Background
Cervical cancer is a common gynecological malignant disease in women [
1‐
4]. The morbidity and mortality of cervical cancer have gradually increased in China, and it has gradually become younger [
5]. In 2020, there were about 604,000 newly diagnosed cases and 342,000 deaths of cervical cancer worldwide [
6]. Human papilloma virus (HPV) infection is a common sexually transmitted disease [
7], and more than 90% of cervical cancer is caused by persistent high-risk HPV (HR-HPV) infection [
8,
9]. Cervical cancer has no obvious symptoms in the early stage of infection, which is easy to be ignored and misdiagnosed. Moreover, cervical cancer has a high degree of malignancy and is prone to metastasis and invasion [
10]. Although the diagnosis and treatment of cervical cancer has been improved, and more than 90% of patients with cervical cancer in the early stage of infection can be cured, the survival rate of patients with advanced cervical cancer is still very low, especially for patients with metastatic cervical cancer [
11]. Tumor invasion and metastasis are still important obstacles to the treatment of cervical cancer [
12].
According to PSORT (
https://psort.hgc.jp/) analysis, TCP11 protein may be mainly distributed in the cytoplasm. TCP11 is evolutionarily conserved in most metazoans and contains an uncharacterized protein domain, the TCP11 domain, that comprises most of the protein [
13].
TCP11 gene is a human homologue of mouse
Tcp11 gene, which can encode fertilization-promoting peptide (FPP) receptor, and there are three splicing products named TCP11a, TCP11b and TCP11c [
14]. It has been reported that TCP11 protein plays a role in sperm capacitation and acrosome reaction, and can regulate the activity of adenylate cyclase cAMP pathway [
15]. In addition, changes in DNA methylation of
TCP11 gene are a characteristic of the sarcoma component of Uterine Carcinosarcoma (UCS) [
16].
TCP11 is overexpressed in bilateral varicocele patients, which can be used as a potential biomarker for bilateral varicocele [
17].
At present, there is no article report on TCP11 in cervical cancer. Interestingly, GEPIA database analysis showed that TCP11 gene was highly expressed in cervical cancer, and its high expression was beneficial to the prognosis of cervical cancer patients. But, the effect of TCP11 gene on the development of cervical cancer remains unclear. The purpose of this study was to evaluate the effect of TCP11 on proliferation, apoptosis and migration of cervical cancer cells. The study revealed that TCP11 may be a potential biomarker of cervical cancer, playing a role in the prevention, treatment and analysis of cervical cancer prognosis.
Materials and methods
The 22 K human genome array of CapitalBio
Four cases of cervical cancer and corresponding normal tissues adjacent to cancer were selected to extract tissue RNA by Trizol (Invitrogen, Gaithersburg, MD, USA) one-step method, and the total RNA was further purified by the NucleoSpin® RNA clean up kit (MACHEREY-NAGEL, Germany), quantified by spectrophotometer, and examined by formaldehyde denaturing gel electrophoresis. Fluorescent labeling of sample RNA (crystal core® CRNA amplification labeling kit). Hybridize and wash, and the labeled DNA is dissolved in 80 µL in the hybridization solution (3×SSC, 0.2% SDS, 5×Denhart’s, 25% formamide), hybridization at 42 ℃, overnight. After hybridization, 0.2% SDS, 2×Wash in liquid of SSC for 5 min, and then wash in 0.2×Wash in SSC at room temperature for 5 min. The slides can be used for scanning after being dried. The chip is scanned by LuxScan 10KA dual channel laser scanner (CapitalBio).
Gene expression profiling interactive analysis (GEPIA) database
GEPIA database (
http://gepia.cancer-pku.cn/index.html), developed by Peking University team, was used for gene expression analysis of 9736 tumors and 8587 normal samples from TCGA and GTEx databases [
18]. In this paper, GEPIA database was used to analyze the expression of
TCP11 gene in cervical cancer and its relationship with survival rate.
