Study On The Roles Of TMPRSS2-ERG Fusion Gene And Circulating MicroRNA In Prostate Cancer

Our study focuses on the basic and clinical research for prostate cancer, try to find the function of TMPRSS2-ERG fusion gene in prostate cancer. At the same time, we also investigate the posibility wheather circulating microRNA can be a new biomarker for prostate cancer.1. Chromosomal rearrangement has been observed recurrently in various cancers. In 2005, Tomlins et al identified and described that an androgen-regulated gene, TMPRSS2 (trans-membrane protease, serine 2), was fused to either ERG or ETV1, members of the ETS family of oncogenes in prostate cancer (CaP). Consequently, this phenomenon was confirmed by multiple independent groups. The proportion of cases that harbour the fusion gene ranged from 50% to 70% in Caucasian patients, and the most frequent fusion type, TMPRSS2-ERG, occurred in about 50% CaP patients. Recent studies have suggested that TMPRSS2-ERG is involved in cell proliferation, cell differentiation and invasion. However, cancer as a complex disease, not only involves uncontrolled cell proliferation but also deregulation of programmed cell death. Therefore, it is of interest to know the function of TMPRSS2-ERG in the regulation of cell death. On the other hand, CaP exhibits racial/ethnic disparities in survival and incidence, and since most of the previous studies were conducted in Caucasian patients, it is not clear the prevalence and diversity of this type of gene rearrangement in Chinese CaP patient population. Thus, in the present study, we first examined the occurrence of such fusion gene in Chinese CaP patients, and then investigated whether TMPRSS2-ERG was involved in the regulation of cell death. It was hypothesized that the frequency of TMPRSS2-ERG occurrence should be different between Chinese and Caucasian CaP patients. And similar to Fli-1, another member of ETS family, TMPRSS2-ERG should also regulate cell death.2. Another focus of this study is the early diagnosis of CaP. Measuring prostate specific antigen (PSA) levels has been a standard procedure for screening CaP, however, it suffers from the low sensitivity and specificity. A better biomarker for prostate cancer with improved sensitivity/specificity is preferred. MicroRNAs (miRNAs) are endogenous noncoding small RNAs that are involved in various physiological and pathological processes, including tumorigenesis and metastasis. Recently, circulating miRNAs have been shown to have the potential as noninvasive diagnosis markers in several types of cancers. Therefore, we also examined the circulating miRNAs in a Chinese CaP patient population in an effort to identify potential biomarkers for the diagnosis of CaP.Part?study on the function of TMPRSS2-ERG fusion gene in prostate cancer Objective: To evaluate the frequency of TMPRSS2-ERG fusion gene in Chinese CaP patients, and investigate whether TMPRSS2-ERG is involved in the regulation of cell death.Methods:1. Total RNA was extracted from fine needle aspiration biopsy (FNAB) of CaP patients by Trizol. Nest PCR was performed to detect TMPRSS2-ETS fusion transcript, then validated by sequencing.2. Stable cell line expressing?ERG was constructed (?ERG cells), then cell migration was determined by scratch assay, cell proliferation and survival was detected by CCK-8 assay. The expression of apoptosis-related proteins and genes was examined by Western blot and real time quantitative PCR, respectively. After double staining with Annexin-V PE and 7-AAD, cell death was evaluated by FACS.Results:1. The occurrence of TMPRSS2-ERG fusion gene in this Chinese CaP population is 22%(6 in 27), while TMPRSS2-ETV1 was not detected. None of the fusion gene was detected in the 5 BPH samples. Interestingly, we also found one case of a new fusion gene TMPRSS2-Egrl.2. There were no significant differences in cell proliferation and migration between?ERG cells and control cells. However, survival rate of?ERG cells was?3 fold higher than that of control after 48 h treatment by cisplatin, with respective apoptotic rate of (25.89±9.66)% and (67.26±9.07)%. In addition, knock-down of?ERG by siRNA increased the apoptotic rate from(41.50±6.70)% to(59.64±3.23)% in AERG cells.3. Caspase-3 and Parp-1 were activated after incubation with cisplatin for 48 h in control cells, but this effect was partially inhibited in AERG cells. Expression levels of BCL-2, BCL-XL, BAX and BIM showed no significant differences between?ERG and control cells. In contrast, ATF-5 was upregulated by 2.5-fold in?ERG cells, while the level of?H2AX protein, an indicator for DNA damage, was downregulated by 1.5-fold in?AERG cells.Conclusions:1. The frequency of TMPRSS2-ERG fusion gene is lower in Chinese CaP group than Caucasian.2. A new type of fusion gene in CaP, namely TMPRSS2-Egrl, was identified.3. T3/E5 fusion gene can encode a truncted ERG protein (?ERG), and the overexpression of?ERG can inhibit cisplatin-induced DNA damage and cell apoptosis. Part?Circulating miRNAs as potential biomarkers for prostate cancerObjective:To identify CaP specific circulating miRNAs and evaluate their potential as diagnostic markers for CaP.METHODS:Illumina's Human v2 miRNA microarray (Illumina, San Diego, CA) was used to analyze microRNAs levels in a small set of patients (25 CaP,17 healthy controls) in an effort to identify CaP-specific miRNAs. The identified miRNAs were further examined by quantitative real time PCR (qRT-PCR) in the same small set of patients. After the training phase of screening and selecting, the candidate miRNAs were validated in a larger independent cohort (80 CaP,44 healthy controls) with qRT-PCR in the verification phase.RESULTS:Eight miRNAs (let-7c, let-7e, miR-25, miR30c, miR-346, miR-622, mir-940, and miR-1285) showed significant differences in plasma concentrations between CaP and control groups from the training sets, and 6 (let-7c, let-7e, miR30c, miR-622, mir-940, and miR-1285) of them were confirmed by qRT-PCR analysis in validation sets. Receiver operating characteristic (ROC) curve analysis showed all 6 miRNAs had diagnostic value with area under curve (AUC) of 0.784,0.805,0.759, 0.755,0.731, and 0.669, respectively. Further principal component analysis indicated component 2 extracted from expression data of the 6 miRNAs could differentiate CaP from controls with a higher diagnosis performance, with an AUC of0.925.CONCLUSIONS:Our data suggested that circulating miRNAs could serve as biomarkers for CaP, and compared to single miRNA, the 6 miRNAs panel can accurately discriminate CaP from healthy controls with high sensitivity and specificity.