Guixi Zheng

MiR-429 is an independent prognostic factor in colorectal cancer and exerts its anti-apoptotic function by targeting SOX2

Emerging evidence has demonstrated that microRNAs (miRNAs) can act as oncogenes or tumor suppressors to participate in cancer development. In this study, we found that miR-429 expression was up-regulated in human colorectal cancer (CRC) tissues, and the high miR-429 expression was significantly associated with tumor size, lymph node metastasis and poor prognosis. Functionally, miR-429 overexpression suppressed cell apoptosis by directly targeting SOX2 in HT-29 cells. Taken together, our data suggest for the first time that miR-429 could play an oncogenic role in the cellular processes of CRC and represent a novel prognostic biomarker for CRC.

Serum microRNA expression signatures identified from genome-wide microRNA profiling serve as novel noninvasive biomarkers for diagnosis and recurrence of bladder cancer.

Recent advantages of serum microRNAs (miRNAs) open a new realm of possibilities for noninvasive diagnosis and prognosis of bladder cancer (BC). The aim of our study was to identify serum miRNA expression signatures in patients with BC and establish new models for the diagnosis of BC and recurrence prediction. We performed genome‐wide serum miRNA analysis by Miseq sequencing followed by evaluations in the training and validation sets with reverse transcription quantitative real‐time PCR assays from serum samples of 250 patients with BC and 240 controls. A six‐miRNA panel (miR‐152, miR‐148b‐3p, miR‐3187‐3p, miR‐15b‐5p, miR‐27a‐3p and miR‐30a‐5p) for the diagnosis of BC was finally developed by multivariate logistic regression model with an area under the receiver operating characteristic curve of 0.899. The corresponding sensitivities of this panel for Ta, T1 and T2–T4 were 90.00, 84.85 and 89.36%, significantly higher than those of urine cytology, which were 13.33, 30.30 and 44.68%, respectively (all at p < 0.001). In addition, Kaplan–Meier analysis showed that patients with nonmuscle‐invasive BC (NMIBC) with high miR‐152 level and low miR‐3187‐3p level had worse recurrence‐free survival (p = 0.023 and 0.043, respectively). In multivariate Cox regression analysis, miR‐152 was independently associated with tumor recurrence of NMIBC (p = 0.028). Our results suggested that a serum miRNA signature may have considerable clinical value in diagnosing BC. Furthermore, expression level of serum miR‐152 could provide information on the recurrence risk of NMIBC.

Identification and Validation of Reference Genes for qPCR Detection of Serum microRNAs in Colorectal Adenocarcinoma Patients

Serum microRNAs (miRNAs) have become a highlighted research hotspot, especially for their great potential as a novel promising non-invasive biomarker in cancer diagnosis. The most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. However, no systematic evaluation and validation of reference genes for normalizing qPCR analysis of serum miRNAs has been reported in colorectal adenocarcinoma. We firstly profiled pooled serum of colorectal adenocarcinoma, colorectal adenoma and healthy controls and selected a list of 13 miRNAs as candidate reference genes. U6 snRNA (U6) and above-mentioned 13 miRNAs were included in further confirmation by qPCR. As a result, 5 miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples and 2 miRNAs (miR-197-3p and miR-26a-5p) were relatively low with median Cq more than 35, and were excluded from further stability analysis. Then variable stability of other 6 miRNAs (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p and miR-191-5p) and U6 were evaluated using two algorithms: geNorm and NormFinder which both identified miR-191-5p as the most stably expressed reference gene and selected miR-191-5p and U6 as the most stable pair of reference genes. After validating in an independent large cohorts and selecting miR-92a-3p as target miRNA to evaluate the effect of reference gene, we propose that combination of miR-191-5p and U6 could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls.

Exosomal long noncoding RNA CRNDE-h as a novel serum-based biomarker for diagnosis and prognosis of colorectal cancer

Cancer-secreted long non-coding RNAs (lncRNAs) are emerging mediators of cancer-host cross talk. The aim of our study was to illustrate the clinical significance of the lncRNA CRNDE-h in exosomes purified from the serum of patients with colorectal cancer (CRC). The study was divided into four parts: (1) The exosome isolated methods and lncRNA detected methods which accurately and reproducibly measure CRC-related exosomal CRNDE-h in serum were optimized in preliminary pilot stage; (2) The stability of exosomal CRNDE-h was evaluated systematically; (3) The origin of exosomal CRNDE-h was explorated in vitro and in vivo; (4) The diagnostic and prognostic value of exosomal CRNDE-h for CRC were validated in 468 patients. In pilot study, our results indicated that exosomal CRNDE-h was detectable and stable in serum of CRC patients, and derived from tumor cells. Then, the increased expression of exosomal CRNDE-h was successfully validated in 148 CRC patients when compared with colorectal benign disease patients and healthy donors. Exosomal CRNDE-h level significantly correlated with CRC regional lymph node metastasis (P = 0.019) and distant metastasis (P = 0.003). Moreover, at the cut-off value of 0.020 exosomal CRNDE-h level of serum, the area under ROC curve distinguishing CRC from colorectal benign disease patients and healthy donors was 0.892, with 70.3% sensitivity and 94.4% specificity, which was superior to carcinoembryogenic antigen. In addition, high exosomal CRNDE-h level has a lower overall survival rates than that for low groups (34.6% vs. 68.2%, P < 0.001). In conclusion, detection of lncRNA CRNDE-h in exosome shed a light on utilizing exosomal CRNDE-h as a noninvasive serum-based tumor marker for diagnosis and prognosis of CRC.

