methylated DNA from HCT 116 cells ranging from 2 to 0.02 ng/μL with a limit of detection of 0.02 ± 0.002 ng/μL (RSD: 9.75%). This method also could discriminate methylated Septin9 in 0.1% HCT 116 cells (RSD: 6.60%), suggesting its high specificity and sensitivity. The feasibility of this assay was further evaluated by clinical plasma samples from 20 colorectal cancer patients and 20 healthy controls, which shows the potential application in simple, low cost, quantitative, and visualized detection of methylated nucleic acids. A dual-modality loop-mediated isothermal amplification (LAMP) integrated with immuno-magnetic beads (IMB) enrichment was proposed for the determination of methylated Septin9 gene in colorectal cancer (CRC). Supplementary Information The online version contains supplementary material available at 10.1007/s00604-021-04979-8.

The increasing threat of Streptococcus gallolyticus subsp. gallolyticus (SGG) infections has gained considerable attention for its strong association with colorectal cancer (CRC). Herein, we proposed real-time fluorescence loop-mediated isothermal amplification (LAMP) as a novel, simple, rapid, and highly sensitive assay for identifying SGG for the first time. This assay was capable of detecting SGG with initial DNA concentrations ranging from 102 to 108 copies per microliter, under isothermal conditions within 30 min via real-time fluorescence monitoring. Our method was tested for specific identification of SGG strains without cross-reaction with other Streptococcus gallolyticus subspecies and Escherichia coli. The developed LAMP shows a superior performance with shorter time and higher sensitivity compared with conventional polymerase chain reaction (PCR). Significantly, this proposed approach was successfully applied for detecting SGG in clinical urine samples, which is non-invasive diagnosis, showing excellent accuracy and reliability to discriminate healthy controls and CRC patients. For comparison, these samples were also tested against PCR assay. These results yielded an analytical sensitivity of 100% and a specificity of 100% for SGG testing using LAMP. The findings suggest LAMP can be employed for detecting SGG infections which is useful for diagnosis and screening of CRC.

Rationale: Colorectal cancer (CRC) is a malignant tumor with the third highest morbidity rate among all cancers. Driven by the host's genetic makeup and environmental exposures, the gut microbiome and its metabolites have been implicated as the causes and regulators of CRC pathogenesis. We assessed human fecal samples as noninvasive and unbiased surrogates to catalog the gut microbiota and metabolome in patients with CRC. Methods: Fecal samples collected from CRC patients (CRC group, n = 50) and healthy volunteers (H group, n = 50) were subjected to microbiome (16S rRNA gene sequencing) and metabolome (gas chromatography-mass spectrometry, GC-MS) analyses. The datasets were analyzed individually and integrated for combined analysis using various bioinformatics approaches. Results: Fecal metabolomic analysis led to the identification of 164 metabolites spread across 40 metabolic pathways in both groups. In addition, there were 42 and 17 metabolites specific to the H and CRC groups, respectively. Sequencing of microbial diversity revealed 1084 operational taxonomic units (OTUs) across the two groups, and there was less species diversity in the CRC group than in the H group. Seventy-six discriminatory OTUs were identified for the microbiota of H volunteers and CRC patients. Integrated analysis correlated CRC-associated microbes with metabolites, such as polyamines (cadaverine and putrescine). Conclusions: Our results provide substantial evidence of a novel interplay between the gut microbiome and metabolome (i.e., polyamines), which is drastically perturbed in CRC. Microbe-associated metabolites can be used as diagnostic biomarkers in therapeutic explorations.

Long noncoding RNAs (lncRNAs) are abnormally expressed in many malignant tumors and involved in regulating the malignant phenotypes of cancer cells. However, the role of LINC00665 in colorectal cancer (CRC) and its regulatory mechanism remain unclear. In this study, real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of LINC00665, miR-9-5p and activating transcription factor 1 (ATF1) mRNA in CRC tissues. The expression of ATF1 in CRC tissues was also detected by immunohistochemistry and Western blot. CCK-8 and colony formation assays were employed to detect cell proliferation. Cell cycle and apoptosis were detected by flow cytometry analysis. Scratch healing assay and Transwell test were exploited to detect cell migration and invasion. The targeting relationships between LINC00665 and miR-9-5p, and miR-9-5p and ATF1 were validated by dual luciferase reporter assay. We found that LINC00665 was significantly overexpressed in CRC tissues, and it was also negatively correlated with the expression of miR-9-5p and positively associated with the expression of ATF1. Besides, LINC00665 promoted the proliferation, migration and invasion of CRC cells, and inhibited cell apoptosis by sponging miR-9-5p. ATF1 was proved to be the downstream target of miR-9-5p and was indirectly regulated by LINC00665. Collectively, it is concluded that LINC00665 contributes to the progression of CRC by regulating miR-9-5p/ATF1 axis.