Sample Collection
The tissue wax blocks, Hematoxylin-Eosin (HE) sections and relevant medical history information of patients in the First Affiliated Hospital of School of Medicine, Shihezi University, Xinjiang from 2017 to 2019 were collected, including 31 normal cervical tissues and 35 cervical cancer tissues. The clinical information can be seen in supplementary Table
S1. All specimens were diagnosed by two experienced pathologists, and all patients provided written informed consent. It has been approved by the Medical Ethics Committee of the First Affiliated Hospital of Shihezi University Medical School (Approval Number: KJ2020-051-01).
Tissue microarray and immunohistochemistry
According to the HE sections of collected cervical tissue wax block, pathological experts marked the site of the cervical epithelium on the sections. Sampling was carried out according to the marker, and tissue microarray were made. The sections were deparaffinized with xylene and hydrated with alcohol, and then treated with citrate buffer for antigen-retrieval. After incubation in the dark in 3% H2O2, they were incubated with TCP11 antibody (Proteintech, China, 1:200) overnight. The next day, the sections were incubated with rabbit/mouse general secondary antibody (ZSGB-BIO, China) for 30 min. After DAB staining, the sections were stained with hematoxylin, dried and sealed, and the results were interpreted by pathological experts.
Cell origin and cell culture
Cervical cancer SiHa cell line and HeLa cell line were purchased from Wuhan Boster Biological Technology Co., Ltd. in 2015. Cervical cancer C33A cell line and immortalized normal epithelial HaCaT cell line were purchased from Wuhan Procell Life Science&Technology Co., Ltd. in 2019. HaCaT cells were cultured on Dulbecco’s Modified Eagle Medium (DMEM) containing 15% Fetal Bovine Serum (FBS) and 1% penicillomycin, and cervical cancer cells were cultured on DMEM containing 10% FBS and 1% penicillomycin. The cells were cultured in a 37 °C incubator with 5% CO2, and passaged at intervals of 2–3 days.
Cell transfection
The lentivirus overexpressing TCP11 gene was designed and constructed by Shanghai Genechem Co., LTD. Lentiviruses were transfected in cervical cancer HeLa and SiHa cells. After 72 h of infection, puromycin (Solarbio, China) was used to select the stably transfected cells. The lentvirus that overexpresses TCP11 gene is built with the transcript NM-001370687 (containing 503 amino acids) as the template. The selected GV341 vector is added with 3 Flag tags (each containing 8 amino acids), and the number of amino acids of TCP11 protein is 527. Therefore, after infection with lentivirus in cervical cancer cells, the molecular weight of TCP11 protein will increase, and the molecular weight size is about 58 kDa. The siRNA that interfered with TCP11 gene was designed and constructed by Shanghai GenePharma Co., Ltd. Cervical cancer HeLa cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, USA) reagent. TCP11 siRNA sequence is as follows:
siRNA-TCP11-1: GCCUGAGAAUUGAGAUUGATT;
siRNA-TCP11-2: GCAGCCUAGUCUCCUUAAUTT;
siRNA-TCP11-3: GCUCUAAGCAGUGAUAAUATT;
siRNA-Negative Control: UUCUCCGAACGUGUCACGUTT.
Western blot
Total cell protein was extracted by using cell lysate buffer (RIPA/ PMSF, Solarbio, China). The protein concentration was detected using BCA kit (Beyotime, China). Proteins were separated by SDS-PAGE electrophoresis, and transfered to PVDF membrane (Immobilon-P, USA). Then, the membrane strips were blocked with blocking solution (5% nonfat dry milk), cut according to different molecular weights of the target strips, and the primary antibody was incubated overnight at 4℃. After washing the membrane strips, the rabbit/mouse secondary antibody (ZSGB-BIO, China) was incubated at room temperature for 2 h. Finally, a chemiluminescence reagent (Thermo, USA) was added dropwise to the membrane strips, and a chemiluminescence instrument (Tanon, China) was used for exposure. Anti-GAPDH (TA-08) and anti-β-actin (TA-09) were purchased from ZSGB-BIO (China). Anti-TCP11 (14606-1-AP) and anti-caspase-3 (8193T) were purchased from Proteintech (China). Anti-cleaved-PARP (BSM-33,138 M), anti-CDK1 (BS-0542R), and anti-CyclinB1 (BS-0572R) were purchased from Bioss (China). Anti-ZO-1 (66470-2-IG), anti-Snail (C15D3), anti-Vimentin (D21H3), anti-β-catenin (D10A8), anti-Claudin-1 (D5H1D) and anti-E-cadherin (3195T) were purchased from Cell Signaling Technology (USA).
