Serum microRNA-92 expression in patients with ovarian epithelial carcinoma

Abstract

Objectives

Studies have reported the significance of circulating microRNAs (miRNAs) as biochemical markers of cancer. Few reports demonstrate the significance of miRNAs in the serum of patients with ovarian epithelial carcinoma (EOC). The present study prospectively investigated the status of miRNA-92 (miR-92) in the serum of EOC patients and healthy controls, and examined the potential usefulness of this molecule as a biomarker for EOC diagnosis.

Methods

Levels of miR-92 in serum samples from EOC patients and controls were measured by quantitative real time reverse transcription–polymerase chain reaction (RT–PCR). Associations between serum miR-92 concentrations and clinical parameters were assessed by Fisher’s exact probability test.

Results

The serum concentration of miR-92 in EOC patients (n = 50) was significantly higher than in controls (n = 50). There were significant correlations between miR-92 expression and regional lymph node involvement and clinical stage of the tumour. There was no significant association between expression of miR-92 and age.

Conclusion

The detection of miR-92 levels in the serum might serve as a new tumour biomarker in the diagnosis and assessment of prognosis of EOC.

Introduction

Ovarian cancer, particularly epithelial ovarian cancer (EOC), which accounts for 90% of all ovarian cancers, continues to be the leading cause of death among gynaecological malignancies. ¹ Due to the asymptomatic nature of early stages of EOC, over two-thirds of cases are not diagnosed until the disease has spread beyond the ovaries. ² Thus, early detection of the disease is essential to improve the survival rate in patients with EOC. As this might be facilitated by identifying novel biomarkers of EOC that enable early tumour detection, the identification of such biomarkers is urgently needed. ³

MicroRNAs (miRNAs) are a class of small (18–24 nucleotide) noncoding RNAs that regulate a variety of cellular processes such as cell differentiation, cycling and apoptosis.^4–6 The expression profiles of miRNA differ between normal and tumour tissues.^7–10 For example, the expression of miRNA-21 in EOC is significantly higher than in benign ovarian tumours and normal ovarian tissue. ¹¹ Circulating biomarkers are a promising means of diagnosis, as serum and plasma samples are easily obtainable. Related studies have shown that human serum contains miRNAs and that their expression patterns can potentially be used to identify different types of cancer.^12–14

To the authors’ knowledge, there are few reports determining whether circulating miRNAs exist in the serum/plasma of patients with EOC. Thus, the present study investigated serum levels of miR-92 in patients with EOC and in healthy controls, and evaluated the relationship between miR-92 status and clinical characteristics of EOC.

Patients and methods

Study population

This was a prospective case-control study carried out in Department of Obstetrics and Gynaecology, The Second Hospital, Jilin University, Changchun, China, between December 2010 and November 2012. This study included consecutive patients diagnosed with EOC and healthy controls. Control subjects were recruited from among individuals who sought a routine health check-up at the Physical Health Examination Centre of Jilin University; the control group was not matched with the patients with EOC, in terms of demographic characteristics. There were no specific exclusion criteria for either the patients or the healthy controls. Clinical and pathological information was collected for the patients with EOC. Tumours were staged according to the tumour–node– metastasis (TNM) staging system of the International Union against Cancer (UICC). ¹⁵

Patients and healthy controls provided informed consent and agreed to participate in the study, which was performed according to protocols approved by the Ethics Committee of The Second Hospital, Jilin University.

Serum collection

Blood samples from EOC patients and healthy controls were collected according to protocols approved by Jilin University. A 5-ml sample of peripheral venous blood was drawn from all study participants after an overnight fast and placed at room temperature for 60 min. Then the blood samples were centrifuged at 1000 g for 10 min at 4℃ to spin down the blood cells. The supernatants (serum) was then transferred into fresh tubes and stored at −80℃ until use.

miRNA extraction

The isolation of miRNA from serum samples was performed with the miRNeasy™ RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, with minor modifications. ¹⁶ Briefly, 200 µl of serum was supplemented with 10 µl of 0.05 µmol/l synthetic nonhuman miRNA (Caenorhabditis elegans miR-54, Takara Bio, Shiga, Japan) as controls, providing an internal reference for normalization of technical variations between samples. After 1 ml of QIAzol® Lysis Reagent (Qiagen) was added and well mixed (by gentle vortexing), the samples were incubated at room temperature for 5 min. Aqueous and organic phase separation was achieved by the addition of chloroform. The aqueous phase was applied to an RNeasy™ spin column and RNeasy™ MinElute™ spin column (Takara Bio). The microRNA was eluted from the column with nuclease-free water.

