*Contributed equally
Cervical cancer (CC) is the fourth ranking gynaecologic tumour in women worldwide, with respect to both incidence and mortality. MUC16 is one of the most frequently mutated genes, which functions as a tumour marker in CC. In the present study, mutation, clinical and RNA-Seq data collected from The Cancer Genome Atlas database were used to investigate the association between MUC16 mutation, immune response and clinical prognosis in CC. mRNA expression levels from the TCGA datasets and the results from the present study demonstrated that MUC16 was overexpressed in CC samples; however, there was no difference between mutant and wild-type CC samples. Furthermore, the results indicated that patients with MUC16-mutant overexpression had a prolonged survival time. In addition, overexpression of MUC16 was associated with immune responses in the microenvironment of MUC16-mutant CC. Immune responses were upregulated in patients with early-stage MUC16-mutant. The results from the present study provided novel biomarkers for potential immunotherapy approaches for CC.
In 2018, it was estimated that there were 570,000 new cases and 311,000 CC-associated mortality cases (
Synthesis of mucin (MUC) is essential for the formation of the mucous barrier, which helps protect the epithelia of most organs, such as the stomach, from physical and chemical damages and infection (
CA125 was first identified ~38 years ago in a screening of antibodies against ovarian cancer antigens. After two decades, it was cloned and characterized as a membrane-bound mucin named MUC16(
Data associated with mutation, clinical parameters, copy number variation (CNV), DNA methylation and RNA-Seq of CC samples were downloaded from the TCGA database. MUC16 RNA-Seq data from the various types of cancer were downloaded from the TCGA database (
Three types of clinical survival and recurrence outcomes were selected in the present study: Overall survival (OS), disease-specific survival (DSS) and progression-free survival (PFS). The outcomes were defined as follows: OS referred to the period of time from the date of diagnosis to the date of mortality from any cause; DSS referred to the period of time from the date of initial diagnosis to the date of last contact or the date of mortality from another cause; and PFS referred to the period from the date of diagnosis to the date of new tumour occurrence (
CC tissues and adjacent normal tissues were obtained from 9 patients; 3 patients used to detect the MUC16 protein expression levels between adjacent normal tissue and CC tissue, 3 patients used to detect the MUC16 protein expression levels in wild-type CC tissues; and 3 patients used to detect the MUC16 protein expression levels in mutant type CC tissues (age range, 44-51 years; median age, 47 years); who underwent radical resection at The First College of Clinical Medical Science, China Three Gorges University (Yichang, China) between March 2019 and July 2019. All samples were stored at -80˚C. The inclusion criteria were as follows: i) All patients were diagnosed with CC, following colposcopy and cervical tissue biopsy; ii) no chemotherapy or radiotherapy was performed prior to surgery, and iii) all patients had complete clinical data. Exclusion criteria: i) Patients with incomplete clinical data; and ii) patients who refused to participate in this study. All experimental procedures were approved by the Ethics Committee of The First College of Clinical Medical Science, China Three Gorges University (Yichang, China). Written informed consent was provided by all patients prior to the study.
Total protein was extracted from CC tissues and adjacent normal tissues using Cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology, China), and the protein concentration was quantified using a BCA Assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology, China). A total of 50 µg protein was added to each sample well, separated by 5% SDS-PAGE prior to being transferred onto polyvinylidene fluoride membranes. Dried, non-fat milk powder was used for blocking, for 1 h at room temperature. The membranes were incubated with a MUC16 monoclonal antibody (cat. no. BM5743; 1:2,000; Wuhan Boster Biological Technology, Ltd.) at 4˚C for 12 h, prior to incubation with a horseradish peroxidase (HRP)-labelled secondary antibody (cat. no. BM2006, 1:1,000; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. ECL luminous liquid A and B (cat. no. P0018S, Beyotime Institute of Biotechnology) were mixed (1:1) and the luminescent droplets were dropped onto the film. Protein bands were visualized using a gel imager (Bio-Rad Laboratories, Inc.). β-actin (cat. no. BA2305; Wuhan Boster Biological Technology, Ltd.) was used as the reference protein.
