Introduction
Cervical cancer is among the most frequently diagnosed malignancies among females [
1,
2]. Cervical cancer affected a total number of 569,847 females in 2018 [
3]. The majority of cervical cancer cases are caused by HPV infections [
4,
5]. Women are more frequently affected by HPV infection due to men being carriers of the virus. With increased understanding of the molecular mechanisms of HPV infection and application of HPV vaccination, the incidence and mortality rates of cervical cancer have significantly dropped during the past decade [
4‐
7]. However, HPV vaccination does not provide benefit to individuals who are already infected. Therefore, it is crucial to have screening programs in place for early cervical cancer diagnosis [
8]. Furthermore, cervical cancer can also impact HPV-negative patients [
9], indicating the intricate molecular pathogenesis of cervical cancer.
The development and progression of cervical cancer entail the involvement of molecular factors [
10]. Understanding of the functions of molecular pathways involved in cervical cancer accelerates the development of novel targeted therapy [
11]. Long non-coding RNAs (lncRNAs) do not encode proteins. However, they regulate gene expression to modulate various aspects of cancer [
12]. Therefore, regulating the expression of lncRNAs may contribute to the treatment of cancers [
13]. WWTR1-AS1 is a recently identified oncogenic lncRNA in head and neck squamous cell carcinoma [
14], while its role in cervical cancer is unknown. Recent studied have shown that microRNA (miR)-136 binds directly to Notch receptor 3 (Notch3) [
15,
16].Our bioinformatics analysis also showed that WWTR1-AS1 might interact with miR-136, which plays tumor suppressive roles by targeting Notch3 to suppress tumor cell stemness [
16]. Therefore, we hypothesized that WWTR1-AS1 upregulated Notch3 through miR-136 to increase cervical squamous cell carcinoma (CSCC) cell stemness. This study was carried out to investigate the interaction among WWTR1-AS1, miR-136 and Notch3 in CSCC, which is the major subtype of cervical cancer. Our findings could have potential advantages for the early diagnosis of CSCC.
Materials and methods
CSCC patients
This study enrolled a total of 60 research subjects who were diagnosed with CSCC at Bishan Hospital Affiliated to Chongqing Medical University, between May 2017 and May 2019. The age of the patients ranged from 40 to 68 years, with a mean age of 53.3 ± 6.0 years. There were 28 male and 32 female patients. The Ethics Committee of this hospital approved this study. All clinicopathological data were shown in Table
1. All 60 patients had complete medical records. All CSCC patients were diagnosed for the first time. Recurrent CSCC cases were excluded from this study. Patients complicated with other clinical disorders or with initiated therapy were also excluded. All patients were diagnosed through histopathological biopsy, during which CSCC and paired non-tumor tissue samples were collected from each patient. Tissues were stored in liquid nitrogen before use. Based on their medical record, the 60 patients included 44 HPV-positive cases (within 6 months before diagnosis) and 16 HPV-negative cases. Based on clinical stage (AJCC), there were 13, 11, 24 and 12 cases at clinical stage I, II, III and IV, respectively. All patients signed the informed consent.
Table 1
Clinicopathological data of 60 CSCC patients
Gender | |
Male | 28 |
Female | 32 |
Age (years, 40–68) | |
≤ 53 | 31 |
>53 | 29 |
HPV | |
HPV positive | 44 |
HPV negative | 16 |
TNM stage | |
I | 13 |
II | 11 |
III | 24 |
IV | 12 |
Specimens and cells
Before therapy, fine needle aspiration was conducted to collect fresh tissues. SiHa (HPV-16 positive) human CSCC cell line (ATCC, USA) was used. SiHa cells were cultivated in EMEM. The cell culture was maintained at 95% humidity, 37 °C and 5% CO2. Cells were harvested when about 85% confluence was reached to perform subsequent experiments.
