The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform
Introduction
In 2008, it is expected that 20,180 women will be diagnosed with ovarian cancer and 15,310 will succumb to the disease [1]. Ovarian cancer is a devastating illness in which only 20% of patients are diagnosed with stage I disease [2]. The poor prognosis associated with ovarian cancer is multi-factorial; a lack of minimally invasive, early detection tests, subtle symptom development and tumor chemo-resistance. Even with the advent of chemo-resistance assays it is still difficult to predict drug resistance and only 10–15% of patients will remain in prolonged remission after initial cytotoxic therapy.
While annual pelvic examination is widely practiced, it lacks the sensitivity to be used a screening strategy for ovarian cancer [3]. Women at high risk for ovarian cancer may typically undergo screening with trans-vaginal ultrasound and serum CA-125. CA-125, however, remains a poor marker for early stage disease with a documented sensitivity of 40% [4], [5]. Additionally, it has been shown that even in a high-risk, screened population, incident cases are still more likely to be advanced stage [6]. The identification of biomarkers that may assist in treatment planning and prediction of chemotherapy outcomes is highly desirable in this population of patients.
There is emerging research about the role of microRNAs in a variety of pathologic conditions; including both solid and hematologic malignancies. MicroRNAs (miRNAs) are small, 22–25 nucleotide non-coding sequences of RNA. These sequences control gene expression either by translational repression or degradation of the messenger RNA transcript after targeting the 3′UTR. Early studies with Caenorhabditis elegans showed that a great number of these sequences are highly conserved across all species, demonstrating the important roles that miRNAs play in cellular differentiation, proliferation and cell cycle control [7]. It is now recognized that miRNAs are frequently de-regulated in malignancy. Under-expressed miRNAs such as let-7 in lung cancer and mirs-15/16 in leukemia, are tumor suppressor genes, suppressing Ras and BCL2 respectively [8], [9]. Over-expressed miRNAs such as mir-21 and the cluster mir-17–92, are oncogenes (oncomirs), targeting tumor suppressors PTEN and E2F1 in solid and hematologic malignancies respectively [10], [11]. While miRNA research in gynecologic malignancies is in its infancy, the miRNA signature profile of ovarian cancer has recently been published [12], [13], [14].
The diagnostic and prognostic utility of circulating RNAs in both benign and malignant conditions has recently been revealed. Placental-associated circulating miRNAs correlate with pregnancy progression [15]. In malignant states, circulating mRNAs in renal cell carcinoma patients [16] as well as miRNAs from the serum of patients with diffuse large B cell lymphoma [17] have been shown to be stable and highly predictive of malignancy as well as survival. Recently, it has been demonstrated that the miRNA signature of circulating tumor exosomes of ovarian cancer patients demonstrates high correlation with miRNA expression of the primary tumor [18]. Ovarian cancer remains a disease for which improved non-invasive, serum screening tests are highly desirable. We offer a description of miRNA extraction from the serum of ovarian cancer patients, the differential expression of a number of these miRNAs between patients and healthy controls as well as a novel real-time PCR microarray detection method.
Section snippets
Methods
Following approval from the Institutional Review Board of The Ohio State University College of Medicine we analyzed serum samples from 28 patients with newly diagnosed ovarian cancer and 15 normal controls. These serum samples were collected at the time of initial consultation, prior to definitive surgical management and/or adjuvant therapy. The serum was obtained as part of a prospective tissue and serum procurement study and was stored at − 80 °C. Fresh serum was obtained from 15 healthy women
Results
Twenty-eight patients with epithelial ovarian cancer were included in this study. Stage breakdown was as follows: stage I-8 (28.5%), stage II-2 (7.1%), stage III-8 (28.5%) stage IV-10 (35.7%). Histologic breakdown was as follows: serous (60%), clear cell (21.2%), endometrioid (12%), mucinous (6%). Median age was 57 years (age range 34–79). Similar to most groups with ovarian cancer, the majority (66%) had stage III or IV disease, and was predominately (60%) serous histology.
Primary miRNA
Discussion
We demonstrate that the extraction of RNA and identification of miRNAs from the serum of individuals diagnosed with ovarian cancer is feasible. We offer the first description of using a real-time PCR, microarray platform to screen large numbers of miRNAs while minimizing the amount of RNA needed. Additionally, we provide evidence that miRNAs may potentially serve as early detection biomarkers in patients with normal CA-125.
With our approach we were able to create a profile that was subsequently
Conflict of interest statement
The authors have no conflicts of interest to declare.
Acknowledgment
This work was supported by the Jacquelyn L. Wells Endowment in Ovarian Cancer Research to D.E.C. and The Ohio State University Targets and Investments in Excellence Award to C.C.
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