Background
Cervical cancer is the third most frequent cancer and the fourth leading cause of cancer death in females worldwide, and more than 85% of these cases and deaths are in developing countries [
1], despite the advances in screening and early diagnostic methods in recent years [
2]. By now, the molecular mechanisms of tumor aggressiveness of cervical cancer still remain to be elucidated and more tumor-specific markers for molecular therapy need to be confirmed. In previous study, we focused on a tumor-related transcription factor FOXM1, and explored its role in cervical cancer metastasis. We found that enforced expression of FOXM1 could increase growth, migration and invasion ability of cervical cancer cells [
3], and clinical retrospective study showed that overexpression of FOXM1 could serve as an independent prognostic factor for poor survival in patients with early-stage cervical cancer [
3]. Therefore, FOXM1 could act as a prognostic marker of cervical cancer, and a promising tumor-specific marker which has potential application value in molecular intervention therapy. However, its upstream regulation pathway and modulation molecule needs to be elucidated.
MicroRNAs are endogenous, single-stranded, small non-coding RNAs that post- transcriptionally modulate gene expression involved in essential cellular processes. They guide the binding of RNA-induced silencing complexes to partially complementary regions located usually within the 3′ untranslated regions of target messenger RNAs (mRNAs), thus resulting in target mRNA degradation and/or translational inhibition [
4‐
6]. Aberrant expression of miRNAs has been found in different types of cancers, and some of them function as tumor suppressor genes (e.g. miR-29c and miR-125b, etc.), whereas some act as oncogenes (e.g. miR-151 and miR-454, etc.) [
4‐
9]. Recently, some microRNAs has been proved to modulate FOXM1 expression in many cancers [
10], including hepatocellular carcinoma [
11], breast cancer [
12], gastric cancer [
13], colorectal cancer [
14], bladder cancer [
15], squamous cell carcinoma [
16], lung cancer [
17,
18], leukemia [
19], etc. But little is known about the miRNAs-FOXM1 signaling pathways that modulate the pathogenesis and progression in cervical cancer patients.
In this study, we detected the miR-216b level in different cervical cancer cell lines, and found that miR-216b level negatively correlated with the FOXM1 expression. Functional assay demonstrated that miR-216b could inhibit the proliferation of cervical cancer cells by down-regulating p-Rb, c-myc and cyclinD1, which were downstream targets or important regulators of FOXM1. Further studies found that miR-216b could bind the 3′-UTR of FOXM1 and inhibit FOXM1 expression. Therefore, we proved that FOXM1 was a direct and functional target of miR-216b, and like FOXM1, miR-216b may act as a prognostic marker of cervical cancer patients.
Methods
Patients
MiR-216b activity and target study enrolled 8 patients, who were diagnosed with early-stage cervical squamous cell carcinoma (SCC) and received radical hysterectomy and lymphadenectomy in the Department of Obstetrics and Gynecology, the First Affiliated Hospital, Sun Yat-sen University from January 2009 to December 2012. The enrollment criteria were SCC patients with no preoperative radiotherapy or chemotherapy and with clinical follow-up data. Clinical stage was determined according to the International Federation of Obstetrics and Gynecology, 2009 (FIGO). Totally 8 fresh cervical SCC samples and their corresponding tumor adjacent tissue samples were collected for determination of relative miR-216b expression level using quantitative polymerase chain reaction (qPCR). Samples of normal cervix from patients undergoing simple hysterectomy because of uterine leiomyomata were obtained as a control in FOXM1 western blotting and miR-216b qPCR analysis of 5 cervical cancer cell lines.
