The potential role of TP63 regulating ZC3H13-mediated HLA-A m6A methylation modification in HR-HPV persistent infection patients

The potential role of TP63 regulating ZC3H13-mediated HLA-A m6A methylation modification in HR-HPV persistent infection patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The potential role of TP63 regulating ZC3H13-mediated HLA-A m6A methylation modification in HR-HPV persistent infection patients Shuiqing Xu, Ming Wang, Jianqing Xu, Yumei Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3362761/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective The aim of this study is to investigate the short-term persistent HPV infection and natural prognosis in patients with IA1 cervical cancer after CKC, and to explore the mechanism of persistent HPV infection leading to cervical lesions based on database analysis. Methods This is a prospective observation cohort study which enrolled the stage IA1 patients who select to receive CKC as the treatment modality in a single center from January 24, 2018, to June 9, 2022. The primary outcome was the persistent infection status and remession rates within two years after the CKC. In addition, the relevant mechanism was explored based on database analysis. The Cancer Genome Atlas (TCGA) database is the source of RNA sequencing data of cervical cancer patients. The gene Expression omnibus (GEO) database was used as the validation set to verify the expression of TP63 mRNA in the process from normal cervical to precancerous lesions, and the difference of TP63 between cancerous lesions and paracarcinoma was verified by Western blot. The limma package of R software, Kaplan-Meier survival curve and Log-rank test were used to screen the genes related to m6A methylation modification affecting the prognosis of cervical cancer. spearman correlation analysis was used to verify the correlation between genes, and Timer2.0 immune database was used to analyze the correlation between the expression level of key genes and the level of immune infiltration. JASPAR and SRAMP open access databases were used to verify the relevant RNA sequence binding sites and m6A methylation modification sites. Results A total of 98 eligible patients were included and the main types of HPV was as following: HPV 16 (76.19%,64/84), HPV 58 (10.71%,9/84), HPV 33 (9.52%,8/84), and unknown type 14.28% (14/98). The HR-HPV negative rates of the whole cohort at 3, 6, 9, 12, 15, 18, 21, and 24 months after CKC were 76% (19/25), 80.95% (34/42), 76.47%(39/51), 76.92%(40/52), 75.93%(41/54), 76.36%(42/55), 75.44%(43/57), and 74.58% (44/59), which was stable within 2 years after surgery. Based on the database analysis, this study proposes the relevant mechanism hypothesis of HPV persistent infection and difficult to clear: TP63 acts on the promoter of ZC3H13 to induce its expression and promote the m6A methylation modification of HLA-A mRNA 3’UTR, which leads to the accelerated degradation of HLA-A mRNA and further inhibits the antigen presentation of HPV viral proteins, leading to HPV escape from CD8 + T cell killing. Conclusion The negative rate of HR-HPV remained stable within 2 years after the CKC.Standardized follow-up after conization is very important for patients with stage IA1 cervical cancer.This study elucidates the mechanism of TP63-ZC3H13-HLA-A axis and provides A therapeutic target for HPV clearance and prevention of recurrence in patients with stage IA cervical cancer after conization. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Cervical carcinomas remain one of the most frequent solid cancers of females in developing countries [ 1 ] , and the stage IA (International Federation of Gynecology and Obstetrics, FIGO) accounts for 25% of cervical cancers, and 85% of stage IA diseases are stage IA1 [ 2 ] . Of note, 50% of patients with stage IA cervical cancer are under 40 years old [ 3 ] . The selection of treatment modality for stage IA1 cervical cancer is based on the results of cone biopsy and whether patients want to preserve their fertility. For patients who desire fertility preservation, conization combined with or without pelvic lymph node dissection is recommended [ 4 ] . After stage IA1 cervical cancer after conization, 16% HSIL and 10.5% invasive lesions were found in the patients who received direct hysterectomy. 5% HSIL and 26% malignancy were found in the patients who received re-conization [ 5 ] . Such recurrence is considered a result of incomplete removal of lesions, incomplete elimination of HPV infection, or new HPV infection. The recurrence of the same HPV genotype as before in treated patients, which is considered type-specific persistent infection (TSPI), is an accurate predictor of residual/recurrent disease [ 6 – 9 ] . Several studies have reported that TSPI of HPV16 and/or 18 is considered a high risk of residual/recurrent disease [ 10 – 11 ] . Therefore, for patients with negative margins, persistent HPV infection is an important predictor of residual or recurrent diseases. Nowadays, previous studies mainly confused on the follow-up of cervical intraepithelial lesions not stage IA1 cervical cancer for which conization is also the major treatment option.At present, previous studies mainly focus on the follow-up of cervical intraepithelial lesions, but the status of HPV infection in patients with IA1 cervical cancer after conization is not clear. TP63, a member of the TP53 gene family, has classical functional domains: trans-activation domain, DNA binding domain and oligomerization domain [ 12 ] . P63 plays an indispensable role in maintaining the proliferation of cervical squamous epithelium and initiating the differentiation of cervical squamous epithelium [ 13 – 14 ] . Wang T. Y. et al. found that p63 expression was positive in 94% of HPV16 and 50% of HPV18 positive cases in cervical squamous cell carcinoma. However, the function of TP63 in the process of cervical cancer is still unclear [ 15 ] . Shirendeb U et al. found that the co-expression of TP63 and HPV16 was verified by double staining identification in cervical squamous cell carcinoma, but the possible mechanism between TP63 expression and HPV infection has not been clarified yet [ 16 ] . In this study, we monitored HPV infection after cervical conization in patients with IA1 cervical cancer, evaluated whether HPV genotyping is helpful in detecting residual/recurrent disease after local treatment, and determined which HPV genotype is a high risk predictor of residual/recurrent disease. Based on the database, the correlation between TP63 expression and HPV persistent infection was analyzed, and the possible mechanism of cervical lesions was explored. Materials and methods 1. Patients Between March 2018 and January 2022, 98 cases with HR-HPV infected cervical cancer stage IA1 undergoing uterine conization were diagnosed in the department of gynecological Oncology at the Beijing Obstetrics and Gynecology Hospital, Capital Medical University. The present study was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (approval number: 2017-KY-026-01). Patients were eligible if they met all of the following inclusion criteria:(1). Age: 18–65; (2). Positive for high-risk HPV infection; (3). Patients with a final histological diagnosis of minimally invasive carcinoma (stage IA1) require uterine preservation. Participants were excluded from patients with severe comorbidities that prevented follow-up and surgery or who had been treated for a previous or newly identified HPV infection. 2. HPV infection status surveillance 2.1 HPV Genotype testing High-risk HPV-positive samples were defined as HPV-positive using fresh cell samples from the cervix for fluorescence quantitative polymerase chain reaction (FQ-PCR) used to detect HPV DNA genotypes. High-risk HPV DNA was detected and typed according to E6/E7 specificity, and 12 high-risk HPV genotypes could be identified by the kit(Hybribio, Guangzhou, China), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68.HPV genotypes were detected before and after cervical conization. 2.2 Follow-up and observation outcome Patients with stage IA1 cervical cancer were followed up at 3, 6, 9, 12, 15, 18, 21, and 24 months after cervical conization, and cytology was also performed. This study focused on HPV infection status and outcome after conization. 2.3 Statistical Analysis Baseline characteristics and laboratory results were summarized utilizing descriptive statistics, including percentage, means ± standard deviation (SD), and 95% CI. Kaplan-Meier analysis was used to construct cumulative risk curves. All data were analyzed by SPSS 23.0 (SPSS, Inc., IBM). 3. Analyze and explore related mechanisms based on database 3.1 Data collection and preprocessing RNAseq data (level3) and corresponding clinical information of cervical squamous cell carcinoma were obtained from the Cancer Genome Atlas (TCGA) database ( https://portal.gdc.com ). Also based on the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ) to select data sets, download data format for MINiML, boxplot drawn through the boxplot. 