Hypoxia-Inducible MiR-210 Is an Independent Prognostic Factor and Contributes to Metastasis in Colorectal Cancer

MicroRNA-210 (miR-210), the master hypoxamir, plays pleiotropic roles in certain cancers; however, its role in the development of human colorectal cancer remains unclear. Herein, we report that miR-210 is frequently up-regulated in colorectal cancer tissues, with high miR-210 expression significantly correlating with large tumor size, lymph node metastasis, advanced clinical stage and poor prognosis. Functionally, miR-210 overexpression promotes the migration and invasion of colorectal cancer cells. Furthermore, miR-210 can be induced by hypoxia and mediates the hypoxia-induced metastasis of colorectal cancer cells. In addition, vacuole membrane protein 1 (VMP1) is identified as the direct and functional target of miR-210. Thus, miR-210 is a useful biomarker for hypoxic tumor cells and a prognostic factor that plays an essential role in colorectal cancer metastasis.

Up-regulation of miR-182 expression in colorectal cancer tissues and its prognostic value.

PurposeAccumulating evidences indicate that dysregulated microRNAs (miRNA) are involved in cancer tumorigenesis and progression. In the present study, we evaluated the expression of miR-182 in colorectal cancer and adjacent noncancerous tissues and explored its associations with clinicopathological characteristics and prognosis.MethodsQuantitative real-time PCR was used to analyze the expression of miR-182 in 148 pairs of colorectal cancer and adjacent noncancerous tissues. The relationship between miR-182 expression and clinicopathological characteristics in colorectal cancer tissues was estimated using Mann–Whitney U test or Kruskal–Wallis test, as appropriate. We calculated the survival curves and prognostic values of each variable by the Kaplan–Meier method and Cox proportional hazards regression analysis, respectively.ResultsThe expression of miR-182 was found up-regulated in colorectal cancer tissues compared with adjacent noncancerous tissues (p < 0.001), and its up-regulation was significantly correlated with large tumor size (p = 0.016), positive regional lymph node metastasis (p = 0.008), and advanced tumor–node–metastasis stage (p = 0.020). Furthermore, Kaplan–Meier analysis demonstrated that high miR-182 expression predicted poor survival (p = 0.001), and Cox proportional hazards risk analysis indicated that miR-182 was an independent prognostic factor for colorectal cancer.ConclusionsMiR-182 was up-regulated in colorectal cancer tissues and correlated with adverse clinical characteristics and poor prognosis, indicating that miR-182 might be involved in colorectal cancer progression and could be used as a potential prognostic biomarker and therapeutic target in the management of colorectal cancer.

The potential role of ORM2 in the development of colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancy in the world. The risk of death is closely correlated to the stage of CRC at the time of primary diagnosis. Therefore, there is a compelling need for the identification of blood biomarkers that can enable early detection of CRC. We used a quantitative proteomic approach with isobaric labeling (iTRAQ) to examine changes in the plasma proteome of 10 patients with CRC compared to healthy volunteers. Enzyme-Linked Immunosorbnent Assay (ELISA) and Western blot were used for further validation. In our quantitative proteomics analysis, we detected 75 human plasma proteins with more than 95% confidence using iTRAQ labeling in conjunction with microQ-TOF MS. 9 up-regulated and 4 down-regulated proteins were observed in the CRC group. The ORM2 level in plasma was confirmed to be significantly elevated in patients suffering from CRC compared with the controls. ORM2 expression in CRC tissues was significantly increased compared with that in corresponding adjacent normal mucous tissues (P<0.001). ITRAQ together with Q-TOF/MS is a sensitive and reproducible technique of quantitative proteomics. Alteration in expression of ORM2 suggests that ORM2 could be used as a potential biomarker in the diagnosis of CRC.

