Diagnostic value and clinical significance of serum miR-4534 combined with transvaginal color Doppler ultrasound in cervical cancer

Abstract

Aim

The purpose of the present study was to evaluate the diagnostic performance of serum miR-4534 combined with Transvaginal Color Doppler Ultrasound (TVCDS) in cervical cancer patients.

Methods

Blood samples from 126 patients with cervical cancer and 83 patients with benign uterine lesions were retrospectively analyzed. Quantitative real time polymerase chain reaction (qRT-PCR) was applied to examine the relative abundances of serum miR-4534 in cervical cancer based on a case–control study. Chi-square test was adopted to analyze the association between serum miR-4534 and other clinicopathological factors. The blood flow of cervix was examined using TVCDS, and the blood flow resistance index (RI) of cervix was summarized. Receiver operating characteristic (ROC) curves were plotted to explore the diagnostic capacity of serum miR-4534 combined with blood flow RI. Logistic regression was employed to analyze the risk factors of cervical cancer.

Results

Serum miR-4534 was distinctly increased in the study group compared with the control group (P < 0.05), while blood flow RI was dramatically decreased (P < 0.05). Moreover, increased miR-4534 was closely associated with lymph node metastasis (P = 0.010), FIGO stage (P = 0.007) and HR-HPV (P = 0.025). ROC curves demonstrated that the area under curve (AUC) of serum miR-4534 combined with the blood flow RI was 0.854, with the sensitivity and specificity of 88.9% and 73.5%, respectively, which displayed a better diagnostic capacity than serum miR-4534 and blood flow RI alone. Logistic regression analysis demonstrated that serum miR-4534 (OR = 8.805, 95% CI = 4.124–18.798; P < 0.001) was a risk factor related to the initiation and formation of cervical cancer, as well as blood flow RI (OR = 0.112; 95% CI = 0.054–0.235; P < 0.001).

Conclusion

Serum miR-4534 was highly expressed in cervical cancer, and associated with the development and metastasis of cervical cancer patients. MiR-4534 combined with TVCDS exhibited a considerable biomarker to detect cervical cancer patients.

Introduction

Cervical cancer is the one of the most frequent female malignancies worldwide, with high incidence rate and mortality [1]. Infection with high-risk human papillomavirus (HR-HPV) infection is considered as the number one risk factor for cervical cancer [2]. HR-HPV infection itself is not enough to cause malignant transformation, but also involves other genetic and epigenetic changes, such as smoking, parity, oral contraceptives, and so on [3]. While HPV detection has a high sensitivity rate, its specificity remains poor. This makes it prone to misdiagnosis. The gold standard remains pathological diagnosis, but its application is limited due to the invasive nature of the procedure [4]. Transvaginal color Doppler ultrasound (TVCDS) is a valuable tool for the determination of the location and size of uterine tumors, as well as the assessment of blood flow status. However, its diagnostic accuracy of the technique is subjective and professional, leading to poor sensitivity and specificity [5]. In order to ensure the more effective clinical application, it is essential to combine with other relevant indicators.

MicroRNAs (miRNAs) are typically 21–25 nucleotides in length. As noncoding RNAs, they act as non-coding RNAs that negatively modulate the expression of their target genes at the post-transcriptional level [6]. They have been observed to participate in a number of different developmental processes, including differentiation, inflammation, apoptosis, and cell cycle modulation [7–9]. It has been established that alterations in miRNA expression can contribute to the onset of multiple tumors, including cervical cancer [10]. These miRNAs can promote or inhibit tumors, leading to tumor formation and the development of cancer. These oncogenic miRNAs are not only therapeutic targets but also important biomarkers for the detection and management of cancer [11]. As a less-studied miRNA, miRNA-4534 represents a typical multifunctional miRNA, which is related to the development of tumors, such as prostate cancer [12], cervical cancer [13].

In the present study, we measured the relative abundance of serum miR-4534 in cervical cancer patients and patients with benign uterine lesions, and recorded the blood flow RI of all subjects via TVCDS. Moreover, this study also evaluated the diagnostic accuracy of serum miR-4534 combined with blood flow RI for distinguishing cervical cancer patients from benign uterine lesions individuals.

