The human papillomavirus (HPV) is a DNA virus that encodes approximately eight genes. It can cause cancers of the antrum, genital tract, upper respiratory tract, and digestive tract (Dunne and Park, 2013). High-risk HPV E6 and E7 proteins reduce immune recognition by disrupting cell adhesion, polarity, and transcription via several pathways, such as the cell adhesion pathway (Oldak et al., 2006; Whiteside et al., 2008; Howie et al., 2009; Läubli and Borsig, 2019; Kombe Kombe et al., 2020). Studies have shown that the cervical squamous epithelium, which is the target of HPV, consists of keratinocytes, immature dendritic cells (DCs), and Langerhans cells (LCs), all of which perform immunosurveillance functions on the epithelium (Schiffman et al., 2007). LCs are the only specialized antigen-presenting cells found in the epidermis, and their abnormal expression affects antiviral immune responses. HPV-associated cervical squamous epithelial neoplasia (SIL) is strongly associated with immature LCs (Habbous et al., 2012). Cell adhesion between cells and the extracellular matrix (ECM) is required for the integrity of the epidermis and the formation of an immune barrier (Honig and Shapiro, 2020). Cell surface adhesion molecules, the major components of cell-to-cell and cell-to-extracellular interactions (Login et al., 2019), are frequently expressed in epithelial (Sumigray and Lechler, 2015), endothelial (Schimmel and Gordon, 2018), and immune (Dustin, 2009) cells. They consist of intracellular and extracellular components (CC) and can be divided into four types, cadherins, integrins, selectins, and immunoglobulins. Many studies have focused on malignant metastasis and cell adhesion (Janiszewska et al., 2020); however, the mechanisms related to interactions among HPV proteins, cell adhesion, and the epithelial immune response remains unclear. Activated leukocyte cell adhesion molecule (ALCAM) (Ferragut et al., 2021) is a cell-surface glycoprotein that has been recognized as a vital prognostic marker for various tumors, including HPV-associated head and neck squamous cell carcinoma (Zhu et al., 2020). Based on current research, ALCAM can regulate adhesion to immune cells (Te Riet et al., 2014) and control disease progression by affecting cell proliferation, migration, and invasion (von Lersner et al., 2019).

Chemokines are the largest subfamily of cytokines (Bule et al., 2021). It is well known that HPV is capable of preventing a robust immune response to the infected cells based on many mechanisms, such as by reducing the expression of chemokines (Attademo et al., 2020; Acevedo-Sánchez et al., 2021). When chemokines are unable to guide immune cells to migrate to the infected site in an orderly fashion, immune tolerance collapses and immune monitoring becomes ineffective (Kolaczkowska and Kubes, 2013).

With the increasing prevalence of Illumina technology in recent years, RNA-sequencing (RNA-Seq) technology has been effectively used in biomedical research (Tao et al., 2021; Wang et al., 2021). The carcinogenetic effect of E6 and E7 is apparent, but molecules that are induced by these proteins in HaCaT cells still need to be researched, and high-throughput sequencing would contribute vastly to this question. In this research, the differential mRNA expression profiles of HPV16E6E7-stably expressing cell lines were determined via RNA-seq. Our results showed that transcriptome levels were significantly different between the two groups of non-transfected control and HPV16E6E7-stably expressing cell lines. Transcriptome data are essential in providing a research target for mechanistic studies aimed at reversing cervical epithelioma and blocking the malignant progression of epithelial cells.

It is well known that E6 and E7 can attach to the ECM of the cervical squamous epithelium. Further, HPV E6 and E7 can be detected in a substantial proportion of women with cervical intraepithelial neoplasia (CIN) (Guan et al., 2012); they also serve as attractive targets for HPV-related diseases (Hoppe-Seyler et al., 2018). Owing to its specific sites and functions in antigen presentation, LCs are considered essential for the immune response against HPV infection (Cunningham et al., 2010). In the epidermis of mice (Kaplan et al., 2005) and female genital tract (Britto et al., 2020), LCs produce effector cytokines to stimulate T lymphocytes and B lymphocytes through interactions with C-type lectin (CLR receptors) and TLRs. Hubert et al. (Hubert et al., 2005) confirmed decreased levels of CDH1 mRNA and numbers of CD1α⁺ LCs in squamous cell carcinoma and SILs compared with those in normal cervical epithelia. According to the high throughput screening of clinical specimens (Britto et al., 2020), the decrease observed in the mRNA levels of CDH1, Claudin 1, Claudin 4, and ZO-1 mRNA and IFN signaling upon HPV infection indicate that it could result in weakness of the epithelial barrier (Aggarwal et al., 2019; Giorgi Rossi et al., 2021). Collectively, these findings suggest that the E6 and E7 oncoproteins are strongly associated with cell adhesion in HPV-induced immunosuppression. The loss of cell adhesion caused by the imbalance in adhesion molecules and chemokines might be another immune-escape strategy that has evolved in HPV.

