A three miRNAs signature predicts survival in cervical cancer using bioinformatics analysis
A three miRNAs signature predicts survival in cervical cancer using bioinformatics analysis
Bin Liang 0
Tianjiao Wang 0
0 Department of Bioinformatics, Key Laboratory of Cell Biology, Ministry of Public Health and Key Laboratory of Medical Cell Biology, Ministry of Education, College of Basic Medical Science, China Medical University , Shenyang
OPEN Growing evidences showed that a large number of miRNAs were abnormally expressed in cervical cancer tissues and played irreplaceable roles in tumorigenesis, progression and metastasis. The aim of the present study was to identify the differential miRNAs expression between cervical cancer and normal cervical tissues by analyzing the high-throughput miRNA data downloaded from TCGA database. Additionally, we evaluated the prognostic values of the differentially expressed miRNAs and constructed a three-miRNA signature that could effectively predict patient survival. According to the cut-off criteria (P < 0.05 and |log2FC| > 2.0), a total of 78 differentially expressed miRNAs were identified between cervical cancer tissues and matched normal tissues, including 37 up-regulated miRNAs and 41 down-regulated miRNAs. The Kaplan-Meier survival method revealed the prognostic function of the three miRNAs (miRNA-145, miRNA-200c, and miRNA-218-1). Univariate and multivariate Cox regression analysis showed that the three-miRNA signature was an independent prognostic factor in cervical cancer. The functional enrichment analysis suggested that the target genes of three miRNAs may be involved in various pathways related to cancer, including MAPK, AMPK, focal adhesion, cGMP-PKG, wnt, and mTOR signaling pathway. Taken together, the present study suggested that three-miRNA signature could be used as a prognostic marker in cervical cancer.
According to the world cancer statistics, cervical cancer is the fourth most common cancer affecting women
globally and the second most common cancer in developing areas1, 2, with an estimated global incidence of 530,000
new cases and 270,000 deaths annually3. The preventive vaccination and organized screening programs are
critical in identifying the cervical cancer before it enters advanced stages. Moreover, the treatments are often less
effective in advanced stages compared with early interventions4. Thus, understanding of the molecular
mechanisms of cervical cancer development and identification of novel biomarkers are required for the early detection
and treatment of cervical cancer.
MicroRNAs (miRNAs), a key component of the noncoding RNA family, are approximately 18?25 nucleotides
that involved in the post-transcriptional regulation of gene expression5. It has been shown that miRNAs are
aberrantly expressed in various types of malignancies and function either as oncogenes or tumor suppressors6.
Accumulating evidence has demonstrated that miRNAs regulated various carcinogenesis processes including cell
maturation7, cell proliferation8, migration, invasion8, autophagy8, apoptosis9, and metastasis10. Therefore,
miRNAs have a large potential to serve as promising markers in the diagnosis, prognosis, and personalized targeted
Although a number of miRNAs have been identified in predicting the clinical outcome in cervical cancer,
there exists inconsistence in previous studies. This may due to the small sample size, heterogeneous histological
subtype, different detection platforms, and various data processing methods. The Cancer Genome Atlas Project
(TCGA) is a National Cancer Institute effort to profile at least 20 different tumor types using genomic platforms
and to make raw and processed data available to all researchers11. The TCGA released a large number of miRNA
sequencing data for cervical cancer patients. The aim of the present study was to identify the differential miRNAs
expression between cervical cancer tissues and matched normal cervical tissues by analyzing the high-throughput
miRNA data downloaded from TCGA database. Additionally, we evaluated the prognostic value of the
differential expressed miRNAs and constructed a three-miRNA signature that could effectively predict patient survival.
Age at diagnosis
Lymph node status
?I + II
?III + IV
?T1 + T2
?T3 + T4
?Squamous Cell Carcinoma
Number of pregnancies
Smoking History Category
Case, n (%)
Furthermore, we analyzed the pathway and function of the target genes of three miRNAs, which may provide
novel insights into understanding the underlying molecular mechanism of cervical cancer.
