MiR-221 represents an innovative bio-marker in cervical carcinoma detection

MiR-221 has been identied to play an important role in tumorigenesis and progression. In the present study, we aimed at to investigate the expression pattern of serum miR-221 and evaluate its diagnostic value in cervical cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression pattern of miR-221 in cervical cancer patients and healthy controls. The association of miR-221 with clinicopathological data was analyzed with χ 2 test. Then receiver operating characteristic (ROC) curve was built to evaluate the diagnostic value of serum miR-221 by calculating the area under the ROC curve (AUC). The results indicated that the miR-221 expression level was statistically elevated in cervical cancer patients compared with healthy individuals. The increased miR-221 expression was signicantly associated with lymph node metastasis (P = 0.026) and FIGO stage (P = 0.028). ROC curve suggested that serum miR-221 had a high diagnostic value in differentiating cervical cancer patients from healthy controls with AUC of 0.932 (95%CI: 0.903–0.960) corresponding with sensitivity of 77.6% and specicity of 94.8%.


Abstract Background
MiR-221 has been identi ed to play an important role in tumorigenesis and progression. In the present study, we aimed at to investigate the expression pattern of serum miR-221 and evaluate its diagnostic value in cervical cancer.

Methods
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression pattern of miR-221 in cervical cancer patients and healthy controls. The association of miR-221 with clinicopathological data was analyzed with χ 2 test. Then receiver operating characteristic (ROC) curve was built to evaluate the diagnostic value of serum miR-221 by calculating the area under the ROC curve (AUC).

Results
The results indicated that the miR-221 expression level was statistically elevated in cervical cancer patients compared with healthy individuals. The increased miR-221 expression was signi cantly associated with lymph node metastasis (P = 0.026) and FIGO stage (P = 0.028). ROC curve suggested that serum miR-221 had a high diagnostic value in differentiating cervical cancer patients from healthy controls with AUC of 0.932 (95%CI: 0.903-0.960) corresponding with sensitivity of 77.6% and speci city of 94.8%.

Conclusions
Taken together, the expression level of miR-221 is increased in cervical carcinoma and it may serve as a promoting bio-marker in the diagnosis of cervical cancer patients.

Background
Cervical cancer is the second most common gynaecological malignancy after breast cancer in the world.
Due to the delayed initial screening about 265,000 deaths from cervical cancer, especially in the developing countries, which representing a healthy threaten for women [1,2]. At present, a series of risk factors have been found to play important roles in the occurrence of cervical cancer, such as life style, hormonal contraceptives, immunosuppression or certain infections, especially human papilloma virus (HPV) [3][4][5][6][7]. However, the morbidity and mortality of cervical cancer are still increasing in the developing countries, and the patients tend to be younger [8]. Screening is the current treatment for early detection of cervical cancer, but those patients who are too young to bene t from screening are usually diagnosed at advanced stage and has poor prognosis. Consequently, identifying novel invasive bio-markers is crucial for early detection of cervical cancer. In recent years, many researches have found that a lot of miRNAs are involved in the initiation and development of cervical cancer.
Aberrantly expression of miRNAs is observed in various cancers that can function as novel biomarkers for diagnosis and potential therapeutic targets [13,14]. As a member of miRNAs, miR-221 was found upregulated in bladder cancer tissues and in uenced T24 cell proliferation and apoptosis [15]. Moreover, the miR-221 was found that modulate proliferation and invasion of cervical cancer cells, but the clinical signi cance of it in the diagnosis of cervical cancer was still unclear [16].
In the present study, we mainly explored the serum expression levels of miR-221 in cervical cancer patients and healthy control and investigated the relationship between miR-221 expression and clinicopathological characteristics of cervical cancer patients. Then the diagnostic value of miR-221 was also estimated.

Patients and specimens
All protocols were approved by the Ethics Committee of the Harrison International Peace Hospital. In total, 125 patients who were diagnosed as cervical cancer before any treatments and 115 healthy control subjects from the Harrison International Peace Hospital, were included in our study. Blood samples from cervical cancer patients and healthy controls were collected during physical examination and then serum was separated and stored at -80℃ for use. The patients with cervical cancer assessment were performed according to histological grade and FIGO staging. The clinical characteristics of the patients were summarized in Table 1. All the participants provided their written informed consent in advance. forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse 5'-AACGCTTCACGAATTTGCGT-3' [18]. The relative expression of miR-221 was calculated and normalized using the 2 −ΔΔCT method relative to U6.

