Serum miR-638 Combined with Squamous Cell Carcinoma-Related Antigen as Potential Screening Biomarkers for Cervical Squamous Cell Carcinoma

Abstract

Aims:

Cervical cancer is the second most common cause of cancer-related deaths in developing nations. Human papillomavirus prophylactic vaccines are not widely available, and there are shortages of gynecologists and cytologists in the already overburdened health care systems. The aim of this study was to identify circulating microRNAs (miRNAs) that could be used as feasible screening tests for cervical cancer in low-resource regions.

Materials and Methods:

Serum expression levels of five miRNAs were measured and validated by quantitative real-time polymerase chain reaction in cervical squamous cell carcinoma (CSCC) patients, cervical intraepithelial neoplasia patients, and healthy individuals. Squamous cell carcinoma-related antigen (SCC-Ag) was also measured in the serum.

Results:

Serum miR-638, miR-203a-3p, miR-1914-5p, and miR-521 levels were downregulated in the CSCC group (p < 0.05). Receiver operating characteristic (ROC) curve analysis indicated that the area under the ROC curve (AUC) values for miR-638 and miR-521 were 0.734 and 0.742, respectively, for discriminating CSCC patients from healthy controls. Furthermore, the combined use of miR-638 and SCC-Ag yielded the best screening performance and increased the AUC value, sensitivity, and specificity to 0.956, 94.87%, and 80.00%, respectively.

Conclusion:

This study suggested that miR-638 and miR-521 have independent screening value and that the combined measurement of miR-638 and SCC-Ag resulted in a better ability to discriminate patients with CSCC from healthy individuals.

Introduction

Cervical cancer is the fourth most common cancer in women, with an estimated 527,600 new cases and 265,700 deaths worldwide in 2012 (Torre et al., 2015). Approximately 85% of the global burden occurs in developing regions, where cervical cancer accounts for almost 12% of all cancers in females (Ferlay et al., 2015). In developing countries such as China, cervical cancer remains a critical problem that threatens the health of women (Shao-Ming and You-Lin, 2015). Compared with urban patients, rural patients have a higher mean age at diagnosis and a lower 5-year overall survival rate in China (Shuang et al., 2013). More than 80% of primary cervical cancers are cervical squamous cell carcinoma (CSCC), most of which contain high-risk human papillomavirus (hr-HPV) DNA (De Sanjose et al., 2010; Li et al., 2013). Cervical intraepithelial neoplasia (CIN) is a premalignant lesion with three stages, CIN1, CIN2, or CIN3, and can progress to cervical cancer if left untreated (WHO, 2013).

Currently, the sensitivity and diagnostic accuracy of cytology-based screening programs can be unsatisfactory due to sampling or interpretation errors (Hee Jung et al., 2010). HPV DNA testing has been considered a promising tool for cervical cancer screening; however, the high cost of the existing HPV DNA test has precluded its use in large-scale screening programs in developing countries with weak, under-resourced health systems (Shi et al., 2011). Squamous cell carcinoma-related antigen (SCC-Ag) is currently used as a tumor marker of cervical cancer, and it is associated with the recurrence and survival of CSCC (Charakorn et al., 2018). However, SCC-Ag is not organ-specific or malignancy-specific, and it is also elevated in the serum of patients with head and neck (Jantharapattana et al., 2018), oral (Yang et al., 2016), and lung cancers (Kulpa et al., 2000). There is uncertainty about the best strategy for the screening of cervical cancer, especially in low-resource settings (Smith et al., 2018).

MicroRNAs (miRNAs) are short noncoding RNAs that influence numerous cancer-related processes, such as cell proliferation, cell cycle control, apoptosis, differentiation, migration, and metabolism (Jansson and Lund, 2012). Recent studies have reported that miRNAs are present in human serum and plasma in a remarkably stable form, and the expression levels can be easily measured by quantitative polymerase chain reaction (qPCR) or miRNA microarrays (Calin and Croce, 2006; Mitchell et al., 2008). miRNAs are differentially expressed in human cancers (Reddy, 2015), and evidence has shown that there is an association between miRNA expression and cervical cancer-related processes (Gonzalez-Quintana et al., 2016). Moreover, miRNA also has predictive value for distinguishing cervical cancer patients from healthy individuals (You et al., 2015). The finding suggests that miRNAs may serve as novel diagnostic or screening biomarkers due to the different expression levels of miRNAs in the blood in cancer patients and healthy individuals (Volinia et al., 2006).

