Jinyao Zhao

MiRNA expression signature for potentially predicting the prognosis of ovarian serous carcinoma.

One of the best prognostic predictors for patients with epithelial ovarian cancer is the Federation of Obstetrics and Gynecology (FIGO) stage at diagnosis. Advanced-stage ovarian serous carcinoma (OSC) generally have poor prognosis. The goal of this study is to develop and validate a miRNA expression profile that can differentiate the OSC at early and advanced stages and study its correlation with the prognosis of OSC. To identify a unique microRNA (miRNA) pattern associated with the progression of OSC at early and advanced stages, a miRNA microarray was performed using Chinese tumor bank specimens of patients with OSC stage I or III in a retrospective analysis. The expression of four dysregulated miRNAs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in an external cohort of 51 cases of OSC samples at stages I and III. Kaplan–Meier analysis was performed to analyze the correlation between the expression of some miRNAs and prognosis. Of the 768 miRNAs analyzed in the microarray, 26 miRNAs were significantly either up- or downregulated, with at least a 2-fold difference, in OSC stage I compared with stage III. The qRT-PCR results showed that miR-510, miR-509-5p, and miR-508-3p were significantly downregulated and that miR-483-5p was upregulated in stage III OSC compared with stage I, which was consistent with the microarray results. Kaplan–Meier analysis showed low miR-510 expression, low miR-509-5p expression, and advanced FIGO stage, and chemotherapy resistance were significantly associated with poorer overall survival (P < 0.05). Our results suggest that miRNAs may play a role in the progression of OSC, and miR-510 and miR-509-5p may be considered novel-candidate clinical biomarkers for predicting OSC outcome.

MiR-770-5p inhibits cisplatin chemoresistance in human ovarian cancer by targeting ERCC2

In this study, we examined the role of the miRNA miR-770-5p in cisplatin chemotherapy resistance in ovarian cancer (OVC) patients. miR-770-5p expression was reduced in platinum-resistant patients. Using a 6.128-fold in expression as the cutoff value, miR-770-5p expression served as a prognostic biomarker and predicted the response to cisplatin treatment and survival among OVC patients. Overexpression of miR-770-5p in vitro reduced survival in chemoresistant cell lines after cisplatin treatment. ERCC2, a target gene of miR-770-5p that participates in the NER system, was negatively regulated by miR-770-5p. siRNA-mediated silencing of ERCC2 reversed the inhibition of apoptosis resulting from miR-770-5p downreglation in A2780S cells. A comet assay confirmed that this restoration of cisplatin chemosensitivity was due to the inhibition of DNA repair. These findings suggest that endogenous miR-770-5p may function as an anti-oncogene and promote chemosensitivity in OVC, at least in part by downregulating ERCC2. miR-770-5p may therefore be a useful biomarker for predicting chemosensitivity to cisplatin in OVC patients and improve the selection of effective, more personalized, treatment strategies.

miRNA expression pattern associated with prognosis in elderly patients with advanced OPSC and OCC

The long-term survival for elderly patients with advanced ovarian papillary serous carcinoma (OPSC) does not exceed 30%, and the incidence and prognosis rise continuously after menopause. The aim of this study was to identify the differences in key miRNAs and their potential regulators through miRNA microarray analysis, functional target prediction, and clinical outcome between the elderly patients with advanced OPSC and ovarian clear cell carcinoma (OCC) who all suffered poor prognosis, to identify the pathogenetic basis, and to improve the understanding of the molecular basis of advanced OPCS in elderly patients. Through microarray analysis, we found 52 unique miRNAs with significant fold‑change in expression levels, of which 9 were upregulated, whereas 43 were downregulated in OCC patients compared to elderly OPSC patients with advanced stage. Among these prediction miRNAs, miR-30a, miR-30e and miR-505 were found to have some common cancer-related targets. Lower expression of these three miRNAs of advanced OPSC in elderly patients, all associated with significantly poorer survival rate, strongly suggesting that they could be critical oncogenes and take important roles in OPSC etiology in elderly patients with advantaged stage. Functional analyses support the above hypothesis. Their targets, ATF3, STMN1 and MYC suggest that OPSC also has aggressive biological behavior when presented with advanced stage, and support the epidemiology results that incidence and mortality of advanced OPSC increases continuously. miR-30a, miR-30e and miR-505 may be important pathogenetic factors for OPSC in elderly patients with advanced stage. Age could be regarded as a continuous covariate in this process. This may improve the understanding of molecular underpinnings of advanced OPSC in elderly patients, and provide improved diagnostic, prognostic and therapeutic approaches.

