Cong Tan

Plasma miR-601 and miR-760 are novel biomarkers for the early detection of colorectal cancer.

Background Colorectal cancer (CRC) is a major cause of death worldwide. Sensitive, non-invasive diagnostic screen methods are urgently needed to improve its survival rates. Stable circulating microRNA offers unique opportunities for the early diagnosis of several diseases, including cancers. Our aim has been to find new plasma miRNAs that can be used as biomarkers for the detection of CRC. Methodology/Principal Findings According to the results of miRNA profiling performed on pooling plasma samples form 10 CRC patients or 10 healthy controls, a panel of miRNAs (hsa-miR-10a, -19a, -22*, -24, -92a, 125a-5p, -141, -150, -188-3p, -192, -210, -221, -224*, -376a, -425*, -495, -572, -601, -720, -760 and hsa-let-7a, -7e) were deregulated in CRC plasma with fold changes >5. After large scale validation by qRT-PCR performed on another 191 independent individuals (90 CRC, 43 advanced adenoma and 58 healthy participants), we found that the levels of plasma miR-601 and miR-760 were significantly decreased in colorectal neoplasia (carcinomas and advanced adenomas) compared with healthy controls. ROC curve analysis showed that plasma miR-601 and miR-760 were of significant diagnostic value for advanced neoplasia. These two miRNAs together yield an AUC of 0.792 with 83.3% sensitivity and 69.1% specificity for separating CRC from normal controls, and yield an AUC of 0.683 with 72.1% sensitivity and 62.1% specificity in discriminating advanced adenomas from normal controls. Conclusions/Significance Plasma miR-601 and miR-760 can potentially serve as promising non-invasive biomarkers for the early detection of CRC.

Circulating CUDR, LSINCT‐5 and PTENP1 long noncoding RNAs in sera distinguish patients with gastric cancer from healthy controls

The examination of circulating nucleic acids (CNAs) is an emerging noninvasive diagnostic technique. However, it is unclear if serum long noncoding RNAs (lncRNAs) represent a novel marker to detect gastric cancer (GC). In this study, we measured 39 candidate cancer‐associated lncRNAs by reverse transcription and quantitative polymerase chain reaction (RT‐qPCR) in sera from 110 patients with GC, 106 age‐ and sex‐matched healthy subjects and 15 patients with gastric peptic ulcer, markers were validated and assessed by RT‐qPCR. The correlation of the expression levels of the candidate serum lncRNAs with clinical parameters of GC patients was performed. A three‐lncRNA signature, including CUDR, LSINCT‐5 and PTENP1, was identified that may be potential diagnostic marker for GC. The areas under the receiver operating characteristic (ROC) curve for this serum three‐lncRNA signature were 0.920 and 0.829 for the two sets of serum samples. Moreover, a risk model for the serum three‐lncRNA signature demonstrated that healthy samples can be distinguished from early GC samples. Three‐lncRNA signature in serum was identified as diagnostic marker for GC. This work may facilitate the detection of GC and serve as the basis for further studies of the clinical value of serum lncRNAs in maintaining surveillance and forecasting prognosis.

A positive feedback loop of lncRNA-PVT1 and FOXM1 facilitates gastric cancer growth and invasion

Purpose: The long, noncoding RNA (lncRNA) PVT1 is an important epigenetic regulator with a critical role in human tumors. Here, we aimed to investigate the clinical application and the potential molecular mechanisms of PVT1 in gastric cancer tumorigenesis and progression. Experimental Design: The expression level of PVT1 was determined by RT-qPCR analysis in 190 pairs of gastric cancer tissues and adjacent normal gastric mucosa tissues (ANT). The biologic functions of PVT1 were assessed by in vitro and in vivo functional experiments. RNA protein pull-down assays and LS/MS mass spectrometry analysis were performed to detect and identify the PVT1-interacting protein FOXM1. Protein–RNA immunoprecipitation assays were conducted to examine the interaction of FOXM1 and PVT1. Chromatin immunoprecipitation (ChIP) and luciferase analyses were utilized to identify the binding site of FOXM1 on the PVT1 promoter. Results: The lncRNA PVT1 was significantly upregulated in gastric cancer tissues compared with ANTs. High expression of PVT1 predicted poor prognosis in patients with gastric cancer. PVT1 enhanced gastric cancer cell proliferation and invasion in vitro and in vivo. PVT1 directly bound FOXM1 protein and increased FOXM1 posttranslationally. Moreover, PVT1 is also a FOXM1-responsive lncRNA, and FOXM1 directly binds to the PVT1 promoter to activate its transcription. Finally, PVT1 fulfilled its oncogenic functions in a FOXM1-mediated manner. Conclusions: Our study suggests that PVT1 promotes tumor progression by interacting with FOXM1. PVT1 may be a valuable prognostic predictor for gastric cancer, and the positive feedback loop of PVT1-FOXM1 could be a therapeutic target in pharmacologic strategies. Clin Cancer Res; 23(8); 2071–80. ©2016 AACR.