In this study, five candidate miRNAs, which included miR-638, miR-203-3p, miR-1914-5p, miR-296-5p, and miR-521, were evaluated to determine whether they were related to cervical cancer in southern Chinese women. miR-203a-3p and miR-296-5p were identified in microarray-based miRNA expression profiles that compared three pairs of cervical cancer tissues and normal tissues (unpublished data). These expression levels were downregulated in cervical cancer tissues (for miR-203a-3p, fold change [FC] = 0.39, p < 0.001; for miR-296-5p, FC = 0.43, p < 0.001). Additionally, it was reported that miR-638, miR-521, and miR-1914-5p contribute to the risk of tumor development and tumor progression (Sohn and Jo, 2010; Tan et al., 2014; Hu et al., 2015). The purpose of this study was to investigate miRNA expression levels in patients with CSCC, patients with CIN, and healthy controls and to evaluate the clinical value of these miRNAs as early screening biomarkers in a southern Chinese population.

Materials and Methods

Study population and serum collection

A total of 121 southern Chinese women were recruited in our study, including 40 patients with CSCC, 40 patients with CIN, and 41 healthy individuals. The patients with CSCC were diagnosed at the Sun Yat-Sen Memorial Hospital from 2017 to 2018. Exfoliated cervical cells from vaginal swabs were collected by a cyto-brush (Qiagen, Valencia, CA) and preserved in 2.5 mL of denaturation buffer. ThinPrep 2000 (Hologic, Inc.) and SurePath liquid-based Pap tests (BD) were used for the ThinPrep cytological test. The cytological smear was read by three clinical cytopathologists; subjects with normal cytological smears who were free of any cervical pathology were recruited as normal controls, and any abnormalities observed were defined as cervical intraepithelial lesions.

A 5 mL peripheral venous blood sample was obtained from each participant by venous puncture with an ordinary EDTA-free tube and stored at 4°C before transport to our laboratory at Jinan University. Peripheral blood was centrifuged at 2000 rpm for 10 min, and the serum layer was carefully removed and then stored at −80°C. Serum samples were collected for the measurement of miRNAs and SCC-Ag.

miRNA extraction, reverse transcription, and qPCR

miRNAs were isolated from the serum samples using an miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Mature miRNAs were reverse-transcribed, and qPCR was performed using an All-in-One miRNA qRT-PCR Detection Kit according to the manufacturer's protocol (GeneCopoeia, Maryland). Reverse transcription reaction mixtures were incubated at 37°C for 60 min and then at 85°C for 5 min before being maintained at 4°C. A quantitative real-time (qRT)-PCR assay was performed using a CFX96 Real-Time PCR System Detector (Bio-Rad, Hercules, CA). miR-39-3p from Caenorhabditis elegans (cel-miR-39-3p) was added to the sample as a reference for miRNA measurements. The FCs were calculated using the 2^−ΔCt method (Livak and Schmittgen, 2000).

Detection of SCC-Ag

SCC-Ag, a clinical diagnostic tumor marker, was measured by KingMed Diagnostics (Guangzhou) using a chemiluminescence microparticle immunoassay (Abbott Architect i2000). An upper limit of 1.5 ng/mL for SCC-Ag was used to define the normal value, as recommended by the manufacturers.

Statistical analysis

Statistical analysis was performed using SPSS 20.0 software (IBM Corporation, New York), GraphPad Prism software version 5.02 (GraphPad, California), and MedCalc 9.0 software (MedCalc Software, Inc., Ostend, Belgium). Data are presented as the mean ± standard error of the mean. The Kruskal-Wallis test was used to compare the miRNA expression levels among CSCC patients, CIN patients, and healthy controls, and the Nemenyi test was used for further multiple comparisons. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of each miRNA and SCC-Ag measurement, and the area under the curve (AUC) was calculated to measure the discriminatory capacity (Hanley and McNeil, 1982). Youden's index (sensitivity + specificity −1) was used to determine the optimal cutoff values for miRNA levels (Schisterman et al., 2005). Logistic regression models were generated by SPSS, and the resulting linear predictor score, which is an index that combines measurements of the independent markers, was evaluated for its differentiating power by ROC analysis (Imbert-Bismut, 2001). The associations between markers and clinical characteristics were analyzed by χ² tests. All statistical tests were two-sided, and a p-value <0.05 was considered statistically significant.