Exosomes have received increasingly significant attention and have shown great clinical value as biomarkers for a number of diseases. However, there is still a lack of a highly sensitive and visualized method for the detection of exosomes in numerous samples simultaneously. Here, we developed a high-throughput, colorimetric and simple method to detect colorectal cancer (CRC) exosomes based on terminal deoxynucleotidyl transferase (TdT)-aided ultraviolet signal amplification. Anti-A33, a CRC exosomal protein marker, was selected as a capture probe, and a facility-prepared EpCAM (CRC exosomal protein) aptamer-Au-primer complex was used as a signal probe. After the CRC exosomes were captured onto the surface of 96-well plates, the primer was extended to the poly(biotin-adenine) chains with the help of TdT, resulting in an increase in the binding amount of avidin-modified horseradish peroxidase (Av-HRP) for H2O2-mediated oxidation of 3,3′,5,5′-tetramethyl benzidine (TMB) in enzyme-linked aptamer-sorbent assay (ELASA). The results showed that the incorporation of ploy(biotin-A) enabled approximately 10.4-fold signal amplification. This approach achieved a linear range of 9.75 × 103–1.95 × 106 particles/μL for CRC cell-derived exosomes. The feasibility of the developed assay was evaluated using clinical serum samples. CRC patients (n = 16) could be clearly and successfully distinguished from healthy individuals (n = 9). Furthermore, this proposed platform holds considerable potential for the detection of different targets, simply by changing the aptamer and antibody

Background/Aims: Recent studies have demonstrated that the manipulation of the gut microbiome represents a promising treatment for inflammatory bowel disease (IBD). We previously identified micro integral membrane protein (MIMP) as the smallest domain of surface layer protein from Lactobacillus Plantarum. However, the therapeutic relevance of MIMP in IBD remains unknown. Methods: We initially employed a dextran sodium sulphate (DSS)-induced colitis model and evaluated the effect of MIMP on the inflammation response, intestinal barrier and gut microbiota using histological examination, Fluorescein isothiocyanate-Dextran detection and pyrosequencing analysis respectively. We then established peripheral blood mononuclear cells (PBMCs) and an epithelial CaCO-2 co-culture model to investigate the regulatory role of MIMP in inflammatory cytokines. The level changes of inflammatory cytokines were detected using Enzyme-linked immunosorbent and real-time polymerase chain reaction assay. The involved regulatory mechanisms were investigated mainly using dual luciferase reporter and chromatin immunoprecipitation assay. Results: In the DSS-induced colitis model, we observed that MIMP intervention effectively improved the body weight loss, increased the colon length and decreased disease activity index. Consistently, the inflammation scores in the MIMP treatment group were significantly lower than those in the DSS treatment group. Furthermore, MIMP intervention was found to successfully neutralize DSS treatment by decreasing the expression of pro-inflammatory cytokines (IFN-γ, IL-17 and IL-23) and increasing the expression of anti-inflammatory cytokines (IL-4 and IL-10). Notably, the permeability assay demonstrated that the MIMP treatment group was remarkably lower than that in the DSS treatment group. We also showed that MIMP improved gut microbiota dysbiosis caused by DSS-induced inflammation. Additionally, in PBMCs and the CaCO-2 co-culture model, MIMP showed an obvious suppressive effect on lipopolysaccharide-induced inflammation in a time- and dose-dependent manner. Furthermore, we revealed that MIMP could modulate inflammatory cytokine expression through the toll-like receptor 4 pathway and histone acetylation. Conclusions: Our results suggested that MIMP showed a significant anti-inflammatory effect through regulating the gut barrier, microbiota and inflammatory cytokines. MIMP may have translational relevance as clinically relevant therapy for IBD patients.