Down-regulation of ncRAN, a long non-coding RNA, contributes to colorectal cancer cell migration and invasion and predicts poor overall survival for colorectal cancer patients

Accumulating evidence has indicated that long non‐coding RNAs (lncRNAs) play critical roles in regulating cellular processes, such as cell growth and apoptosis, as well as cancer progression and metastasis. ncRAN (non‐coding RNA expressed in aggressive neuroblastoma) was previously shown to be dramatically up‐regulated and associated with poor prognosis in human neuroblastoma. This lncRNA also plays an important role in bladder cancer growth and invasion. Colorectal cancer (CRC) progression typically follows a complex cascade from primary malignancy to distant metastasis, but whether the aberrant expression of ncRAN in CRC is associated with malignancy, metastasis or prognosis remains unknown. In this study, we demonstrated that ncRAN expression is significantly down‐regulated in tumor tissue and CRC cell lines compared with adjacent normal tissue and a normal intestinal mucous cell line. Reduced expression of ncRAN was detected in poorly differentiated or undifferentiated tumors and in tumors with liver metastases. Kaplan–Meier analysis indicated that patients with lower ncRAN expression have a worse overall survival. Moreover, multivariate analysis revealed that decreased expression of ncRAN is an independent predictor of overall survival. Our experimental data indicated that ncRAN mediates the in vitro migration and invasion of CRC cells. Together, these results suggest that ncRAN might represent a novel prognostic indicator, a biomarker for the early detection of metastasis and a target for gene therapy in CRC.

Reciprocal repression between TUSC7 and miR-23b in gastric cancer

Recently, long noncoding RNAs (lncRNAs) were demonstrated to play important regulatory roles in biological processes and cancer biology. However, the overall pathophysiological contribution of lncRNAs to gastric cancer (GC) remains largely unknown. In this study, differentially expressed lncRNAs in GC and paired adjacent normal tissue samples were identified by microarray and were validated using quantitative real‐time polymerase chain reaction (qRT‐PCR). One particular lncRNA, tumour suppressor candidate 7 (TUSC7), was analyzed in sequential large cohorts, and the Kaplan–Meier method with the log‐rank test for comparisons was used to analyse the survival data. The results indicated that TUSC7 was downregulated in GC samples and was an independent prognostic indicator of disease‐free survival (DFS) and disease‐specific survival (DSS) in GC patients. Applying loss‐of‐function and gain‐of‐function approaches, we determined that TUSC7 suppressed tumour cell growth in vitro and in vivo. Furthermore, we showed that TUSC7 was a direct transcriptional target of p53 via interaction of p53 with the putative p53‐response element in the upstream region of TUSC7. Finally, we demonstrated reciprocal repression between TUSC7 and miR‐23b; in contrast to TUSC7, miR‐23b promoted cell growth. The results indicated that TUSC7 is a p53‐regulated tumour suppressor that acts in part by repressing miR‐23b and that TUSC7 may be a key regulatory hub in GC.