Results

Characteristics of the subjects

The clinical characteristics of the 121 enrolled subjects are listed in Table 1. The mean age of all subjects was 45.5 ± 0.84 years, and the ages ranged between 25 and 70 years. Among the enrolled subjects, no one had a family history of cervical cancer. No significant differences were observed in age, body mass index, or other physical characteristics among CSCC patients, CIN patients, and healthy individuals.

Table 1.

Characteristics of Subjects with Cervical Squamous Cell Carcinoma, Subjects with Cervical Intraepithelial Neoplasia, and Healthy Controls

Characteristic	CSCC (n = 40)	CIN (n = 40)	HCs (n = 41)
Age, years	50.39 ± 1.62	43.88 ± 1.30	42.71 ± 1.17
BMI	23.66 ± 4.54	22.28 ± 0.60	22.26 ± 0.67
The age of menarche, years	14.79 ± 1.60	15.30 ± 1.86	15.29 ± 1.87
The age of first sexual life, years	23.44 ± 2.32	22.38 ± 0.47	21.88 ± 0.42
The age of first pregnancy, years	22.56 ± 0.85	23.55 ± 0.44	23.54 ± 0.61
Number of pregnancies
0	2	0	0
1-2	8	11	17
>2	30	29	24
FIGO stage, n (%)
I B1-I B2	26 (65.0)	—	—
II A1-II B2	12 (30.0)	—	—
Missing	2 (5.0)
Tumor size, cm, n (%)
≤4	28 (70.0)	—	—
>4	6 (15.0)	—	—
Missing	6 (15.0)
Metastasis, n (%)
Yes	9 (22.5)	—	—
No	25 (62.4)	—	—
Missing	6 (15.0)
SCC-Ag	5.65 ± 0.7	—	0.99 ± 0.06

—, not applicable; BMI, body mass index; CIN, cervical intraepithelial neoplasia; CSCC, cervical squamous cell carcinoma; FIGO, International Federation of Gynecology and Obstetrics; HCs, healthy controls; SCC-Ag, squamous cell carcinoma-related antigen.

Serum miRNA expression of the subjects

To investigate the serum miRNA levels in subjects, we performed qPCR assays with candidate miRNAs in CSCC patients, CIN patients, and healthy controls. There were significant differences in the serum levels of miR-638 (p < 0.001), miR-203a-3p (p = 0.03), miR-1914-5p (p = 0.01), and miR-521 (p < 0.001) among the three groups. However, no evidence was found suggesting that miR-296-5p level was significantly different among the three groups (p = 0.05).

As shown in Figure 1, the levels of four of the miRNAs, namely miR-638, miR-203a-3p, miR-1914-5p, and miR-521, were found to be markedly decreased in the CSCC group compared with the CIN group. In addition, the relative expression levels of miR-638, miR-521, and miR-203a-3p were low in patients with CSCC compared with the levels in the healthy controls (p < 0.05, Fig. 1).

FIG. 1.

The relative expression of miR-638, miR-521, miR-203-3p, miR-1914-5p, and miR-296 in patients with CSCC, patients with CIN, and healthy controls. *p < 0.05, **p < 0.01, ***p < 0.001. CIN, cervical intraepithelial neoplasia; CSCC, cervical squamous cell carcinoma.