Background Emerging evidence suggests that PIWI-interacting RNAs (piRNAs) may be important epigenetic regulators of gene expression in human cancers; however, their functional and clinical significance in colorectal cancer (CRC) remains unknown. Methods We performed piRNA expression profiling in paired cancer and normal tissues through small RNA-sequencing. The clinical significance of candidate piRNAs was investigated, and independently validated in 771 CRC patients from three independent cohorts. The biological function of piRNAs was characterized in cell lines, followed by identification and validation of downstream target genes in CRC tissues. Results We identified piR-1245 as a novel and frequently overexpressed noncoding RNA in CRC, and its expression significantly correlated with advanced and metastatic disease. Patients with high piR-1245 expression experienced significantly shorter overall survival, and multivariate analysis identified its expression to serve as an independent prognostic biomarker in CRC. Functionally, piR-1245 acts as an oncogene and promotes tumor progression, and gene expression profiling results identified a panel of downstream target-genes involved in regulating cell survival pathway. Based upon piRNA:mRNA sequence complementarity, we identified a panel of tumor suppressor genes (ATF3, BTG1, DUSP1, FAS,NFKBIA, UPP1, SESN2, TP53INP1 and MDX1) as direct targets of piR-1245, and successfully validated an inverse correlation between their expression and piR-1245 in CRC. Conclusions We for the first time have identified the role for a PIWI-interacting noncoding RNA, piR-1245, as a novel oncogene and a potential prognostic biomarker in colorectal cancer. Electronic supplementary material The online version of this article (10.1186/s12943-018-0767-3) contains supplementary material, which is available to authorized users.

Background Emerging evidence indicates that small nucleolar RNAs (snoRNAs) play a central role in oncogenesis. Herein, we systematically evaluated expression profiles of snoRNAs in colorectal cancer (CRC) and investigated their clinical and functional role in this malignancy. Methods We compared expression levels of snoRNAs between cancer and normal tissues using publicly available datasets and identified the most differentially expressed and commonly upregulated snoRNAs in CRC. These results were examined in 489 colorectal tissues to assess their clinical significance, followed by a series of in vitro and in vivo experiments to evaluate the functional role of candidate snoRNAs. Results Using multiple RNA profiling datasets, we identified consistent overexpression of SNORA21 in CRC. In the clinical validation cohorts, the expression level of SNORA21 was upregulated in colorectal adenomas and cancers. Furthermore, elevated SNORA21 emerged as an independent factor for predicting poor survival. Both in vitro and in vivo experiments revealed that CRISPR/Cas9-mediated inhibition of SNORA21 expression resulted in decreased cell proliferation and invasion through modulation of multiple cancer related pathways. Conclusions We systematically identified SNORA21 as a key oncogenic snoRNA in CRC, which plays an important role in cancer progression, and might serve as an important prognostic biomarker in CRC., Highlights • Systematic and comprehensive analysis revealed that SNORA21 was significantly upregulated in CRC. • Elevated SNONA21 expression was related to metastasis and predicted poor prognosis in CRC patients. • SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC. Emerging evidence indicates that small nucleolar RNAs (snoRNAs) might play a central role in oncogenesis; however, the clinical significance and functional roles of snoRNAs in colorectal cancer (CRC) remain unclear. Herein, using multiple publicly available datasets, we systematically assessed expression profiles of snoRNAs in CRC and elucidated that the high expression of SNORA21 was associated with distant metastasis and poor overall survival. A series of in vitro and in vivo experiments revealed oncogenic role for SNORA21. Our findings indicate that SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC.

Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients. Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings. Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae (P = 0.0018), and its overexpression was associated with poor patient survival (P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918–28. ©2017 AACR.

Albumin is a significant prognostic marker in diabetic nephropathy and cardiovascular disease. In this work, we developed a rapid, sensitive and inexpensive method for albumin detection based on the polydopamine (PDA) bi-functionalized glass chip and surface-enhanced Raman scattering (SERS) tag. Anti-albumin antibodies were encapsulated on the glass chip and SERS tag through dopamine self-polymerization in alkaline conditions and the immunoaffinity was highly maintained to form the sandwich structure with the addition of target microalbuminuria. Under the optimum experimental conditions, this SERS immunoassay showed the wide linear range of 10–300 mg/L with the limit of detection of 0.2 mg/L. High specificity and selectivity of this SERS method were demonstrated by performing with some common proteins include human hemoglobin protein (Hgb) and human immunoglobulin G (IgG) in urine. This method was also applied to detect albumin in urine samples from 17 patients and 5 healthy control and the results were highly consistent with those obtained from clinical standard immunoturbidimetric method. The volume requirement of urine samples is only 2 μL. Compared with traditional dipstick and immunoturbidimetric methods used in clinic, SERS-based method has higher accuracy, lower sensitivity and less sample consumption.

