Serum Of Colorectal Cancer Patients Research Articles

Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level.

Long non-coding RNA (LncRNA) telomerase RNA component (TERC) has telomerase-dependent and independent activity in numerous cancer types. The present study purposes to demonstrate the role of lncRNA TERC as a diagnostic serum biomarker in colorectal cancer (CRC) patients and the molecular mechanism of lncRNA TERC in inducing tumor in CRC cell lines. PCR array was performed to examine lncRNAs dysregulated in CRC. LncRNA TERC expression level was evaluated in 70 CRC patients and 35 control subjects using RT-qPCR. Then transfection was performed to build down-expression models of lncRNA TERC. ROC curve analysis was applied to assess the diagnostic value of serum LncRNA CRC. In addition, RT-qPCR was used to detect expression level of lncRNA TERC and β-catenin mRNA. Moreover, ELISA and Western blot were used to detect the level of β-catenin protein in sera of CRC patients and cell lines. The biological functions such as cell growth and migration of CRC cells were assessed using a wound healing assay. Cell cycle analysis and apoptosis analysis were performed using flow cytometry. The lncRNA TERC is overexpressed in the sera of CRC patients with high diagnostic and stage discrimination accuracy. Furthermore, lncRNA TERC expression was upregulated in CRC cell lines and lncRNA TERC silencing induced cell arrest and apoptosis and inhibited cell migration. Furthermore, inhibition of lncRNA TERC reduces β-catenin protein levels. The lncRNA TERC could be considered as an early stages CRC diagnostic biomarker with a good ability to discriminate between CRC stages. lncRNA TERC induces CRC by promoting cell migration and evading apoptosis by elevating the level of β-catenin protein.

Fusobacterium nucleatum promotes colorectal cancer metastasis by excretion of miR-122-5p from cells via exosomes

Fusobacterium nucleatum (Fn) infection and microRNAs (miRNAs) are closely associated with colorectal cancer (CRC) development, but the mechanism by which Fn regulates tumor-suppressive miRNAs via exosomes and facilitates CRC metastasis remains unclear. Here, we identified that Fn infection significantly increased exosomal miR-122-5p levels in the serum of CRC patients and CRC cell culture supernatants through two miRNA panels of high-throughput sequencing and RT-qPCR analysis. In Fn-infected patients, the serum exosomal levels of miR-122-5p were negatively associated with their expression levels of tissues. Downregulated miR-122-5p was demonstrated to enhance the migration, invasion, and metastasis abilities of CRC cells invivo and invitro. Secretion of miR-122-5p into exosomes is mediated by hnRNPA2B1. Mechanistically, Fn activated the TGF-β1/Smads signaling pathway to promote EMT by regulation of the miR-122-5p/FUT8 axis. In conclusion, Fn infection may stimulate CRC cells to excrete exosome-wrapped miR-122-5p, and activate the FUT8/TGF-β1/Smads axis to promote metastasis.

Isobutyric acid promotes colorectal cancer metastasis through activating RACK1.

Colorectal cancer (CRC) metastasis plays a crucial role in disease progression, yet the regulatory mechanisms underlying metastasis remain incompletely understood. Isobutyric acid (IBA), a short-chain fatty acid found at high levels in serum of CRC patients, has been shown to be a critical metabolite influencing CRC proliferation. However, its role in tumor metastasis remains unknown. Here, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, we found that levels of IBA were significantly higher in patients with distant organ metastasis of CRC than in those without. Furthermore, IBA promoted CRC metastasis both in vitro and in vivo. Mass spectrometry, immunofluorescence, and cellular thermal shift assay revealed that IBA interacts with RACK1. Mechanistically, IBA binding to and activating RACK1 promotes regulation of downstream Akt and FAK signaling and CRC metastasis. Collectively, our study highlights the critical interplay between IBA and RACK1 and its impact on tumor metastasis. This study suggests that targeting the IBA-RACK1 signaling axis may be an effective therapeutic strategy for controlling CRC metastasis.