Materials and methods

Patients and serum samples

One hundred and twenty-six cervical cancer patients pathologically confirmed were selected as the study group from The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen. 83 patients with benign uterine lesions were selected as the control group in this case–control study. Whole blood samples were collected in serum separator tubes from cervical cancer patients and patients with benign uterine lesions before operation. The collected serum was placed at 4 ℃, and centrifuged to remove the residual blood cells. Then, all the serum samples were preserved in − 80 ℃ refrigerator for further analysis.

Criterial for inclusion: postoperative diagnosis confirmed by pathological diagnosis; preoperative TVCDS examination and complete examination data; control group in good mental state with no other acute and chronic infectious diseases. Criterial for extraction: combined with other malignant tumors or severe cardiovascular and cerebrovascular diseases; serious functional abnormalities of the liver, kidneys and other major organs; combined with blood-borne infectious diseases.

All participants have signed informed consents. The Ethics Committee of The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen have reviewed and supported the application for serum samples.

HR-HPV examination

Blood DNA from the subjects was extracted using a whole genome kit, and amplified using the PCR kit. HR-HPV infection was examined using a hybridization kit and a medical nucleic acid molecular rapid hybridizer. Viral load was measured as the ratio of relative light units (RLU) to CutOff (CO), and an RLU/CO ratio ≥ 1.0 was regarded to be positive.

Quantitative real time polymerase chain reaction (QRT-PCR) assay

Total RNA from serum specimens was extracted using TRIzol Reagent (Invitrogen) in accordance with the direction provided by the manufacturer. Quantitative miRNA-4534 of serum samples were analyzed using qRT-PCR through the standard SYBR-Green PCR instructions (Applied Biosystems). RNU6B was employed to be an internal reference for normalization. The relative expression level of miR-4534 was determined using the equation of 2^−△△CT, △CT = CT_miR-4534-CT_{internal reference}. Each rection was done in triplicate.

TVCDS examination

After admission, transverse, longitudinal and oblique multi-directional scanning was performed with a transvaginal probe at a frequency of 5–9 MHz using a color Doppler ultrasound machine (GE Voluson E8). Resistance index (RI) is recorded by pulse Doppler ultrasound, and the sampling line was parallel to the direction of blood flow as far as possible, and the clamping angel kept below 30 degrees, and the average value was calculated from three consecutive measurements.

Statistical analysis

All statistical analysis was carried out using SPSS 23.0 software (IBM, USA) and GraphPad Prism 9.0 (CA, USA). The continuous data was assessed using Student’s t test with the percentage (n, %). Chi-square test was applied to the assessment of categorical data between two groups. The relevance between serum miR-4534 expression and blood flow RI was assessed using Pearson’s correlation analysis. Receiver operating characteristic (ROC) curves were constructed to determine the area under the curve (AUC) to evaluate the diagnostic efficiency of serum miR-4534 and blood flow RI. Logistic regression analysis was adopted to analyze the risk factors affecting the occurrence of cervical cancer. P values were two-tailed and a P value less than 0.05 was recognized to be a statistical significance.

Results

Baseline information of all the subjects

Table 1 indicated basic data of cervical cancer patients and the control group. The average ages were 51.11 ± 3.04 and 50.83 ± 3.87 in cervical cancer patients and control group, respectively. The mean BMI (body mass index) was 22.55 ± 2.39 in cervical cancer patients, while 22.37 ± 2.07 in control group. 15 of 126 (11.90%) cervical patients have smoking history, while 13 of 83 (15.66%) in patients with benign uterine lesions. There were 37 patients with menopausal (29.37%) in 126 cervical cancer patients, while 21 cases (25.30%) with menopausal in 83 control individuals. 10 cases with incidence of other sexually transmitted diseases (STDs) (7.94%) existed in 126 cervical cancer patients, while 6 of 83 (7.23%) in patients with benign uterine lesions. There were no significant association in age, BMI, smoking history, menopausal and STDs (all P > 0.05) between the study group and the control group. The cervical cancer patients with HR-HPV were 31 cases (24.60%), while 9 cases (10.84%) with HR-HPV in patients with benign uterine lesions. There was a dramatic association between the incidence of HR-HPV in the study group and the control group (P = 0.013) (Table 1).