3.2 Differential gene expression screening, verification and prognostic analysis The limma package of R software (version: 3.40.2) was used to study the differential expression of mRNA based on the database. Adjusted p-values were analyzed in TCGA or GEO to correct for false positive results. "Adjusted P 1 or log2(fold change) < -1" was defined as a screen for differential expression of threshold mrnas. Log-rank was used to test KM survival analysis to compare survival differences between two or more of the above groups, and for Kaplan-Meier curves, p values and hazard ratios (HR) with 95% confidence intervals (CI) were derived by log-rank test and univariate Cox regression. All the analytic methods and R packages described above were executed with the use of R software, version 4.0.3 (R Foundation for Statistical Computing, 2020). A p value of less than 0.05 was considered statistically significant. Fresh tumor tissues and adjacent tissues from 4 patients with cervical cancer (3 cases of squamous cell carcinoma and 1 case of adenocarcinoma) who underwent surgery in our hospital were selected. Patients who underwent preoperative radiotherapy and chemotherapy or combined with other systemic diseases were excluded. All patients were pathologically diagnosed. In addition, the human protein atlas database can be used to verify the immunohistochemical expression in different tissues. 3.3 Gene correlation analysis and immune cell infiltration analysis Spearman's correlation analysis was used to describe the correlation between quantitative variables that did not have a normal distribution. A p-value of less than 0.05 was considered statistically significant, and the two-gene correlation map was implemented using the R (v4.0.3) package pheatmap. TIMER is a comprehensive resource database for systematic analysis of immune infiltration. It uses RNA-Seq profiling data to detect immune cell infiltration in different tumor tissues (Li et al., 2017). The correlation between the expression level of key genes and the abundance of immune infiltration was analyzed by the QUNTISEQ algorithm of the Timer2.0 immune database. 3.4 RNA sequence associated binding sites Using JASPAR ( https://jaspar.genereg.net/ ), SRAMP open access database ( http://www.cuilab.cn/sramp ), verify the relevant RNA sequences binding sites and m6A methylation modification site. Results 1. Study population and baseline characteristics Of the 730 patients receiving conization for cytology HSIL, 115 patients were diagnosed with post-conization stage IA1 cervical cancer, and 98 patients were included in the study. 17 of them were excluded from the study for the following reasons: 10 patients received subsequent hysterectomy, 5 with lymph node vascular invasion, and 2 women with extensive VaIN III( see Fig. 1 )... The mean age of the cohort was 36 years (range 32–39) and 94.90% of patients were premenopausal. 27.55% of patients were nulliparous, 52.04% had given birth once and 21.42% were multiparous. In total, HPV genotypes were available for 85.71%(84/98) patients before surgery, and HPV genotypes were unknown in 14.28%(14/98) patients. Of the 84 patients with HPV genotypes available, the most common genotypes were HPV 16 (68.09%, 64/94), HPV 58(9.57%, 9/94), and HPV 33 (8.51%,8/94). Besides, 24.47%(23/94) of these patients were mixed infections. For the details of HPV genotypes distribution see Table 1 . Table 1 Baseline characteristics of patients with IA1 cervical cancer (n = 98) Characteristics IA1 cervical cancer (n = 98) Age (years), median (IQR) 36(32–39) Gravida, medium (IQR) 2(1–3) Parity, medium (IQR) 1(0–1) Cervical cytology, n (%) HSIL 51(52.04) ASC-H 1 (1.02) LSIL 10 (10.20) ASC-US 13(13.26) NILM or unknown 23 (23.47) HPV infection, n (%) HPV16 64/94 (68.09) Hpv-16 49 HPV16 and other types 15 HPV18 3/94(3.19) HPV 18 2 HPV and other types(except for HPV16 ) 1 HPV16 and/or HPV18 3/94(3.19) HPV 33 7/94(7.45) HPV 33 3 HPV 33 and other types(except for HPV 16/18) 4 HPV-58 6/94 (6.38) HPV58 5 HPV58 and other types(except for HPV 16/18/33) 1 Other HR-HPV or unknown types 13/94 (33.6) HPV negative 1/94(1.06) Unknown 4 (4.08) Abbreviations: NILM (Negative for intraepithelial lesion or malignancy); ASC-US (atypical squamous cells undetermined significance); ASC-H (atypical squamous cells cannot exclude a high-grade squamous intraepithelial lesion); LSIL (low grade squamous intraepithelial lesion); HSIL (high-grade squamous intraepithelial lesions). The cytology of patients before conization showed 52.04% (51/98) high-grade squamous intraepithelial lesions(HSIL), 1.02% (1/98) atypical squamous cells cannot exclude HSIL(ASC-H), 10.20% (10/98) low-grade squamous intraepithelial lesions(LSIL), 13.26% (13/98)Atypical squamous cells of undetermined significance (ASCUS), and 23.47% (23/98) normal/unknown results. 2. Remission of HPV infection The negative rates of HR-HPV at 3, 6, 9, 12, 15, 18, 21, and 24 months after surgery were 76% (19/25), 80.95% (34/42), 76.47%(39/51), 76.92%(40/52), 75.93%(41/54),76.36%(42/55), 75.44%(43/57), and 74.58% (44/59), respectively, which was basically stable within 2 years. Among them, the patients who were still positive for HPV 3 months after surgery were all initially treated with HPV16 infection. From 6 to 9 months after surgery, 83.3% (5/6) of the HPV-positive patients were initially infected with HPV16, but only 27.7% (1/6) of the HPV-positive patients were HPV16 at 9 months after surgery. Among the patients who were retested positive 21 months after surgery, there was one unknown type. Among the known types, 60% (3/5) were initially treated with HPV16 positive, 20% (1/5) were initially treated with HPV18 positive, and 20% (1/5) were initially treated with HPV 58 positive. Only 1 case (1/5, 20%) was HPV16 positive at the initial treatment. 66.67%(2/3) of the patients who were retested positive at 24 months after surgery were HPV16 positive at the first treatment and none of the HPV-positive patients at 24 months was HPV16 type. There was no patient with persistent abnormal cytology (HSIL/ASC-H) after surgery. For the details of the remission of HPV infection see figure 2. 3. Mechanism of the TP63-ZC3H13-HLA-A axis 3 .1 The expression of TP63 increased gradually during the process of cervical epithelial carcinogenesis In this study, tumor tissues and adjacent tissues from four patients with cervical cancer (three squamous cell carcinomas and one adenocarcinoma) were selected. It was found that the mRNA expression level of TP63 in cervical squamous cell carcinoma tissues was higher than that in adjacent tissues (Figure 3A), while the protein level of TP63 in tumor tissues was higher than that in adjacent tissues (Figure 3B). We further found in GEO database (GSE75132) that the mRNA expression level of TP63 gradually increased in HPV16-infected cervical tissues from normal to precancerous lesions (see Figure 3C). 3 .2 m6A methylation-related gene ZC3H13 may be involved in the carcinogenesis of cervical cancer By comparing the expression of m6A methylation-related genes in cervical cancer and adjacent tissues in TCGA database, it was found that several molecules such as METTL3, METTL14, ZC3H13, and YTHDC1 were highly expressed in cervical cancer tissues compared with adjacent tissues (FIG. 4A), and the relationship between abnormal expression molecules and the prognosis of cervical cancer was further compared. It was found that patients with high expression of the m6A methylation-related molecule ZC3H13 had significantly decreased OS and PFS (Figure 4B-C). Meanwhile, immunohistochemistry showed that ZC3H13 was significantly overexpressed in cervical cancer compared with normal cervical tissues (Figure 4D-E). 3.3 TP63 molecule is associated with low expression of HLA-A receptor and low level of CD8+T cell infiltration in cervical cancer cells We analyzed the infiltration levels of CD4+T cells, CD8+T cells, Treg cells, B cells, dendritic cells, M1 macrophages, and M2 macrophages by the Timer2.0 immune database QUNTISEQ algorithm and found that the infiltration level of CD8+T cells was significantly decreased in patients with high TP63 expression (Figure 5A-G). Moreover, patients with low CD8+T cell infiltration had a worse survival prognosis (Figure 5H). To understand whether the decreased CD8+T cell infiltration was associated with the down-regulated expression of HLAs molecules, bioinformatics analysis predicted that patients with high TP63 expression in the TCGA cervical cancer database had down-regulated expression of human leukocyte associated antigen HLAA, B, and C receptors (FIG. 5I). 3 .4 TP63 may affect the infiltration of CD8+T cells by regulating ZC3H13 Subsequently, the relationship between ZC3H13 and cervical cancer immune cell infiltration was further predicted by bioinformatics, and it was found that patients with high expression of ZC3H13 had a statistically significant reduction in CD8+T cell immune infiltration (Figure 6A). Correlation analysis showed that ZC3H13 was positively correlated with TP63 in cervical squamous cell carcinoma (FIG. 6B). The JASPAR website predicted that there were two binding sites of TP63 in the promoter region of ZC3H13 (Figure 6C), so it was speculated that the reduction of CD8+T cells caused by TP63 may be related to its effect on the expression of ZC3H13. 3 .5 ZC3H13 binding sites in the 3 '-UTR of HLA-A mRNA Through SRAMP website prediction, we found that there were two high-scoring m6A binding sites in the 3 '-UTR of HLA-A pre-mRNA, one of which was the classical binding motif "GGACU" of ZC3H13. Therefore, it was speculated that ZC3H13 might promote HLA-A mRNA 3 '-UTR degradation through m6A methylation (Figure. 