Clinical Significance of MMP7 Levels in Colorectal Cancer Patients Receiving FOLFOX4 Chemotherapy Treatment.

Various studies have shown an association between the anti-cancer drug 5-fluorouracil and matrix metalloproteinase 7 (MMP7). The expression of MMP7 in the serum of colorectal cancer patients, as well as their sensitivity to chemotherapy, were examined using the FOLFOX4 chemotherapy treatment. Serum samples were taken from 216 colorectal cancer patients who had undergone four cycles of gemcitabine and cisplatin treatment. The sera of 216 healthy persons were used as controls. MMP7 levels in the serum were measured by ELISA. Demographic and survival data were collected. MMP7 levels were not associated with sex, age, peritoneal dissemination, liver metastasis, lymph node metastasis, lymphatic invasion, or venous invasion in CRC patients, but were associated with histological grade, tumor size, TNM stage, and depth of tumor invasion. Patients' serum MMP7 expression reduced after treatment. MMP7 expression was significantly lower chemotherapy-sensitive patients compared with chemotherapy-resistant patients. Elevated MMP7 expression was associated with worse prognosis and chemotherapy-sensitive patients had markedly better overall survival compared with chemotherapy-resistant patients. MMP7 expression was potentially associated with the development of colorectal cancer and elevated levels were associated with chemoresistance in CRC patients. Serum MMP7 levels can be used to screen for drug resistance during FOLFOX4 chemotherapy treatment.

Serum N-Glycosylation RPLC-FD-MS Assay to Assess Colorectal Cancer Surgical Interventions.

A newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography-fluorescence detection-mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored.

IL-13/IL-13RA2 signaling promotes colorectal cancer stem cell tumorigenesis by inducing ubiquitinated degradation of p53

Cancer stem cells (CSCs) are considered tumor-initiating cells and the main drivers of disease progression. Targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the up-regulation of IL-13RA2 expression in colorectal cancer (CRC) tissues and spheroid cells. The expression of IL-13RA2 was positively correlated with canonical stemness markers in CRC. We further demonstrated that the level of IL-13 was up-regulated in the serum of CRC patients. Biologically, recombinant IL-13 (rIL-13) stimulation promoted the sphere formation, proliferation, and migration of CRC cells in vitro and enhanced tumorigenesis in vivo. This phenotype could be reversed by knocking down IL-13RA2. Mechanistically, IL-13 activated autophagy by inducing LC3I/LC3II transformation in CRC-CSCs, which was crucial for the biological functions of IL-13. We further demonstrated that IL-13RA2 acted as a modular link of the E3 ligase UBE3C and the substrate p53 protein, enhancing the interaction of UBE3C and p53, thereby inducing the K48-linked ubiquitination of p53. In conclusion, the IL-13/IL-13RA2 signaling cascade promotes CRC-CSC self-renewal and tumorigenesis by inducing p53 ubiquitination, adding an important layer to the connection between IL-13 and p53, which can be translated into novel targeted therapies.

Abstract 6709: Utility of RNA sequencing for transcriptome analysis of small extracellular vesicles derived from blood sera of colorectal cancer patients