Figure S1. The peak map of DNA sequencing about the constructions of wild-type and mutant BIRC3 promoters. (PDF 712 kb)

Piwi-interacting RNAs (piRNAs) are a very recently discovered class of small non-coding RNAs (ncRNAs), with approximately 20,000 piRNA genes already identified within the human genome. These short RNAs were originally described as key functional regulators for the germline maintenance and transposon silencing. However, due to our limited knowledge regarding their function, piRNAs were for a long time assumed to be the "dark matter" of ncRNAs in our genome. However, recent evidence has now changed our viewpoint of their biological and clinical significance in various diseases, as newly emerging data reveals that aberrant expression of piRNAs is a unique and distinct feature in many diseases, including multiple human cancers. Furthermore, their altered expression in cancer patients has been significantly associated with clinical outcomes, highlighting their important biological functional role in disease progression. Functionally, piRNAs maintain genomic integrity by silencing transposable elements, and are capable of regulating the expression of specific downstream target genes in a post-transcriptional manner. Moreover, accumulating evidences demonstrates that analogous to other small ncRNAs (e.g. miRNAs) piRNAs have both oncogenic and tumor suppressive roles in cancer development. In this article, we discuss emerging insights into roles of piRNAs in a variety of cancers, reveal new findings underpinning various mechanisms of piRNAs-mediated gene regulation, and highlight their potential clinical significance in the management of cancer patients.

ABSTRACT Colorectal carcinogenesis represents a sequential progression of normal colonic mucosa from adenoma to carcinoma. It has become apparent that miRNA deregulation contributes to the initiation and progression of colorectal cancer (CRC). These oncogenic or tumor-suppressive miRNAs interact with intracellular signaling networks and lead to alteration of cell proliferation, apoptosis, metastasis and even response to chemotherapeutic treatments. This article aims to review the cutting edge progress in the discovery of the role of novel mechanisms for miRNAs in the development of CRC. We will also discuss the potential use of miRNAs as biomarkers for early diagnosis and prognosis of CRC. Furthermore, with advancements in RNA delivery technology, it is anticipated that manipulation of miRNAs may offer an alternative therapy for CRC treatment.

Background We evaluated the diagnostic value of serum-CD166 in patients with hepatocellular carcinoma (HCC). Methods Tissue-CD166 was measured using immunohistochemistry. Cell proliferation and migration were evaluated using MTT and Transwell assays, respectively. Serum-CD166 was examined using ELISA and western blotting. Results CD166 was up-regulated in HCC compared to those in normal liver tissues. Cell proliferation was positively correlated and cell migration was negatively correlated with endogenous CD166 expression in HCC cells. CD166 inhibition using specific shRNA decreased cell proliferation but increased cell migration. Serum CD166 concentrations were much higher in HCC than in colon cancer, hepatitis B, hepatitis C, cirrhosis, gastric cancer, breast cancer, lung cancer and healthy individuals. Serum CD166 also decreased dramatically after curative surgery. A positive correlation was found between serum CD166 and AFP (R = 0.7141, p = 0.000). Serum CD166 was also positively correlated with γ-GT, bile acid, ALT, AST, and ALP but was negatively correlated with Alb and pre-Alb. The area under the receiver operating characteristic curve for serum-CD166 was 0.9860, which was better than AFP (AUC-ROC, 0.9354) for the differentiation of HCC patients from healthy individuals, with a cut-off of 261 ng/ml (sensitivity: 100.00%, specificity: 89.41%). Conclusion Serum CD166 is a novel diagnostic tumor marker for HCC.

