Cervical cancer stands as one of the prevailing gynecological malignancies, marked by a heightened mortality rate [1]. Human papillomavirus (HPV) infection emerges as the primary instigator of cervical cancer, with the pivotal role played by the E6 and E7 genes of HPV16 in instigating the malignant transformation of host cells, thereby precipitating cervical epithelial lesions [2]. Recent investigations reveal that HPV E6/E7 mRNA detection, alongside HPV-DNA detection, exhibits commensurate sensitivity, commendable stability, and heightened specificity in screening cervical lesions [3].

DNA methylation, a prominent epigenetic mechanism, has been identified as a major contributor to various diseases, including cancer, through aberrant methylation of host genes. Hyper-methylation of the CpG sites within tumor suppressor genes represents a recurrent anomaly in tumour cells, leading to the silencing of these genes and ensuing tumorigenesis. HPV may precipitate cellular malignant transformation by directly or indirectly modulating gene DNA methylation levels [4].

Long non-coding RNAs (lncRNAs), particularly the recently discovered lncRNA MAGI2-AS3 of WW and PDZ domain 2, have garnered attention in recent years. A mounting body of evidence underscores the pivotal role of lncRNAs in the progression of cervical cancer, as they contribute to enhanced cell proliferation, migration, and invasion, thereby influencing the onset and advancement of cervical cancer [5]. lncRNA MAGI2-AS3, recently identified as a tumour suppressor in various cancer types, is subjected to scrutiny in this study [6]. Accordingly, our investigation delves into elucidating the role of lncRNA MAGI2-AS3 in cervical cancer and unravelling the specific mechanism through which HPV infection modulates the malignant transformation of cervical epithelial cells by regulating MAGI2-AS3 expression levels through DNA methylation. This inquiry not only provides novel molecular targets but also establishes a theoretical foundation for the diagnosis, treatment, and prevention of cervical cancer.

This paper is an experimental study, laboratory based, using cell lines.

2.6 Exploration of siRNA Transfection Conditions and siRNA Screening

Exploring the Conditions of siRNA Transfection

(1) Cell Plank: SiHa cells, exhibiting a robust growth state with a confluence rate of 90%, were subjected to trypsin digestion. A single-cell suspension was prepared with an antibiotic-free medium at a concentration of 5 $\times$ 10${}^4$ cells/mL. This suspension was then inoculated into a 24-well plate, allocating 500 µL per well, and cultivated in a 37 ℃, saturated humidity, and 5% CO${}_2$ cell incubator for 24 hours.

(2) siRNA/Lipofectamine RNAiMAX Complex Configuration: Three 1.5 mL EP tubes were designated as A, B, and C tubes. In A tube, 25 µL of serum-free and antibiotic-free medium, along with 0.25 µL of 20 µM Block-iTAlexa Fluor Red reagent (final concentration 0.01 µM), were added and gently mixed. The B tube received 2.5 µL of 20 µM Block-iTAlexa Fluor Red reagent (final concentration 0.1 µM) and was gently mixed. For the C tube, 50 µL of serum-free and antibiotic-free medium, along with 1.5 µL of Lipofectamine RNAiMAX reagent, were added and gently mixed. Subsequently, 25 µL from the C tube was transferred to both A and B tubes, mixed gently, and left to stand for 10 minutes at room temperature.

(3) Transfection: The combined siRNA/Lipofectamine RNAiMAX complex was added to the wells of the 24-well culture plate containing cells at a rate of 50 µL per well. The cell culture plate was gently shaken, and the cells were incubated in a 37 ℃, saturated humidity, and 5% CO${}_2$ incubator for 24 and 48 hours. Fluorescence photos post-transfection were captured to ascertain the optimal transfection conditions.

(4) Determination of Optimal Transfection Conditions: To establish the optimum transfection conditions for siRNA, control siRNA, labelled with varying concentrations of red fluorescence, was used to transfect HPV-positive cervical cancer cells (SiHa). After 48 hours, the red fluorescence within the cells was observed using a fluorescence microscope. Experimental outcomes revealed that siRNA achieved the best transfection efficiency at a concentration of 0.1 µM and a transfection time of 48 hours (refer to Fig. 1). Consequently, the transfection concentration of 0.1 µM siRNA and a transfection time of 48 hours were chosen as the optimal conditions for subsequent experiments.

Fig. 1.

Transfection fluorescence picture (100$\mathrm{\times }$).

(5) Determination of Transfection Conditions: The optimal transfection parameters were established, with a siRNA concentration of 0.1 µM and a transfection duration of 48 hours selected as the most efficacious conditions for subsequent experiments.