7). Discussion 1. Persistent HPV infection is the most important risk factor for recurrence, and different HPV types help to predict disease recurrence Cervical conization is the major treatment modality of stage IA1 cervical cancer in patients with fertility needs. Persistent or recurrent HPV infection is the major reason for postoperative relapse or disease residuality of IA1 cervical cancer. The study followed HPV outcomes in high-risk HPV patients who underwent cervical cone resection, which showed that with time, the cumulative HR-HPV clearance rate after cervical conization is stable to 74%-80% within 2 years. New HPV infection and type-specific persistent infection were two mechanisms of postoperative persistent HPV. Persistent HPV infection after treatment, with or without residual HSIL/cancer, is the most important established risk factor for recurrent disease [17] . In this study, among the patients with known HPV types, the proportion of patients who were initially treated with HPV16 was the most serious, which was the main infection type (76.19%,64/84). Among the patients who were still HPV positive at 3, 6-9, 9, 21, and 24 months after conization, the proportion of patients with HPV16 infection was 100%, 100% (6/6), 83.3% (5/6), 27.7% (1/6), 60% (3/5) and 66.67% (2/3), respectively. No HPV type 16 was detected at 24 months. Patients with HPV16 infection take a longer time to clear HPV after conization, are at high risk of recurrence, and are more likely to be infected with other new HPV types. One retrospective study reported that six months after LEEP, HPV 16 positive was associated with a 37% increase in the absolute risk of CIN 2+2 years, twice as much as HPV 18 (18.5%), and three times as much as other types of oncogenes (10.8%) [18-19] . Didem Egemen et al. [20] has reported on the new risk-based guidelines present recommendations for the management of abnormal screening test and histology results, and meaningfully indicated the impact of HPV test results on the immediate and 5-year risks of CIN 3+. HPV positivity was a significant predictor of immediate risks of CIN 3+ in patients monitored for patients with previous HSIL [20] . Therefore, HPV typing is necessary to evaluate whether HPV infection is persistent or new after treatment and to study the relationship of different typing to predict disease recurrence. Studies have shown that persistent infection with HPV16, 18, 31, 33, 52, and 58 is a high-risk predictor of residual/recurrent disease [21-23] . 2. HPV immune escape is the fundamental cause of persistent infection and cervical lesions In this study, we found that the persistent infection rate of HPV was stable after conization in patients with stage IA1 (microscopic early invasion) cervical cancer who requested fertility preservation. However, some patients do not turn negative due to conization, and during infection, HPV subtypes can gradually change to other subtypes due to antigenic drift to avoid immune attack. The genome of HPV virus is composed of a circular double-stranded DNA of about 8000 base pairs, which contains three genomic regions, namely, early coding region (E), late coding region (L) and long control region (LCR) [24] . The E gene includes the E6 and E7 genes, which are responsible for cell transformation and are the main causes of cervical lesions. The E6 molecule binds p53 and promotes ubiquitination and degradation of p53, and the E7 molecule binds to the unphosphorylated retinoblastoma (RB) protein, both of which allow the viral DNA to replicate without circulation. It controls G1 and S phases of the cell cycle [25-26] .However, the occurrence of cervical lesions requires not only the inhibition of tumor suppressor genes such as P53 and RB by HPV E6 and E7 genes, but also the persistent infection of HPV to activate other protooncogenes in the cervical epithelium. HPV evades the immune clearance of the body, which is the premise of HPV causing persistent infection and activating other proto-oncogenes. HPV infection is confined to the cervical keratinized squamous epithelial cells. The keratinized epithelial cells have the function of antigen presentation, Th1 and Th2 cytokines secretion, and can induce the cytotoxic effect of CD4+ and CD8+ memory T cells. human leukocyte antigens (HLAs) are expressed on the surface of human nucleated cells and are the main pathway for the presentation of endogenous antigens, including viruses and tumors. The antigen produced by the virus in the cytoplasm first binds to ubiquitin, and the ubiquitinated protein enters the proteasome in a linear manner to be degraded to produce endogenous antigen. The transporter associated with antigen processing (TAP) assists in the transport of antigen peptides from the cytosol to the endoplasmic reticulum. With the assistance of molecular chaperones calreticulin, calreticulin and TAP, endogenous antigens bind to HLA class I molecules. It is then transported to the cell membrane by the Golgi apparatus and recognized by CD8+T cells, which kill infected cells (see FIG. 8 for details) [26] . HPV can evade the damage of the immune system and carry on persistent infection and replication by affecting multiple links of the innate and adaptive immune response. The main mechanisms are: a) The replication cycle of HPV is closely related to the differentiation of keratinocyte. With the virus replication, epithelial cells become keratinized and exfoliated, and infected cells are not lysed, which cannot produce a strong inflammatory response to trigger natural immunity [27] ; b) HPV E6 and E7 proteins can negatively regulate the expression of HLAs in cervical cells to escape the recognition of immune cells [28, 29] ; c) HPV can promote the change of Th1 pro-inflammatory cytokines to Th2 anti-inflammatory cytokines in the cervical epithelial microenvironment, such as the reduction of proinflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, IL-18, CCL2, CCL20, CXCL9 and type I interferon. To inhibit the increased expression of inflammatory cytokines such as IL-4,IL-10 and IL-17 to escape the innate immune response and adaptive immunity induced by cytokines; d) recruitment of immune cells that suppress immune response or inhibit the recruitment and activation of killer T cells. In the cervical epithelial microenvironment, the ratio of CD8+T cells /Treg cells decreases, and the generation of appropriate killer T cells cannot be induced to kill virus-infected cells [30-32] . In conclusion, before the carcinogenesis of cervical epithelial cells, occult HPV replicates and exfoliates with the keratinization of cervical epithelium, which cannot produce sufficient inflammatory response to induce the body to kill the infected keratinized epithelial cells, and then evade immunity. Therefore, it is necessary to further reveal how the expression of HLAs molecules caused by HPV infection confined in keratinized cervical squamous epithelium affects the mechanism of CD8+T cells to clear HPV-infected cells. 3. Hpv persistent infection through TP63-ZC3H13-HLA-A axis leads to cervical lesions 3.1 m6A demethylase affects mRNA expression and affects tumorigenesis N6-methyladenosine (m6A) is one of the most abundant RNA modifications, with an estimated 3-5 m6A sites on each mRNA molecule. m6A is another important epigenetic modification pathway newly discovered after DNA methylation [33] . At the level of transcriptional regulation, m6A can regulate various processes of mRNA molecules, including alternative splicing, maturation, nuclear export, translation, degradation and stability [33] . In terms of biological effects, m6A can regulate stem cell differentiation, animal growth and development, DNA damage repair, tumor occurrence and development, and immune response [34-35] . The m6A modification on RNA is determined by the methyltransferase complex and the demethylase, and the function of m6A is performed by the protein that specifically recognizes it [36] . The methyltransferase complex METTL3/METTL14/WTAP catalyzes the formation of m6A. METTL3 can bind SAM and catalyze the formation of m6A, and METTL14 mainly provides a platform for substrate binding. WTAP is responsible for recruiting METTL3/METTL14 complex to the nuclear plaques in the nucleus to perform the catalytic function of methylation [37] .Demethylases FTO and ALKBH5 remove the methylation of m6A [38-39] . m6A readers, such as YTH N6-methyladenosine RNA binding protein 1/2/3 (YTHDF1/2/3), bind to m6A modified mRNA to determine the fate of m6A modified mRNA. m6A regulators such as YTHDF1, METTL3 and ALKBH5 have been intensively studied in cancer. m6A methylation can affect the function of downstream genes through several mechanisms: for example, the modification of introns affects the alternative splicing of target gene mRNA;Upregulation of mRNA has been reported to be associated with poor prognosis in various cancer types, and YTHDF1 promotes tumorigenesis and cancer metastasis [40-44] .ZC3H13 is another important m6A methylating protein, which can promote the formation of methylation at the m6A site of the "GGACU" motif in the 3 'untranslated region of the downstream mRNA by forming a complex with WTAP, Virilizer and Hakai [45] . However, the formation of m6A methylation sites in mRNA 3 '-UTR can promote the degradation of mRNA [46] . Our previous bioinformatics prediction found that the expression of m6A methylated genes in cervical cancer lesions was significantly up-regulated compared with adjacent tissues, among which only ZC3H13 expression significantly affected the prognosis of patients, and the survival outcome of patients with high expression of ZC3H13 was worse, and the binding site of ZC3H13 was predicted in the 3 '-UTR of HLA-A mRNA. Therefore, it is speculated that ZC3H13 may promote HLA-A mRNA degradation through m6A methylation modification during cervical epithelial lesions. 