Real-time quantitative PCR (qRT-PCR)
Trizol lysate buffer (Ambion, USA) was used to extract total cell RNA. Using the reverse transcription kit (TaKaRa, Japan), 1ug of each group of RNA was reversely transcribed into cDNA, and then PCR amplification was performed with GAPDH as internal reference. The amplification conditions were 95℃ for 30s, 95℃ for 3s, 60℃ for 30s, 40 cycles. The mRNA relative expression of target gene was calculated by 2
−ΔΔCt method. See Supplementary Table
S2 for primer sequences.
Thiazole blue (MTT) assay
Cervical cancer cells transfected with lentivirus or siRNA for 24 h were digested with trypsin and counted. Each well of 96-well plates was seeded with 1000 cells, and 5 replicates were set up for each group. MTT reagent (Solarbio, China) was added into wells, and incubated in the incubator for 3 h. The liquid in the wells was carefully discarded, and added with DMSO (MACKLIN, China). The 96-well plate was incubated on the shaker for 15 min in darkness. Cell proliferation was measured at 490 nm with BIO-RAD (USA) at 0, 24, 48, 72 and 96 h, respectively.
Cervical cancer cells transfected with lentivirus or siRNA for 24 h were digested with trypsin and counted. Six-well plates were seeded with 500 cells/well. The culture was continued for about 2 weeks, and the medium was changed every 3–4 days. When the number of colony cells was > 50, or the cell colonies were visible to the naked eye, the culture was stopped. After washing the cells with PBS, they were fixed with 4% paraformaldehyde (Biosharp, China) for 30 min and stained with 0.1% crystal violet (Solarbio, China) for 30 min. Finally, the difference of colony number was analyzed by photographing and counting.
Cell drop slice
Cervical cancer cells with stable infection of lentivirus were collected, fixed with 4% paraformaldehyde for 20 min and then incubated with 0.1% Triton X-100 (Solarbio, China) for 15 min to increase cell permeability. After the cells were washed with PBS, an appropriate amount of PBS was added for resuspension, and the trace suspension was absorbed and added to the 12-well slides soaked in poly-lysine, so that the cells presented a monolayer and a density of about 70%. After drying, 12-well slides were stored at -20℃ for using in subsequent experiment. After the slides were immunochemically stained (Ki67, ZSGB-BIO, 1:400), 4 areas were randomly selected, and Image Pro Plus software was used to measure the integrated optical density (IOD) value and area value. Mean density = IOD/area was calculated, which could reflect the protein expression level.
Transwell transfer assay
Cell migration was measured using a 24-well plate with Transwell chambers (Corning Costar, USA). The cells were suspended in serum-free medium, and 200 µL cell suspension (100 cells/ml) was added into the upper chamber, and 600 µL DMEM medium containing 10% FBS was added into the lower chamber. After incubating for 36 h in an incubator at 37℃ and 5% CO2, they were fixed with 4% paraformaldehyde, stained with crystal violet, washed with water and dried. The number of transmembrane cells was counted in 9 fields of view (×400) randomly taken from each chamber membrane under a high magnification microscope.
Cell scratch test
4 × 105 cells transfected with lentivirus were collected and inoculated in 6-well plates for culture. When the cells have grown to about 80% of the cell density, the cells were scratched in a straight using 200 µL pipette tip perpendicular to the bottom. The inverted microscope was used to take pictures at 0, 24 and 48 h, trying to keep the same position.
Cell cycle assay
Cervical cancer cells stably infected with lentivirus were collected and immobilized with pre-cooled 70% ethanol according to the instructions of the Cell Cycle Detection Kit (LIANKE, China). On the day of detection, 1mL DNA Staining Solution was added and let stand for 30 min avoiding light at room temperature. The cell cycle distribution was detected and analyzed by flow cytometry, and > 10,000 cells were collected per sample.