Profile of differentially expressed miRNAs in high-grade serous carcinoma and clear cell ovarian carcinoma, and the expression of miR-510 in ovarian carcinoma

Improved insight into the molecular and genetic profile of different types of epithelial ovarian cancer (EOC) is required for understanding the carcinogenesis of EOC and may potentially be exploited by future targeted therapies. The aim of the present study was to identify a unique microRNA (miRNA) patterns and key miRNAs, which may assist in predicting progression and prognosis in high-grade serous carcinoma (HGSC) and clear cell carcinoma (CCC). To identify unique miRNA patterns associated with HGSC and CCC, a miRNA microarray was performed using Chinese tumor bank specimens of patients with HGSC or CCC in a retrospective analysis. The expression levels of four deregulated miRNAs were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in an external cohort of 42 cases of HGSC and 36 cases of CCC. Kaplan-Meier analysis was performed to analyze the correlation between the expression levels of the four miRNAs and patient prognosis. Among these validated miRNAs, miR-510 was further examined in another cohort of normal ovarian tissues, as well as the HGSC, low-grade serous carcinoma (LGSC) and CCC specimens using RT-qPCR and in situ hybridization. The results revealed that, of the 768 miRNAs analyzed in the microarray, 33 and 50 miRNAs were significantly upregulated and downregulated, respectively, with at least a 2-fold difference in HGSC, compared with CCC. The quantitative analysis demonstrated that miR-510 and miR-129-3p were significantly downregulated, and that miR-483-5p and miR-miR-449a were significantly upregulated in CCC, compared with HGSC (P<0.05), which was consistent with the microarray results. Kaplan-Meier analysis revealed low expression levels of miR-510 and low expression levels of miR-129-3p, advanced International Federation of Gynecology and Obstetrics (FIGO) stage, lymphatic metastasis and that HGSC was significantly associated with the poorer overall survival rates (P<0.05). The expression of miR-510 was significantly higher in the LGSC and CCC tissues, compared with the HGSC and normal ovarian tissues. The results of the present study suggested that different subtypes of EOC have specific miRNA signatures, and that miR-510 may be involved differently in HGSC and CCC. Thus, miR-510 and miR-129-3p may be considered as potential novel candidate clinical biomarkers for predicting the outcome of EOC.

Expression and activation of EGFR and STAT3 during the multistage carcinogenesis of intrahepatic cholangiocarcinoma induced by 3′-methyl-4 dimethylaminoazobenzene in rats

The purpose of this study was to investigate whether the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription-3 (STAT3) signal pathway contributes to the carcinogenesis of intrahepatic cholangiocarcinoma (ICC) induced by 3’-methyl-4 dimethylaminoazobenzene (3’Me-DAB) in rats. EGFR, TGFα, STAT3 and p-STAT3 in different stages of carcinogenesis were detected by immunohistochemistry (IHC). In situ hybridization (ISH) was applied to investigate the expression of STAT3 mRNA. Oval cells were verified by the immunohistochemical staining of alpha-fetoprotein (AFP), CD133 and epithelial cell adhesion molecules (EpCAM). Sequential development of necrosis, oval cell proliferation, cholangiofibrosis (CF) and ICC was observed in the liver of rats administered 3’Me-DAB. Oval cells showed positive expression of AFP, CD133 and EpCAM. The expression of EGFR was significantly higher in the ICC than in oval cells, CF or normal bile ducts (p<0.05), but there was no difference in EGFR expression between the other groups. The highest expression of p-STAT3 and TGFα was observed in CF. The expression of these two molecules in the ICC and oval cells was significantly higher than in normal bile ducts (p<0.05). Elevation of STAT3 mRNA was detected during carcinogenesis as shown by ISH, strong intensity was observed in the ICC and moderate intensity was observed in oval cells and CF. These observations suggest that the EGFR and STAT3 signal pathway contributes to the carcinogenesis of ICC. High activity of STAT3 during the carcinogenesis of ICC may be the result of high activity of EGFR triggered by TGFα.