Cervical cancer stands as one of the prevalent malignant tumours in gynecology, with HPV infection serving as a pivotal risk factor in its onset and progression. In recent years, cytological screening has facilitated the early identification of cervical cancer and precancerous lesions, leading to enhanced treatment outcomes and prognoses. However, gene methylation screening exhibits superior sensitivity and specificity in this context [7]. DNA methylation involves the binding of cytosine to methyl groups within the CpG dinucleotides of the genome, inducing alterations in gene expression levels without modifying the DNA sequence. CpG islands, situated in the human genome, typically remain unmethylated in normal cells. Notably, hypermethylation of the CpG regions within tumour suppressor genes is a prevalent occurrence in tumour cells, resulting in the silencing of these genes and the initiation of tumorigenesis [8]. Recent investigations suggest that HPV DNA methylation, along with host DNA methylation, constitutes a crucial carcinogenic mechanism of HPV [9]. Methylation serves as a dependable marker for diagnosing, monitoring disease progression, and predicting the prognosis of cervical cancer. Furthermore, the modulation of methylation status can inform the development of effective treatment strategies, imparting profound guidance in the treatment of cervical cancer [10]. lncRNA is an RNA molecule exceeding 200 bp in length [11]. A substantial body of evidence indicates that lncRNA is intricately involved in various biological processes, encompassing tumorigenesis, differentiation, and metastasis [12]. Within hepatocellular carcinoma cells, lncRNA-H19 orchestrates the activation of the CDC42/PAK1 pathway by targeted miR-15b modulation, thereby fostering cell proliferation, migration, and invasion [13]. Growing evidence underscores the pivotal role of lncRNA in the progression of cervical cancer [14]. These molecules enhance cell proliferation, migration, and invasion, significantly contributing to the initiation and advancement of cervical cancer [5]. In the context of cervical cancer, lncRNA-CTS promotes epithelial-mesenchymal transition and cancer cell metastasis through the regulation of the miR-505/ZEB2 axis [15]. The lncRNA ZNF667-AS1 pathway mitigates the reduction of miRNA-93-3p-dependent PEG3 in cervical cancer, thereby restraining tumour cell invasion [16]. In recent years, the newly discovered lncRNA MAGI2-AS3 has emerged as closely associated with tumour occurrence and progression, regulating malignant biological behaviours such as tumour proliferation, apoptosis, invasion, and metastasis [17]. MAGI2-AS3, located within the chromosomal region 79452957-79471208 of human genome number 7, has been implicated in various cancers [17].

In murine models, hepatoma cells rely on lncRNA MAGI2-AS3 to inhibit cell viability and metastasis through the miR-374b/SMG1 signalling pathway [6]. In non-small cell lung cancer, lncRNA MAGI2-AS3 inhibits cell proliferation and invasion through the miRNA-23a-3p/PTEN axis [18]. Furthermore, MAGI2-AS3 has been found to promote the progression of gastric and colorectal cancer [19]. A study has identified an association between lncRNA MAGI2-AS3 and poor prognosis in bladder cancer [20]. Interestingly, MAGI2-AS3 exhibits a tumour-inhibitory role in high-grade serous ovarian and breast cancers [21]. However, the functional mechanisms of MAGI2-AS3 in cervical cancer remain unclear. This study employed qRT-PCR to assess the expression levels of the MAGI2-AS3 gene in HPV-negative cervical cancer cells (C33A) and HPV-positive cervical cancer cells (SiHa). The findings revealed a significantly lower expression of MAGI2-AS3 in HPV-positive cervical cancer cells (SiHa) compared to HPV-negative cervical cancer cells (C33A), suggesting a correlation between reduced MAGI2-AS3 expression and the onset and progression of cervical cancer. High methylation level is easy to cause HPV virus infection, which is easy to lead to cervical cancer. Concurrently, MassARRAY was utilised to evaluate the DNA methylation levels in the promoter region of the target gene MAGI2-AS3 in HPV-negative cervical cancer cells (C33A) and HPV-positive cervical cancer cells (SiHa). Results showed a notable increase in the methylation rate of these sites in HPV-positive cervical cancer cells (SiHa) compared to HPV-negative cervical cancer cells (C33A), highlighting a potential link between heightened methylation rates and the development of cervical cancer. The role of MAGI2-AS3 as a tumour suppressor and a biomarker for clinical diagnosis in cervical cancer cells warrants further investigation.

In conclusion, our hypothesis posits a close association between HPV infection and elevated methylation levels coupled with diminished expression of MAGI2-AS3. The regulatory influence of HPV infection on MAGI2-AS3 expression levels is postulated to occur through DNA methylation mechanisms, thereby influencing the malignant transformation of cervical epithelial cells.

In adherence to the journal’s stipulations, we commit to providing our data to facilitate the reproducibility of this study upon approval from our institution.

JZ: conceptualization, data curation, formal analysis, investigation, methodology, writing — original draft, writing - review & editing. QX: participate in the completion of writing — original draft, interpretation of data. QZ: conception, funding acquisition, resources. All authors contributed to editorial changes in the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors read and approved the final manuscript.

All subjects gave their informed consent for inclusion before they participated in the study. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xinjiang Medical University (Ethics Approval Number: 2022H024).

Not applicable.

This study was funded by Natural Science Foundation of Xinjiang Uygur Autonomous Region (2023D01C122) and Department of Gynecology, Second Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia (SKL-HIDCA-2022-GJ4), Open topic of the Key Laboratory of Neurological Diseases in Xinjiang (XJDX1711-2260) and the ”Tianshan Talents” medical and health high-level personnel training plan (TSYC202301B128).

The authors declare no conflict of interest.