The survival and prognosis study of miR-216b enrolled 150 cervical cancer samples randomly collected from 2009 to 2012. The enrollment criteria were all the cervical cancer patients with pathological biopsy confirmation and clinical follow-up data, irrespective of the stage and patient age. Patients from Ia2 to IIa1 received radical hysterectomy and concomitant chemo-radiotherapy according to their risk factors. Patients with IIa2 and higher stage receive concomitant chemo- radiotherapy, or only follow-up. Of the collected cases, 121 were SCC and 29 were other types. The 150 samples were detected for their miR-216b expression using qRT-PCR. The results showed that among them, 75 were relatively miR-216b high level and 75 were miR-216b low. The mean age of these patients was 55.0 ± 10.3 (ranging from 29 to 75), and no age differences existed between miR-216b-high and miR-216b-low patients (
P > 0.05). The last follow-up was carried out in December 2015, with the mean observation period of 41 months (1–60 months), and there were 95 cancer-related deaths. Prior written consent of each patient for the use of clinical materials for research purposes was signed, and approval from the Institutional Ethical Board (IRB) in the First Affiliated Hospital of Sun Yat-sen University was obtained. The clinical information of patients in survival analysis was summarized in Table
1.
Table 1
Clinicopathological characteristics and expression of miR-216 in studied cervical cancer patients
Age (years) |
≤ 55 | 76 | 50.6 |
> 55 | 74 | 49.7 |
FIGO stage |
I/II | 51 | 34 |
III/IV | 99 | 66 |
Histology |
Squamous | 121 | 80.7 |
Others | 29 | 19.3 |
Survival status |
Alive | 55 | 36.7 |
Dead | 95 | 63.3 |
Expression of miR-216 |
Low expression | 75 | 50 |
High expression | 75 | 50 |
Cell lines and cell transfection
The cervical cancer cell lines HCC94 (Cat no. YB-ATCC-5495, FOXM1-low [
3]), HeLa (Cat no. CCL-2), SiHa (Cat no. HTB-35, FOXM1-high [
3]), Ca Ski (Cat no. CRL-1550) and C33A (Cat no. HTB-31) cell lines were obtained from the Department of Anatomy, the Zhongshan School of Medicine, Sun Yat-sen University, and cultured in RPMI 1640 medium (Gibco BRL, Rockville, MD). Media were supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Rockville, MD) and 1% antibiotics mixture (100 U/ml penicillin and 100 μg/ml streptomycin) in a 5% CO2 humidified atmosphere at 37 °C [
3,
20]. Medium was changed every 2 days. These five cell lines were all from American Type Culture Collection (ATCC, MD, USA).
MiR-216b mimics and mimics negative control (NC), miR-216b inhibitors (miR-216b-in) and negative control inhibitors (NC-in), mutant miR-216b and FOXM1-siRNAs were all synthesized by RiboBio. (RiboBio Co. Guangzhou, China). The concentration of miR-216b mimics and inhibitors was 20 nM, and in transfection, 2 μl/well of mimics/inhibitors or control mimics/inhibitors were added. Cells were inoculated into 6 well culture plate (Corning, NY, USA) at the concentration of 5 × 105/ml the day before transfection, and cells were cultured in 2 ml/well of complete medium until 90% confluence. Transfection was performed by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 48 h after transfection, total RNAs were prepared and used for qRT-PCR and the proteins were extracted for Western blotting immediately, or stored at −80 °C for future use.
RNA extraction and qRT-PCR
Quantitative RT-PCR was used for the analysis of miR-216b expression level, and cyclinD1, myc and LEF1 (lymphoid enhancer-binding factor 1) mRNA level as described elsewhere [
18‐
24]. Briefly, total RNA was extracted using TRIZOL Reagent (Invitrogen, CA, USA) from cultured cells following the manufacturer’s instructions. qRT-PCR was performed using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The miR-216b primers were synthesized by RiboBio Co., Guangzhou. The qRT-PCR procedure used to detect the miR-216b level was: cycle 1, 95 °C for 2 min; cycle 2 through 40, 95°Cfor 30 s, 60 °C for 35 s, and fluorescence signal was detected at the end of each cycle. Melting curve analysis was drawn to confirm the specificity. U6 snRNA level was used as an internal control for normalization. The primers used in
cyclinD1,
myc and
LEF1 mRNA detection were shown as follows. CyclinD1 forward: 5′-AACTACCTGGACCGCTTCCT-3′, reverse: 5′-CCACTTGAGCTTGTTCAC CA-3′. MYC forward: 5′-TCAAGAGGCGAACACACAAC-3′, reverse: 5′-GGCCTTTTCATTGTTTTCCA-3′. LEF1 forward: 5′-CACTGTAAGTGATGA GGGGG-3′, reverse: 5′-TGGATCTCTTTCTCCACCCA-3′. β-actin forward: 5′-TGGCACCCAGCACAATGAA-3′, reverse: 5′-CTAAGTCATAGTCCGCCTA GAAGCA-3′. Detection of each sample was repeated 3 times and the results were analyzed by Bio-Rad CFX96 Manager software.