Abstract Introduction: Currently used molecular diagnostic tests for colorectal cancer (CRC) are underperforming and more sensitive, non-invasive biomarkers are needed. Long non-coding RNAs (lncRNA) and microRNA (miRNA) have shown potential as diagnostic biomarkers. Unfortunately, the identification of non-coding RNA circulating biomarkers in blood serum is significantly burdened by abundant RNA specimens from disrupted blood cells. Recently, small extracellular vesicles (sEVs) emerged as potential reservoirs of clinically relevant biomarkers, including lncRNAs and miRNAs. In theory, sEVs protect RNAs from degradation and might serve as a source of intact RNA for further analyses. However, there is a lack of evidence supporting the superior quality of RNA extracted from sEVs over RNA from whole blood serum. This study aimed to analyze the RNA content of blood serum and the sEVs derived from the blood serum of CRC patients and healthy controls using RNA sequencing. Moreover, small RNA sequencing was used to evaluate the difference in miRNA profiles of sEVs and corresponding blood sera of CRC patients and healthy controls. Methods: Spin-column chromatography (Exiqon), precipitation-based method (Norgen), and size-exclusion chromatography (iZON) were used to extract sEVs from blood sera. The concentration of sEVs was measured by dynamic light scattering (DLS), the size was evaluated by electron microscopy (EM), and sEV-specific content was analyzed by western blot and qRT-PCR. RNA was extracted using the column-based method. Next-generation sequencing (NGS) analyses of blood serum and sEVs extracted from blood serum included samples from 10 CRC patients and 10 healthy controls for RNAseq, and 5 CRC patients and 5 healthy controls for small RNAseq. Differential expression analysis was carried out in R using DESeq2 package. Results: DLS and EM showed that size-exclusion chromatography yielded the purest population of sEVs characterized according to ISEV recommendations. Extraction of sEVs and subsequent RNA extraction and sequencing library preparation from ultra-low input samples were optimized. Over 30k different RNAs were identified in the sEVs derived from blood sera of CRC patients and healthy controls, including lncRNAs, miRNAs, and protein-coding RNAs. A detailed comparison of the transcriptome of blood sera and corresponding sEVs is a part of the poster. Conclusion: sEVs could serve as a source of RNA biomarkers; however, proper characterization and optimal methodology are necessary. This work was supported by the Ministry of Health of the Czech Republic grant No. NU20-03-00127, by The project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU, by the project BBMRI-CZ, nr. LM2018125, and in co-operation with CEMCOF, CEITEC MU (CIISB) supported by MEYS CR, LM2018127. Citation Format: Tana Machackova, Petra Vychytilova-Faltejskova, Marie Madrzyk, Karolina Trachtova, Marketa Pavlikova, Jan Kotoucek, Jana Halamkova, Dagmar Al Tukmachi, Jiri Sana, Petra Pokorna, Milana Sachlova, Ondrej Slaby. Utility of RNA sequencing for transcriptome analysis of small extracellular vesicles derived from blood sera of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6709.

Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies (C3Mab-6 and C3Mab-7).

One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15 and Glu16 are essential for C3Mab-7 binding.

Exosomal Circ_FMN2 Derived from the Serum of Colorectal Cancer Patients Promotes Cancer Progression by miR-338-3p/MSI1 Axis.

Colorectal cancer (CRC) is a common malignancy of the gastrointestinal tract with high incidence and mortality. Exosomal circular RNA (circRNA) has been shown to be associated with the malignant progression of cancers, including CRC. Circ_0005100 (named as circ_FMN2) has been shown to promote CRC cell proliferation and migration. However, whether exosomal circ_FMN2 participated in CRC progression remains unclear. Exosomes were isolated from the serum of CRC patients and then identified using transmission electron microscope. Western blot assay was used to test the protein levels of exosome markers, proliferation-related marker, metastasis-related markers and musashi-1 (MSI1). The expression levels of circ_FMN2, microRNA (miR)-338-3p and MSI1 were detected by qPCR. Flow cytometry, colony formation assay, MTT assay, and transwell assay were employed to measure cell cycle, apoptosis, colony formation ability, viability, migration and invasion. Dual-luciferase reporter assay was performed to assess the interaction between miR-338-3p and circ_FMN2 or MSI1. BALB/c nude mice was used to conduct animal experiments. Circ_FMN2 was overexpressed in the exosomes of CRC patient's serums and CRC cells. Overexpressed exosomal circ_FMN2 could promote CRC cell proliferation, metastasis, and suppress apoptosis. Circ_FMN2 acted as miR-338-3p sponge. MiR-338-3p overexpression reversed the promotion effect of circ_FMN2 on CRC progression. MSI1 was found to be a target of miR-338-3p, and its overexpression revoked the inhibitory effect of miR-338-3p on CRC progression. Furthermore, exosomal circ_FMN2 overexpression also could facilitate CRC tumor growth in vivo. Exosomal circ_FMN2 accelerated CRC progression through miR-338-3p/MSI1 axis, revealing that exosomal circ_FMN2 might be a target for CRC treatment.

