Background
Cervical cancer is the fourth most common cancer in women worldwide after cancer of the breast, colon, and lung [
1]. According to the World Health Organization (WHO), each year approximately 528,000 new cases are recorded, and 266,000 deaths occur due to cervical cancer [
2]. Cervical cancer is mainly caused by infection with high-risk human papillomavirus (HR-HPV) genotypes [
3]. With the development of screening and prevention methods of cervical cancer such as HPV co-testing and HPV vaccination, early diagnosis programs of cervical dysplasia and cancer lead to decrease the incidence, morbidity, and mortality of cervical cancer [
4].
However, nearly 50% out of all cervical cancer patients worldwide are still diagnosed with stages IB2 to IVA according to International Federation of Gynecology and Obstetrics (FIGO), whereas about one-third of patients diagnosed with cervical cancer in Korea presented locally advanced stage of the disease [
5]. Currently, the standard treatment guideline for patients with advanced FIGO stage IIB and more than stage IIB is radiotherapy combined with platinum-based chemotherapy [
6]. Unfortunately, these patients have a higher recurrence rate and a worse survival rate in the first 5 years [
5]. Therefore, it is of great importance to identify a novel biomarker that can reliably detect cervical cancer and improve clinical monitoring.
microRNAs (miRs or miRNAs), small non-coding RNAs consisting of approximately 22 nucleotides, regulate gene expression by binding to partially or fully complementary sequences in target mRNAs, resulting in translational inhibition or mRNA degradation, respectively [
5]. miRNAs have been suggested to function as oncogenes or tumor suppressors based on their function of inhibiting the expression of tumor suppressive or oncogenic target mRNAs, respectively [
6‐
8]. Numerous studies have shown that dysregulation of miRNAs plays an essential role in cell proliferation, cell cycle regulation, differentiation, and apoptosis and is related to various tumors such as colon, gastric, breast, lung, and cervical cancers [
9‐
13].
miR-944 is located in the intron of the tumor protein p63 gene (
TP63) mapped to chromosome 3q28. Previous reports show that miR-944 functions as an oncogene in a number human cancers, including cervical cancer, endometrial cancer, breast cancer, and lung cancer by promoting cell migration, proliferation, and invasion [
14‐
17]. However, some studies have reported that miR-944 can function as a tumor suppressor, inhibiting migration in colorectal cancer, gastric cancer and breast cancer [
18‐
20]. In cervical cancer, miR-944 promotes cell proliferation and migration in CaSki and HeLa cervical cancer cell lines [
16,
21]. To our knowledge, there are no other further studies on the clinical relevance of miR-944 in cervical cancer, and specifically in terms of prognostic value.
Human papillomavirus infection is known to one of the most significant risk factors for cervical cancer [
22]. Several studies suggested that the sustained expression of the two oncogenic genes
E6 and
E7 of HPV is involved in cervical cancer progression by degradation of p53 and deactivation of retinoblastoma protein (pRB) [
23‐
27]. Recently, miRNA sequencing data from The Cancer Genome Atlas (TCGA) was reported that miRNA clusters such as miR-205-5p, miR-944, miR-200a-5p, miR-30a-5p, miR-338-3p, miR-224-5p, and miR-193b-3p were associated with cervical cancer and especially miR-944 was shown the significant difference between HPV positive and HPV negative cervical cancer [
28]. However, there is no further study of the relationship between miR-944 and HPV E6/E7 expression in cervical cancer.
In this study, the prognostic value of miR-944 was investigated using 116 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissues and normal tissues. Furthermore, the association between miR-944 and HPV E6/E7 mRNA-positive or -negative was explored.
Methods
Study population
A total of 66 FFPE cervical cancer tissues and 50 FFPE normal cervical tissues were collected between January 2010 and December 2014. A total of 66 cervical cancer cases data on age, tumor size, FIGO stage, lymph node metastasis, and HPV infection were retrospectively reviewed from patient electrical medical records. The 50 FFPE normal cervical tissues consisted of 44 patients with non-cervical, benign, uterine disease and six cervical cancer patients with tumor-matched non-cancerous tissues (Table
1). This study was approved by the Institutional Ethics Committee of Yonsei University Wonju Severance Christian Hospital (approval no. CR315052), and all subjects provided written informed consent.
Table 1
Clinical characteristics
Total, n (%) | 66 (100.0) | 50 (100.0) |
Age |
< 45 years | 20 (30.3) | 20 (40.0) |
≥ 45 years | 46 (69.7) | 30 (60.0) |
Histological type |
ADC | 7 (10.6) | |
SCC | 59 (89.4) | |
Tumor size |
< 4 cm | 30 (45.5) | |
≥ 4 cm | 36 (54.5) | |
FIGO stage |
IA-IIA | 29 (43.9) | |
IIB-IVB | 37 (56.1) | |
Lymph node metastasis |
No | 35 (53.0) | |
Yes | 31 (47.0) | |
HPV E6/E7 expression |
Negative | 12 (18.2) | |
Positive | 54 (81.8) | |
Survival |
Alive | 52 (78.8) | |
Died | 14 (21.2) | |
Cell culture
Cervical cancer cell lines C33A (HPV-negative), SiHa (HPV 16), Caski (HPV 16), HeLa (HPV 18), and ME180 (HPV 18, 68) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea) and the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) was used to maintain the cells in tissue culture and the five cell lines were maintained at 37 °C with 5% CO2.
