MiR-150-5p靶向TP53对结直肠癌细胞侵袭和增殖的影响

摘要 目的：探索miR-150-5p靶向调控TP53基因对结直肠癌（colorectal cancer, CRC）在临床和生物学的相关性,研究miR-150-5p调控TP53基因在结直肠癌细胞增殖和侵袭病变中的作用。方法：收集临床手术切除并病理证实的结直肠癌患者的手术及血浆样本（结直肠癌和癌旁组织）60例,另选取癌旁正常粘膜10例,腺瘤30例。根据瘤体直径分为肿瘤＞5 cm（n=30）和≤5 cm（n=30）。qRT-PCR法测定样本中miR-150-5p表达,荧光素酶活性测定以确定TP53是否为miR-150-5p的靶基因。使用SW480细胞株,进行Transwell小室检测细胞侵袭能力,CCK-8检测细胞增值能力,对miR-150-5p进行生物信息学分析。结果：TP53是miR-150-5p的下游基因。癌组织及血浆中miR-150-5p表达量低于癌旁组织,直径＞5 cm瘤体中的miR-150-5p表达量显著低于直径≤5 cm瘤体,Ⅰ-Ⅱ期结直肠癌组织中的miR-150-5p表达显著高于Ⅲ-Ⅳ期（P＜0.05）。上调miR-150-5p后,细胞中TP53表达下降,下调miR-150-5p后,TP53表达升高;CCK-8增殖试验显示细胞中miR-150-5p过表达组抑制细胞增殖（P＜0.05）。结论：MiR-150-5p在结直肠癌组织和细胞中显著低表达,miR-150-5p通过靶向调节TP53抑制人CRC细胞的侵袭和增殖,有望成为结直肠癌治疗的新靶点。     

CircPRMT5 circular RNA promotes proliferation of colorectal cancer through sponging miR-377 to induce E2F3 expression

CircPRTM5 is associated with cell proliferation and migration in many kinds of malignancies. However, the functions and mechanisms of CircPRTM5 in CRC progression remain unclear. We explored the role and the mechanisms of CircPRTM5 in the development of CRC. Tissues of CRC patients and matched adjacent non-tumour tissues were collected to evaluate the expression of CircPRTM5. The expression of CircPRTM5 in CRC tissues was significantly higher than that in adjacent tissues. The biological functions of CircPRTM5 in CRC were determined by overexpression and down-regulation of CircPRTM5 in CRC cells in vitro and in vivo. The results indicate that knockdown of CircPRTM5 can significantly inhibit the proliferation of CRC cells. The potential mechanisms of CircPRTM5 in CRC development were identified by RT-qPCR, Western blotting analysis and luciferase reporter assay. CircPRTM5 competitively regulates the expression of E2F3 by capillary adsorption of miR-377. CircPRMT5 regulates CRC proliferation by regulating the expression of E2F3, which affects the expression of the cell cycle-associated proteins cyclinD1 and CDK2. CircPRTM5 exerts critical regulatory role in CRC progression by sponging miR-377 to induce E2F3 expression.     

MicroRNA‐93‐5p expression in tumor tissue and its tumor suppressor function via targeting programmed death ligand‐1 in colorectal cancer