Identification of differentially expressed miRNAs in cervical cancer. In the present study, a total of
254 samples were enrolled in this study, including 251 cervical cancer tissues and 3 matched normal tissues. The
detailed clinical characteristics include diagnosis at age, metastasis, lymph node status, stage, T stage, histological
type, pregnancy numbers, and smoking history category (Table?1). According to the cut-off criteria (P < 0.05
and |log2FC| > 2.0), a total of 78 differentially expressed miRNAs were identified between cervical cancer tissues
and matched normal tissues, including 37 up-regulated and 41 down-regulated miRNAs (Table?S1). In order to
prove the P value and |log2FC| whether conform to logic with different test, we present the result as Volcano plot
(Fig.?1). Unsupervised hierarchic cluster analysis revealed that cervical cancer tissues could be distinguished from
normal tissues based on differentially expressed miRNAs patterns (Figure?S1).
Identification of three miRNAs associated with OS in cervical cancer. To identify the miRNAs
which would be potentially associated with overall survival of cervical cancer patients, we evaluated the
association between miRNAs expression and patients? survival using Kaplan-Meier curve and Log-rank test. The results
showed that one miRNA (miR-200c) was negatively correlated with overall survival (OS), and two miRNAs
(miR145 and miR-218-1) were positively related to OS (Fig.?2). The association between three miRNAs and clinical
features was evaluated in cervical cancer patients (Table?2). The results showed that miR-145 was significantly
associated with metastasis (P = 0.033) and T stage (P < 0.001); miR-218-1 was associated with stage (P = 0.004),
T stage (P = 0.001), and histological type (P < 0.001). No significant difference was found between miR-200c and
clinical features (P > 0.05).
Prognostic value of three miRNAs signature risk score in cervical cancer. We constructed a
prognostic signature by integrating the expression profiles of three miRNAs and corresponding estimated regression
coefficient. Then, we calculated a risk score for each patient, and ranked them according to increased score. Thus,
a total of 251 patients were classified into a high risk group (n = 125) and a low risk group (n = 126) according to
the median risk score. Survival analysis was performed using the Kaplan-Meier method with a Log-rank
statistical test. The result showed that patients in high risk group have significantly worse OS than patients in low risk
group (P < 0.001, Fig.?3).
Taking into account the following clinical features: age, metastasis, lymph node status, stage, T stage,
histological type, pregnancy number, and smoking history category, univariate and multivariate Cox regression analysis
were used to test the effect of the three-miRNA signature (high risk vs. low risk) on OS. In univariate analysis,
age (HR = 0.562, P = 0.037), lymph node status (HR = 2.567, P = 0.010), stage (HR = 2.511, P = 0.001), T stage
(HR = 4.640, P < 0.001), and three-miRNA signature (HR = 2.574, P < 0.001) were associated with OS in cervical
cancer patients. In multivariate analysis, the three-miRNA signature (HR = 2.183, P = 0.028) was showed to be
an independent prognostic factor in cervical cancer patients (Table?3).
Target prediction and function analysis. The target genes of three miRNAs (miR-145, miR-200c, and
miR-218-1) were predicted using TargetScan, miRDB, PicTar, and miRanda online analysis tools. A total of 67
overlapping genes of miR-145, 126 overlapping genes of miR-200c, and 5 overlapping genes of miR-218-1 were
identified (Fig.?4). Then, enrichment analysis was performed to elucidate the biological function of consensus
target genes. The KEGG pathways were significantly enriched in MAPK signaling pathway, AMPK signaling
pathway, focal adhesion, cGMP-PKG signaling pathway, wnt signaling pathway, and mTOR signaling pathway.
In addition, the GO biological process (BP) terms were mainly enriched in signal transduction, regulation of cell
migration, and regulation of transcription.
With the introduction of vaccination and screening programs, the incidence of mortality associated with cervical
cancer in developed areas have dramatically declined in recent decades, but the mortality in developing countries
remains high, up to 87% of cervical cancer deaths12?15. The cervical cancer patient prognosis would be improved
considerably if tumor behavior could be predicted reliably at the time of initial diagnosis. Therefore,
understanding the molecular mechanisms of cervical cancer development and identification of novel biomarkers are needed.
In the present study, a total of 78 differentially expressed miRNAs were identified, and three of them were
associated with overall survival in cervical cancer patients. The three-miRNA (miR-145, miR-200c, and miR-218-1)
signature was established and was identified to be an independent prognostic factor for cervical cancer patients.
Moreover, we screened the target genes of these three miRNAs, and predicted the enrichment pathways and
biological functions of target genes using bioinformatics methods.