Transient transfections
WWTR1-AS1 and Notch3 expression vectors were prepared using pcDNA3.1 as the backbone vector. Negative control (miR-NC mimics, Sigma-Aldrich), miR-136 mimic and miR-134-inhibitor were purchased from Sigma-Aldrich (USA). The sequences for miR-NC, miR-136 mimics, miR-136 inhibitors and inhibitor NC were: 5’-GUCCCAC-UUCGACCGUGCUUCCA-3’, 5’-ACUCCAUUUGUUUUGAUGAUGGA-3’, 5’-CAUCAUCGUCUCAAAUGAGUCU-3’, and 5’-UGUGCCUAUGUGAUGAUGUAC-3’, respectively. SiHa cells at 50–70% confluence were transfected with 10 nM expression vector and (co-transfection)/or 40 nM miRNA using lipofectamine 2000 (Invitrogen) following the instruction of manufacturers. Untransfected cells were used as the Control (C) cells. NC cells were miR-NC mimics - or empty vector-transfected cells. Subsequent experiments were performed 48 h later.
Dual luciferase activity assay
Dual-luciferase reporter assay was performed as previously described [
17]. The WWTR1-AS1 wild-type (WT) and mutant (MUT) luciferase system were constructed using pGL3 as the vector backbone. Lipofectamine 2000 (Invitrogen) was used to co-transfect WWTR1-AS1 (WT/MUT) + miR-136 mimic (miR-136 group) or WWTR1-AS1 (WT/MUT) + NC miRNA (C group) into SiHa cells. Luciferase activity was measured and compared 48 h later using a Dual-luciferase Reporter Assay System (Promega).
RNA preparation
RNA was extracted from paired tissue samples and in vitro-cultured cells, followed by quantification using a NanoDrop spectrophotometer (NanoDrop Technologies). Subsequently, DNA removal was carried out using gDNA eraser (Takara Bio).
RT-qPCR
Total RNA reverse transcriptions and qPCR reactions were conducted using BlazeTaq™ One-Step SYBR Green RT-qPCR Kit (Genecopoeia) to detect the expression of WWTR1-AS1, miR-136 AND Notch3 mRNA with GAPDH as the endogenous control. Primer used were as follows: GAPDH forward: 5’-GGTGAAGGTCGGAGTCAACG-3’, reverse: 5’-CAAAG TTGTCATGGATGHACC-3’; WWTR1 AS1 forward: 5’-GATGCCTCCTCGCCAGACCA-3’, reverse: 5’-TACTTAGTGGCT- CAGGTCTC-3’; Notch3 forward: 5’-GTCTTCCAGATTCTCATCC-3’, reverse: 5’-ATCCACAGCATTGACATC-3’; miRNA-136 forward: 5’-ACUCCAUUUGUUUUGAUGAUGGA-3’, reverse: 5’-UCCAUCAUCAAAACAAAUGGAGU-3’; U6 forward: 5’-GCTTCGGCAGCACATATACTAAAA-3’, reverse: 5’-CGCTTCACGAATTTGCGTGTCA-3’.
Western blot analysis
In vitro cultivated cells were subjected to total protein isolation using RIPA buffer (Invitrogen), and the protein concentration was quantified using a BCA assay (Invitrogen). Following denaturation at 95 °C for 10 min, SDS-PAGE gel (10%) was used to separate proteins, and separated proteins were transferred to PVDF membranes. Blocking was performed in PBS containing 5% non-fat milk, followed by incubation with rabbit primary antibodies of Notch3 (1:1,000, ab23426, Abcam) and GAPDH (1: 1,000, ab8245, Abcam) at 4 °C for 15 h. After that, secondary antibody incubation was performed. Signals were normalized using Image J V 1.6 software.
Cell stemness assay
SiHa cells collected at 48 h post-transfection and washed with cold PBS, followed by incubation with immunoglobulin (Ig) G1-PE (Miltenyi Biotec) or phycoerythrin (PE)-conjugated CD133, CD44 or CK17 (Biosciences) at 4 °C for 25 min. Following that, CD133 + cells were separated by performing flow cytometry.
Statistical analysis
Data from 3 independent replicates were presented as the mean ± standard deviation (SD). Paired t-tests were employed to compare paired tissue samples, while unpaired t-tests were utilized for comparing two independent groups. For the comparison of multiple groups, a one-way ANOVA and Tukey’s test were employed. Linear regression and Pearson’s analysis were used to analyze correlations. Kaplan-Meier curves were generated using SPSS, and P-values were calculated using the log-rank test. P < 0.05 was considered as statistically significant.
