Discussion
Cervical cancer remains a prevalent malignancy among women ( Hernández-Silva et al., 2024 ), and reliance on existing therapeutic strategies continues to irreversibly impact patients’ life span and quality of life. Consequently, early diagnosis and further exploration into the pathogenic mechanisms of cervical cancer are paramount. Proteins, being executors of biological activities, offer a more intuitive insight into the proteomic expression spectrum of cervical cancer cells through the measurement and analysis of protein levels compared to genomic studies. Currently, mass spectrometry technology finds extensive application in clinical practice ( Keevil, 2013 ), enabling further investigation into protein alterations throughout the cervical cancer pathogenic process using proteomic methods.

The mass spectrometry analytical method used in this experiment is the label-free quantification (label free) technique. This approach allows the analysis of an almost unlimited number of samples without introducing any labels, thereby maintaining low cost and minimizing sample preparation steps. This method is highly desirable for biomarker research. However, its poor reproducibility may necessitate analysis of numerous technical replicates, possibly leading to low accuracy in quantitative measurements. Despite this, compared to labeled strategies, the label-free method has been proven to possess the greatest dynamic range and highest proteome coverage ( Rozanova et al., 2021 ).

In our research, we use the label-free quantification (label free) technique to obtain the proteomic spectrum of cervical squamous cell carcinoma, achieving comprehensive analysis of differentially expressed proteins through GO/KEGG analysis and gene set enrichment analysis (GSEA). Our analysis reveals 562 up-regulated or down-regulated proteins compared to the control. Based on the results of proteomics and the latest reported literature, we selected three different proteins from the 20 up-expressed protein molecules for further analysis. The proteins DSP, PPP1R13L and ANXA8 are selected for further analysis. Immunohistochemistry and western blotting of cervical squamous cell carcinoma tissue slices with antibodies against DSP, PPP1R13L and ANXA8 is conducted, hypothesizing these proteins’ fundamental roles in the regulation and progression of cervical cancer cells.

Desmoplakin (DSP) is involved in the organization of the desmosomal cadherin-plakoglobin complexes into discrete plasma membrane domains and in the anchoring of intermediate filaments to the desmosomes ( Andrei et al., 2024 ). DSP serves as an obligate component of desmosomes that function as intercellular adhesion junctions and sites for intermediate filament attachment ( Boyden et al., 2016 ). Besides maintaining the integrity of desmosomes, DSP also plays an important role as tumor suppressor ( Sano et al., 2021 ) by regulating various signaling pathways in cancer cells. DSP has a tumor suppressive function through inhibition of Wnt/-catenin signalling pathway in human lung cancer ( Wang et al., 2019 ). PPP1R13L gene, also known as iASPP (inhibitory member of the ASPP family, iASPP), is located in 19q13 ( Nexø et al., 2008 ). As the third member of the TP53 apoptosis stimulating protein family, its role is to inhibit the anti-tumor effect of the other two members (ASPP1 and ASPP2), leading to abnormal cell proliferation and carcinogenesis ( Li et al., 2024 ). Accordingly, it was generally believed that PPP1R13L as a novel oncogene highly expressed in a variety of tumor cells ( Yagudin et al., 2021 ). Protein Phosphatase 1 Regulatory Subunit 13 Like (PPP1R13L) is a newly identified oncoprotein previously reported to inhibit the transcriptional activity of SP1 via a direct protein-protein interaction) ( Zhang et al., 2022 ). The other study showed that High iASPP expression in the tumor cell cytoplasm is associated with poor outcomes of OSCC patients in terms of recurrence and survival, suggesting a role for iASPP as a novel biomarker and therapeutic target for oral cavity squamous cell carcinomas (OSCC) ( Kim et al., 2015 ).

ANXA8 is a member of the annexin family and belongs to the superfamily of Ca 2+ /phospholipid-binding proteins ( Zhou et al., 2021 ). ANXA8 plays a crucial role in the principal communication pathways between Ca 2+ signal transduction and Ca 2+ -regulated cell membrane dynamics in all eukaryotic organisms ( Lee et al., 2009 ). These biological entities are closely associated with various diseases, such as cancer, diabetes, and autoimmune disorders. ANXA8 has been demonstrated to be intimately connected with various types of malignant cancers ( Zhang & Han, 2021 ). According to proteomic Gene Ontology (GO) analysis predictions, ANXA8’s primary functions in cervical cancer might include signal transduction, positive regulation of apoptosis processes, negative regulation of coagulation, protein oligomerization, and negative regulation of calcium ion chelation, among others ( Zhou et al., 2021 ). Cancer-related articles have corroborated ANXA8’s involvement in signal transduction functions. ANXA8 regulates the proliferation of human non-small cell lung cancer cells A549 through the EGFR-AKT-mTOR signaling pathway ( Yuan et al., 2021 ). Over-expression of miR-140-3p, targeting ANXA8, inhibits bladder cancer cell proliferation, migration, invasion, and EMT ( Lee et al., 2009 ). Downregulation of ANXA8 in the EGF-FOXO4 signaling pathway is involved in cholangiocarcinoma cell migration and tumor metastasis ( Hata, Tatemichi & Nakadate, 2014 ). Regarding cervical cancer, reports demonstrate that ANXA8 is significantly expressed in squamous cell carcinoma but not evidently in adenocarcinoma, suggesting its potential as a molecular marker to differentiate between cervical squamous cell carcinoma and adenocarcinoma ( Chao et al., 2006 ). This proteomic data, derived from cervical squamous cell carcinoma cells, indeed detected the upregulation of ANXA8, hypothesizing that ANXA8, as an up-regulated protein, plays a significant role in cervical squamous cell carcinoma.

In summary, the above three molecules DSP, PPP1R13L and ANXA8 may play important roles in related tumors, but their role in cervical cancer is still unclear. Our group research found that DSP as well as PPP1R13L and ANXA8’ expression were up-regulated by IHC method. Further study showed that the western blotting results showed that they were also overexpressed in cervical cancer tissues compared with the paracancer tissues, and further statistical analysis shows significant differences between the two groups. Thus results indicated that the three proteins DSP, PPP1R13L and ANXA8, studied above may play important roles in the occurrence or metastasis of cervical cancer, which can provide important theoretical supplements for the occurrence, development, and clinical transformation of cervical cancer. To further verify the validity of the data, we used GEPIA database to validate the expression of DSP, PPP1R13L and ANXA8 in human cancers and normal cervix. We found that the transcription levels of DSP, PPP1R13L and ANXA8 increased significantly.

This study employs label-free technology to explore the proteomics of cervical cancer tissue. In comparison with normal cervical tissue, differential proteins were identified, and further research was conducted on the molecular functions possessed by these differential proteins, their roles in biological processes, and their participation in molecular pathways. Additionally, three differential proteins, DSP, PPP1R13L and ANXA8, were selected, and their possible promoting roles in the occurrence and development of cervical cancer were speculated upon.