Down-regulation of HLA class I antigen in human papillomavirus type 16 E7 expressing HaCaT cells: correlate with TAP-1 expression

Objectives: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer, and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein of high-risk HPV types disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting a role for E7 in tumor immune evasion. We previously reported that HPV-16 E7 expression and down-regulation of HLA class I was highly correlated in cervical lesions. This study was aimed to determine whether HPV-16 E7 oncoprotein could down-regulate surface HLA class I antigen in HPV-16 E7-transfected cells, and whether it had correlation with the expression of the transporter associated with antigen processing (TAP). Methods: The HPV-16 E7 open reading frame was transfected into HaCaT cells. After G418 selection, resistant colonies were individually picked and expanded into clonal cell lines. Using the fluoresence-activated cell sorting analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 and TAP-2 expressions were detected. Results Compared with the empty vector control, a statistical significant decrease of approximately 50% in cell surface HLA class I expression was observed in HPV-16 E7 expressing HaCaT cells (P < 0.001). Moreover, the expression of HPV-16 E7 in HaCaT cells resulted in decreased expression of TAP-1 that was essential for HLA class I expression at the cell surface, a statistical significant decrease of approximately 40% compared with that with the empty vector control (P < 0.001). Conclusions: Our finding demonstrates that HPV-16 E7 down-regulates surface HLA class I antigen, which in part correlates with the decrease of TAP-1.

Krüppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer

AIM To investigate Krüppel-like factor 8 (KLF8) expression in gastric cancer and its relationship with angiogenesis and prognosis of gastric cancer. METHODS One hundred and fifty-four patients with gastric cancer who underwent successful curative resection were retrospectively enrolled in the study. Fifty tumor-adjacent healthy gastric tissues (≥ 5 cm from the tumor margin) obtained during the original resection were randomly selected for comparative analysis. In situ expression of KLF8 and CD34 proteins were examined by immunohistochemistry. The intratumoral microvessel density (MVD) was determined by manually counting the immunostained CD34-positive endothelial cells in three consecutive high-magnification fields (× 200). The relationship between differential KLF8 expression and MVD was assessed using Spearmans correlation coefficient test. χ² test was performed to evaluate the effects of differential KLF8 expression on clinicopathologic factors. Kaplan-Meier and multivariate Cox survival analyses were used to assess the prognostic value of differential KLF8 expression in gastric cancer. RESULTS Significantly higher levels of KLF8 protein were detected in gastric cancer tissues than in the adjacent non-cancerous tissues (54.5% vs 34.0%, P < 0.05). KLF8 expression was associated with tumor size (P < 0.001), local invasion (P = 0.005), regional lymph node metastasis (P = 0.029), distant metastasis (P = 0.023), and tumor node metastasis (TNM) stage (P = 0.002), as well as the MVD (r = 0.392, P < 0.001). Patients with KLF8 positive expression had poorer overall survival (P < 0.001) and cancer-specific survival (P < 0.001) than those with negative expression. Multivariate analysis demonstrated that KLF8 expression independently affected both overall and cancer-specific survival of gastric cancer patients (P = 0.035 and 0.042, respectively). CONCLUSION KLF8 is closely associated with gastric tumor progression, angiogenesis and poor prognosis, suggesting it may represent a novel prognostic biomarker and therapeutic target for gastric cancer.

Detection of circulating Bmi-1 mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancer

Bmi‐1 is overexpressed in uterine cervical cancer (UCC) and is found to be associated with adverse clinical characteristics and poor prognosis. However, little information is available on the status of circulating Bmi‐1 mRNA in UCC. Because circulating cell‐free nucleic acids have emerged as a novel class of markers for cancer detection, our research aims to address this question by detecting the circulating Bmi‐1 mRNA and to assess its diagnostic and prognostic potential in UCC. Reverse transcription quantitative real‐time PCR method was established to detect the circulating Bmi‐1 mRNA in plasma of 109 patients with UCC, 138 patients with cervical intraepithelial neoplasia (CIN) and 80 healthy volunteers, and found that it was significantly increased in UCC compared with CINs and healthy controls (all at p < 0.001). Moreover, its high level was significantly correlated with advanced clinical stage (p < 0.001) and positive lymph nodes metastasis (p = 0.002). The area under the receiver operating characteristic curve (AUC) was 0.881, and the optimal cut‐off value was 0.057, providing a sensitivity of 69.7% and a specificity of 95.9%. The AUC for circulating Bmi‐1 mRNA showed higher diagnosis capability than that for SCC‐Ag (p = 0.035) or CA125 (p < 0.001) currently utilized. Kaplan–Meier analysis demonstrated a correlation between increased circulating Bmi‐1 mRNA level and reduced disease‐free survival (DFS) (p = 0.001) and overall survival (OS) (p = 0.015). And, Cox analysis indicated that it was an independent prognostic factor for DFS and OS. We conclude that circulating Bmi‐1 mRNA may be a potential noninvasive molecular marker for diagnosis and prognosis of UCC.