Table 1.

Baseline information of cervical cancer group and the control group

Factors	Cervical cancer (n = 126)	Control (n = 83)	P value
Age (years)	51.11 ± 3.04	50.83 ± 3.87	0.580
BMI (kg/m2)	22.55 ± 2.39	22.37 ± 2.07	0.560
Smoking history (n, %)	15 (11.90%)	13 (15.66%)	0.435
Menopausal (n, %)	37 (29.37%)	21 (25.30%)	0.521
HR-HPV	31 (24.60%)	9 (10.84%)	0.013
STDs	10 (7.94%)	6 (7.23%)	0.851

BMI: Body Mass Index; HR-HPV: high risk Human Papilloma Virus; STDs: incidence of other sexually transmitted diseases

P < 0.05 means significant difference

Relative abundances of serum miR-4534 and the values of blood flow RI in subjects

To investigate the role of serum miR-4534 in cervical cancer, qRT-PCR assay was conducted. It was suggested that serum miR-4534 levels were distinctly elevated in cervical cancer patients in comparison to patients with benign uterine lesions (P < 0.05, Fig. 1A). According to TVCDS examination, relative to the control group, the blood flow RI of cervical cancer patients was distinctly reduced (P < 0.05, Fig. 1B).

Fig. 1.

Serum miR-4534 expression in cervical cancer patients and the control group. A: Serum miR-4534 expression was distinctly increased in cervical cancer patients compared with controls (P < 0.05); B: The blood flow RI was significantly diminished in cervical cancer patients compared with control group (P < 0.05); C: Serum miR-4534 was negatively correlated with blood flow RI value between two groups via Pearson’s correlation analysis (r = − 0.6073; P < 0.001)

To explore the relevance of serum miR-4534 and blood flow RI, Pearson correlation analysis was carried out. As indicated in Fig. 1C, there was a negative correlation between serum miR-4534 and blood flow RI (r = − 0.6073, P < 0.001).

Correlation between serum miR-4534 expression and clinicopathological variables

In order to assess the relevance of serum miR-4534 with clinical factors, Chi-square was conducted. Based on the median value of serum miR-4534 level, all cervical cancer patients were classified into two groups, such as low miR-4534 expression group and high miR-4534 expression group. As summarized in Table 2, increased miR-4534 expression was notable associated with lymph node metastasis (LNM) (P = 0.010), FIGO stage (P = 0.007) and HR-HPV (P = 0.025). Nevertheless, no obvious correlations were found between serum miR-4534 expression and other clinicopathological factors, such as age, tumor size and differentiation (all P > 0.05).

Table 2.

Associations of serum miR-4534 expression and clinicopathological parameters

Factors	NO. (n = 126)	MiR-4534 expression		χ2	P value
		Low expression (n = 58)	High expression (n = 68)
Age (years)				0.090	0.764
< 50	32	14	18
≥ 50	94	44	50
Tumor size (cm)				0.753	0.385
< 4	73	36	37
≥ 4	53	22	31
Differentiation				1.759	0.185
Well-Moderate	68	35	33
Poor	58	23	35
LYM				6.570	0.010
Negative	76	42	34
Positive	50	16	34
FIGO stage				7.397	0.007
I–II	82	45	37
III–IV	44	13	31
HR-HPV				5.000	0.025
No	95	49	45
Yes	31	9	22

LYM: lymph node metastasis; FIGO: The Federation International of Gynecology and Obstetrics; HR-HPV: high risk human papillomavirus

P < 0.05 means significant difference

Analysis of risk factors related to cervical cancer

Moreover, the influence of clinical factors on cervical cancer was analyzed by logistic regression. Univariate logistic analysis indicated that HR-HPV (P = 0.016; OR = 2.683; 95% CI = 1.203–5.983), blood flow RI (P < 0.001; OR = 0.139; 95% CI 0.074–0.259) and miR-4534 (P < 0.001, OR = 6.970; 95% CI = 3.680–13.204) were tightly associated with the initiation and formation of cervical cancer. Additionally, multivariate logistic analysis suggested that serum miR-4534 (OR = 8.805, 95% CI = 4.124–18.798; P < 0.001) was an independent risk factor for cervical cancer, together with blood flow RI (OR = 0.112; 95% CI = 0.054–0.235; P < 0.001) (Table 3).