3.2 Mechanism of TP63 promoting cervical lesions caused by HPV infection through ZC3H13-mediated HLA-A m6A methylation In this study, it was verified by molecular level and database analysis that TP63 promotes the occurrence of cervical squamous cell carcinoma and can be used as a meaningful therapeutic target. Moreover, TIMER 2.0 database showed that TP63 affected the expression of HLA-A receptor and inhibited the activation and proliferation of CD8+T cells. In addition, TP63 expression was positively correlated with the above-mentioned ZC3H13 expression in cervical squamous cell carcinoma, and negatively correlated with HLA-A expression. JASPAR website predicted that there was a TP63 binding site in the upstream of the promoter region of ZC3H13 and SRAMP database predicted that there was a binding site of ZC3H13 in the 3 '-UTR of HLA-A mRNA. Therefore, this study proposes hypotheses: TP63 up-regulates the expression of ZC3H13 by acting on the promoter of ZC3H13, and ZC3H13 further promotes the m6A methylation of HLA-A mRNA 3’UTR, leading to accelerated degradation of HLA-A mRNA, thereby inhibiting the antigen presentation of HPV viral proteins. As a result, HPV escapes from the killing effect of CD8+T cells (see Figure 9), making high-risk HPV persistent infection and difficult to clear. In addition, the possible mechanism of TP63-ZC3H13-HLA-A axis may provide A therapeutic target for HPV clearance and prevention of recurrence in patients with stage IA cervical cancer after conization. Declarations Acknowledgment 1.Ethics approval and consent to participate：All procedures performed in studies involving human participants following the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The Institutional Review Board approved the study (approval number: 2017-KY-026-01). Informed consent was unavailable in the study. 2.Consent for publication：All authors approved the final manuscript and the submission to the journal. 3.Availability of data and materials：The datasets generated during and/or analyzed during the current study are publicly available. 4.Competing interests：The authors declare that they have no conflict of interest. 5.Funding：This study is supported by Beijing Hospitals Authority Sailing plan (grant number: ZYLX201705). 6.Authors' contributions：X.S.Q collected the data, followed the patients, completed the experimental section, and wrote the paper. W.M collected the data, completed the data analysis and wrote the paper. X.J.Q collects data. W.Y.M supervised the conduct of the study, revised the article, and made suggestions. References Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. 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Zhao BS, Wang X, Beadell AV, et al. m(6)A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition [J]. Nature. 2017;542(7642):475–8. Additional Declarations No competing interests reported. Supplementary Files Figure3BwesternblotKeratinandGADPHUncroppedimages.jpg Figure3BwesternblotTP63.Uncroppedimages.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3362761","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":238804248,"identity":"2da17415-9fb7-4dab-ae23-f6466688d74f","order_by":0,"name":"Shuiqing Xu","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuiqing","middleName":"","lastName":"Xu","suffix":""},{"id":238804249,"identity":"89574594-a474-4500-9c80-9ca7b7b41b8d","order_by":1,"name":"Ming Wang","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Wang","suffix":""},{"id":238804250,"identity":"2fa3fb1e-530a-417c-be4f-1729973bdb42","order_by":2,"name":"Jianqing Xu","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianqing","middleName":"","lastName":"Xu","suffix":""},{"id":238804251,"identity":"74257fa7-bc4c-4646-887d-e53f21dec559","order_by":3,"name":"Yumei Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYHAC9g8f/0nw8LM3QPkHCGthY5zBZiEj2QNTSowWZh62ChuDGwlEajG4kZ32mIdHgkdy5uOHn262Mcjx3Uhg/FyAV0vudsM5EkC/SKcZS+e2MRhL3khglp6BR4vZjdwNEm8MgLbMzmFjBmpJ3HAjAehUQlp4EiR4DG6eAWupJ0bLNkmeA0AtN3jAWhIMCGmxP/N2s+HMBqDDeoB+yTknYTjzzMNmaXxaJNtzNz742FBnz89++OHnnDIbeb7jyQc/49PCIJCAwpUAYsYGfBoYGPgP4JcfBaNgFIyCUcAAAJunSuxEACIPAAAAAElFTkSuQmCC","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yumei","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2023-09-17 08:29:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3362761/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3362761/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44524046,"identity":"eaa192ca-f701-4d74-a0cf-9af75b01419c","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe flow diagram of the study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/261ba3e59737c6ac99b375d9.png"},{"id":44524045,"identity":"21a56a3d-8add-4a84-b459-12a361e67906","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePersistent High-risk HPV Infection after Surgery for stage IA1 Cervical Cancer\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.PersistentHighriskHPVInfectionafterSurgeryforstageIA1CervicalCancer.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/e6c7f5e60760b7c8021d1e62.jpg"},{"id":44524048,"identity":"5aae067b-1843-43aa-be77-a3ce5874423f","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of P63 Molecules in Cervical Cancer and Precancerous Lesions:\u003c/strong\u003e (A) TP63 mRNA expression levels in cervical cancer and paracancerous tissues detected by RT-PCR; (B) Western blot was used to detect the protein expression of TP63 in cervical cancer tissues and adjacent tissues (Case 1-3 was cervical squamous cell carcinoma, Case 4 was cervical adenocarcinoma); (C) GEO database (GSE75132) showed that TP63 mRNA expression level gradually increased in HPV16-infected cervical tissues from normal to precancerous lesions (G1 was HPV16-positive and histopathologically negative; G2 was HPV16 positive and histopathology was HSIL).\u003c/p\u003e","description":"","filename":"Figure3.ExpressionofP63MoleculesinCervicalCancerandPrecancerousLesions.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/d4493864ece1c618de66bbaa.jpg"},{"id":44524055,"identity":"f2efefd7-09ae-4602-83b1-6331fdc260f6","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":283976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential m6A genes in the carcinogenesis of cervical squamous cell carcinoma:\u003c/strong\u003e Figure (A) expression changes of m6A methylation-related genes in cervical cancer and adjacent tissues in TCGA database; Figure (B-C) Relationship between abnormal expression of ZC3H13 and OS and PFS of cervical cancer in TCGA database;Figure (D-E) the immunohistochemical expression changes of ZC3H13 in cervical cancer and adjacent tissues in the human protein atlas database.\u003c/p\u003e","description":"","filename":"Figure4.Differentialm6Agenesinthecarcinogenesisofcervicalsquamouscellcarcinoma.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/abaad9dd81ce2b360df33066.jpg"},{"id":44524432,"identity":"6b02ecbc-e725-4bad-ae6e-132d4bbfd27b","added_by":"auto","created_at":"2023-10-12 16:56:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":803896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between TP63 molecules and cellular immune infiltration in cervical cancer: \u003c/strong\u003e(A-G) TP63 expression and infiltration levels of CD4+T cells, CD8+T cells, Treg cells, B cells, dendritic cells, M1 macrophages, and M2 macrophages; (H) the relationship between CD8+T cell infiltration level and patient survival. (I) The expression of HLA-A, B and C receptors was down-regulated in patients with high TP63 expression\u003c/p\u003e","description":"","filename":"Figure5.RelationshipbetweenTP63moleculesandcellularimmuneinfiltrationincervicalcancer.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/fb28a9bf5f81015a93f38bf5.jpg"},{"id":44525684,"identity":"b202a77f-c958-47cc-a37e-e26ca3c151a4","added_by":"auto","created_at":"2023-10-12 17:04:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTP63 may affect CD8+T cell infiltration by regulating ZC3H13: \u003c/strong\u003e(A) Relationship between ZC3H13 expression and immune cell infiltration; (B) correlation between TP63 and ZC3H13 expression in TCGA database; (C) Two TP63 binding sites were predicted in the promoter region of ZC3H13 by JASPAR website.\u003c/p\u003e","description":"","filename":"Figure6.TP63mayaffectCD8TcellinfiltrationbyregulatingZC3H13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/b1661fa796199b89052a27ce.jpg"},{"id":44524051,"identity":"a2214834-e17b-49f7-8f0c-333ea8df445c","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":35148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe presence of a classical binding site for ZC3H13 in the 3 '-UTR region of HLA-A mRNA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.ThepresenceofaclassicalbindingsiteforZC3H13inthe3UTRregionofHLAAmRNA..jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/29308ac13b61c18e167054df.jpg"},{"id":44524431,"identity":"ec79c095-4f9b-460b-b5e1-c4a36af59aaa","added_by":"auto","created_at":"2023-10-12 16:56:39","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":40426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic Diagram of HPV Immune clearance.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.MechanisticDiagramofHPVImmuneclearance..jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/87b3286f65712420cfbf0683.jpg"},{"id":44524049,"identity":"af847e55-0c81-4e63-9842-b88ae0a1c621","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":48526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanistic diagram of HPV immune escape.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure9.MechanisticdiagramofHPVimmuneescape..jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/2dfc62bab33834192e03ec45.jpg"},{"id":48976091,"identity":"06a95497-ff37-4760-b0d7-1c1fa8159d6b","added_by":"auto","created_at":"2023-12-29 16:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1486873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/9c32f02e-da6b-4a3e-86ad-d4751f147bae.pdf"},{"id":44524044,"identity":"2eb9a2f3-6bab-4c1c-88d9-4b40eaa3dbe3","added_by":"auto","created_at":"2023-10-12 16:48:39","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":65437,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3BwesternblotKeratinandGADPHUncroppedimages.