In the last decade, MiRNAs, as the master modulators of multiple biological and pathological processes,
are a hot research topic in the field of cancer development. Mounting evidence has demonstrated that
miRNAs established a complex combinatorial system of gene expression and pathway regulation, as well as
prognostic indicators and therapeutic targets in different cancers, including cervical cancer16, 17. Previous studies have
demonstrated that many miRNAs are crucial for the initiation, progression and metastasis of cervical cancer
by regulating various processes, including cancer cell proliferation, differentiation, apoptosis, adhesion, cell
cycle arrest, migration and invasion18. To date, several studies had identified a number of miRNAs with
prognostic values, such as miR-15519, miR-425-5p20, miR-196a21, miR-50322, miR-26b23, miR-33524, miR-325,
miR21526, miR-22427, and so on. However, previous studies were based on small sample size, sample types, different
detection platforms, various assay methods, and relatively limited numbers of miRNAs. In the present study, we
Age (?60 vs. <60)
Mestasis (M1 vs. M0)
Lymph node status (N1?2 vs. N0)
Clinical stage (III+IV vs. I + II)
T stage (T3+T4 vs. T1+T2)
Histology type (SCC vs. Adenocarcinoma)
Pregnancy (>5 vs. ?5)
HR (95% CI)
analyzed high-throughput data, and identified that two down-regulated miRNAs (miR-145and miR-218-1) and
one up-regulated miRNA (miR-200c) were associated with clinical outcome of cervical cancer patients. Zhou X,
et al. reported miRNA-145 inhibits tumorigenesis and invasion of cervical cancer stem cells by inducing cancer
stem cell (CSC) differentiation through down-regulation of the stem cell transcription factors that maintain CSC
pluripotency28. Sathyanarayanan A, et al. suggested that miRNA-145 modulates epithelial-mesenchymal
transition (EMT) and suppresses proliferation, migration and invasion by targeting SIP1 in human cervical cancer
cells29. Previous article has also reported human papillomaviruses (HPV) oncoproteins E6 and E7 could suppress
miR-145 expression30. Moreover, miR-218, as a tumor suppressor, was strongly down-expressed and related to
proliferation, apoptosis and invasion in cervical cancer31. Yamamoto N, et al. demonstrated that miR-218, acting
as a tumor suppressor in cervical cancer, inhibited cancer cell migration and invasion by targeting focal adhesion
pathways in cervical squamous cell carcinoma32. In addition, HPV16 E6 promoted EMT and invasion in cervical
cancer via the repression of miR-218, while miR-218 inhibited EMT and invasion in cervical cancer by
targeting Scm-like with four MBT domains 1 (SFMBT1) and defective in cullin neddylation 1, domain containing 1
(DCUN1D1)33. MiR-200c, a member of miR-200 family, located on chromosome 12p1334. MiR-200c was
confirmed to be down-regulated in human breast cancer stem cells, and up-regulated in ovarian cancer, lung cancer,
gastric cancer, pancreatic cancer, colorectal cancer, and gastric cancer35, suggesting its complexity role in cancer
as it can act either as oncogene or tumor suppressor depending on the origin of cancer. Our results showed that
miR-200c was up-regulated in cervical cancer, and may be as an oncogene in development of cervical cancer.
Furthermore, miR-145 was significantly associated with metastasis and T stage; miR-218-1 was associated with
stage, T stage, and histological type, indicating miR-145 and miR-218-1 were involved in the progression of
cervical cancer. But, no significant difference was found between miR-200c and clinical features. TCGA database does
not provide completed cell differentiation (Grade), HPV infection, CIN stage, FIGO stage, and so on. Maybe,
miRNA-200c was related to other factors. The future study will focus on this point, and investigate the function
of miRNA-200c in cervical cancer.
In the present study, we found that miR-145, miR-200c, and miR-218-1 were differentially expressed, and
significantly associated with overall survival in cervical cancer patients. While efficacy of a single marker was
limited, multi-markers based model may provide more powerful information for the prognosis prediction of
patients. We constructed three-miRNA signature, and the results suggested that the three-miRNA signature (high
risk and low risk) predicted survival well, and was an independent prognostic factor in cervical cancer.
To gain a deep insight into the molecular functions of three miRNAs, we predicted the target genes and
analyzed the related pathways and GO annotations. Abnormal signaling pathways play crucial roles in the
pathogenesis and progression of cervical cancer. We found that three miRNAs could regulate several key signaling
pathways, including MAPK signaling pathway, AMPK signaling pathway, focal adhesion, cGMP-PKG signaling
pathway, wnt signaling pathway, and mTOR signaling pathway. Accumulating evidence has demonstrated that
activation of MAPK signaling pathway is important in cervical cancer progression, invasion, and metastasis36.