Emerging studies have revealed that Malat1 is overexpressed in many malignant diseases, including liver cancer, and contributes to enhancing cell migration or facilitating proliferation. However, the mechanism underlying its regulation has largely remained elusive. Here, we characterised the oncoprotein Yes-associated protein (YAP), which up-regulated metastasis-associated lung adenocarcinoma transcript 1 (Malat1) expression at both transcriptional and post-transcriptional levels, whereas serine/arginine-rich splicing factor 1 (SRSF1) played an opposing role. SRSF1 inhibited YAP activity by preventing its co-occupation with TCF/β-catenin on the Malat1 promoter. In contrast, overexpression of YAP impaired the nuclear retention of both SRSF1 and itself via an interaction with Angiomotin (AMOT). This effect removed the inhibitory role of SRSF1 on Malat1 in the nucleus. Furthermore, higher expression of YAP was consistent with a lower SRSF1 nuclear accumulation in human liver cancer tissues. We also revealed that overexpression of YAP combined with a knockdown of SRSF1 resulted in conspicuously enhanced transwell cell mobility, accelerated tumour growth rate, and loss of body weight in a tail vein-injected mouse models. Taken together, these data provided a novel mechanism underlying the balance between SRSF1, YAP and Malat1 and uncovered a new role of YAP in regulating long non-coding RNA (lncRNA). Thus, disrupting the interaction between YAP and SRSF1 may serve as a crucial therapeutic method in liver cancer.

Renal cell carcinoma (RCC) is a common urological malignancy. It remains unclear, however, whether Yin Yang 1 (YY1) plays a functional role in the development of human RCC. In the present study, we demonstrated that levels of YY1 were significantly increased in primary RCC tissues when compared to these levels in the matched healthy tissues. YY1 knockdown inhibited cell growth, migration and invasion of RCC cells. Additionally, we highlighted a positive feedback-loop pathway resulting in YY1 upregulation. We observed that overexpression of YY1 caused repression of C/EBPα and the inhibition of C/EBPα led to the suppression of miR-34a. Since YY1 is a direct target of miR-34a, the low level of miR-34a increased the expression of YY1, promoting the aggressiveness of RCC cells. Furthermore, this feedforward mechanism was found in RCC tissues. We observed that miR-34a was downregulated in the pools of cancer tissues when compared to that of the normal tissues. The expression of miR-34a displayed an inverse correlation with YY1, but a positive correlation with C/EBPα. In conclusion, our study highlights the importance of a novel regulatory circuitry for YY1 activation in maintaining the aggressive phenotypes of RCC.

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. The increased incidence rates of CRC places a considerable burden on individuals and society in Asian countries. Although surgical resection remains the standard treatment for CRC patients, accumulating evidences showed advanced patients derived an additional benefit from molecular targeted agents. Therefore, there is a great need to identify novel cellular targets in CRC that can be exploited for therapeutic benefit. Trefoil factor 3 (TFF3), is a member of a family of small secretory proteins characterized by three intrachain disulfide bonds constituting the trefoil motif. TFF3 was initially found as a mediator of mucosal healing and play a critical role in epithelial restitution. Recent studies showed that TFF3 may contribute to tumorigenesis. However, its role in CRC remains unclear. Herein, the protein expression of TFF3 was measured in colorectal adenoma polyps (PLP, n=20), cancer tissues (n=20) and matched adjacent healthy tissues (n=20) by immunohistochemical (IHC) assay. We further evaluated serum and urine level of TFF3 from healthy volunteers (n=40), PLP patients (n=40) and CRC patients (n=40) by using ELISA assay. To investigate the functional or mechanistic role of TFF3 in CRC, we conducted several in vivo and in vitro approaches. The IHC showed that TFF3 was frequently overexpressed in cancer tissues (12/20, P Note: This abstract was not presented at the meeting. Citation Format: Ying Long, Tsuyoshi Ozawa, Hanhua Li, Weijie Pan, Wenhao Weng. Novel evidence for Trefoil Factor 3 as an oncogenic mediator of disease progression, and a potential predictor for neoadjuvant chemotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3161.