Gene set enrichment analysis (GSEA) was performed to determine the correlation between the immune response in CC and MUC16 expression in patients with mutated and wild-type MUC16. Furthermore, genes were arranged from high to low according to their correlation with MUC16 expression. Genes and R values were determined and subsequently assessed via Kyoto Encyclopaedia of Genes and Genomes and Gene Ontology (GO) analyses within clusterProfiler v3.10.0 (
Analysis of mutations was performed using R software version 2.5.0(
The top 10 mutated genes in CC included TTN (32%), PIK3CA (29%), MUC4 (28%), KMT2C (20%), MUC16 (17%), KMT2D (15%), FLG (13%), DMD (13%), EP300 (13%) and SYNE1 (13%;
MUC16 expression levels in different types of cancer were analyzed within the TCGA datasets (
For the purpose of evaluating the clinical significance of MUC16 in the survival of patients with CC and to determine the association between MUC16 mutational status and survival, MUC16 expression was assessed in 286 CC clinical samples (≤4,000 days of follow-up data) from TCGA for OS, DSS and PFS (
CSCC accounts for 70-80% of CC and antigen levels are associated with early-stage CC (
MUC16 mutant patients with CC with MUC16 overexpression exhibited a longer OS, DSS and PFS time, which indicates that overexpression of MUC16 may be involved in the progression and tumorigenesis of MUC16-mutant CC. Previous studies have reported that MUC16 functions as a suppressor of the immune response (
In order to investigate the association between immune responses, MUC16 mutation and early-stage CC, an analysis of the differences between early-stage and stages I-IV CC samples was performed (
CC is the fourth most common cancer in women worldwide (
The immune response is of great significance for the development and progression of CC (
In conclusion, the results from the present study suggested that overexpression of MUC16 may specifically predict favourable survival and prognosis in patients with MUC16-mutant CC. Furthermore, MUC16 mutational status was associated with the immune response in CC, particularly in patients at early stage of disease. MUC16 mutations are a key marker in immunotherapy for CC patients. MUC16 overexpression is not only significantly associated with the prognosis of MUC16-mutant patients with CC, but also lead to the enrichment of associated immune activity, which can be used to identify which patients may benefit from immunotherapy in CC. Taken together, the findings from the present study provided novel and promising approaches for immunotherapy in CC; however, further studies are required to investigate these methods.
Not applicable.
No funding was received.
The datasets generated and analyzed during the current study are available in the TCGA dataset [
HW and CY conceptualized the study, designed the research and performed the bioinformatics analysis. HW performed the experiments. HW and HY analyzed and interpreted the data. HW wrote and edited the manuscript, and HY supervised the project. All authors read and approved the final manuscript.
All experimental procedures were approved by the Ethics Committee of The First College of Clinical Medical Science, China Three Gorges University (Yichang, China). Written informed consent was provided by all patients prior to the study.
Not applicable.
The authors declare that there have no competing interests.
Landscape of MUC16 mutations in CC. (A) An OncoPlot of the top 10 mutated genes in CC samples from TCGA database. The upper bar plot indicates the number of genetic mutations/patient, while the bar plot on the right indicates the number of genetic mutations/gene. The CC pathology types and mutation types are represented as annotations at the bottom. (B) A lollipop plot of MUC16 mutations in CC samples from TCGA database. Amino acid axis labelled for domain. (C) Proportions of MUC16 mutations between endocervical adenocarcinoma and cervical squamous cell carcinoma. (D) Proportions of different types of single nucleotide variants of MUC16 in CC samples from TCGA database. (E) Numbers of different types of single nucleotide variants of MUC16 in CC samples from TCGA database. CC, cervical cancer; TCGA, The Cancer Genome Atlas; TTN, Titin; PIK3CA, phosphatidylinositol 3; MUC4, mucoprotein 4; KMT2C, histone lysine methyltransferase 2C; MUC16, mucoprotein 16; KMT2D, histone lysine methyltransferase 2D; FLG, filaggrin; DMD, duchenne muscular dystrophy; EP300, E1A binding protein p300; SYNE1, spectrin repeat-containing nuclear envelope protein 1.
Landscape of MUC16 expression in CC. (A) Bar plot of FPKM for LINC00265 in different types of cancer from TCGA database. Data are presented as the mean ± SEM. (B) Left panel: Bar plot depicting MUC16 expression in CC and normal samples from the GSE9750 dataset. Right panel: Expression levels of MUC16-mutated and MUC16-wild-type samples of CC from TCGA database. Data are presented as the mean ± SEM. (C) MUC16 protein levels were determined via western blotting for normal and CC samples (top), and mutant and wild-type CC samples (bottom). (D) Association between CNV level and MUC16 expression in MUC16-mutated and MUC16-wild-type samples. (E) Association between DNA methylation level and MUC16 expression in MUC16-mutated and MUC16-wild-type samples. CC, cervical cancer; FPKM, fragments per kilobase million; TCGA, The Cancer Genome Atlas; SEM, standard error of the mean; CNV, copy number variation.
Overexpression of MUC16 was associated with favourable prognosis of patients with CC and mutant MUC16. Kaplan-Meier plots depicting the OS, DSS and PFS time in patients with (A) MUC16-mutant, (B) MUC16-wild-type and (C) all patients with CC from TCGA database. Kaplan-Meier plots depicting the OS, DSS and PFS time in patients with (D) MUC16-mutant, (E) MUC16-wild-type and (F) all patients with cervical squamous cell carcinoma from TCGA database. CC, cervical cancer; OS, overall survival; DSS, disease-specific survival; PFS, progression-free survival; TCGA, The Cancer Genome Atlas; HR, hazard ratio.
MUC16 mutations were involved in immune responses. (A) Volcano plot depicting the NES and negative logarithmically transformed P-value of immune response-associated gene sets in MUC16-mutant and MUC16-wild-type CC samples from TCGA database. Enrichment of (B) B cell-associated and (C) T cell-associated gene sets with overexpression of MUC16, in MUC16-wild-type and MUC16-mutant CC samples from TCGA database. (D) Bar plots depicting the activity level of multiple immune responses in different disease stages between MUC16-mutant and MUC16-wild-type samples of CC from TCGA database. Data are presented as the mean ± standard error of the mean. NES, normalized enrichment score; CC, cervical cancer; TCGA, The Cancer Genome Atlas.