Integrated analysis of serum untargeted metabolomics and targeted bile acid metabolomics for identification of diagnostic biomarkers for colorectal cancer

To identify potential diagnostic biomarkers of colorectal cancer (CRC) using serum metabolomic technology for minimally invasive and efficient screening for CRC. Serum samples from 79 healthy individuals and 82 CRC patients were analyzed by metabolomics using ultra-high-performance liquid chromatography-tandem highresolution mass spectrometry (UHPLC-HRMS). The differential metabolites between the two groups were analyzed using principal component analysis and orthogonal partial least squares discriminant analysis (OPLS-DA). Receiver operating characteristic curve (ROC) analysis was performed to identify the differential metabolites with good diagnostic performance (AUC>0.80) for CRC, and targeted bile acid metabolomics was used to verify the selected bile acids as biomarkers. Serum metabolic profiles differed significantly between the healthy individuals and CRC patients, and a total of 82 differential metabolites (mostly fatty acids and glycerophospholipids) were selected. ROC analysis identified 10 differential metabolites, including adenine, bilirubin, ACar 12:0, ACar 10:1, ACar 9:0, PC 18:2e, deoxycholic acid, chenodeoxycholic acid, ACar 14:1 and palmitoylcarnitine. One of these metabolites was significantly up-regulated and 9 were down-regulated in the serum of CRC patients (P < 0.05). Multivariate ROC analysis with support vector machine algorithm showed that the biomarker panel consisting of 7 differential metabolites had an AUC of 0.94 for CRC diagnosis. The results of targeted bile acid metabolomics were consistent with those of untargeted metabolomics. The serum levels of deoxycholic acid and chenodeoxycholic acid were significantly down-regulated in patients with CRC as compared with the healthy individuals (P < 0.05). Metabolic disorders of fatty acids and glycerophospholipids are closely related wigh tumorigenesis of CRC. Ten differential metabolites show good performance for CRC diagnosis, and the panel consisting 7 of these metabolites has important diagnostic value for CRC. Deoxycholic acid and chenodeoxycholic acid may serve as potential diagnostic biomarkers of CRC.

Exosomal circTUBGCP4 promotes vascular endothelial cell tipping and colorectal cancer metastasis by activating Akt signaling pathway

BackgroundExosome is crucial mediator and play an important role in tumor angiogenesis. Tip cell formation is a prerequisite for persistent tumor angiogenesis which causes tumor metastasis. However, the functions and underlying mechanisms of tumor cell-derived exosomes in angiogenesis and tip cell formation remain less understood.MethodsExosomes derived from serum of colorectal cancer (CRC) patients with metastasis/non-metastasis and CRC cells were isolated by ultracentrifugation. CircRNAs in these exosomes were analyzed by circRNA microarray. Then, exosomal circTUBGCP4 was identified and verified by quantitative real-time PCR (qRT–PCR) and in situ hybridization (ISH). Loss- and gain-of-function assays were performed to explore the effect of exosomal circTUBGCP4 on vascular endothelial cell tipping and colorectal cancer metastasis in vitro and in vivo. Mechanically, bioinformatics analysis, biotin-labeled circTUBGCP4/ miR-146b-3p RNA pulldown, RNA immunoprecipitation (RIP), and luciferase reporter assay were used to confirm the interaction among circTUBGCP4, miR-146b-3p, and PDK2.ResultsHere, we showed that exosomes derived from CRC cells enhanced vascular endothelial cell migration and tube formation via inducing filopodia formation and endothelial cell tipping. We further screened the upregulated circTUBGCP4 in serum of CRC patients with metastasis compared to non-metastasis. Silencing circTUBGCP4 expression in CRC cell-derived exosomes (CRC-CDEs) inhibited endothelial cell migration, tube formation, tip cell formation, and CRC metastasis. Overexpression of circTUBGCP4 had opposite results in vitro and in vivo. Mechanically, circTUBGCP4 upregulated PDK2 to activate Akt signaling pathway by sponging miR-146b-3p. Moreover, we found that miR-146b-3p could be a key regulator for vascular endothelial cell dysfunction. Exosomal circTUBGCP4 promoted tip cell formation and activated the Akt signaling pathway by inhibiting miR-146b-3p.ConclusionsOur results suggest that colorectal cancer cells generate exosomal circTUBGCP4, which causes vascular endothelial cell tipping to promote angiogenesis and tumor metastasis by activating Akt signaling pathway.