Quantitative real-time RT–PCR analysis

To carry out quantitative real time reverse transcription–polymerase chain reaction (RT–PCR) of serum miR-92 levels, cDNA was synthesized from miRNA with a Mir-X™ miRNA First Strand Synthesis and SYBR® green quantitative RT-PCR Kit (Takara Bio) according to the manufacturer’s instructions. Quantitative real-time RT–PCR was carried out using the Thermal Cycler Dice TP800 (Takara Bio) with primers and cDNA templates mixed with the SYBR® premix. The primer sequence of the miR-92 was designed based on miRBase (http://www.mirbase.org): 5′-TATTGCACTTGTCCCGGCCTG-3′. This primer was prevalidated to generate single amplicons. DNA was amplified for 50 cycles of denaturation for 5 s at 95℃ and annealing for 20 s at 60℃. Analysis of relative miRNA and mRNA expression was performed using the ΔΔC_T method, ¹⁷ with miR-16 as endogenous controls, following the manufacturer’s guidelines. Data analysis was performed using ABI Prism® 7300 SDS software (Applied Biosystems, Foster City, CA, USA).

Statistical analyses

Statistical analyses were performed with SPSS® software, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows®. An independent-samples t-test was used to compare differences in continuous data between the two groups. A receiver operating characteristic (ROC) curve was drawn to evaluate the diagnosis value of plasma miR21 for EOC, and Fisher’s exact probability test was used to compare differences in categorical data. A P-value < 0.05 was considered to be statistically significant.

Results

A total of 50 patients with EOC (mean ± SD age, 54.5 ± 2.4 years) and 50 healthy controls (mean ± SD age, 51.4 ± 3.2 years) were included. Using miR-16 to normalize samples, serum levels of miR-92 were shown to be significantly higher in the patients than in the healthy controls (P < 0.05) (Figure 1).

Figure 1.

Relative expression of microRNA (miR)-92, detected using quantitative real time reverse transcription–polymerase chain reaction in serum from patients with epithelial ovarian cancer (EOC) and healthy controls (control). The relative amount of miR-92 was calculated using the 2^{−ΔΔ C} _T method. ¹⁷ Data points are values for individual patients or controls; heavy black bars represent mean values. *P < 0.05 for EOC group versus control group, independent-samples t-test.

The ROC curve analysis revealed that serum miR-92 was a valuable biomarker for differentiating between patients with EOC and healthy controls, with an area under the ROC curve (AUC) of 0.803 (95% CI: 0.735, 0.871) (Figure 2).

Figure 2.

Receiver operating characteristics (ROC) curve analysis using serum microRNA (miR)-92 levels to discriminate patients with epithelial ovarian cancer (EOC) from healthy controls. Serum miR-92 yielded an area under the ROC curve (AUC) of 0.803 (95% confidence intervals: 0.735, 0.871) with 80.7% sensitivity and 75% specificity.

Analyses of the relationships between serum miR-92 levels and various clinical and pathological characteristics (such as patient age, presence of lymph node metastasis and clinical stage) showed significantly higher miR-92 levels in patients with regional lymph node involvement than in those with no lymph node involvement ,and in patients with clinical stage III–IV disease compared with those with clinical stage I–II disease (P < 0.05; Table 1). ¹⁵ There was no association between serum miR-92 levels and age.

Table 1.

Correlations between the relative expression of microRNA (miR)-92 levels in serum, detected using quantitative real time reverse transcription–polymerase chain reaction in patients with epithelial ovarian cancer (EOC), and clinical and pathological characteristics of EOC.

Characteristic	n	Relative miR-92 expression a	Statistical significance b
Mean age, years
<45	20	3.478 ± 0.530	NS
≥45	30	3.675 ± 0.648
Regional lymph node involvement
No	26	3.187 ± 0.496	P < 0.001
Yes	24	5.246 ± 0.892
Clinical stage 15
I–II	35	3.342 ± 0.219	P < 0.001
III–IV	15	5.769 ± 1.102

Data presented as mean ± SD.

a

Relative amount of miR-92 calculated using 2^{−ΔΔ C} _T method. ¹⁷

b

Independent-samples t-test. NS, not statistically significant (P ≥ 0.05).

Discussion

Some miRNAs have been reported to play important roles in vivo, such as in cancer development, angiogenesis and the immune response.^18,19 Additionally, certain unique patterns of microRNA expression have been associated with prognostic factors and disease progression in several cancers.^20–22 Thus, it was speculated that the serum miRNA level may also be a marker for EOC.

The present study showed that serum levels of miR-92 level were upregulated in patients with EOC compared with healthy controls, and may be useful in discriminating between such patients and healthy controls. Furthermore, in our study, overexpression of miR-92 was closely correlated with advanced clinical stage and lymph node metastasis, which are the main prognostic factors for ovarian cancer. ² Such findings imply that miR-92 might be involved in the development and metastasis of cancer, and might have prognostic significance in EOC. This is consistent with studies showing that microRNA can act as biomarker in the diagnosis of ovarian cancer.^23,24

A limitation of the present study was the small number of patients examined. Further studies, with larger sample numbers, are needed to confirm whether miR-92 can be used as a prognostic marker for EOC.

In conclusion, higher concentrations of miR-92 were observed in serum from patients with EOC compared with healthy controls, and miR-92 levels increased with lymph node involvement and clinical stage of EOC. miR-92 could therefore be a useful biomarker for diagnosis and assessment of disease progression in EOC.