Cell apoptosis assay
Cervical cancer cells stably infected with lentivirus were collected. According to the instructions of apoptosis Detection Kit (LIANKE, China), apoptosis was detected by flow cytometry using Annexin V-FITC and PI double staining.
Statistical analysis
All data input was organized by Excel software, processed and analyzed by SPSS 22.0. Statistics of measurement data were expressed as \({\bar{{\rm X}}}\) ± s, differences between groups were tested by t test, and tissue microarray results were tested by χ2 test. P < 0.05 was considered to be statistically significant.
Discussion
According to Uniprot database (
https://www.uniprot.org/),
TCP11 gene is related to cell differentiation, signal transduction, multicellular biological development and protein kinase A signal transduction. In this study, through GEPIA database and tissue microarray assay, we demonstrated that the expression of
TCP11 gene was higher in cervical cancer tissues than in normal cervical tissues. It is worth noting that the
TCP11 had low expression in testicular germ cell tumor in GEPIA database, which was contrary to the high expression in cervical cancer. This may be because
TCP11 gene is a testicle-specific gene product and enriched in normal testicle, but the expression of
TCP11 is reduced when the testis becomes cancerous, for reasons that are not clear. Interestingly, cervical cancer patients with high expression of
TCP11 had higher survival rates. This suggests that
TCP11 may be a prognostic indicator for cervical cancer patients. Furthermore, we found that
TCP11 overexpression significantly inhibited the proliferation and migration of cervical cancer HeLa and SiHa cells. Conversely, the low expression of
TCP11 promoted the cell proliferation in HeLa and SiHa cells. Knocking down the expression level of
TCP11 gene can promote the cell migration in HeLa cells.
TCP11 gene can affect the proliferation and migration of cervical cancer cells, possibly by affecting the expression of cell cycle related molecules and EMT related molecules. Therefore, we next analyzed the characteristics of the
TCP11 gene acting on these molecules.
It has been shown that the disorder of cell cycle regulation is one of the main factors leading to the proliferation of malignant cells [
19]. We found that overexpression of
TCP11 arrested the cell cycle of cervical cancer cells, HeLa cells were arrested in G2/M phase, SiHa cells were arrested in S phase. Cyclin B1 is a cyclin-related protein of the M phase, which can bind to CDK1 and activate CDK1 to realize the cell cycle transition from G2 phase to M phase [
20,
21]. The results showed that
TCP11 overexpression inhibited the protein and mRNA expressions of CDK1 and Cyclin B1. We speculate that
TCP11 gene may block the cell cycle by regulating the expression of CDK1/Cyclin B1, thereby inhibiting the proliferation of cervical cancer cells. In addition, we also explored the effect of
TCP11 on apoptosis of cervical cancer cells.
TCP11 gene may induce cell apoptosis by activating caspase-3, further activating its substrate PARP. EMT is the key to migration and invasion of malignant tumor cells [
22,
23]. To further investigate whether
TCP11 inhibits the migration of cervical cancer cells by regulating EMT, we detected the protein and mRNA expressions of EMT-related molecules ZO-1 and E-cadherin. The results showed that
TCP11 overexpression can inhibit the EMT process by increasing the protein and mRNA expressions of ZO-1 and E-cadherin, thereby inhibiting the migration of cervical cancer cells. Further research is needed on the proliferation and migration effects of
TCP11 gene on cervical cancer cells.
According to UniProt database analysis,
TCP11 gene can regulate the activity of cAMP pathway of adenylyl cyclase. cAMP as a second messenger can activate cAMP-dependent protein kinase (PKA) [
24,
25]. cAMP-PKA signaling pathway can regulate many cellular responses, including proliferation, apoptosis, migration and metabolism [
26‐
28]. It has been shown that cAMP/PKA signaling pathway can inhibit the migration of cervical cancer HeLa cells [
29].
TCP11 has been reported to be correlated with PKA signal transduction [
30]. Therefore, we speculated whether
TCP11 gene could inhibit the progression of cervical cancer cells by regulating the activity of cAMP/PKA signaling pathway. However, further studies are needed to verify this hypothesis.
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