Low serum level of 25-OH vitamin D relates to Th17 and treg changes in colorectal cancer patients.

BackgroundSerum 25‐hydroxyvitamin D [25(OH)D] level alters in colorectal cancer (CRC) development. Regulatory T (Treg) cells and T‐ helper type 17 (Th17) cells are involved in immune response. Th17‐mediated proinflammatory responses contribute to tumorigenesis, and Treg plays different roles in different periods of CRC. Vitamin D deficiency is associated with significant variations in peripheral immune cells. This study investigated the relationship between Th17 and Treg cells and 25(OH)D level in CRC.MethodsNinety‐five CRC patients were included, as well as 80 healthy controls during the same period at the Affiliated Hospital of Jiangnan University. 25(OH)D level was analyzed through electrochemiluminescence (ECLIA). Th17 and Treg levels were evaluated through flow cytometry. Serum levels of interleukin (IL)‐10, IL‐17, IL‐23, and transforming growth factor‐β (TGF‐β), were analyzed through commercial enzyme‐linked immunoassay (ELISA) kits.Results25(OH)D levels were downregulated in the serum of CRC patients. Decreased 25(OH)D level contributed to CRC pathogenesis. Decreased 25(OH)D level in CRC correlated with increased Treg and Th17 cell ratios and TGF‐β1, IL‐10, IL‐17, and IL‐23 levels in peripheral blood.ConclusionDecreased 25(OH)D level in the serum of CRC patients had negative correlation with Treg and Th17 ratios and relative cytokines levels.

Evaluation of the diagnostic value of serum-based proteomics for colorectal cancer.

BACKGROUNDColorectal cancer (CRC) is a highly malignant cancer with a high incidence and mortality in China. It is urgent to find a diagnostic marker with higher sensitivity and specificity than the traditional approaches for CRC diagnosis.AIMTo provide new ideas for the diagnosis of CRC based on serum proteomics.METHODSSpecimens from 83 healthy people, 62 colon polyp (CRP) patients, and 101 CRC patients were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The diagnostic value of the profiles of differentially expressed proteins was then analyzed.RESULTSCompared with the healthy control group, CRC patients had elevated expression of 5 proteins and reduced expression of 14 proteins. The area under the curve (AUC) for a differentially expressed protein with a mass-to-charge ratio of 2022.34 was the largest; the AUC was 0.843, which was higher than the AUC of 0.717 observed with carcinoembryonic antigen (CEA), and the sensitivity and specificity of this identified marker were 75.3% and 79.5%, respectively. After cross-validation, the accuracy of diagnosis using levels of this differentially expressed protein was 82.37%. Compared with the CRP group, the expression of 3 proteins in the serum of CRC patients was elevated and 11 proteins were expressed at reduced levels. Proteins possessing mass-to-charge ratio values of 2899.38 and 877.3 were selected to establish a classification tree model. The results showed that the accuracy of CRC diagnosis was 89.5%, the accuracy of CRP diagnosis was 81.6%, and the overall accuracy of this approach was 86.3%. The overall sensitivity and specificity of diagnosis using the proteomics approach were 81.8% and 66.75%, respectively. The sensitivities and specificities of diagnoses based on CEA and carbohydrate antigen 19-9 expression were 55.6% and 91.3% and 65.4% and 65.2%, respectively.CONCLUSIONWe demonstrated that serum proteomics may be helpful for the detection of CRC, and it may assist clinical practice for CRC diagnosis.