Discrimination capability of the miRNAs and SCC-Ag

ROC curves for each miRNA were generated to evaluate their potential value for screening, and the AUC, sensitivity, and specificity for each miRNA are presented in Tables 2 and 3. For single miRNAs, ROC curve analysis indicated that miR-1914-5p and miR-521 could be used to differentiate the CSCC group from the CIN group, with AUC values of 0.717 and 0.716, respectively (p < 0.01, Table 2). Serum miR-638 and miR-521 had AUC values of 0.737 and 0.742, respectively, when used to differentiate CSCC patients and healthy controls (p < 0.001, Table 3). However, the five miRNAs had poor screening capabilities when used to distinguish CIN patients from healthy controls, and all AUC values for these miRNAs were <0.70 (data not shown). Therefore, we evaluated the expression level of SCC-Ag in CSCC patients and healthy controls. The AUC value of SCC-Ag was 0.872 for differentiating between CSCC patients and healthy controls when the cutoff value was 1.50. No significant differences were found in the AUC values for miR-638, miR-521, and SCC-Ag (p > 0.05).

Table 2.

The Power of microRNAs for Differentiating Patients with Cervical Squamous Cell Carcinoma and Patients with Cervical Intraepithelial Neoplasia

	AUC	Cutoff value	Sensitivity (%)	Specificity (%)	p
miR-638	0.681	≤0.93	85.00	46.15	0.005^**
miR-203a-3p	0.651	≤0.25	42.50	82.05	0.025^*
miR-1914-5p	0.717	≤0.35	82.50	61.54	<0.001 ^***
miR-296-5p	0.644	≤0.03	84.62	52.63	0.018^*
miR-521	0.716	≤0.39	62.50	76.92	0.001 ^**

^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Bold represents that the AUC value of miRNAs was over 0.70.

AUC, area under the curve.

Table 3.

The Power of microRNAs and Squamous Cell Carcinoma-Related Antigen for Differentiating Cervical Intraepithelial Neoplasia Patients from Healthy Controls

	AUC	Cutoff value	Sensitivity (%)	Specificity (%)	p
miR-638	0.734	≤0.82	80.00	60.98	<0.001^***
miR-203a-3p	0.660	≤0.70	75.00	53.66	0.008^**
miR-1914-5p	0.626	≤0.30	85.00	43.90	0.044^*
miR-296-5p	0.623	≤0.02	71.79	58.54	0.043^*
miR-521	0.742	≤0.74	80.00	65.85	<0.001^***
SCC-Ag	0.872	>1.50	74.36	92.68	<0.001^***

^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Bold represents that the AUC value of miRNAs was over 0.70.

Next, we performed ROC analysis for the combinations of SCC-Ag and miRNAs with AUC values over 0.70 for differentiating CSCC patients and healthy individuals (Fig. 2). Different combinations of miR-638, miR-521, and SCC-Ag, according to a linear predictor score calculated by logistic regression models, were used to determine their differentiation capabilities. The results indicated that, compared with single markers and other two-marker combinations, a two-parameter combination (miR-638 + SCC-Ag) improved the ability to differentiate between CSCC patients and healthy controls (AUC = 0.956, 95% confidence interval [CI]: 0.888-0.990, Fig. 2). A cutoff value of >0.20 for the predictor score was used for the combination of miR-638 and SCC-Ag, with a sensitivity and specificity of 94.87% and 80.00%, respectively. Furthermore, a three-parameter combination (miR-638 + miR-521 + SCC-Ag) resulted in an AUC, sensitivity, and specificity of 0.958, 87.18%, and 92.50%, respectively (AUC = 0.958, 95% CI: 0.891-0.991, Fig. 2). This AUC was not markedly different from that for the combination of miR-638 and SCC-Ag (p = 0.57). Collectively, the results suggested that the combination of miR-638 and SCC-Ag has high sensitivity and specificity for the discrimination of CSCC patients from healthy individuals.

FIG. 2.

The ROC curve analysis for discriminant ability between CSCC cases and healthy individuals by the combination of miRNAs and SCC-Ag. miRNAs, microRNAs; ROC, receiver operating characteristic; SCC-Ag, squamous cell carcinoma-related antigen.

Association between serum biomarkers level and clinical characteristics in CSCC patients

CSCC patients were classified into two groups according to their miRNAs expression level. Patients whose miR-638, miR-521, and SCC-Ag levels were higher than the cutoff values were placed in the high group, whereas patients with levels that were lower than the cutoff values were placed in the low group. No association was found between biomarkers level and patient characteristics, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor differentiation, and lymph node metastasis (p > 0.05, Supplementary Table S1).