This work aimed to investigate miR‐93‐5p expression in tumor tissue and its in vitro effects in colorectal cancer (CRC) by targeting programmed death ligand‐1 (PD‐L1). MiR‐93‐5p and PD‐L1 expression was detected in CRC and adjacent normal tissues by quantitative real‐time polymerase chain reaction and immunohistochemistry. The correlation between miR‐93‐5p and PD‐L1 was validated by a dual‐luciferase reporter assay. HCT116 and SW480 cells were divided into blank, miR‐NC, miR‐93‐5p mimics, miR‐93‐5p inhibitor, PD‐L1 small interfering RNA (siRNA) and miR‐93‐5p inhibitor + PD‐L1 siRNA groups, and wound‐healing and transwell assays were performed to detect cell migration and invasion, respectively. Protein expression was measured by western blotting. The secretion of cytokines was detected in the CRC cell/T coculture models. MiR‐93‐5p was downregulated in CRC tissues with upregulated PD‐L1. In PD‐L1‐negative patients, miR‐93‐5p expression was increased compared with that in PD‐L1‐positive patients. MiR‐93‐5p and PD‐L1 expression levels were associated with the tumor differentiation, lymphatic metastasis, TNM, Duke's stage, and prognosis of CRC. PD‐L1 siRNA weakened the migration and invasion abilities via decreased expression of matrix metalloproteinase‐1 (MMP‐1), ‐2, and ‐9, and these effects were abolished by the miR‐93‐5p inhibitor. Additionally, anti‐PD‐L1 upregulated the expressions of interleukin‐2 (IL‐2), tumor necrosis factor‐α (TNF‐α), and interferon γ (IFN‐γ) in the coculture of T cells with CRC cells, but downregulated the expressions of IL‐1β, IL‐10, and TGF‐β. However, these changes were partially reversed by miR‐93‐5p inhibition. miR‐93‐5p is expected to be a novel target for CRC treatment since it decreases the migration and invasion, as well as the immune evasion, of CRC cells via targeting PD‐L1.     

Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1 mM sulindac over 16 h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433–451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1 mM sulindac treatment for 8 h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30 K, 3 K and 1 K). Proteins isolated in the > 30 K and 3–30 K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3 K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS–MS. Collectively, our data show that LIM1215 cells treated with 1 mM sulindac for 8 h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5 AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8 h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1 mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell–cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

A new method for post Genome-Wide Association Study (GWAS) analysis of colorectal cancer in Taiwan

Recently, single nucleotide polymorphisms (SNPs) located in specific loci or genes have been identified associated with susceptibility to colorectal cancer (CRC) in Genome-Wide Association Studies (GWAS). However, in different ethnicities and regions, the genetic variations and the environmental factors can widely vary. Therefore, here we propose a post-GWAS analysis method to investigate the CRC susceptibility SNPs in Taiwan by conducting a replication analysis and bioinformatics analysis. One hundred and forty-four significant SNPs from published GWAS results were collected by a literature survey, and two hundred and eighteen CRC samples and 385 normal samples were collected for post-GWAS analysis. Finally, twenty-six significant SNPs were identified and reported as associated with susceptibility to colorectal cancer, other cancers, obesity, and celiac disease in a previous GWAS study. Functional analysis results of 26 SNPs indicate that most biological processes identified are involved in regulating immune responses and apoptosis. In addition, an efficient prediction model was constructed by applying Jackknife feature selection and ANOVA testing. As compared to another risk prediction model of CRC for European Caucasians population, which performs 0.616 of AUC by using 54 SNPs, the proposed model shows good performance in predicting CRC risk within the Taiwanese population, i.e., 0.724 AUC by using 16 SNPs. We believe that the proposed risk prediction model is highly promising for predicting CRC risk within the Taiwanese population. In addition, the functional analysis results could be helpful to explore the potential associated regulatory mechanisms that may be involved in CRC development.     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.     

Inhibition of polypeptide N-acetyl-α-galactosaminyltransferases is an underlying mechanism of dietary polyphenols preventing colorectal tumorigenesis