Circulating Long RNAs in Serum Extracellular Vesicles: Their Characterization and Potential Application as Biomarkers for Diagnosis of Colorectal Cancer

Background: Long noncoding RNA (lncRNA) and mRNAs are long RNAs (≥200 nucleotides) compared with miRNAs. In blood, long RNAs may be protected by serum extracellular vesicles, such as apoptotic bodies (AB), microvesicles (MV), and exosomes (EXO). They are potential biomarkers for identifying cancer. Methods: Sera from 76 preoperative colorectal cancer patients, 76 age- and sex-matched healthy subjects, and 20 colorectal adenoma patients without colorectal cancer were collected. We investigated the distribution of long RNAs into the three vesicles. Seventy-nine cancer-related long RNAs were chosen and detected using qPCR. Results: The quantity of long RNA has varying distribution among three subtypes of extracellular vesicles in serum. Most mRNA and lncRNA genes had higher quantity in EXOs than that in ABs and MVs, whereas MVs contain lowest quantity. We investigated 79 long RNAs chosen from The Cancer Genome Atlas and the LncRNADisease database in the sera of healthy patients, and those with colorectal cancer. In the training and test sets, the AUCs were 0.936 and 0.877, respectively. The AUC of total serum RNA was lower (0.857) than that of exosomal RNA in the same samples (0.936). Conclusion: The present study shows that exosomal mRNAs and lncRNAs in serum could be used as biomarkers to detect colorectal cancer. Impact: Among three types of vesicles in sera, EXOs were the richest reservoir for almost all measured long RNAs. The combination of two mRNAs, KRTAP5-4 and MAGEA3, and one lncRNA, BCAR4, could be potential candidates to detect colorectal cancer. Cancer Epidemiol Biomarkers Prev; 25(7); 1158–66. ©2016 AACR.

MicroRNA-202-3p Inhibits Cell Proliferation by Targeting ADP-Ribosylation Factor-like 5A in Human Colorectal Carcinoma

Purpose: MicroRNAs (miRNA) that are strongly implicated in carcinogenesis have recently reshaped our understanding of the role of non–protein-coding RNAs. Here, we focused on the function and molecular mechanism of miR-202-3p and its potential clinical application in colorectal cancer. Experimental Design: miR-202-3p expression was determined by quantitative reverse transcriptase PCR (qRT-PCR) in 94 colorectal cancer tissues and corresponding noncancerous tissues (NCT). Cell proliferation and colony formation assays in vitro and xenograft experiments in vivo were used to evaluate the effect of miR-202-3p on colorectal cancer cell proliferation. Luciferase assay and Western blot analysis were performed to validate the potential targets of miR-202-3p after the preliminary screening by online prediction and microarray analysis. The mRNA and protein levels of target genes were detected by qRT-PCR and immunohistochemical staining. The copy number of pre-miR-202 was measured by quantitative PCR. Results: First, miR-202-3p was significantly downregulated in 46.7% colorectal cancer samples compared with NCTs. The overexpression of miR-202-3p inhibited colorectal cancer cell growth in vitro and repressed tumorigenesis in nude mice. Then, miR-202-3p downregulated ADP-ribosylation factor-like 5A (ARL5A) protein level by binding to its 3′ untranslated region, and knockdown of ARL5A phenocopied the proliferation inhibition effect of miR-202-3p. Furthermore, both of ARL5A mRNA and protein levels were upregulated in colorectal cancer samples compared with NCTs and high ARL5A protein levels predicted a poor prognosis. Conclusions: miR-202-3p might function as a tumor suppressor in colorectal cancer, and ARL5A, the functional target of miR-202-3p in colorectal cancer, is a potential prognostic factor for colorectal cancer. Clin Cancer Res; 20(5); 1146–57. ©2013 AACR.

Long Non-Coding RNA LSINCT5 Predicts Negative Prognosis and Exhibits Oncogenic Activity in Gastric Cancer