Galanin Receptors (GalR1, GalR2, and GalR3) Expression in Colorectal Cancer Tissue and Correlations to the Overall Survival and Poor Prognosis of CRC Patients.

Colorectal cancer (CRC) is the second most common cause of cancer in women and the third in men. The postoperative pathomorphological evaluation of patients with CRC is extremely important for future therapeutic decisions. Although our previous studies demonstrated high galanin (GAL) presence within tumor tissue and an elevated concentration of GAL in the serum of CRC patients, to date, there is a lack of data regarding GAL receptor (GalR) protein expression in CRC cells. Therefore, the aim of this study was to evaluate the presence of all three types of GalRs (GalR1, GalR2 and GalR3) within epithelial cells of the human colon and CRC tissue with the use of the immunohistochemical method and to correlate the results with the clinical-pathological data. We found stronger immunoreactivity of GalR1 and GalR3 in CRC cells compared to epithelial cells of the unchanged mucosa of the large intestine. No differences in the GalR2 protein immunoreactivity between the studied tissues were noted. We also found that the increased immunoexpression of the GalR3 in CRC tissue correlated with the better prognosis and longer survival (p < 0.0079) of CRC patients (n = 55). The obtained results suggest that GalR3 may play the role of a prognostic factor for CRC patients. Based on data from the TCGA-COAD project deposited in the GDC Data Portal, we also found that GalR mRNA in cancer samples and the adjacent normal tissue did not correlate with immunoexpression of the GalR proteins in CRC cells and epithelial cells of the unchanged mucosa.

A panel of platelet-associated circulating long non-coding RNAs as potential biomarkers for colorectal cancer

Evidence has suggested the potential of tumor-educated platelets as a biomarker trove for cancer diagnostics, but the difficulty in isolation limits its application. Since most of the circulating RNAs are derived from platelets, the change of RNA profile in platelets may lead to altered RNA expression in serum. Here, we identified a panel of platelet-associated long non-coding RNAs (lncRNAs) and evaluated its diagnostic capacity in serum of colorectal cancer (CRC) patients. Four lncRNAs, LNCAROD, SNHG20, LINC00534, and TSPOAP-AS1, were upregulated in both platelets and serum of CRC patients. A binary logistic model derived from them has validated area under roc curve of 0.78 indicating great performance. Furthermore, the expression levels of LNCAROD and TSPOAP-AS1 were correlated with cancer staging and tumor location. Together, our results add novel lncRNA biomarkers to the list of blood tests for CRC diagnostics and provide molecular evidence for the cross-talk between CRC platelets and serum.

Isolation and Identification of Long Non-Coding RNAs in Exosomes Derived from the Serum of Colorectal Carcinoma Patients.