Ellagitannin-derived ellagic acid (EA) and colonic metabolite urolithins are functional dietary ingredients for cancer prevention, but the underlying mechanism need elucidation. Mucin-type O-glycosylation, initiated by polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts), fine-tunes multiple biological processes and is closely associated with cancer progression. Herein, we aim to explore how specific tannin-based polyphenols affect tumor behavior of colorectal cancer cells (CRC) by modulating O-glycosylation. Utilizing HPLC-based enzyme assay, we find urolithin D (UroD), EA and gallic acid (GA) potently inhibit ppGalNAc-Ts. In particular, UroD inhibits ppGalNAc-T2 through a peptide/protein-competitive manner with nanomolar affinity. Computational simulations combined with site-directed mutagenesis further support the inhibitors’ mode of action. Moreover, lectin analysis and metabolic labelling reveal that UroD can reduce cell O-glycans but not N-glycans. Transwell experiments prove that UroD inhibits migration and invasion of CRC cells. Our work proves that specific tannin-based polyphenols can potently inhibit ppGalNAc-Ts activity to reduce cell O-glycosylation and lead to lowering the migration and invasion of CRC cells, suggesting that disturbance of mucin-type O-glycosylation is an important mechanism for the function of dietary polyphenols.     

Circulating exosomal CPNE3 as a diagnostic and prognostic biomarker for colorectal cancer

Exosomal proteins are emerging as relevant diagnostic and prognostic biomarkers for cancer. This study was aimed at illustrating the clinical significance of exosomal Copine III (CPNE3) purified from the plasma of colorectal cancer (CRC) patients. The CPNE3 expression levels in CRC tissues were analyzed by real-time PCR, western blot, and immunohistochemistry. Plasma exosomes were isolated to examine the CPNE3 level using ELISA. Pearson’s correlation analysis was performed to investigate the CPNE3 levels between CRC tissues and matched plasma samples. Receiver operating characteristic curve analysis was developed to measure the diagnostic performance of exosomal CPNE3. The Kaplan–Meier method and Cox's proportional hazards model were utilized to determine statistical differences in survival times. CPNE3 showed increased expressions in the CRC tissues. A moderately significant correlation was found between CPNE3 expression in CRC tissues by immunohistochemistry and matched serum exosomal CPNE3 expression by ELISA (r = 0.645,(r = 0.645, p < 0.001). < 0.001). Exosomal CPNE3 yielded a sensitivity of 67.5% and a specificity of 84.4% in CRC at the cutoff value of 0.143 pg per 1ug1 ug exosome. Combined data from carcinoembryonic antigen and exosomal CPNE3 achieved 84.8% sensitivity and 81.2% specificity as a diagnostic tool. CRC patients with lower exosomal CPNE3 levels had substantially better disease-free survival (hazard ratio [HR], 2.9; 95% confidence interval [CI]: 1.3–6.4; p = 0.009) = 0.009) and overall survival (HR, 3.4; 95% CI: 1.2–9.9; p = 0.026) = 0.026) compared with those with higher exosomal CPNE3 levels. Exosomal CPNE3 show potential implications in CRC diagnosis and prognosis.     

Epigenetic-based therapy for colorectal cancer: Prospect and involved mechanisms

Epigenetic modifications are heritable variations in gene expression not encoded by the DNA sequence. According to reports, a large number of studies have been performed to characterize epigenetic modification during normal development and also in cancer. Epigenetics can be regarded more widely to contain all of the changes in expression of genes that make by adjusted interactions between the regulatory portions of DNA or messenger RNAs that lead to indirect variation in the DNA sequence. In the last decade, epigenetic modification importance in colorectal cancer (CRC) pathogenesis was demonstrated powerfully. Although developments in CRC therapy have been made in the last years, much work is required as it remains the second leading cause of cancer death. Nowadays, epigenetic programs and genetic change have pivotal roles in the CRC incidence as well as progression. While our knowledge about epigenetic mechanism in CRC is not comprehensive, selective histone modifications and resultant chromatin conformation together with DNA methylation most likely regulate CRC pathogenesis that involved genes expression. Undoubtedly, the advanced understanding of epigenetic-based gene expression regulation in the CRC is essential to make epigenetic drugs for CRC therapy. The major aim of this review is to deliver a summary of valuable results that represent evidence of principle for epigenetic-based therapeutic approaches employment in CRC with a focus on the advantages of epigenetic-based therapy in the inhibition of the CRC metastasis and proliferation.