AbstractLong non-coding RNAs (lncRNAs) are recently discovered RNA transcripts that are aberrantly expressed in many tumor types. Numerous studies have suggested that lncRNAs can be utilized for cancer diagnosis and prognosis. LSINCT5 (long stress-induced non-coding transcript 5) is dramatically upregulated in breast and ovarian cancer and affects cellular proliferation. However, the expression pattern of LSINCT5 in gastrointestinal cancer and the association between aberrant expression of LSINCT5 in gastrointestinal cancer and malignancy, metastasis, or prognosis remain unknown.LSINCT5 expression was detected in gastrointestinal cancer and paired adjacent normal tissue samples or cell lines using reverse transcription quantitative PCR (RT-qPCR). We also investigated the potential relationship between tumor LSINCT5 levels and clinicopathological features of gastrointestinal cancer. Finally, we assessed whether LSINCT5 influences in vitro cell proliferation.The expression of LSINCT5 is significantly upregulated in gastrointestinal cancer tissues and cell lines relative to their normal counterparts. In addition, increased LSINCT5 expression was correlated with a larger tumor size, deeper tumor depth, and advanced clinical stage. Kaplan–Meier analysis indicated that gastric cancer (GC) and colorectal cancer (CRC) patients with higher LSINCT5 expression levels have worse disease-free survival (DFS) and disease-specific survival (DSS) rates. Moreover, multivariate analysis revealed that increased expression of LSINCT5 is an independent predictor of DFS and DSS rates in GC patients. The ectopic expression of LSINCT5 in gastrointestinal cancer cell lines resulted in an increase in cellular proliferation; conversely, knock down of LSINCT5 significantly inhibited proliferation.These results suggest that LSINCT5 may represent a novel prognostic indicator and a target for gene therapy in gastrointestinal cancer.

DIXDC1 activates the Wnt signaling pathway and promotes gastric cancer cell invasion and metastasis

DIXDC1 (Dishevelled‐Axin domain containing 1) is a DIX (Dishevelled‐Axin) domain‐possessing protein that promotes colon cancer cell proliferation and increases the invasion and migration ability of non‐small‐cell lung cancer via the PI3K pathway. As a positive regulator of the Wnt/β‐catenin pathway, the biological role of DIXDC1 in human gastric cancer and the relationship between DIXDC1 and the Wnt pathway are unclear. In the current study, the upregulation of DIXDC1 was detected in gastric cancer and was associated with advanced TNM stage cancer, lymph node metastasis, and poor prognosis. We also found that the overexpression of DIXDC1 could promote the invasion and migration of gastric cancer cells. The upregulation of MMPs and the downregulation of E‐cadherin were found to be involved in the process. DIXDC1 enhanced β‐catenin nuclear accumulation, which activated the Wnt pathway. Additionally, the inhibition of β‐catenin in DIXDC1‐overexpressing cells reversed the metastasis promotion effects of DIXDC1. These results demonstrate that the expression of DIXDC1 is associated with poor prognosis of gastric cancer patients and that DIXDC1 promotes gastric cancer invasion and metastasis through the activation of the Wnt pathway; E‐cadherin and MMPs are also involved in this process.

Role of MUC20 overexpression as a predictor of recurrence and poor outcome in colorectal cancer

BackgroundColorectal cancer (CRC) remains one of the most common cancers worldwide. We observed that MUC20 was significantly up-regulated in CRC patients with poor prognosis based on the microarray analysis. However, little is known about the role of MUC20 in CRC.MethodsMicroarray experiments were performed on the Affymetrix U133 plus 2.0 GeneChip Array. The protein and mRNA levels of MUC20 were examined by immunohistochemistry (IHC) and Real-Time quantitative PCR (RT-qPCR) in CRC tissues and adjacent noncancerous tissues (ANCT). ShRNA and overexpression plasmids were used to regulate MUC20 expression in CRC cell lines in vitro; wound healing, Transwell migration assays, and Western blotting were used to detect migration and invasion changes.ResultsMUC20 was one of the up-regulated genes in CRC patients with poor prognosis by microarray. Using IHC and RT-qPCR, we showed that MUC20 expression was significantly higher in CRC tissues than in ANCT (P < 0.05). We further showed that MUC20 overexpression was correlated with recurrence and poor outcome (P < 0.05). The Kaplan-Meier survival curves indicated that disease-free survival (DFS) and overall survival (OS) were significantly worse in CRC patients with MUC20 overexpression. The Cox multivariate analysis revealed that MUC20 overexpression and TNM stage were independent prognostic factors. Elevated expression of MUC20 in cells promoted migration and invasion, whereas ShRNA-mediated knockdown inhibited these processes. In addition, Western blotting demonstrated that MUC20-induced invasion was associated with MMP-2, MMP-3, and E-cadherin.ConclusionsCumulatively, MUC20 may serve as an important predictor of recurrence and poor outcome for CRC patients. MUC20 overexpression could enhance migration and invasion abilities of CRC cells. Translation of its roles into clinical practice will need further investigation and additional test validation.