Table 3.

Logistic analysis of clinical features related to cervical cancer

Factor	Univariate analysis		Multivariate analysis
	OR (95% CI)	P	OR (95% CI)	P
Age	1.025 (0.944–1.112)	0.559	–	–
BMI	1.038 (0.917–1.174)	0.558	–	–
Smoking	0.728 (0.327–1.621)	0.436	–	–
Menopausal	1.227 (0.656–2.295)	0.521	–	–
HR-HPV	2.683 (1.203–5.983)	0.016	2.313 (0.842–6.350)	0.104
STDs	1.106 (0.386–3.169)	0.851	–	–
RI	0.139 (0.074–0.259)	<0.001	0.112 (0.054–0.235)	<0.001
MiR-4534	6.970 (3.680–13.204)	<0.001	8.805 (4.124–18.798)	<0.001

BMI: Body Mass Index; HR-HPV: high risk Human Papilloma Virus; STDs: incidence of other sexually transmitted diseases; RI: resistance index; miR-4534: microRNA-4534; OR: odds ratio; CI: confidence interval

P < 0.05 means significant difference

Diagnostic performance of serum miR-4534 and blood flow RI

ROC curves were plotted to evaluate the diagnostic capacity of miR-4534 and blood flow RI in cervical cancer. The AUCs for distinguishing cervical cancer from benign uterine lesions were 0.811 for serum miR-4534 (Fig. 2A), and 0.787 for blood flow RI (Fig. 2B). The sensitivity and specificity of serum miR-4534 were 85.7% and 72.3%, respectively; while those of blood flow RI were 79.4% and 75.9%, respectively. Based on the cut-off value of 1.255 for serum miR-4534, 126 patients with cervical cancer contained 108 true positives and 18 false negatives, while 60 true negatives and 23 false positives in controls. The positive predictive rate was 82.44% (108/108 + 23), and the negative predictive rate was 76.92% (60/60 + 18). Then, in order to test whether combining the two could improve the diagnostic performance, a diagnostic model was constructed using logistic regression. ROC was draw following the prediction probability of the combined index determined by regression equation. It was demonstrated that the combination brought out in a superior capacity to distinguish between cervical cancer from patients and benign uterine lesions with an AUC of 0.854. Correspondingly, the sensitivity and specificity were 88.9% and 73.5%, respectively (Fig. 2C).

Fig. 2.

ROC curve of serum miR-4534, RI and the combination for diagnosing cervical cancer. A AUC value of serum miR-4534 was 0.811 with the sensitivity of 85.7% and specificity of 72.3% to discriminate the cervical cancer patients from controls. B The AUC value of RI value was 0.787 with the sensitivity of 79.4% and 75.9% to distinguish between cervical cancer patients and controls. C The sensitivity and specificity were 88.9% and 73.5%, respectively, when combining miR-4534 and RI, to detect the cervical cancer

Discussion

Cervical cancer is one of the most frequent female malignant tumors with a high morbidity and mortality rate in China [14, 15]. The early symptoms of the malignant tumor are atypical, and the current screening approaches have defects in sensitivity and specificity as well as operability, so it is easy to miss the best opportunity for diagnosis and treatment [16]. Non-invasive diagnostic serum biomarkers have been widely used for the early detection of numerous types tumors, such as AFP for hepatocellular carcinoma and PSA for prostate cancer [17, 18]. In the context of cervical cancer, certain serum indicators were also examined for the purpose of screening for tumors, including glycoantigen (CA125) and carcinoembryonic antigen (CEA) [19]. However, the exploration of serum biomarkers with considerable specificity and sensitivity has not yet been achieved. Thus, it is of great importance to identify potential indicator of cervical cancer at the early stage.