Discussion
CSCC treatments traditionally include surgery, chemotherapy, and radiation. Despite these interventions, overall survival rates have not seen substantial improvements, highlighting the need for novel approaches. Immunotherapy, which has demonstrated effectiveness in various cancer types, has emerged as a promising avenue. Recent developments in immunotherapy have expanded the possibilities, including the potential for therapeutic vaccines and other tailored treatment strategies in cervical cancers. These advancements contribute to the pursuit of more precise and effective therapies [
21]. The molecular mechanisms unveiled in this study provide insights into the enhancement of CSCC screening and potentially refining treatment approaches.
The present study investigated the interplay among WWTR1-AS1, miR-136 and Notch3 in CSCC. We observed significant alterations in the expressions of WWTR1-AS1 and miR-136 in CSCC. Notably, WWTR1-AS1 was identified as a sponge for miR-136, leading to the upregulation of Notch3 and consequently enhancing cell stemness. The function of WWTR1-AS1 has only been investigated in head-neck squamous cell carcinoma, in which WWTR1-AS1 is upregulated and promotes cancer cell proliferation, invasion and migration [
14]. We observed the upregulation of WWTR1-AS1 in CSCC, and overexpression of WWTR1-AS1 increased cancer cell stemness. Therefore, WWTR1-AS1 is likely an oncogenic lncRNA in CSCC. It is known that HPV infections may promote the development of CSCC by regulating the expression of lncRNAs [
22]. In this study, we observed no significant differences in the expression levels of WWTR1-AS1 in CSCC tissues between HPV-positive and HPV-negative patients. This suggests that WWTR1-AS1 may be involved in CSCC through an HPV-independent mechanism.
MiR-136 suppresses several types of cancers including CSCC [
23,
24]. It has been reported that miR-136 is downregulated in cervical cancer, suppresses tumor cell apoptosis and induces cell apoptosis by targeting E2F1 [
24]. In a recent study, miR-136 was reported to suppress cell stemness [
16]. Additionally, Zong et al. [
15]. demonstrated that miR-136 could target the NOTCH3 pathway. However, there is no there is no prior study investigating the specific mechanism of the miR-136/Notch3 interaction in cervical squamous cell carcinoma in cervical squamous cell carcinoma.
Interestingly, in this study, we showed that WWTR1-AS1 and miR-136 could interact with each other. However, there is no significant correlation between them in both CSCC and non-tumor tissues. Furthermore, WWTR1-AS1 and miR-136 do not mutually regulate each other’s expression. A similar lack of regulatory influence between SNHG10 and miR-543 has been documented [
25]. In our study, we similarly observed an interaction between WWTR1-AS1 and miR-136, yet neither of them exhibited regulatory control over the expression of the other, and they did not display a significant correlation. Therefore, WWTR1-AS1 was unlikely a target of miR-136. However, WWTR1-AS1 reduced the effects of miR-136 on the expression of Notch3 and cancer cell stemness. Furthermore, considering that WWTR1-AS1 directly binds to miR-136 and miR-136 directly targets Notch [
15,
16], our hypothesis was that WWTR1-AS1 promotes Notch3 expression by acting as a miR-136 sponge. Indeed, we observed an upregulation of WWTR1-AS1 expression in CSCC tissues and predicted its interaction with miR-136. While the correlation analysis indicated no significant correlation between WWTR1-AS1 and miR-136, we observed that the overexpression of WWTR1-AS1 increased the expression levels of Notch3, a target of miR-136. Our study demonstrated that miR-136 could also target Notch3 to decrease cancer cell stemness in CSCC. In summary, although we have provided initial insights into the relationship between WWTR1-AS1 and miR-136 and have shown that the lncRNA WWTR1-AS1 upregulates Notch3 through miR-136 to enhance cancer cell stemness in CSCC, further evidence is required to hold the reliability of our findings. For instance, additional analysis involving stemness markers such as Nanog, OCT-4, and SOX-2 is needed to further elucidate the impact of WWTR1-AS1 on cancer cell stemness.
In conclusion, WWTR1-AS1 was upregulated, and miR-136 was downregulated in CSCC. WWTR1-AS1 may sponge miR-136 to upregulate Notch3, thereby increasing cell stemness.
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