Deparaffinization of FFPE tissue and total RNA extraction
For total RNA extraction from FFPE cervical tissue, three to four 10-μm-thick sections were transferred to 1.5 mL tube each. Before extracting RNA, the paraffin was removed from the tissue sections by adding 160 μL of deparaffinization solution (Qiagen, Hilden, Germany) followed by incubation for 3 min at 56 °C. Total RNA extraction was extracted according to the manufacturer’s protocol (Qiagen RNeasy FFPE kit, Qiagen). Next, the concentration of the total RNA was measured with an Infinite 200 spectrophotometer (Tecan, Salzburg, Austria). Total RNA was stored at − 80 °C until used.
microRNA RT-qPCR analysis
We used the TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) to synthesize cDNA according to the manufacturer’s instructions. Reverse transcription reactions were performed using 10 ng of total RNA and specific reverse transcription primers (Life Technologies) for Homo sapiens (hsa)-miR-944 (assay ID 002189), and the internal reference RNU6B (assay ID 001093). Reverse transcription was performed at 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min.
After the reverse transcription, quantitative PCR reaction was performed using the TaqMan microRNA assay (Applied Biosystems) according to the manufacturer’s instructions. Briefly, the initiation step of PCR cycling conditions at 95 °C for 10 min, followed by an amplification step of 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The miR-944 expression levels were calculated via the comparative cycle threshold (CT) method. The CT values of miR-944 were normalized to the level of RNU6B.
HPV E6/E7 mRNA RT-qPCR assay
M-MLV reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) was used for complementary DNA (cDNA) synthesis. Briefly, add 0.25 μg random hexamers, 1 μL 10 mM dNTP mix, and 5 μL DEPC-treated water and 10 μL total RNA. The mixture was then incubated at 65 °C for 5 min and quickly cooled on ice. After that, 4 μL of 5x Buffer, 2 μL of 0.1 M dithiothreitol (DTT) and 1 μL of M-MLV Reverse Transcriptase (RT) were added to the first mix. The reverse transcriptase reaction was carried out at 25 °C for 10 min, at 37 °C for 50 min, and at 70 °C for 15 min.
Detection of HPV E6/E7 mRNA in FFPE cervical tissues was performed by OPTIMYGENE HPV E6/E7 mRNA RT-qDx assay kit (Optipharm M&D, Osong, Republic of Korea), according to the manufacturer’s instructions. The reverse transcription quantitative PCR (RT-qPCR) assay was carried out using the CFX96 system (Bio-Rad, Hercules, CA). Real-time PCR amplification for HPV E6/E7 mRNA was performed in a total volume of 20 μL, containing 10 μL of 2x Thunderbird probe qPCR mix (Toyobo, Osaka, Japan), 5 μL primer and TaqMan probe mixture, 3 μL distilled water and 2 μL template cDNA. Positive and negative controls were contained per each procedure. Reaction conditions of real-time PCR were as follows: 95 °C for 3 min, and 40 cycles of 95 °C for 3 s and 55 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and confirmation of mRNA degradation.
The Cancer genome atlas (TCGA) analysis
Raw data for miR-944 and clinical information in cervical cancer were extracted from the TCGA open source repository website (
http://firebrowse.org/) on 01/28/2016. Variables, such as follow-up times, tumor stage, race, and reads per million of miR-944 in 180 cervical cancer patients were included. Information of HPV E6/E7-positive and HPV E6/E7-negative cervical cancers matched with patients from TCGA open source repository website was obtained from the Banister et al. study [
29]. The HPV E6/E7 expression were classified as HPV alpha-9, HPV alpha-7, and HPV negative cervical cancer based on HPV E6/E7 expression. The reads per million of miR-944 according to HPV E6/E7 expression were analyzed by student t-test.
Statistical analysis
All statistical analyses were performed using GraphPad Prism version 6 software (La Jolla, CA, USA) and SPSS version 21.0 software (IBM, Armonk, NY, USA). Wilcoxon matched-pairs singed rank test was performed for comparison between the paired cervical normal and cancer tissues. Student’s t-test were conducted for comparison between normal and cancer tissues. To identify whether miR-944 is affected by HPV E6/E7 mRNA positive or negative cervical cancer tissues, the expression levels of miR-944 was compared by student’s t-test. To determine the clinical cut-off value of miR-944 between cervical cancer and normal tissues, receiver operator characteristic (ROC) curve analysis were performed. Based on the clinical cut-off value, cases were divided into two groups according to the expression level of miR-944 (high miR-944 vs. low miR-944). Potential associations between the expression of miR-944 and various prognostic parameters were analyzed by the Chi-square test. Survival was estimated by the Kaplan-Meier method and evaluated by the log-rank test. Multivariate analyses of prognostic values were evaluated using the Cox proportional hazards model. In all analyses, a P-value of less than 0.05 was considered statistically significant.