Construction of FOXM1 3′-UTR-PsiCHECK2 vector
The 3′ untranslating region (3′-UTR) of
FOXM1 containing putative miR-216b target binding sites was amplified by PCR from FOXM1 high-expression HeLa cells. The sequence of the
FOXM1 3′-UTR forward primer was: 5′- CCGCTCGAGGGACTGTTCTGCTCCTCATAG-3′; and the reverse primer was: 5′- ATAAGAATGCGGCCGCTGGCAGTCTCTGGATAATGATC-3′. The primers contained
Xho I and
Not I restriction sites, respectively. The amplified 3′-UTR region was then subcloned into the
Xho I/
Not I sites of the PsiCHECK2 vector (Promega, Madison, WI, USA) behind the start codon and identified by sequencing, as described elsewhere [
18,
23,
25]. The PCR procedure was: 94 °C 4 min, 1 cycle, 94 °C 30s, 62 °C 30s, 72 °C 30s, 35 cycles, 72 °C, 7 min.
Western blotting analysis
Western blotting analysis was performed with standard techniques, as described previously [
3]. Cell proteins were extracted by a modified RIPA buffer containing 0.5% sodium dodecyl sulfate (SDS) in the presence of a proteinase inhibitor cocktail (Roche, IN, USA). Polyacrylamide gel electrophoresis (PAGE) was performed to separate cell lysate proteins and then fractionated proteins were transferred onto a PVDF membrane (Amersham Biosciences, NJ, USA). Immonodetection was performed using antibodies including rabbit anti-FOXM1 polyclonal antibody, anti-cyclinD1, anti-p21, anti-LEF1, anti-c-myc, anti-Rb, anti- phosphorylated –Rb, and β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA) at the dilution ratio of 1:1000. The membrane was then incubated with HRP labeled goat anti-rabbit secondary antibody (BosterBio, CA, USA) at the dilution ratio of 1:6000. Anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) served as an internal control. Signals were detected by exposure to films with SuperSignal West Pico Chemoluminescent substrate (Thermo Fisher Scientific, MA, USA).
Luciferase assay
For luciferase reporter assays, 5 × 105 HeLa cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in 24-wells culture plates, with 5 pmol of miR-216b (or mimics negative control, or miR-216b-mut), and 100 ng of firefly luciferase reporter vector in the transfection mixture. MiR-216b mimics negative control served as a negative control (NC) and microRNA inhibitor control served as NC-in control. Cells were harvested 48 h after transfection, and then the luciferase activity was measured using a dual luciferase reporter assay system (Promega, WI, USA) according to the manufacturer’s instructions. Three independent experiments were performed and the data were presented as the mean ± SD.
MTT assay
Cell proliferation assay was performed using 3- (4, 5-dymethyl-2-thiazolyl) -2, 5- diphenyl-2H-tetrazolium bromide (MTT) assay, as described elsewhere [
18,
23,
25]. Briefly, different groups of 2 × 10
3 cultured HeLa cells were seeded into U-bottom 96-well plates per well (Corning, NY, USA) and cultured with miR-216b mimics and negative control (NC), miR-216b inhibitors (miR-216b-in) and negative control inhibitors (NC-in), mutant miR-216b and FOXM1-siRNAs respectively in 200 μl per well culture medium. Totally 4 duplicate plates were inoculated. Cultures were maintained for 7 days at 37 °C, 5%CO
2 in a humidified atmosphere. Cell proliferation was detected on day 0–5 by MTT method and each group was analyzed in triplicate wells. MTT solution of 5 mg/ml (Sigma, MO, USA) was added at 20 μl per well during the final 4 h of culture. The medium was then replaced with 150 μl dimethyl-sulfoxide (DMSO) and vortexed for 10 min. The optimal density (OD) was read at a wavelength of 490 nm on a Tecan Sunrise microplate reader. Relative MTT absorbance was counted by: average OD
exp on day X/average OD
NC on day 1.