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/c9cab4264d0e4208955194f2.jpg"},{"id":44524429,"identity":"648029e3-fae2-4d8d-b218-eff6720dda82","added_by":"auto","created_at":"2023-10-12 16:56:39","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24111,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3BwesternblotTP63.Uncroppedimages.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3362761/v1/8fe91461db4c642df3ed7f0d.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"The potential role of TP63 regulating ZC3H13-mediated HLA-A m6A methylation modification in HR-HPV persistent infection patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical carcinomas remain one of the most frequent solid cancers of females in developing countries\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, and the stage IA (International Federation of Gynecology and Obstetrics, FIGO) accounts for 25% of cervical cancers, and 85% of stage IA diseases are stage IA1\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Of note, 50% of patients with stage IA cervical cancer are under 40 years old\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The selection of treatment modality for stage IA1 cervical cancer is based on the results of cone biopsy and whether patients want to preserve their fertility. For patients who desire fertility preservation, conization combined with or without pelvic lymph node dissection is recommended\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter stage IA1 cervical cancer after conization, 16% HSIL and 10.5% invasive lesions were found in the patients who received direct hysterectomy. 5% HSIL and 26% malignancy were found in the patients who received re-conization\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Such recurrence is considered a result of incomplete removal of lesions, incomplete elimination of HPV infection, or new HPV infection. The recurrence of the same HPV genotype as before in treated patients, which is considered type-specific persistent infection (TSPI), is an accurate predictor of residual/recurrent disease\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Several studies have reported that TSPI of HPV16 and/or 18 is considered a high risk of residual/recurrent disease\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, for patients with negative margins, persistent HPV infection is an important predictor of residual or recurrent diseases. Nowadays, previous studies mainly confused on the follow-up of cervical intraepithelial lesions not stage IA1 cervical cancer for which conization is also the major treatment option.At present, previous studies mainly focus on the follow-up of cervical intraepithelial lesions, but the status of HPV infection in patients with IA1 cervical cancer after conization is not clear.\u003c/p\u003e \u003cp\u003eTP63, a member of the TP53 gene family, has classical functional domains: trans-activation domain, DNA binding domain and oligomerization domain \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. P63 plays an indispensable role in maintaining the proliferation of cervical squamous epithelium and initiating the differentiation of cervical squamous epithelium \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Wang T. Y. et al. found that p63 expression was positive in 94% of HPV16 and 50% of HPV18 positive cases in cervical squamous cell carcinoma. However, the function of TP63 in the process of cervical cancer is still unclear \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Shirendeb U et al. found that the co-expression of TP63 and HPV16 was verified by double staining identification in cervical squamous cell carcinoma, but the possible mechanism between TP63 expression and HPV infection has not been clarified yet \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we monitored HPV infection after cervical conization in patients with IA1 cervical cancer, evaluated whether HPV genotyping is helpful in detecting residual/recurrent disease after local treatment, and determined which HPV genotype is a high risk predictor of residual/recurrent disease. Based on the database, the correlation between TP63 expression and HPV persistent infection was analyzed, and the possible mechanism of cervical lesions was explored.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Patients\u003c/h2\u003e \u003cp\u003eBetween March 2018 and January 2022, 98 cases with HR-HPV infected cervical cancer stage IA1 undergoing uterine conization were diagnosed in the department of gynecological Oncology at the Beijing Obstetrics and Gynecology Hospital, Capital Medical University. The present study was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (approval number: 2017-KY-026-01).\u003c/p\u003e \u003cp\u003ePatients were eligible if they met all of the following inclusion criteria:(1). Age: 18\u0026ndash;65; (2). Positive for high-risk HPV infection; (3). Patients with a final histological diagnosis of minimally invasive carcinoma (stage IA1) require uterine preservation. Participants were excluded from patients with severe comorbidities that prevented follow-up and surgery or who had been treated for a previous or newly identified HPV infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2. HPV infection status surveillance\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1 HPV Genotype testing\u003c/h2\u003e \u003cp\u003eHigh-risk HPV-positive samples were defined as HPV-positive using fresh cell samples from the cervix for fluorescence quantitative polymerase chain reaction (FQ-PCR) used to detect HPV DNA genotypes. High-risk HPV DNA was detected and typed according to E6/E7 specificity, and 12 high-risk HPV genotypes could be identified by the kit(Hybribio, Guangzhou, China), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 68.HPV genotypes were detected before and after cervical conization.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Follow-up and observation outcome\u003c/h2\u003e \u003cp\u003ePatients with stage IA1 cervical cancer were followed up at 3, 6, 9, 12, 15, 18, 21, and 24 months after cervical conization, and cytology was also performed. This study focused on HPV infection status and outcome after conization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical Analysis\u003c/h2\u003e \u003cp\u003eBaseline characteristics and laboratory results were summarized utilizing descriptive statistics, including percentage, means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and 95% CI. Kaplan-Meier analysis was used to construct cumulative risk curves. All data were analyzed by SPSS 23.0 (SPSS, Inc., IBM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3. Analyze and explore related mechanisms based on database\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.1 Data collection and preprocessing\u003c/h2\u003e \u003cp\u003eRNAseq data (level3) and corresponding clinical information of cervical squamous cell carcinoma were obtained from the Cancer Genome Atlas (TCGA) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.com\u003c/span\u003e\u003cspan address=\"https://portal.gdc.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Also based on the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to select data sets, download data format for MINiML, boxplot drawn through the boxplot.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Differential gene expression screening, verification and prognostic analysis\u003c/h2\u003e \u003cp\u003eThe limma package of R software (version: 3.40.2) was used to study the differential expression of mRNA based on the database. Adjusted p-values were analyzed in TCGA or GEO to correct for false positive results. \"Adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2(fold change)\u0026thinsp;\u0026gt;\u0026thinsp;1 or log2(fold change) \u0026lt; -1\" was defined as a screen for differential expression of threshold mrnas. Log-rank was used to test KM survival analysis to compare survival differences between two or more of the above groups, and for Kaplan-Meier curves, p values and hazard ratios (HR) with 95% confidence intervals (CI) were derived by log-rank test and univariate Cox regression. All the analytic methods and R packages described above were executed with the use of R software, version 4.0.3 (R Foundation for Statistical Computing, 2020). A p value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003eFresh tumor tissues and adjacent tissues from 4 patients with cervical cancer (3 cases of squamous cell carcinoma and 1 case of adenocarcinoma) who underwent surgery in our hospital were selected. Patients who underwent preoperative radiotherapy and chemotherapy or combined with other systemic diseases were excluded. All patients were pathologically diagnosed. In addition, the human protein atlas database can be used to verify the immunohistochemical expression in different tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Gene correlation analysis and immune cell infiltration analysis\u003c/h2\u003e \u003cp\u003eSpearman's correlation analysis was used to describe the correlation between quantitative variables that did not have a normal distribution. A p-value of less than 0.05 was considered statistically significant, and the two-gene correlation map was implemented using the R (v4.0.3) package pheatmap. TIMER is a comprehensive resource database for systematic analysis of immune infiltration. It uses RNA-Seq profiling data to detect immune cell infiltration in different tumor tissues (Li et al., 2017). The correlation between the expression level of key genes and the abundance of immune infiltration was analyzed by the QUNTISEQ algorithm of the Timer2.0 immune database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RNA sequence associated binding sites\u003c/h2\u003e \u003cp\u003eUsing JASPAR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.genereg.net/\u003c/span\u003e\u003cspan address=\"https://jaspar.genereg.