Discussion
In this study, we investigated miR-944 expression levels in FFPE cervical cancer and normal tissues by RT-qPCR methods, and the result showed poor prognosis in patients with high miR-944 expression levels and in an advanced stage of cervical cancer. In addition, we demonstrated that there was an association between high expression levels of miR-944 and expression levels of HPV oncogene E6/E7 in HPV-positive cervical cancer cells by analysis of FFPE cervical cancer tissues and TCGA data.
The expression levels of miR-944 were significantly higher in cervical cancer tissues as compared to those in paired non-cancerous tissues (
n = 6) (Fig.
1), which suggested that miR-944 plays a role in tumorigenesis through clinical cervical cancer tissues. Xie et al. (2015) also had shown that high miR-944 expression levels are associated with tumorigenesis of cervical cancer, based on analysis of cervical cancer cell lines (Caski and HeLa) [
16]. We further examined 66 cervical cancer and 50 normal FFPE samples and showed that the expression of miR-944 in cervical cancer was up-regulated which was in line with previous reports [
16].
To gain insight into the clinical significance and prognostic value of miR-944, we investigated the relationship between miR-944 expression levels and the clinicopathological characteristics of cervical cancer patients and found that expression of miR-944 was firmly related to the tumor size, FIGO stage, and lymph node metastasis (Table
2). Ma et al. reported that up-regulation of miR-944 has also been associated with lymph node metastasis and advanced stage of lung squamous cell carcinoma [
30].
Furthermore, survival analysis of cervical cancer patients showed that high expression levels of miR-944 were associated with poor survival prognosis (Fig.
3a). As a prognostic marker, FIGO stage and high miR-944 expression levels were shown useful prognostic indicators of poor survival (Table
3). Notably, in advanced cervical cancer, high miR-944 expression levels were significantly poorer prognosis than low miR-944 expression levels (Fig.
3b).
Previous studies have attempted to identify the mRNA target of miR-944 in cervical cancer, and Xie et al. showed that miR-944 has two target genes, HECT domain ligase W2 (HECW2), which are known to regulate p63 stabilization, and S100P binding protein (S100PBP), which is known to reduce adhesion and invasion. miR-944 was predicted to play an oncogenic role in cervical cancer malignancy by repressing these two genes (HECW2 and S100PBP) that function as a tumor suppressors [
16].
Several studies have reported that the expression levels of some miRNAs are associated with poor prognosis in cervical cancer, such as downregulation of miR-335 from Wang et al. [
31], down-regulation of miR-145 and up-regulation of miR-9 from Azizmohammadi et al. [
32]. Recently, Jiang et al. suggested that down-regulation of circulating miR-101 is associated with poor prognosis of cervical cancer [
33]. Our study also suggests that high expression of miR-944 could be of clinical relevance for poor prognosis. The miR-944 expression has been analyzed in colorectal, bladder, and breast cancer. High miR-944 expression levels have been associated with tumor recurrence in colorectal cancer [
34] and chemo-resistance in bladder cancer [
35]. He et al. found that miR-944 is significantly up-regulated in the blood and tumor tissues of breast cancer patients [
36]. Like our study, miR-944 is remarkable tumor-associated microRNA.
Interestingly, in our study, miR-944 expression in HPV E6/E7 mRNA-positive cervical cancer was higher than that in HPV E6/E7 mRNA-negative cervical cancer (
P = 0.02; Fig.
2). Meanwhile, among five cervical cancer cell lines, the expression of miR-944 in cervical cancer cell lines with HPV infection (SiHa and HeLa, Caski, and ME180) was significantly higher than that in the cervical cancer cell line without HPV infection (C33A) (Additional file
1) as well as TCGA data (Additional file
3), analyzed by using individual miR-944 reads and HPV E6/E7 expression status. It suggests that up-regulation of miR-944 is associated with HPV infection and the carcinogenic processes of the
E6 and
E7 genes. Recently, microRNA heat map categorized by HPV infection and cervical cancer subtypes from TCGA projects also showed that the levels of miR-944, miR-767-5p, and miR-105-5p between HPV-positive and negative cancer were distinctively expressed [
28]. Taken together, our results supported that miR-944 expression levels are closely related to HPV infection.
HPV E6 protein was found to bind p53 and inhibit cell apoptosis. HPV E7 proteins binding to retinoblastoma protein (pRb) interferes with cell cycle regulation [
36,
37]. We found that miR-944 expression was associated with HPV E6/E7 mRNA. However, the relationship between miR-944 and p53 or pRb, which showed a relationship with HPV E6/E7, in cervical cancer progression was not evaluated. Further studies are needed to evaluate the functional role of miR-944 with p53 or pRb to understand the progression of cervical cancer.
The limitation of this study was the small sample size with single institution. Nevertheless, this study was valuable that miR-944 was shown potential marker complementing conventional clinical prognostic parameters and the relation to HPV E6/E7 positive cervical cancer tissues. It may be worthwhile to conduct replication to clarify our findings with sufficient clinical sample set.