CXC motif chemokine ligand 10 (CXCL10), CC motif chemokine ligand 2 (CCL2), CCL3, and CCL5 are pro-inflammatory chemokines that actively participate in the inflammatory response after viral exposure (Kolaczkowska and Kubes, 2013). CCL2 (Nakamura et al., 1995; Kunstfeld et al., 1998; Hacke et al., 2010) is a key chemokine that attracts cells, including T lymphocytes, eosinophil granulocytes, macrophages, mast cells, and monocytes, to inflammatory sites. In general, CCL2 expression is activated after viral infection and recruits Langerhans and DCs, respectively. However, co-expression of the E6 and E7 proteins inhibits CCL2 expression in primary epithelial cells of the female genital tract (Hacke et al., 2010). An in vitro study (Nakamura et al., 1995) has suggested that HPV E7 interacts with interferon regulatory factor 1 (IRF-1), a significant regulator of cellular immune responses, to suppress CCL2 expression. This partly explains the almost undetectable activation of potent anti-tumor immune responses in primary HPV-infected epithelial tissues. It has been reported that humans lack a robust inflammatory response against primary HPV infection and progressively lose immune cells in the cervical stroma during CIN (Kleine-Lowinski et al., 2003; Hacke et al., 2010; Garrido et al., 2021). CIN is strongly associated with CCL2 expression in the tumor microenvironment. Habbous et al. (2012) suggested that as CIN progresses, levels of many hosts immune response markers, including the chemokine CCL2, are reduced. However, the specific mechanisms involved remain unclear. As epithelial dysplasia progresses to cervical cancer, macrophages, T cells, and LCs of the cervical mucosa are depleted. Restoring CCL2 expression can specifically improve the anti-tumor immunity of the host and block disease progression. Therefore, exploring the selective loss of CCL2 expression after E6/E7 transduction is crucial to blocking disease progression. However, in cell adhesion, the role of chemokines still needs to be explored in many ways. CXCL10 (Halle et al., 2017; Takeuchi and Saito, 2017) is associated with increased quantities of tumor-infiltrating CD8⁺ T cells, inducing the release of cytotoxic molecules (such as granzyme B and perforin) and apoptosis, which are associated with lower levels of cancer metastasis and improved patient survival in ovarian and colon cancer. CCL5 (Bruand et al., 2021) expression was found to be positively correlated with CD8⁺ T cell infiltration. T cell infiltration reportedly requires the secretion of CCL5 by cells in both epithelial cancer cell models and animal models. Zhang et al. (2020) suggested that CCL7 promotes anti-PD-1 therapy (CDC1) in a mouse model by recruiting conventional DCs to the tumor microenvironment to promote T cell proliferation. In addition, chemokines contribute to anti-tumor immunity by recruiting tumor-associated antigen-presenting cells, promoting their binding to T cells, and influencing tumorigenesis by interacting with tumor stem cell-like cells and stromal cells (Kombe Kombe et al., 2020). In summary, different lymphocyte subsets are recruited into the tumor microenvironment through different chemokine–chemokine receptor signaling pathways. The restricted expression of chemokines directly or indirectly leads to the failure of anti-tumor immunity.