Simple SummaryTreatment regimens for patients with advanced disease are limited and the mortality rate is high in these patients. A better understanding on pathogenesis and progression of cancer is critical for the development of new treatment strategies. In colorectal cancer (CRC), exosomes (secreted vesicles from cells) and long non-coding RNAs (lncRNAs) have been shown to play significant roles in disease development and progression. Long non-coding RNAs (lncRNAs) are present in the exosomes of serum and their profiles may potentially be useful as novel biomarkers for CRC patients and may provide a new insight in the pathogenesis and progression of CRC. Here, we compared the expression profiles of exosomal lncRNAs between non-cancer individuals and patients with colorectal carcinoma. The relative expression level of LINC00152 was found to be significantly lower in exosomes from sera of CRC patients as compared to non-cancer individuals whereas lncRNA H19 was significantly up-regulated in advanced-stages (stage III and IV) of CRC as compared to early-stages (stage I and II). Our data suggest that LINC00152 and H19 may play important roles in pathogenesis and progression of CRC.Long non-coding RNAs (lncRNAs) are non-coding RNAs consisting of more than 200 nucleotides in length. LncRNAs present in exosomes may play a critical role in the cellular processes involved in cancer pathogenesis and progression including proliferation, invasion, and migration of tumor cells. This paper aims to identify the differential expression of exosomal lncRNAs derived from the sera of non-cancer individuals and patients diagnosed with colorectal carcinoma. These differentially-expressed exosomal serum lncRNAs may provide an insight into the pathogenesis and progression of colorectal cancer (CRC). Serum exosomes and exosomes from SW480-7 cell culture supernatants were isolated and viewed by transmission electron microscope (TEM). The particle size distribution and protein markers of exosomes derived from SW480-7 were further analyzed using the Zetasizer Nano S instrument and western blotting technique. TEM showed that exosomes derived from serum and SW480-7 cells were round vesicles with sizes ranging from 50–200 nm. The exosomes derived from SW480-7 had an average diameter of 274.6 nm and contained the exosomal protein, ALIX/PDCD6IP. In our clinical studies, six lncRNAs, namely GAS5, H19, LINC00152, SNHG16, RMRP, and ZFAS1 were detected in the exosomes from sera of 18 CRC patients. Among these six lncRNAs, the expression level of LINC00152 was found to be significantly lower in CRC patients as compared to non-cancer individuals (p = 0.04) while lncRNA H19 was significantly up-regulated in advanced-stages (stage III and IV) of CRC (p = 0.04) as compared to early-stages (stage I and II). In conclusion, the detection of lower LINC00152 in exosomes of sera from CRC patients versus non-cancer individuals and H19 upregulation in advanced stages suggests that they may play important roles in pathogenesis and progression of CRC.

The HSF1/miR-135b-5p axis induces protective autophagy to promote oxaliplatin resistance through the MUL1/ULK1 pathway in colorectal cancer.

Oxaliplatin (oxa) is widely used in the treatment of colorectal cancer (CRC), but the development of oxaliplatin resistance is a major obstacle to the therapeutic efficacy in patients. MicroRNAs (miRNAs), endogenous noncoding RNAs measuring between 22 and 24 nucleotides, have been shown to be involved in the development of CRC drug resistance. However, the mechanism by which differentially expressed miRNAs induce chemotherapy resistance in CRC has not been fully elucidated to date. Here, we showed the differentially expressed miRNAs in oxaliplatin-sensitive and oxaliplatin-resistant CRC cells through miRNA microarray technology and found that miR-135b-5p was significantly increased in oxaliplatin-resistant cells. And miR-135b-5p was increased in the serum of colorectal cancer patients. More importantly, the miR-135b-5p level in the serum of oxaliplatin-resistant patients was further increased compared to that of oxaliplatin-sensitive patients. Recent studies have shown that protective autophagy is an important mechanism that promotes drug resistance in tumors. The potential role of miR-135b-5p in inducing protective autophagy and promoting oxaliplatin resistance was evaluated in two stable oxaliplatin-resistant CRC cell lines and their parental cells. We further identified MUL1 as a direct downstream target of miR-135b-5p and showed that MUL1 could degrade the key molecule of autophagy, ULK1, through ubiquitination. Mouse xenograft models were adopted to evaluate the correlation between miR-135b-5p and oxaliplatin-induced autophagy in vivo. Furthermore, we also investigated the regulatory factors for the upregulation of miR-135b-5p in CRC cells under oxaliplatin chemotoxicity. These results indicated that miR-135b-5p upregulation in colorectal cancer could induce protective autophagy through the MUL1/ULK1 signaling pathway and promote oxaliplatin resistance. Targeting miR-135b-5p may provide a new treatment strategy for reversing oxaliplatin resistance in CRC.

The Effectiveness of Glutathione Redox Status as a Possible Tumor Marker in Colorectal Cancer.