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), SRAMP open access database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cuilab.cn/sramp\u003c/span\u003e\u003cspan address=\"http://www.cuilab.cn/sramp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), verify the relevant RNA sequences binding sites and m6A methylation modification site.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e1. Study population and baseline characteristics\u003c/h2\u003e \u003cp\u003eOf the 730 patients receiving conization for cytology HSIL, 115 patients were diagnosed with post-conization stage IA1 cervical cancer, and 98 patients were included in the study. 17 of them were excluded from the study for the following reasons: 10 patients received subsequent hysterectomy, 5 with lymph node vascular invasion, and 2 women with extensive VaIN III(\u003cb\u003esee\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)...\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean age of the cohort was 36 years (range 32\u0026ndash;39) and 94.90% of patients were premenopausal. 27.55% of patients were nulliparous, 52.04% had given birth once and 21.42% were multiparous. In total, HPV genotypes were available for 85.71%(84/98) patients before surgery, and HPV genotypes were unknown in 14.28%(14/98) patients. Of the 84 patients with HPV genotypes available, the most common genotypes were HPV 16 (68.09%, 64/94), HPV 58(9.57%, 9/94), and HPV 33 (8.51%,8/94). Besides, 24.47%(23/94) of these patients were mixed infections. For the details of HPV genotypes distribution see Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of patients with IA1 cervical cancer (n\u0026thinsp;=\u0026thinsp;98)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIA1 cervical cancer (n\u0026thinsp;=\u0026thinsp;98)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (years), median (IQR)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36(32\u0026ndash;39)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGravida, medium (IQR)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(1\u0026ndash;3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eParity, medium (IQR)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCervical cytology, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51(52.04)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.02)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (10.20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13(13.26)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNILM or unknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (23.47)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHPV infection, n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64/94 (68.09)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHpv-16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV16 and other types\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/94(3.19)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV 18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV and other types(except for HPV16 )\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV16 and/or HPV18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/94(3.19)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV 33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/94(7.45)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV 33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV 33 and other types(except for HPV 16/18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV-58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6/94 (6.38)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV58 and other types(except for HPV 16/18/33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther HR-HPV or unknown types\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13/94 (33.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPV negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/94(1.06)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (4.08)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eAbbreviations: NILM (Negative for intraepithelial lesion or malignancy); ASC-US (atypical squamous cells undetermined significance); ASC-H (atypical squamous cells cannot exclude a high-grade squamous intraepithelial lesion); LSIL (low grade squamous intraepithelial lesion); HSIL (high-grade squamous intraepithelial lesions).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe cytology of patients before conization showed 52.04% (51/98) high-grade squamous intraepithelial lesions(HSIL), 1.02% (1/98) atypical squamous cells cannot exclude HSIL(ASC-H), 10.20% (10/98) low-grade squamous intraepithelial lesions(LSIL), 13.26% (13/98)Atypical squamous cells of undetermined significance (ASCUS), and 23.47% (23/98) normal/unknown results.\u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2. Remission of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHPV\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;negative rates of HR-HPV at 3, 6, 9, 12, 15, 18, 21, and 24 months after surgery were 76% (19/25), 80.95% (34/42), 76.47%(39/51), 76.92%(40/52), 75.93%(41/54),76.36%(42/55), 75.44%(43/57), and 74.58% (44/59), respectively, which\u0026nbsp;was basically stable within 2 years. Among them, the patients who were still positive for HPV 3 months after surgery were all initially treated with HPV16 infection. From 6 to 9 months after surgery, 83.3% (5/6) of the HPV-positive patients were initially infected with HPV16, but only 27.7% (1/6) of the HPV-positive patients were HPV16 at 9 months after surgery. Among the patients who were retested positive 21 months after surgery, there was one unknown type. Among the known types, 60% (3/5) were initially treated with HPV16 positive, 20% (1/5) were initially treated with HPV18 positive, and 20% (1/5) were initially treated with HPV 58 positive. Only 1 case (1/5, 20%) was HPV16 positive at the initial treatment. 66.67%(2/3) of the patients who were retested positive at 24 months after surgery were HPV16 positive at the first treatment and none of the HPV-positive patients at 24 months was HPV16 type. There was no patient with persistent abnormal cytology (HSIL/ASC-H) after surgery.\u0026nbsp;For the details of the remission of HPV infection see\u0026nbsp;figure 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Mechanism of the TP63-ZC3H13-HLA-A axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.1 The expression of TP63 increased gradually during the process of cervical epithelial carcinogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, tumor tissues and adjacent tissues from four patients with cervical cancer (three squamous cell carcinomas and one adenocarcinoma) were selected. It was found that the mRNA expression level of TP63 in cervical squamous cell carcinoma tissues was higher than that in adjacent tissues (Figure 3A), while the protein level of TP63 in tumor tissues was higher than that in adjacent tissues (Figure 3B). We further found in GEO database (GSE75132) that the mRNA expression level of TP63 gradually increased in HPV16-infected cervical tissues from normal to precancerous lesions (see Figure 3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.2 m6A methylation-related gene ZC3H13 may be involved in the carcinogenesis of cervical cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy comparing the expression of m6A methylation-related genes in cervical cancer and adjacent tissues in TCGA database, it was found that several molecules such as METTL3, METTL14, ZC3H13, and YTHDC1 were highly expressed in cervical cancer tissues compared with adjacent tissues (FIG. 4A), and the relationship between abnormal expression molecules and the prognosis of cervical cancer was further compared. It was found that patients with high expression of the m6A methylation-related molecule ZC3H13 had significantly decreased OS and PFS (Figure 4B-C). Meanwhile, immunohistochemistry showed that ZC3H13 was significantly overexpressed in cervical cancer compared with normal cervical tissues (Figure 4D-E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTP63 molecule is associated with low expression of HLA-A receptor and low level of CD8+T cell infiltration in cervical cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed the infiltration levels of CD4+T cells, CD8+T cells, Treg cells, B cells, dendritic cells, M1 macrophages, and M2 macrophages by the Timer2.0 immune database QUNTISEQ algorithm and found that the infiltration level of CD8+T cells was significantly decreased in patients with high TP63 expression (Figure 5A-G). Moreover, patients with low CD8+T cell infiltration had a worse survival prognosis (Figure 5H). To understand whether the decreased CD8+T cell infiltration was associated with the down-regulated expression of HLAs molecules, bioinformatics analysis predicted that patients with high TP63 expression in the TCGA cervical cancer database had down-regulated expression of human leukocyte associated antigen HLAA, B, and C receptors (FIG. 5I).