Yung M. M., et al. reported that activation of AMP-activated protein kinase (AMPK), a metabolic sensor,
hampers cervical cancer cell growth through blocking the Wnt/?-catenin signaling activity37. The transformation of
HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve
as a screening tool in HPV-positive populations to detect malignant progression38. Moreover, it has been well
established that the PI3K/Akt/mTOR signaling pathway plays a crucial role in cervical cancer development39,
and inhibition of mTOR kinase activity suppress tumor growth40. Therefore, further molecular investigations are
needed to confirm these predictions, and it can provide new therapeutic interventions in cervical cancer.
Taken together, we identified three-miRNA signature as a potential prognostic predictor for cervical cancer
patients. Further studies are needed to validate our findings in large sample size, and further function
investigation are also required to explore the molecular mechanism of these miRNAs in cervical cancer progression.
Materials and Methods
Data processing. The raw sequencing data and clinical information were downloaded from TCGA database
(https://cancergenome.nih.gov/). The inclusion criteria were set as follows: (1) the sample with both miRNA
sequencing data and clinical information; (2) the sample with prognosis information. Finally, a total of 254
samples were enrolled in this study, including 251 cervical cancer tissues and 3 matched normal tissues. The detailed
clinical characteristics and differentially expressed miRNAs were list in Table?S2. The miRNA sequencing data
were processed using R language package. The differentially expressed miRNAs between cervical cancer and
normal tissues were analyzed by limma package in R. The fold changes (FCs) in the expression of individual
miRNA were calculated and differentially expressed miRNAs with log2|FC| > 2.0 and P < 0.05 were considered
to be significant.
Association of differentially expressed miRNAs and patient prognosis. The differentially
expressed miRNA profiles were normalized by log2 transformed. The prognostic value of each differentially
expressed miRNA was evaluated using Kaplan-Meier curve and Log-rank method. The miRNAs that were
significantly associated with overall survival were identified as prognostic miRNAs, and then subjected to a binary
logistic regression analysis. Subsequently, a prognostic miRNA signature was constructed, and the miRNA signature
could calculate a risk score for each cervical cancer patient. With the miRNA signature, cervical cancer patients
were classified into high risk and low risk groups using the median risk score. Then, the differences in patients?
survival between the high risk group and low risk group were evaluated by Kaplan-Meier method.
The target gene prediction of prognostic miRNA signature. The target genes of prognostic miR
NAs were predicted using TargetScan (http://www.targetscan.org/), miRDB (http://www.mirdb.org/miRDB/),
PicTar (http://pictar.mdc-berlin.de/), and miRanda (http://www.microrna.org/) online analysis tools. To
further enhance the bioinformatics analysis reliability, the overlapping target genes were identified using Venn
diagram. Then, the overlapping genes were analyzed by The Database for Annotation, Visualization and Integrated
Discovery (DAVID) bioinformatics tool (https://david.ncifcrf.gov/). DAVID is a web-based online bioinformatics
resource that aims to provide a comprehensive set of functional annotation tools for the investigators to
understand the biological mechanisms associated with large lists of genes/proteins41. Gene Ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were then performed for the target
genes. The P-value< 0.05 and gene count ? 3 were set as the cut-off criteria.
Statistical analysis. The data were expressed as mean? standard deviation (SD). The expression levels of
miRNAs in cervical cancer and matched normal tissues were analyzed by unpaired t test. The chi-square and t
tests were performed to assess the relationship between miRNA expression and clinical features. Kaplan-Meier
survival analysis and univariate/multivariate Cox proportional hazard regression analysis were carried out to
compare each miRNA (low vs. high level) and prognostic miRNA signature (low vs. high risk). P value less than
0.05 was considered as statistical significant. The statistical analysis was performed using IBM SPSS Statistics
software program version 22.0 (IBM Corp., NY, USA).
The study was supported and funded by the National Science Foundation of China (No. 81301835). We
acknowledge the staff members in Bioinformatics Department in China Medical University.
Liang B. wrote the main manuscript and prepared all figures, Liang B. and Li Y. collect the data and performed the
statistical analysis, and Wang T. performed the bioinformatics analysis.
Supplementary information accompanies this paper at doi:10.1038/s41598-017-06032-2
Competing Interests: The authors declare that they have no competing interests.
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