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: Applications in clinical and basic research

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.

Abstract 4615: Key miRNAs and co-targets in patients of OPSC and OCC

Objectives: To identify the key microRNAs (miRNAs) and their top co-targets in patients of elderly ovarian papillary serous carcinoma (OPSC) patients with advanced stage and ovarian clear cell carcinoma (OCC), for the insight of possible regulation gene and pathway of epithelial ovarian cancer (EOC) prognosis. Methods: The study was opened to patients with primary, previously untreated EOC referred to member institutions. Pathologic diagnosis was confirmed by central review. High-throughput analysis of the miRNA profile in a panel of OCC and OPSC cells was assessed using a microarray platform. The miRNA-targets and possible pathway were predicted by TargetScans, MicroCosm Targets version 5, miRBase and Ingenuity. Finally, the downstream predicated co-target, activating transcription factor 3 (ATF3) was validated by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and immunohistochemical (IHC) assay. Results: Of the 667 miRNAs analyzed from human, we identified 83 as enriched in the OPSC, and 15 in OCC (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4615. doi:1538-7445.AM2012-4615

Abstract C154: microRNA and targets in the poor prognosis of ovarian cancer patients.

Epithelial ovarian cancer (EOC) is the most important cause of gynecologic malignancy-related mortality in women. Ovarian clear cell carcinoma (CC) has a poor survival rates and ovarian papillary serous carcinoma (PSC) has a high incidence in the EOC. To get the insight of possible regulator and pathway that may involved in the EOC prognosis and progress, patient samples of CC, PSC and normal ovarian or oviduct tissues were employed in the study. Total 48 human tumor samples and 6 normal ovarian and oviduct tissues were obtained from the patients who were surgically treated for ovarian cancer or other disease at the Obstetrics and Gynecology Hospital, Dalian China. The tissue was further fixed by formalin and embedded by paraffin as routine. Total RNA that enriched miRNAs was extracted from the FFPE tissue by using the Ambion mirVana microRNA isolation kit (Ambion, Austin TX). The quality of total RNA was assessed using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara CA). Quantitative Real-Time polymerase chain reaction (qRT-PCR) assays were formatted into a TaqMan low-density array (TLDA; Applied Biosystems), which was performed at the Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services, Dana Farber Cancer Institute, Harvard Clinic and Translational Science Center. The normalized microarray data were managed and analyzed by Statminer version 3.0. The targets of miRNA were predicted by using computational approaches, the MicroCosmTargets version 5 and miRBase. The pathway was analyzed by commercial available Ingenuity software. Finally the target predicted was validated by qRT-PCR and imunohistochemistry. The results showed that 101 miRNAs are differentially expressed in the patients of CC compared with PSC, of which 86 (85%) miRNAs were down-regulated in CC patients, suggesting a majority of regulators in the poor prognosis of CC are tumor suppressors. The key miRNAs that were down-expressed in CC patients are miR-9, miR-202, miR-488, miR-509–5p and up-expressed are miR-217, and miR-550. Hundred of gene targets and protein target on the pathways were predicted, but the top targets are enhancer of polycomb homolog 1 (EPC1) and mycophyta (c-MYC), which were validated by qRT-PCR assay at mRNA level and confirmed by immunohistochemistry at molecules level. We identified the key miRNAs that differentially expressed in patients of CC and PSC tumors, predicted and validated the most popular targets regulated by the key miRNAs, indicating the possible regulation gene and pathway for EOC prognosis. This work was supported by the Ministry of Science and Technology of China, Program No. 2008DFA30720 to Shiying Cui. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C154.