Mouse embryonic fibroblasts (MEFs) differentiate into fully functional chondrocytes in response to bone morphogenetic protein-2 (BMP-2). However, the comprehensive proteomic aspect of BMP-2-induced chondrogenesis remains unknown. We took advantage of quantitative proteomic analysis based on isobaric tag for relative and absolute quantitation (iTRAQ) and on-line 2D nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify proteins differentially expressed during BMP-2-induced chondrogenic differentiation of MEFs. We found 85 downregulated proteins, and ingenuity pathways analysis (IPA) revealed a protein-protein network with chromodomain-helicase-DNA-binding protein 4 (Chd4) in the center. Chromatin immunoprecipitation (ChIP) and nuclease hypersensitivity assays showed that Chd4, interacting with Hdac1/2, cooperates with its related proteins Kap1 and Cbx1 to bind at -207/-148 of the Sox9 promoter. We also provided evidence that let-7a targets the 3'UTR of Chd4 to promote chondrogenesis of MEFs. Together, our findings indicate that BMP-2 induced the upregulation of let-7a, targeting Chd4 and positively controlling the chondrogenic differentiation of MEFs. These findings illustrate epigenetic regulation of the chondrogenic differentiation process and also expand the understanding of the involved intracellular mechanisms.

Yes-associated protein (YAP), the downstream effecter of the Hippo-signaling pathway as well as cyclic adenosine monophosphate response element-binding protein (CREB), has been linked to hepatocarcinogenesis. However, little is known about whether and how YAP and CREB interact with each other. In this study, we found that YAP-CREB interaction is critical for liver cancer cell survival and maintenance of transformative phenotypes, both in vitro and in vivo. Moreover, both CREB and YAP proteins are highly expressed in a subset of human liver cancer samples and are closely correlated. Mechanistically, CREB promotes YAP transcriptional output through binding to −608/−439, a novel region from the YAP promoter. By contrast, YAP promotes protein stabilization of CREB through interaction with mitogen-activated protein kinase 14 (MAPK14/p38) and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC). Gain-of-function and loss-of-function studies demonstrated that phosphorylation of CREB by MAPK14/p38 at ser133 ultimately leads to its degradation. Such effects can be enhanced by BTRC through phosphorylation of MAPK14/p38 at Thr180/Tyr182. However, YAP negatively controls phosphorylation of MAPK14/p38 through inhibition of BTRC expression. Conclusion: There is a novel positive autoregulatory feedback loop underlying the interaction between YAP and CREB in liver cancer, suggesting that YAP and CREB form a nexus to integrate the protein kinase A, Hippo/YAP, and MAPK14/p38 pathways in cancer cells and thus may be helpful in the development of effective diagnosis and treatment strategies against liver cancer. (Hepatology 2013;53:1011–1020)

Background & Aims Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice. Methods Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate-buffered saline (PBS [control]) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL adenomatous polyposis coli min/+ , C57BL miR21a −/− , and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane and dextran sodium sulfate to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by enzyme-linked immunosorbent assay. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. Fusobacterium nucleatum DNA in 90 tumor and matched nontumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times. Results Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. Adenomatous polyposis coli min/+ mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F, interleukin 21, and interleukin 22, and MIP3A). We found 50 miRNAs to be significantly up-regulated and 52 miRNAs to be significantly down-regulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (>4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a −/− mice had a later appearance of fecal blood and diarrhea after administration of azoxymethane and dextran sodium sulfate, and had longer survival times compared with control mice. The colorectum of miR21a −/− mice had fewer tumors, of smaller size, and the miR21a −/− mice survived longer than control mice. We found RASA1 , which encodes an RAS GTPase, to be one of the target genes consistently down-regulated in cells that overexpressed miR21 and up-regulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor−κB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues) compared with non-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 had shorter survival times than patients whose tumors had lower amounts. Conclusions We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. Fusobacterium nucleatum activates Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor−κB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes.

Purpose: Colorectal cancer ranks as the third most frequent cancer type, and its incidence continues to rise gradually worldwide, highlighting the need to identify previously unrecognized molecular events that propel development of this malignancy. Recent evidence suggests that dysregulated expression of FOX family of transcription factors may be critical in various genetic disorders as well as cancer; however, the functional and clinical significance of this pathway in colorectal cancer remains unclear. Experimental Design and Results: Herein, we performed a systematic and comprehensive discovery step by evaluating the expression of FOX family members, and identified that FOXM1 and FOXQ1 are frequently overexpressed in colorectal cancer. We subsequently confirmed these findings in two large testing cohorts (n = 550) and an independent clinical validation cohort (n = 134), in which high expression of FOXM1 and FOXQ1 emerged as an independent prognostic factor in colorectal cancer patients. We corroborated these findings by performing functional assays in which knockdown of FOXM1 and FOXQ1 resulted in inhibited cell proliferation and suppressed migration and invasion in colorectal cancer cells. Furthermore, using bioinformatic approaches, we identified miR-342 as a novel regulator of both FOXM1 and FOXQ1. Overexpression or inhibition of miR-342 modulated the expression of both genes and contributed to phenotypic alterations in colorectal cancer cells, which was subsequently validated in a xenograft animal model. Conclusions: Collectively, we have firstly identified FOXM1 and FOXQ1 as promising prognostic biomarkers in colorectal cancer patients, and provided novel evidence that therapeutic targeting of these genes or miR-342 may be a potential treatment approach in colorectal cancer patients. Clin Cancer Res; 22(19); 4947–57. ©2016 AACR.