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.4 TP63 may affect the infiltration of CD8+T cells by regulating ZC3H13\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, the relationship between ZC3H13 and cervical cancer immune cell infiltration was further predicted by bioinformatics, and it was found that patients with high expression of ZC3H13 had a statistically significant reduction in CD8+T cell immune infiltration (Figure 6A). Correlation analysis showed that ZC3H13 was positively correlated with TP63 in cervical squamous cell carcinoma (FIG. 6B). The JASPAR website predicted that there were two binding sites of TP63 in the promoter region of ZC3H13 (Figure 6C), so it was speculated that the reduction of CD8+T cells caused by TP63 may be related to its effect on the expression of ZC3H13.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.5 ZC3H13 binding sites in the 3 \u0026apos;-UTR of HLA-A mRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough SRAMP website prediction, we found that there were two high-scoring m6A binding sites in the 3 \u0026apos;-UTR of HLA-A pre-mRNA, one of which was the classical binding motif \u0026quot;GGACU\u0026quot; of ZC3H13. Therefore, it was speculated that ZC3H13 might promote HLA-A mRNA 3 \u0026apos;-UTR degradation through m6A methylation (Figure. 7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003e1. Persistent HPV infection is the most important risk factor for recurrence, and different HPV types help to predict disease recurrence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCervical conization is the major treatment modality of stage IA1 cervical cancer in patients with fertility needs. Persistent or recurrent HPV infection is the major reason for postoperative relapse or disease residuality of IA1 cervical cancer. The study followed HPV outcomes in high-risk HPV patients who underwent cervical cone resection, which showed that with time, the cumulative HR-HPV clearance rate after cervical conization is stable to 74%-80% within 2 years.\u0026nbsp;New HPV infection and type-specific persistent infection were two mechanisms of postoperative persistent HPV. Persistent HPV infection after treatment, with or without residual HSIL/cancer, is the most important established risk factor for recurrent disease\u003csup\u003e[17]\u003c/sup\u003e.\u0026nbsp;In this study, among the patients with known HPV types, the proportion of patients who were initially treated with HPV16 was the most serious, which was the main infection type (76.19%,64/84). Among the patients who were still HPV positive at 3, 6-9, 9, 21, and 24 months after conization, the proportion of patients with HPV16 infection was 100%, 100% (6/6), 83.3% (5/6), 27.7% (1/6), 60% (3/5) and 66.67% (2/3), respectively. No HPV type 16 was detected at 24 months. Patients with HPV16 infection take a longer time to clear HPV after conization, are at high risk of recurrence, and are more likely to be infected with other new HPV types. One retrospective study reported that six months after LEEP, HPV 16 positive was associated with a 37% increase in the absolute risk of CIN 2+2 years, twice as much as HPV 18 (18.5%), and three times as much as other types of oncogenes (10.8%)\u003csup\u003e[18-19]\u003c/sup\u003e. Didem Egemen et al. \u003csup\u003e[20]\u003c/sup\u003ehas reported on the new risk-based guidelines present recommendations for the management of abnormal screening test and histology results, and meaningfully indicated the impact of HPV test results on the immediate and 5-year risks of CIN 3+. HPV positivity was a significant predictor of immediate\u0026nbsp;risks of CIN 3+ in patients monitored for\u0026nbsp;patients with previous HSIL\u003csup\u003e[20]\u003c/sup\u003e. Therefore, HPV typing is necessary to evaluate whether HPV infection is persistent or new after treatment and to study the relationship of different typing to predict disease recurrence. Studies have shown that persistent infection with HPV16, 18, 31, 33, 52, and 58 is a high-risk predictor of residual/recurrent disease\u003csup\u003e[21-23]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. HPV immune escape is the fundamental cause of persistent infection and cervical lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we found that the persistent infection rate of HPV was stable after conization in patients with stage IA1 (microscopic early invasion) cervical cancer who requested fertility preservation. However, some patients do not turn negative due to conization, and during infection, HPV subtypes can gradually change to other subtypes due to antigenic drift to avoid immune attack. The genome of HPV virus is composed of a circular double-stranded DNA of about 8000 base pairs, which contains three genomic regions, namely, early coding region (E), late coding region (L) and long control region (LCR) \u003csup\u003e[24]\u003c/sup\u003e. The E gene includes the E6 and E7 genes, which are responsible for cell transformation and are the main causes of cervical lesions. The E6 molecule binds p53 and promotes ubiquitination and degradation of p53, and the E7 molecule binds to the unphosphorylated retinoblastoma (RB) protein, both of which allow the viral DNA to replicate without circulation. It controls G1 and S phases of the cell cycle\u003csup\u003e\u0026nbsp;[25-26]\u003c/sup\u003e.However, the occurrence of cervical lesions requires not only the inhibition of tumor suppressor genes such as P53 and RB by HPV E6 and E7 genes, but also the persistent infection of HPV to activate other protooncogenes in the cervical epithelium. HPV evades the immune clearance of the body, which is the premise of HPV causing persistent infection and activating other proto-oncogenes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPV infection is confined to the cervical keratinized squamous epithelial cells. The keratinized epithelial cells have the function of antigen presentation, Th1 and Th2 cytokines secretion, and can induce the cytotoxic effect of CD4+ and CD8+ memory T cells. human leukocyte antigens (HLAs) are expressed on the surface of human nucleated cells and are the main pathway for the presentation of endogenous antigens, including viruses and tumors. The antigen produced by the virus in the cytoplasm first binds to ubiquitin, and the ubiquitinated protein enters the proteasome in a linear manner to be degraded to produce endogenous antigen. The transporter associated with antigen processing (TAP) assists in the transport of antigen peptides from the cytosol to the endoplasmic reticulum. With the assistance of molecular chaperones calreticulin, calreticulin and TAP, endogenous antigens bind to HLA class I molecules. It is then transported to the cell membrane by the Golgi apparatus and recognized by CD8+T cells, which kill infected cells (see FIG. 8 for details)\u003csup\u003e\u0026nbsp;[26]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPV can evade the damage of the immune system and carry on persistent infection and replication by affecting multiple links of the innate and adaptive immune response. The main mechanisms are: a) The replication cycle of HPV is closely related to the differentiation of keratinocyte. With the virus replication, epithelial cells become keratinized and exfoliated, and infected cells are not lysed, which cannot produce a strong inflammatory response to trigger natural immunity\u003csup\u003e\u0026nbsp;[27]\u003c/sup\u003e; b) HPV E6 and E7 proteins can negatively regulate the expression of HLAs in cervical cells to escape the recognition of immune cells \u003csup\u003e[28, 29]\u003c/sup\u003e; c) HPV can promote the change of Th1 pro-inflammatory cytokines to Th2 anti-inflammatory cytokines in the cervical epithelial microenvironment, such as the reduction of proinflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, IL-18, CCL2, CCL20, CXCL9 and type I interferon. To inhibit the increased expression of inflammatory cytokines such as IL-4,IL-10 and IL-17 to escape the innate immune response and adaptive immunity induced by cytokines; d) recruitment of immune cells that suppress immune response or inhibit the recruitment and activation of killer T cells. In the cervical epithelial microenvironment, the ratio of CD8+T cells /Treg cells decreases, and the generation of appropriate killer T cells cannot be induced to kill virus-infected cells\u003csup\u003e\u0026nbsp;[30-32]\u003c/sup\u003e. In conclusion, before the carcinogenesis of cervical epithelial cells, occult HPV replicates and exfoliates with the keratinization of cervical epithelium, which cannot produce sufficient inflammatory response to induce the body to kill the infected keratinized epithelial cells, and then evade immunity. Therefore, it is necessary to further reveal how the expression of HLAs molecules caused by HPV infection confined in keratinized cervical squamous epithelium affects the mechanism of CD8+T cells to clear HPV-infected cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Hpv persistent infection through TP63-ZC3H13-HLA-A axis leads to cervical lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1 m6A demethylase affects mRNA expression and affects tumorigenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN6-methyladenosine (m6A) is one of the most abundant RNA modifications, with an estimated 3-5 m6A sites on each mRNA molecule. m6A is another important epigenetic modification pathway newly discovered after DNA methylation\u003csup\u003e\u0026nbsp;[33]\u003c/sup\u003e. At the level of transcriptional regulation, m6A can regulate various processes of mRNA molecules, including alternative splicing, maturation, nuclear export, translation, degradation and stability\u003csup\u003e\u0026nbsp;[33]\u003c/sup\u003e. In terms of biological effects, m6A can regulate stem cell differentiation, animal growth and development, DNA damage repair, tumor occurrence and development, and immune response\u003csup\u003e\u0026nbsp;[34-35]\u003c/sup\u003e. The m6A modification on RNA is determined by the methyltransferase complex and the demethylase, and the function of m6A is performed