Discussion

In this study, we evaluated the screening potential of five miRNAs by investigating their serum expression levels in CSCC patients, CIN patients, and healthy controls. Our work suggested that miR-638 and miR-521 are independent biomarkers that can be used to screen for CSCC and that the simultaneous assessment of miR-638 and SCC-Ag results in a significantly better capability to discriminate CSCC patients from healthy individuals.

The search for noninvasive tumor markers in cancer is a rapidly growing area of research (Chen et al., 2008). SCC-Ag is an existing molecular biomarker that is used for clinical cancer diagnoses in China, but it is not a tumor-specific biomarker (Jantharapattana et al., 2018). Aberrations in miRNA expression levels might be linked to the onset and progression of various types of cancer (Ferlay et al., 2015). Circulating miRNAs have received increasing attention as potential novel biomarkers for the early screening and diagnosis of cancer (Kosaka et al., 2010). miRNAs are released in human serum or plasma, are present in a stable and protected form, and are much smaller than that miRNAs in a typical epithelial cell (Mitchell et al., 2008).

miR-638 was shown to be correlated with the progression of several types of tumors (Tan et al., 2014; Yu et al., 2017), whereas its potential value as a screening biomarker for CSCC has not been further investigated. Similarly, some studies have shown that miR-521 may be related to ovarian (Delfino and Rodriguez-Zas, 2013) and prostate cancers (Josson et al., 2008), but its involvement in cervical cancer has not been reported. Our work suggested that miR-638 has significant value for discriminating patients with CSCC and healthy controls, whereas miR-521 can be used to discriminate among CSCC patients, CIN patients, and healthy individuals. In addition, the combination of miR-638 and SCC-Ag significantly improved the differentiation capability compared with those of the individual biomarkers. The three-marker combination, miR-638 + miR-521 + SCC-Ag, achieved similar screening power compared with the combination of miR-638 + SCC-Ag. Considering cost-effectiveness, a two-marker combination might be a better option for cervical cancer screening tests. Furthermore, detection based on serum measurements is more convenient and readily accepted and less invasive, which makes it ideal for the early detection of cancer (Stefanescu et al., 2018). Therefore, miR-521 and miR-638 might be regarded as potential screening biomarkers for cervical cancer. Further studies are needed to validate the mechanism underlying the involvement of miR-521 and miR-638 in cervical cancer and to determine the screening capability of miR-638 combined with SCC-Ag in participants from a large multicenter population.

Previously, it was reported that the serum level of miR-203 was significantly increased in cervical cancer patients (Zhao et al., 2013), but few studies regarding miR-1914-5p in cervical cancer have been conducted. Lv and Wang (2017) reported that miR-296 was significantly downregulated in cervical cancer tissues and cell lines. In the present study, we found that the expression levels of miR-296-5p were downregulated in cervical cancer tissues compared with normal cervical tissues based on microarrays, but we did not detect a difference in the expression level in the serum. Differences between previous research results differ from our results on miR-203a-3p and miR-296-5p, which might be due to differences in the characteristics of the subjects, in terms of age, clinical stage, or pathological type.

There are also some limitations to our study. The sample size was not large, which may have reduced the statistical power. In addition, all candidate miRNAs in this study had a poor ability to differentiate between CIN patients and healthy controls. Therefore, more samples or other markers are needed to differentiate patients with a cervical precancerous risk from healthy controls.

In conclusion, serum miR-638 combined with SCC-Ag showed high sensitivity and specificity. SCC-Ag detection utilizes mature clinical technology, and miRNA detection in the serum with qRT-PCR is a convenient, accurate, and noninvasive approach to screening. Our work suggested that serum miR-638 combined with SCC-Ag may be valuable supplement markers of screening CSCC in Chinese population.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported in part by Training Program of the Major Research Plan of the National Natural Science Foundation of China (Grant number: 91543132), the National Natural Science Foundation of China (Grant numbers: 81541070, 81101267, and 30901249), Guangdong Natural Science Foundation (Grant numbers: 2018A030313601, 10151063201000036, S2011010002526, and 2016A030313089), Guangdong Province Medical Research Foundation (Grant numbers: A2014374 and A2015310), and Project from Jinan University (Grant numbers: 21612426, 21615426, JNUPHPM2016001, and JNUPHPM2016002).

Supplementary Material

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