Purpose: Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. But the process is still unknown. Here, we analyzed genetic differences between primary tumors associated with liver metastasis and those without liver metastasis and predicted clinical significance. Experimental design: Eight primary tumors associated with liver metastasis and twelve without liver metastasis of CRC were analyzed by whole-exome sequencing. The functional characterisation for the role of metastatic-related genes was investigated through in vitro migration and invasion assays. For clinical relevance, TMAs were used for immunohistochemistry assay. Results: The mutation spectrum was similar to that of previous studies. TP53, APC and KRAS were the three most frequently mutated genes, with no significant difference of mutation frequency between primary tumors from patients with liver metastasis and those from other patients. Notably, four recurrently mutated genes (FBXW7, EDIL3, KCNK9 and PCDHGA7) were identified exclusively in the tumors associated with liver metastasis. Furthermore, in vitro analyses showed that EDIL3 and KCNK9 overexpression resulted in enhanced cell migration and invasion, while CRISPR-mediated deletion of FBXW7 and PCDHGA7 exhibited enhanced migratory and invasive properties as well. For clinical relevance, highly expressed EDIL3 and KCNK9 was significantly associated with liver metastasis, whereas the expression of FBXW7 and PCDHGA7 negatively correlated with it in our cohorts. Pathway enrichment analysis also indicated a role for cell adhesion signaling in liver metastasis of CRC patients. Conclusion: Our study identified candidate genes, which might response to the initiation of metastasis, exclusively mutated in tumors associated with liver metastasis. These findings also provided potential therapeutic targets for personalized treatment. Note: This abstract was not presented at the meeting. Citation Format: Yanlei Ma, Wenhao Weng, Yongzhi Yang, Qing Wei, Sanjun Cai. Whole exome sequencing analyses of colorectal cancers associated with liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2711. doi:10.1158/1538-7445.AM2017-2711

The association between B-cell lymphoma 2 (Bcl-2) polymorphism and cancer is under debate and remains elusive. This meta-analysis was performed to evaluate the relationships of Bcl-2 -938 C>A polymorphism (rs2279115) with susceptibility and prognosis of cancer. Odds ratios (ORs) were used to measure the association between Bcl-2 polymorphisms and cancer risk. Hazard ratios (HRs) were used to measure the association between Bcl-2 polymorphisms and cancer prognosis. On the basis of 26 studies about Bcl-2 -938C>A polymorphism and cancer, we found Bcl-2 -938 C>A polymorphism was significantly associated with increased cancer risk in dominant model (OR = 1.12, 95%CI: 1.00–1.25, P = 0.04), recessive model (OR = 1.38, 95%CI: 1.11–1.71, P = 0.004), allelic model (OR = 1.15, 95%CI: 1.04–1.28, P = 0.007) and homozygote comparison(OR = 1.44, 95%CI: 1.11–1.87, P = 0.006). Furthermore, Bcl-2 -938 C>A polymorphism was significantly associated with increased cancer risk in Asians but not in Caucasians. Moreover, Bcl-2 -938 C>A polymorphism was not significantly associated with the prognosis of cancer (AA vs CA: OR = 0.99, 95%CI: 0.77–1.27, P = 0.93; AA vs CC: OR = 0.92, 95%CI: 0.65–1.30, P = 0.63; AC vs CC: OR = 0.94, 95%CI: 0.80–1.11, P = 0.48; CC vs AA+CA: OR = 1.21, 95%CI: 0.69–2.13, P = 0.50; AA vs CC+CA: OR = 0.99, 95%CI: 0.48–2.04, P = 0.97). Studies with larger samples and gene-environment interactions are needed to validate our findings.