by the protein that specifically recognizes it \u003csup\u003e[36]\u003c/sup\u003e. The methyltransferase complex METTL3/METTL14/WTAP catalyzes the formation of m6A. METTL3 can bind SAM and catalyze the formation of m6A, and METTL14 mainly provides a platform for substrate binding. WTAP is responsible for recruiting METTL3/METTL14 complex to the nuclear plaques in the nucleus to perform the catalytic function of methylation\u003csup\u003e\u0026nbsp;[37]\u003c/sup\u003e.Demethylases FTO and ALKBH5 remove the methylation of m6A\u003csup\u003e\u0026nbsp;[38-39]\u003c/sup\u003e. m6A readers, such as YTH N6-methyladenosine RNA binding protein 1/2/3 (YTHDF1/2/3), bind to m6A modified mRNA to determine the fate of m6A modified mRNA. m6A regulators such as YTHDF1, METTL3 and ALKBH5 have been intensively studied in cancer. m6A methylation can affect the function of downstream genes through several mechanisms: for example, the modification of introns affects the alternative splicing of target gene mRNA;Upregulation of mRNA has been reported to be associated with poor prognosis in various cancer types, and YTHDF1 promotes tumorigenesis and cancer metastasis\u003csup\u003e\u0026nbsp;[40-44]\u003c/sup\u003e.ZC3H13 is another important m6A methylating protein, which can promote the formation of methylation at the m6A site of the \u0026quot;GGACU\u0026quot; motif in the 3 \u0026apos;untranslated region of the downstream mRNA by forming a complex with WTAP, Virilizer and Hakai\u003csup\u003e\u0026nbsp;[45]\u003c/sup\u003e. However, the formation of m6A methylation sites in mRNA 3 \u0026apos;-UTR can promote the degradation of mRNA\u003csup\u003e\u0026nbsp;[46]\u003c/sup\u003e. Our previous bioinformatics prediction found that the expression of m6A methylated genes in cervical cancer lesions was significantly up-regulated compared with adjacent tissues, among which only ZC3H13 expression significantly affected the prognosis of patients, and the survival outcome of patients with high expression of ZC3H13 was worse, and the binding site of ZC3H13 was predicted in the 3 \u0026apos;-UTR of HLA-A mRNA. Therefore, it is speculated that ZC3H13 may promote HLA-A mRNA degradation through m6A methylation modification during cervical epithelial lesions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Mechanism of TP63 promoting cervical lesions caused by HPV infection through ZC3H13-mediated HLA-A m6A methylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, it was verified by molecular level and database analysis that TP63 promotes the occurrence of cervical squamous cell carcinoma and can be used as a meaningful therapeutic target. Moreover, TIMER 2.0 database showed that TP63 affected the expression of HLA-A receptor and inhibited the activation and proliferation of CD8+T cells. In addition, TP63 expression was positively correlated with the above-mentioned ZC3H13 expression in cervical squamous cell carcinoma, and negatively correlated with HLA-A expression. JASPAR website predicted that there was a TP63 binding site in the upstream of the promoter region of ZC3H13 and SRAMP database predicted that there was a binding site of ZC3H13 in the 3 \u0026apos;-UTR of HLA-A mRNA. Therefore, this study proposes hypotheses: TP63 up-regulates the expression of ZC3H13 by acting on the promoter of ZC3H13, and ZC3H13 further promotes the m6A methylation of HLA-A mRNA 3\u0026rsquo;UTR, leading to accelerated degradation of HLA-A mRNA, thereby inhibiting the antigen presentation of HPV viral proteins. As a result, HPV escapes from the killing effect of CD8+T cells (see Figure 9), making high-risk HPV persistent infection and difficult to clear.\u003c/p\u003e\n\u003cp\u003eIn addition, the possible mechanism of TP63-ZC3H13-HLA-A axis may provide A therapeutic target for HPV clearance and prevention of recurrence in patients with stage IA cervical cancer after conization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1.Ethics approval and consent to participate：All procedures performed in studies involving human participants following the ethical standards of the institutional\u0026nbsp;and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The Institutional Review Board approved the study (approval number: 2017-KY-026-01). Informed consent was unavailable in the study.\u003c/p\u003e\n\u003cp\u003e2.Consent for publication：All authors approved the final manuscript and the submission to the journal.\u003c/p\u003e\n\u003cp\u003e3.Availability of data and materials：The datasets generated during and/or analyzed during the current study are publicly available.\u003c/p\u003e\n\u003cp\u003e4.Competing interests：The authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5.Funding：This study is supported by Beijing Hospitals Authority Sailing plan (grant number: ZYLX201705).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6.Authors\u0026apos; contributions：X.S.Q collected the data, followed the patients, completed the experimental section, and wrote the paper. W.M collected the data, completed the data analysis and wrote the paper. X.J.Q collects data. W.Y.M supervised the conduct of the study, revised the article, and made suggestions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeol HJ, Ki KD, Lee JM. Epidemiologic characteristics of cervical cancer in Korean women. J Gynecol Oncol. 2014;25:70\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagase S, Inoue Y, Umesaki N, et al. Evidence-based guidelines for the treatment of cervical cancer in Japan: Japan Society of Gynecologic Oncology (JSGO) 2007 edition. Int J Clin Oncol. 2010;15:117\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Comprehensive Cancer Network. 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Nature. 2017;542(7642):475\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3362761/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3362761/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe aim of this study is to investigate the short-term persistent HPV infection and natural prognosis in patients with IA1 cervical cancer after CKC, and to explore the mechanism of persistent HPV infection leading to cervical lesions based on database analysis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis is a prospective observation cohort study which enrolled the stage IA1 patients who select to receive CKC as the treatment modality in a single center from January 24, 2018, to June 9, 2022. The primary outcome was the persistent infection status and remession rates within two years after the CKC. In addition, the relevant mechanism was explored based on database analysis. The Cancer Genome Atlas (TCGA) database is the source of RNA sequencing data of cervical cancer patients. The gene Expression omnibus (GEO) database was used as the validation set to verify the expression of TP63 mRNA in the process from normal cervical to precancerous lesions, and the difference of TP63 between cancerous lesions and paracarcinoma was verified by Western blot. The limma package of R software, Kaplan-Meier survival curve and Log-rank test were used to screen the genes related to m6A methylation modification affecting the prognosis of cervical cancer. spearman correlation analysis was used to verify the correlation between genes, and Timer2.0 immune database was used to analyze the correlation between the expression level of key genes and the level of immune infiltration. JASPAR and SRAMP open access databases were used to verify the relevant RNA sequence binding sites and m6A methylation modification sites.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA total of 98 eligible patients were included and the main types of HPV was as following: HPV 16 (76.19%,64/84), HPV 58 (10.71%,9/84), HPV 33 (9.52%,8/84), and unknown type 14.28% (14/98). The HR-HPV negative rates of the whole cohort at 3, 6, 9, 12, 15, 18, 21, and 24 months after CKC were 76% (19/25), 80.95% (34/42), 76.47%(39/51), 76.92%(40/52), 75.93%(41/54), 76.36%(42/55), 75.44%(43/57), and 74.58% (44/59), which was stable within 2 years after surgery. Based on the database analysis, this study proposes the relevant mechanism hypothesis of HPV persistent infection and difficult to clear: TP63 acts on the promoter of ZC3H13 to induce its expression and promote the m6A methylation modification of HLA-A mRNA 3\u0026rsquo;UTR, which leads to the accelerated degradation of HLA-A mRNA and further inhibits the antigen presentation of HPV viral proteins, leading to HPV escape from CD8\u0026thinsp;+\u0026thinsp;T cell killing.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe negative rate of HR-HPV remained stable within 2 years after the CKC.Standardized follow-up after conization is very important for patients with stage IA1 cervical cancer.This study elucidates the mechanism of TP63-ZC3H13-HLA-A axis and provides A therapeutic target for HPV clearance and prevention of recurrence in patients with stage IA cervical cancer after conization.\u003c/p\u003e","manuscriptTitle":"The potential role of TP63 regulating ZC3H13-mediated HLA-A m6A methylation modification in HR-HPV persistent infection patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-12 16:48:34","doi":"10.21203/rs.3.rs-3362761/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2006392e-b2de-474e-a421-ce5be5d20578","owner":[],"postedDate":"October 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-29T16:14:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-12 16:48:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3362761","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3362761","identity":"rs-3362761","version":["v1"]},"buildId":"-D5TCW68w8eVRRLyjaTIo","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}