The role of oxidative stress (OS) in cancer is a matter of great interest due to the implication of reactive oxygen species (ROS) and their oxidation products in the initiation of tumorigenesis, its progression, and metastatic dissemination. Great efforts have been made to identify the mechanisms of ROS-induced carcinogenesis; however, the validation of OS byproducts as potential tumor markers (TMs) remains to be established. This interventional study included a total of 80 colorectal cancer (CRC) patients and 60 controls. By measuring reduced glutathione (GSH), its oxidized form (GSSG), and the glutathione redox state in terms of the GSSG/GSH ratio in the serum of CRC patients, we identified significant changes as compared to healthy subjects. These findings are compatible with the effectiveness of glutathione as a TM. The thiol redox state showed a significant increase towards oxidation in the CRC group and correlated significantly with both the tumor state and the clinical evolution. The sensitivity and specificity of serum glutathione levels are far above those of the classical TMs CEA and CA19.9. We conclude that the GSSG/GSH ratio is a simple assay which could be validated as a novel clinical TM for the diagnosis and monitoring of CRC.

Rearrangements of Blood and Tissue Fatty Acid Profile in Colorectal Cancer - Molecular Mechanism and Diagnostic Potential.

Colorectal cancer (CRC) is often diagnosed at an advanced stage due to the invasiveness of colonoscopy; thus, non-invasive CRC diagnostics are desirable. CRC is associated with lipid alterations. We aimed to verify whether fatty acid (FA) profiles in CRC patients may serve as a potential diagnostic tool for CRC diagnosis. FA profiles were assayed by GC-MS in cancer tissue, paired normal mucosa and serum from CRC patients and healthy controls. The levels of very long FAs – VLCFAs (26:0, 28:0 and 26:1) were the most highly increased FAs in cancer tissue compared to normal colon mucosa. Moreover, these FA were present in serum of CRC patients, they were absent in the serum of healthy subjects, or present in only trace amounts. To verify if cancer cells are the source of small amounts of these VLCFAs in the serum of patients we performed experiment in HT-29 CRC cells, which proved that CRC cells can produce and release VLCFAs into the blood. Most importantly, we defined a panel of FAs that may be assayed in a single analysis that definitely distinguishes CRC patients and healthy subjects, which was confirmed by PLS-DA and multivariate ROC analysis (AUC = 0.985). This study shows that selected FA panel may serve as a diagnostic marker for CRC.

Exosomal circ_IFT80 Enhances Tumorigenesis and Suppresses Radiosensitivity in Colorectal Cancer by Regulating miR-296-5p/MSI1 Axis.

BackgroundExosomal circular RNAs (circRNAs) can act as biomarkers and play crucial roles in colorectal cancer (CRC) and radiosensitivity. The aim of this study was to explore the functions and regulatory mechanism of exosomal circRNA intraflagellar transport 80 (circ_IFT80) in tumorigenesis and radiosensitivity of CRC.MethodsExosomes were detected using transmission electron microscopy (TEM). Protein levels were determined by Western blot assay. The expression of circ_IFT80, microRNA-296-5p (miR-296-5p) and musashi1 (MSI1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell cycle distribution, cell apoptosis, and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Colony formation assay was used to determine the radiosensitivity of cells. The interaction between miR-296-5p and circ_IFT80 or MSI1 was verified by dual-luciferase reporter assay. A xenograft tumor model was established to explore the role of exosomal circ_IFT80 in vivo.ResultsCirc_IFT80 was upregulated in exosomes derived from CRC patient serum and CRC cells. Exosomal circ_IFT80 or circ_IFT80 overexpression facilitated tumorigenesis by increasing cell proliferation and reducing apoptosis, and inhibited radiosensitivity via promoting colony formation and inhibiting apoptosis. Additionally, circ_IFT80 acted as a sponge of miR-296-5p, and miR-296-5p reversed the effects of circ_IFT80 on tumorigenesis and radiosensitivity. Moreover, MSI1 was a direct target of miR-296-5p. Furthermore, miR-296-5p overexpression inhibited tumorigenesis and promoted radiosensitivity by downregulating MSI1. Exosomal circ_IFT80 also accelerated tumor growth in vivo.ConclusionExosomal circ_IFT80 promoted tumorigenesis and reduced radiosensitivity by regulating miR-296-5p/MSI1 axis, which might provide a novel avenue for treatment of CRC.