Qinghua Xu

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.

Identification and Validation of Long Noncoding RNA Biomarkers in Human Non–Small-Cell Lung Carcinomas

Introduction: Dysregulation of long noncoding RNAs (lncRNAs) has been regarded as a primary feature of several human cancers. However, the genome-wide expression and functional significance of lncRNAs in non–small-cell lung carcinomas (NSCLC) remains unclear. The aim of this study was to identify novel lncRNAs that may play an important role in contributing to NSCLC pathogenesis. Methods: We performed an integrative analysis of two NSCLC microarray datasets comprising 165 and 90 patients, respectively. The candidate lncRNAs were identified using the GSE19188 dataset, and then confirmed in the GSE18842 dataset. In addition, an independent cohort of 73 clinical samples was analyzed to validate the selected lncRNAs by quantitative real-time polymerase chain reaction analysis. Results: With microarray gene expression analysis, we identified and validated a list of 64 lncRNAs significantly dysregulated in NSCLC tumors compared with normal lung tissues; and a panel of 181 lncRNAs that were specific to histological subtypes of NSCLC (adenocarcinoma, large-cell carcinoma, and squamous cell carcinoma). The quantitative real-time polymerase chain reaction analysis of six selected lncRNAs in clinical samples further confirmed the results of microarray analysis. Conclusions: We have identified and validated multiple novel lncRNAs associated with tumorigenesis and histological differentiation in human NSCLC. These lncRNAs could be further exploited for the development of useful biomarkers in diagnosis, prognosis, and treatment of NSCLC.

Comparison of Hepatic Resection and Radiofrequency Ablation for Small Hepatocellular Carcinoma: A Meta-Analysis of 16,103 Patients

We performed a meta-analysis to evaluate the therapeutic effects of radiofrequency ablation (RFA) and surgical hepatic resection (HR) in the treatment of small hepatocellular carcinoma (HCC). Thirty-one studies were included in the analysis. A total of 16,103 patients were involved: 8,252 treated with RFA and 7,851 with HR. Compared to the RFA group, the 3, 5-year overall and disease-free survival rates in the HR group were significantly higher. On the other hand, complications were significantly fewer and hospital-stay was significantly shorter in the RFA group than in the HR group. In subgroup analyses, the overall and disease-free survival in the HR group were also significantly higher than those in the RFA group for HCCs ≤ 3 cm, whereas there were no significant differences between the two groups for HCCs ≤ 2 cm. Our analysis showed that although HR was associated with higher complication rate and longer hospital-stay, HR is proposed as the first-line treatment rather than RFA for patients with HCCs larger than 2 cm. For patients with HCCs of 2 cm or less, RFA may be an alternative to HR because of their comparable long-term efficacy.

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.

Investigation of Variation in Gene Expression Profiling of Human Blood by Extended Principle Component Analysis

Background Human peripheral blood is a promising material for biomedical research. However, various kinds of biological and technological factors result in a large degree of variation in blood gene expression profiles. Methodology/Principal Findings Human peripheral blood samples were drawn from healthy volunteers and analysed using the Human Genome U133Plus2 Microarray. We applied a novel approach using the Principle Component Analysis and Eigen-R 2 methods to dissect the overall variation of blood gene expression profiles with respect to the interested biological and technological factors. The results indicated that the predominating sources of the variation could be traced to the individual heterogeneity of the relative proportions of different blood cell types (leukocyte subsets and erythrocytes). The physiological factors like age, gender and BMI were demonstrated to be associated with 5.3% to 9.2% of the total variation in the blood gene expression profiles. We investigated the gene expression profiles of samples from the same donors but with different levels of RNA quality. Although the proportion of variation associated to the RNA Integrity Number was mild (2.1%), the significant impact of RNA quality on the expression of individual genes was observed. Conclusions By characterizing the major sources of variation in blood gene expression profiles, such variability can be minimized by modifications to study designs. Increasing sample size, balancing confounding factors between study groups, using rigorous selection criteria for sample quality, and well controlled experimental processes will significantly improve the accuracy and reproducibility of blood transcriptome study.