Our study revealed 1,644 upregulated mRNAs and 1,261 downregulated mRNAs after E6/E7 overexpression. According to RNA-seq analysis, there is a tight connection between DEGs and cell adhesion, focal adhesion, leukocyte–endothelial migration, tight junctions, and Hippo and Wnt signaling pathways. KEGG analysis revealed that these DEGs were highly focused in cancer proliferation-related pathways, including the MAPK, Hippo, Wnt, and cell adhesion signaling pathways. It has been suggested that E6/E7 might exhibit crosstalk with these signaling pathways, affecting cell adhesion and limiting the immune response. CXCR4 (Zou et al., 1998; Goïta and Guenot, 2022) is a chemokine G protein-coupled transmembrane receptor released by stromal cells. Stromal cell-derived factor-1 (SDF-1; CXCL12), a ligand of CXCR4, induces B-cell proliferation and T-cell recruitment by activating CXCR4-expressing cells. Studies have demonstrated that the immune infiltration of B cells in the tumor microenvironment is associated with a survival advantage in patients with cervical (Abdul Rahman et al., 2020), breast (Hassel et al., 2021), and high-grade serous ovarian (Montfort et al., 2017) cancer; B cells are recruited through the binding of CXCL12 and CXCR4 (Greenbaum et al., 2013). Furthermore, CXCL12/CXCR4 enhances Src phosphorylation by activating MAPK or mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) kinases (Kasina and Macoska, 2012). CXCR4-induced signaling cascades are commonly referred to as the “CXCR4–SDF-1 axis” and are frequently associated with lymphocyte trafficking and homing. To participate in the immune response (Peled et al., 1999), CXCL12 binds to CXCR4 to stimulate the firm adhesion of leukocytes by increasing integrin stability, which binds to intracellular adhesion molecules (ICAM-1) and vascular CAMs (VCAM-1). Researchers (Zheng et al., 2017) have used the CRISPR-Cas9 system in a cellular model to knock out CXCR4 and observed that colorectal cancer cell lines showed reduced adhesion to the ECM and endothelium, further demonstrating that CXCR4 is closely related to the Akt and type 1 insulin-like growth factor receptor (IGF1R) signaling pathways. In mouse models (Aggarwal et al., 2019), the CXCL12/CXCR4 axis enhances the function of M1-type macrophages, stimulates the production of inflammatory cytokines, and increases the phagocytosis of pathogens. Furthermore, it has been shown that CXCL12 primes IL-1 production in M2 macrophages by inducing its receptor CXCR4 and directly mediating the activation of CD4⁺T cells (Yu et al., 2020). Macrophages, an essential component of the innate immune system, are highly heterogeneous and malleable, and the reduced expression of CXCR4 might affect the dysregulation of M1/M2 phenotypes (Beneduce et al., 2019). This could play a vital role in the immune escape mediated by E6 and E7. These results suggest that CXCL12/CXCR4 is a potential therapeutic target for treating autoimmune diseases, including human immunodeficiency virus infection, cancer, warts, and immunodeficiency (Comba et al., 2020).

The expression of E6 and E7 resulted in the detection of many DEGs using RNA-seq and RT-qPCR. Fourteen of these hub genes were linked to cell adhesion (Chute, 2006; Teicher and Fricker, 2010; Paolillo and Schinelli, 2019; Marotta et al., 2021), leukocyte–endothelial migration (Yakovlev et al., 2019; Morein et al., 2020; Yan et al., 2021), stem cell signaling (Davidson et al., 2007; Bijlmakers, 2009), and epithelial–mesenchymal transition (Pastushenko et al., 2021; Wang et al., 2021). Based on these experimental and bioinformatics analyses, we hypothesized that HPV16 E6 and E7 signaling causes differential mRNA expression through direct or indirect mRNA pathways that regulate cell adhesion and chemokine secretion.

In this study, we used RNA-seq, RT-qPCR, and bioinformatics to validate the HPV-associated signaling pathways and possible downstream signaling molecules of the E6 and E7 proteins in human keratinocytes. This study provides a new direction and basis for further elucidation of the mechanism of E6/E7-mediated cell adhesion suppression. However, on the one hand, our study was restricted to in vitro conditions and the mode of interaction between CCL2 and hub genes is unclear in keratinocytes immortalized with both HPV E6 and E7 genes. The detailed mechanism will be investigated profoundly in our future studies. On the other hand, although some studies have indicated that FGF stimulation produces phosphorylation of E-cadherin and β-catenin on tyrosine residues, as well as increased E-cadherin localization to the cytoplasmic membrane and association with FGFR1 demonstrable by co-immunoprecipitation in the human pancreatic adenocarcinoma cell lines (BxPc3, T3M4 and HPAF), more direct interactions between hub genes need to be verified by co-immunoprecipitation or immunoblastic hybridization in our cellular models. The levels of mRNA only respond to the transcriptional aspect, while the process of post-translational modifications is highly variable, with different modifications resulting in different protein expression. Such differences may be influenced by post-transcriptional mechanisms such as methylation and acetylation. A previous study revealed that the level of m6A-modified mRNA is increased in cancer cells during the epithelial-mesenchymal transition of hepatocellular carcinoma cell metastasis (Lin et al., 2019). Although several studies have confirmed that the mRNA levels of certain hub genes such as CHD1, CDH2 (Castillo et al., 2016; Wang et al., 2020), IL-1β, EGF (Fu et al., 2020), BDNF (McDole et al., 2015), SOX2 (Scapin et al., 2019), and TLR4 (Jin et al., 2017) were consistent with the protein levels, it has not been validated in HaCaT-HPV16E6E7 cells. Considering this special feature, we will focus on discovering and exploring the differences in hub gene at the mRNA and protein levels in our future studies. It is undeniable that lentiviral vectors have an effect on gene expression, and we chose parental HaCaT as a negative control in our study, which makes it difficult to exclude the differential gene changes that would result from null loading at this time.

The data presented in this study are deposited in the SRA repository, accession number: PRJNA850539 can be found in the article/Supplementary material.

RD and QR conceived and designed the experiments and revised the manuscript. RD and XL performed the experiments. RT carried out RNA-seq. RD, RT, SZ, TS, and QR analyzed the data. RD wrote the manuscript. All authors have read and approved the final manuscript.