Colony formation assay was performed as described elsewhere [
18,
23,
25]. Briefly, different groups of 1 × 10
3 HeLa cells were seeded into 6-well plates (Corning, NY, USA) per well and cultured with miR-216b mimics and negative control (NC), miR-216b inhibitors (miR-216b-in) and negative control inhibitors (NC-in) in 2 ml in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were cultured for 7–10 days and colonies were observed everyday. The medium was removed and washed by PBS for 3 times. Cells were fixed by methanol for 10 min and stained with 0.1% crystal violet for 10 min. The numbers of colonies with more than 50 cells were counted manually.
Statistical analysis
All statistical analyses were performed using SPSS 16.0 software package (SPSS Inc. IL, USA). The measurement data are expressed as mean ± standard error (Mean ± SD). The relationships between FOXM1 expression and miR-216b expression level were determined by correlation analysis and expressed as correlation coefficient (r). Differences of measurement data were assessed by Student’s t test. The clinicopathological differences between miR-216b-high and miR-216b-low patients were assessed using Pearson’s χ2 test. Survival curves were estimated using the Kaplan-Meier method. A two-sided value of P < 0.05 was considered statistically significant.
Discussion
FOXM1 is involved in multiple biological process including cell proliferation, differentiation, growth, migration and invasion [
26,
30,
31]. Its overexpression is implicated to play an important role in pathogenesis, progression and metastasis of many types of cancers [
29‐
34], including cervical cancer as we have confirmed in our previous study [
3]. Although there is report that miR-342-3p could suppress the proliferation and metastasis in cervical cancer by targeting FOXM1 [
23], the FOXM1- microRNAs modulation pathways in cervical cancer cells remains to be discovered. Based on our previous study outcomes, to further elucidate the regulation pathway of FOXM1 in cervical cancer, in this study, we screened possible FOXM1 upstream modulator microRNAs using online miRNA target prediction databases including Targetscan and
microRNA.org and presumed that miR-216b may regulate FOXM1 expression.
MiR-216b gene has been reported to be involved in several cancers including nasopharyngeal carcinoma, medulloepitheliomas, breast cancer, etc. [
21,
22,
35‐
37]. Although recently it has been reported that miR-216b could inhibit cell growth by targeting FOXM1 in hepatocellular carcinoma and melanoma [
38,
39], the role of miR-216b and the relationship between miR-216b and FOXM1 in cervical cancer remains unclear. In the present study, we first screened the expression level of miR-216b in cervical cancer cell lines and clinical samples, and found that miR-216b was down-regulated, suggesting that miR-216b is involved in cervical cancer and may be a tumor suppressor miRNA. We also found that miR-216b level in these cervical cell lines showed a reverse trend of FOXM1 expression (Fig.
1a), indicating that miR-216b may regulate FOXM1 expression. Because the involvement of FOXM1 in tumorigenesis is mainly related to its role in cell-cycle progression and proliferation, and migration and invasion of cervical cancer cells [
3], we further explored the cervical cancer cell cycle, cell proliferation and invasion ability after miR-216b overexpression and inhibition. The results showed that overexpression of miR-216b could significantly inhibit cell proliferation in HeLa cells (Fig.
2) and decrease the ratio of cells in S period (Additional file
1: Figure S1), but its effect on tumor invasion and migration was not obvious (data not shown), indicating that miR-216b may regulate FOXM1-related cell proliferation factors, but not FOXM1-related metastasis factors, and there may be other microRNA-FOXM1 pathways involved in FOXM1 related cell metastasis. Further studies will be needed to explore more micorRNA-FOXM1 links that are involved in cervical cancer carcinogenesis.