Relatively conservative endogenous RNAs, miRNAs are able to regulate post-transcriptional genes. Research have shown that a large number miRNA genes are located in tumor-associated gene regions and different RNAs play different roles in the cancer initiation, metastasis and invasion [20]. Abnormal miRNAs played a central role in the development and progression of tumor by targeting downstream genes [21]. MiRNAs are abundant and stable in peripheral blood and they are potential serological detectors of tumors [22, 23]. Due to their high metabolic rate, malignant tumor cells require a richer blood supply to meet their proliferative needs, leading to abnormal blood flow [24]. Doppler ultrasound blood flow parameters can evaluate the blood richness and blood flow status of tumor blood vessels, and it is a commonly used diagnostic tool in the clinic [25]. In this study, we explored the value of serum miR-4534 combined with blood flow RI in the diagnosis of cervical cancer. It could provide an efficient and non-invasive method for the screening and diagnosis of cervical cancer.

We discovered a high HR-HPV infection rate in cervical cancer patients compared with benign uterine lesions patients. Moreover, serum miR-4534 levels were notably up-regulated in cervical cancer patients in comparison to benign uterine lesions patients. Nip et al. reported that miR-4534 was increased both prostate cancer tissues and cells, and silencing miR-4534 could inhibit cell growth and induce cell apoptosis, which provided a potential target for treatment of prostate cancer [12]. Relevant research had shown that miR-4534 was up-regulated based on the microarray experiments, displaying promising prognosis for cervical cancer patients with positive LNM  at an early stage [13]. These findings are consistent with what we found, and suggest that miR-4534 was a tumor promoter in the development of cervical cancer.

On the other hand, blood flow RI values were distinctly reduced in the study group relative to the control group. Pearson’s correlation analysis showed that a negative correlation was exhibited between serum miR-4534 expression and blood flow RI value. In addition, increased expression of miR-4534 was closely related to LNM, FIGO stage and HR-HPV. Furthermore, multivariate logistic regression indicated that blood flow RI and serum miR-4534 were the risk factors to influence the initiation and formation of cervical cancer, indicating that the above factors reflect the risk of cervical cancer to some extent. Serum miR-4534 and blood flow RI yielded a greater degree of influence on the occurrence of cervical cancer, suggesting that these two indicators have the potential to diagnose cervical cancer patients. Therefore, based on clinical experience, the diagnostic performance of serum miR-4534 and blood flow RI was deeply studied in this study.

In this study, the ROC curve analysis showed that the AUC of miR-4534 for diagnosing cervical cancer was 0.811, and the sensitivity and specificity were 85.7% and 72.3%, which suggests that serum miR-4534 has a certain diagnostic performance for cervical cancer. The AUC of serum miR-4534 combined with blood flow RI in the diagnosis of cervical cancer was 0.854, and the sensitivity (88.9%) and specificity (73.5%) were better than those of single biomarker. Low blood flow RI and high levels of serum miR-4534 were the independent risk factors affecting cervical cancer. Published research claimed that serum miR-18a, miR-130a and miR-92a combined with TVCDS exhibited a considerable diagnostic capacity for the detection of cervical cancer [26].

To sum up, serum miR-4534 was up-regulated and blood flow RI was reduced in patients with cervical cancer. The detection of blood flow RI by TVCDS combined with serum miR-4534 exhibited a considerable diagnostic capacity, which is used to distinguish cervical cancer patients from patients with benign uterine lesions. The combination diagnostic approach exhibited easy operation, and could improve the diagnostic rate of CC at an early stage.

However, due to the sample size and working conditions of the research, the relationship between HR-HPV infection and blood flow RI and serum miR-4534, and its molecular mechanism remain unexplored, which require further investigation. This study still needs multi-center cooperation to carry out a large-scale epidemiological investigation and research, and at the same time cooperate with the basic laboratory to explore the theoretical basis of serological index changes.

Author contribution

Xiyan Shao, Lu Bai, Jinlan Liang made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and draft of the manuscript. Ming Li revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Funding

No funding was received for conducting this study.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Code availability

Not applicable.

Declarations

Ethics approval and consent to participate

The protocol was approved by The Second Affiliated Hospital, School of Medicine, The Chinese University of Hong Kong, Shenzhen in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.