Statistical analysis
Statistical analysis was performed using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as the mean ± standard deviation (SD) with at least three independent experiments. Student's t-test was used to analyze differences between tumor and normal groups, and χ 2 test was used to analyze the correlation between miR-221 expression and clinicopathological factors of cervical cancer patients. Receiver-operating characteristic (ROC) curve and the area under the ROC curve (AUC) were applied to assess the serum miR-221 diagnosis value in cervical cancer. Differences were considered statistically signi cant when P-value less than 0.05 (*P < 0.05 and **P < 0.01).

Results
The expression of miR-221 was increased in cervical cancer We used qRT-PCR analysis to measure the serum miR-221 expression in cervical cancer patients and healthy individuals. As shown in Fig. 1, the expression of serum miR-221 was strongly up-regulated in cervical cancer patients compared to healthy individuals (P < 0.01).
Correlation of miR-221 expression with clinicopathological characteristics of cervical cancer patients To assess to association between miR-221 expression and the clinicopathological parameters, the 125 patients were divided into high-expression and low-expression groups according to the average level of miR-221. Our nding showed that high expression of miR-221 was signi cantly related to lymph node metastasis (P = 0.026) and FIGO stage (P = 0.028), but no relationship was found with other clinicopathological features, including age, tumor size, histological grade and differentiation (all P > 0.05, Table 1).

Diagnostic potential of miR-221 in cervical cancer
To investigate the correlations between the miR-221 dysexpression and diagnosis in cervical cancer patients, we used ROC curve analysis. As shown in Fig. 2, the result showed that miR-221 had a relatively high accuracy in differentiating cervical cancer patients from healthy individuals based on AUC of 0.932 (95%CI: 0.903-0.960 ) with the sensitivity of 77.6% and speci city of 94.8% at the optimal cut-off value of 2.155.

Discussion
Cervical cancer is one of the most common malignancies in women all over the world. Among the risk factors, the infection of HPV, especially the high risk type HPV virus, is the main cause of cervical cancer, and with the longer of carcinogenic infection lasting, showing the greater of risk [19,20]. Most women with early-stage cervical tumors can be cured. Although bene t from the development of early detection and diagnostic technique, cervical cancer having a good prognosis, there are still many patients appeared tissues in ltration and metastasis and the morbidity and mortality of this cancer are still increasing in the developing countries [8]. Thus, the accurate bio-markers are meaningful for the diagnosis and prognosis of cervical cancer.
To date, molecular markers have been identi ed to be involved in the tumorigenesis, development and progression of cancers, including cervical cancer [21]. For instance, Azizmohammadi S et al.
demonstrated that miRNA-145 and miR-9 were both up-regulated in cervical cancer and may be as potential prognostic markers in patients suffering from cervical cancer [22]. SHI et al. suggested that secreted protein acidic and rich in cysteine (SPARC) may be a potential therapeutic option for cervical cancer patients [23]. Liliana Alvarado-Ruiz et al. found that normal cervical cancer from women without cervical lesions expression HOXA9 but controlling HOXA9 expression appears to be a necessary step during cervical cancer development [24]. These data suggested the crucial roles of cancer related molecules in cervical cancers. In the present study, we aimed at to identify a novel accurate diagnostic biomarker for patients with cervical cancer. serum miR-221 expression levels has been indicated that signi cantly higher in patients with cutaneous malignant melanoma (CMM) and it has prognostic value in CMM patients [28]. Previous study has found the aberrant expression of miR-221 in the progression of cervical cancer [16]. But clinical diagnostic value of miR-221 in cervical cancer has not been investigated. In the present study, we sought to assess serum miR-221 expression pattern as well as its diagnostic role in patients with cervical cancer.
In this study, we measured the expression of miR-221 and the association between miR-221 with clinicopathological features in cervical cancer patients. The result of qRT-PCR revealed the expression of miR-221 in cervical cancer was increased compared with healthy control, which suggests that miR-221 may therefore function as a tumor oncogene. The elevated expression of miR-221 was correlated with lymph node metastasis and FIGO stage, which suggested that miR-221 was involved in the development of cervical cancer. Furthermore, the diagnostic value of miR-221 has also been investigated in our study using ROC curve analysis. The high AUC value, sensitivity and speci city values suggested that miR-221 may be a valuable diagnosis biomarker for cervical cancer detection.

Conclusions
In conclusion, serum miR-221 was signi cantly up-regulated in cervical cancer in this study. Moreover, the elevated miR-221 expression might play as a non-invasive diagnostic bio-marker for detection of cervical cancer patients from healthy individuals. Further studies with larger sample sizes are still needed to enhance the accuracy and potential of miR-221 in cervical cancer.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Relative miR-221 expression in cervical cancer patients and healthy controls. The serum expression of miR-221 in cervical cancer patients was signi cantly higher than that in healthy individuals (P < 0.01).