To elucidate miR-216b targets and to further verify the relationships between miR-216b and FOXM1, we explored the effect of miR-216b on expression of FOXM1 related cell division genes, including the CDK (cyclin-dependent kinases) inhibitor p21 which can be negatively regulated by FOXM1, Rb and p-Rb that could indirectly regulate FOXM1 activity as an upstream regulator of FOXM1, and the CDK regulator cyclin D1 which can be positively regulated by FOXM1, and also the mRNA level of Wnt/β-catenin downstream target genes,
cyclinD1,
myc and
LEF1, as FOXM1 has been previously proved to be a downstream component of Wnt/β-catenin signal pathway [
29]. The results proved our deduction. We observed that p21 was up-regulated and Wnt/β-catenin downstream targets including cyclinD1, myc and LEF1 were down-regulated in miR-216b overexpression cells, and up-regulated in miR-216b inhibited cells. Coincident with altered expression of cell-cycle regulators, the phosphorylation level of Rb, a downstream target protein level of CDK and an upstream regulator of FOXM1, was significantly decreased in miR-216b transfected cells and obviously increased in miR-216b inhibited cells (Fig.
3a), further confirming that miR-216b can affect the proliferation of cervical cancer cells by regulating FOXM1 related cell division factors. Using dual luciferase reporter system, we proved that miR-216b directly bind to FOXM1 3′- UTR. Therefore, FOXM1 is a direct target of.miR-216b. Further analysis confirmed that miR-216b inhibits cell proliferation by repressing endogenous FOXM1 in cervical cancer cells and tissues, and negative correlation existed between miR-216b and FOXM1 with coefficient
r − 0.805 (Fig.
5a). These findings indicate that dysregulation of FOXM1 by miR-216b may be an important mechanism underlying cervical cancer tumorigenesis, and future studies should address the detailed molecular mechanisms behind the role of miR-216b-FOXM1 link in the tumorigenesis of cervical cancer. However, the interaction of microRNAs and transcription factors in tumors contains very complicated networks, and the relationship of miR-216b-FOXM1 is only been reported recently [
38,
39], and not been included in the recent published work of systematic -omic evaluation of cervical cancer by the Cancer Genome Atlas Project (TCGA) [
40]. The reason may lies in different control and hierarchical clustering. In our study, the relative T/ANT (cancer tissues/adjacent non-cancer tissues) ratio of FOXM1 [
3] and miR-216b expression was examined in cervical cancer tissues, whereas in TCGA studies, cancer tissues/normal controls were compared. The change of p21, myc expression and Wnt/β-catenin signal pathway in cervical cancer were also revealed in TCGA study, using squamous/adenocarcinomas and HPV positive/negative hierarchy, whereas in our study, most of the cancer tissues were squamous and HPV positive. We believe our study will further enrich and helps to understand the molecular modulation mechanism of tumor associated genes and factors in cervical cancer.
FOXM1 has been proved to be a prognostic factor for poor survival in patients with early-stage cervical cancer [
3]. Since miR-216b targets and suppresses FOXM1, it may also be related to the prognosis of cervical cancer patients. We then evaluated the role of miR-216b in the prognosis of 150 patients (121 SCC and 29 other types) using Kaplan-Meier survival analysis. It was confirmed that as a contrast to FOXM1, high expression of miR-216b was related to earlier FIGO stage, better histological type and better survival status in cervical cancer patients (Table
2, Fig.
6). However, the overall mortality rate was relatively higher than that in FOXM1 study [
3], because more late-stage patients who receive only chemo- radiotherapy or no treatment were enrolled in this study (Table
1). One reason that more late-stage patients were collected was that their lesions were clearer and tumor specimen was easier to obtain for miR-216b detection. And with more late-stage cancer tissues, the trend that miR-216b expression decreased with the FIGO stage was more obvious, consistent with reports of other tumors [
21,
35‐
37]. Moreover, more late-stage specimens can better display the value of miR-216b in cervical cancer development and prognosis as a biomarker. Despite of the high mortality, we still found that higher level of miR-216b was associated with both better overall survival and better disease-free survival than miR-216b low level patients (
P < 0.01, Fig.
6). As far as we know, this is the first time that miR-216b is reported to be correlated with better prognosis of cervical cancer patients. High level of miR-216b in cervical cancer patients indicated not only longer survival time but also longer disease-free time. Therefore, miR-216b may also be a potential prognostic marker for cervical cancer.