PPAR signaling pathway may be an important predictor of breast cancer response to neoadjuvant chemotherapy

PurposeNeoadjuvant chemotherapy for advanced breast cancer may improve the radicality for a subset of patients, but others may suffer from severe adverse drug reactions without any benefit. To predict the responses to chemotherapy, we performed a phase II trial of neoadjuvant chemotherapy using a weekly PCb [paclitaxel (Taxol) plus carboplatin] regimen for stage II/III breast cancer and assessed the correlation between baseline gene expression and the tumor response to treatment.MethodsA total of 61 patients with stage II-III breast cancer were included and administered four cycles of preoperative PCb. We performed a gene expression analysis using Affymetrix HG-U133 Plus 2.0 GeneChip arrays in 31 breast cancer tissues. Differentially expressed genes (DEGs) were identified by the significance analysis of microarrays (SAM) program using a false discovery rate of 0.05. The Functional Annotation Tool in the DAVID Bioinformatics Resources was used to perform the gene functional enrichment analysis. The other 30 patients (15 pCR and 15 non-pCR patients) were available as an independent validation set to test the selected DEGs by quantitative real-time PCR analysis (qRT-PCR).ResultsBy analyzing six pathological complete response (pCR) patients and 25 patients with non-pCR, 300 probes (231 genes) were identified as differentially expressed between pCR and residual disease by the SAM program when the fold change was >2. The gene functional enrichment analysis revealed 15 prominent gene categories that were different between pCR and non-pCR patients, most notably the genes involved in the peroxisome proliferator-activated receptor (PPAR), DNA repair and ER signal pathways and in the immune-related gene cluster. The qRT-PCR analysis results for the genes in the PPAR pathway (LPL, SORBS1, PLTP, SCD5, MMP1 and CSTA) in independent validation set were consistent with the results from the microarray data analysis.ConclusionIn the present study, we identified a number of gene categories pertinent to the therapeutic response. We believe that the PPAR pathway may be an important predictor of genes that are involved in the chemotherapy response.

Identification and validation of a blood-based 18-gene expression signature in colorectal cancer

Purpose: The early detection of colorectal cancer (CRC) is crucial for successful treatment and patient survival. However, compliance with current screening methods remains poor. This study aimed to identify an accurate blood-based gene expression signature for CRC detection. Experimental Design: Gene expression in peripheral blood samples from 216 patients with CRC tumors and 187 controls was investigated in the study. We first conducted a microarray analysis to select candidate genes that were significantly differentially expressed between patients with cancer and controls. A quantitative reverse transcription PCR assay was then used to evaluate the expression of selected genes. A gene expression signature was identified using a training set (n = 200) and then validated using an independent test set (n = 160). Results: We identified an 18-gene signature that discriminated the patients with CRC from controls with 92% accuracy, 91% sensitivity, and 92% specificity. The signature performance was further validated in the independent test set with 86% accuracy, 84% sensitivity, and 88% specificity. The area under the receiver operating characteristics curve was 0.94. The signature was shown to be enriched in genes related to immune functions. Conclusions: This study identified an 18-gene signature that accurately discriminated patients with CRC from controls in peripheral blood samples. Our results prompt the further development of blood-based gene expression biomarkers for the diagnosis and early detection of CRC. Clin Cancer Res; 19(11); 3039–49. ©2013 AACR.

Decrease in Natural Killer cell associated gene expression as a major characteristic of the immune status in the bloodstream of colorectal cancer patients

Early detection and stratification of patients with colorectal cancer (CRC) are major challenges, particularly in the context of the development of new therapies. Several screening strategies are already in place in various countries, but compliance remains a major issue, mainly due to logistics or discomfort for the patients. In this study, we hypothesized that transcriptional signatures associated with leukocytes in peripheral blood can be informative to the identification of CRC patients. Gene expression was studied using RNA extracted from whole blood samples collected in PAXgene tubes and DNA microarrays. Analyzing 119 CRC patients and 101 colonoscopy-negative control (CNC) samples, we observed 327 differentially expressed genes (DEG), mostly associated with immune cell activation and trafficking. Natural Killer (NK) cell signaling and cytotoxicity associated genes appeared to undergo major changes in CRC peripheral blood samples. These changes were more pronounced in the advanced stages of the disease. A summarizing score of the expression of 10 genes related to NK cells interestingly revealed a marked heterogeneity within the CRC Stage IV group, suggesting possible further stratification of the patients. This study shows the potential of transcriptomics in peripheral blood to discover biomarkers and provides new insight on the immune response in colorectal cancer. In addition to preparing a possible alternative to current screening modalities, these results also show that the expression analysis of genes like those related to NK cells should allow the stratification of patients with colorectal cancer, opening the door to personalized medicine.