INTRODUCTION:Human papillomavirus (HPV) integration can induce gene expression dysregulation by destroying higher-order chromatin structure in cervical cancer. OBJECTIVES:We established a 13q22 site-specific HPV16 gene knock-in cell model to interrogate the changes in chromatin structure at the initial stages of host cell malignant transformation. METHODS:We designed a CRISPR-Cas9 system with sgRNA targeting 13q22 site and constructed the HPV16 gene donor. Cells were cotransfected, screened, and fluorescence sorted. The whole genome sequencing (WGS) was used to confirm the precise HPV16 gene integration site. Western blot and qRT-PCR were used to measure gene expression. In vitro and in vivo analysis were performed to estimate the tumorigenic potential of the HPV16 knock-in cell model. Combined Hi-C, chromatin immunoprecipitation and RNA sequencing analyses revealed correlations between chromatin structure and gene expression. We performed a coimmunoprecipitation assay with anti-PIBF1 antibody to identify endogenous interacting proteins. In vivo analysis was used to determine the role of PIBF1 in the tumor growth of cervical cancer cells. RESULTS:We successfully established a 13q22 site-specific HPV16 gene knock-in cell model. We found that HPV integration promoted cell proliferation, invasion and stratified growth in vitro, and monoclonal proliferation in vivo. HPV integration divided the affected topologically associated domain (TAD) into two smaller domains, and the progesterone-induced blocking factor 1 (PIBF1) gene near the integration site was upregulated, although PIBF1 was not enriched at the domain boundary by CUT-Tag signal analysis. Moreover, PIBF1 was found to interact with the cohesin complex off chromatin to reduce contact domain formation by disrupting the cohesin ring-shaped structure, causing dysregulation of tumorigenesis-related genes. Xenograft experiments determined the role of PIBF1 in the proliferation in cervical cancer cells. CONCLUSION:We highlight that PIBF1, a potential chromatin structure regulatory protein, is activated by HPV integration, which provides new insights into HPV integration-driven cervical carcinogenesis.

Clinical trials have shown that immunotherapy based on Vγ9Vδ2 T cells (Vδ2 T cells) is safe and well-tolerated for various cancers including cervical cancer (CC), but its overall treatment efficacy remains limited. Therefore, exploring the mechanisms underlying the suboptimal efficacy of Vδ2 T cell-based cancer immunotherapy is crucial for enabling its successful clinical translation. Tumor samples from CC patients and CC cell line-derived xenograft (CDX) mice were analyzed using flow cytometry to examine the exhausted phenotype of tumor-infiltrating Vδ2 T cells. The interrelationship between BTN3A1 expression and Vδ2 T cells in CC, along with their correlation with patient prognosis, was analyzed using data from The Cancer Genome Atlas (TCGA) database. CC cell lines with BTN3A1 knockout (KO) and overexpression (OE) were constructed through lentivirus transduction, which were then co-cultured with expanded Vδ2 T cells, followed by detecting the function of Vδ2 T cells using flow cytometry. The pathways and transcription factors (TFs) related to BTN3A1-induced Vδ2 T cells exhaustion and the factors affecting BTN3A1 expression were identified by RNA-seq analysis, which was confirmed by flow cytometry, Western Blot, and gene manipulation. Tumor-infiltrating Vδ2 T cells exhibited an exhausted phenotype in both CC patients and CDX mice. BTN3A1 expressed in CC is highly enhancing exhaustion markers, while reducing the secretion of effector molecules in Vδ2 T cells. Blocking TCR or knocking down nuclear receptor subfamily 4 group A (NR4A) 2/3 can reverse BTN3A1-induced exhaustion in Vδ2 T cells. On the other hand, IFN-γ secreted by Vδ2 T cells promoted the expression of BTN3A1 and PD-L1. Through binding γδ TCRs, BTN3A1 expressed on tumor cells, which is induced by IFN-γ, can promote Vδ2 T cells to upregulate the expression of TFs NR4A2/3, thereby affecting their activation and expression of exhaustion-related molecules in the tumor microenvironment (TME). Therefore, targeting BTN3A1 might overcome the immunosuppressive effect of the TME on Vδ2 T cells in CC.

IHC stainings of PCNA, Caspase-3 and CD31 in the non-necrosis area of xenografts.

The amino acid sequences of FokI variants.

Target sequences and construction approaches of ZFNs used in this manuscript.

Recent studies have shown that prostate cancer-associated long non-coding RNA, PRNCR1, plays crucial roles in the development of multiple human cancers. However, its role in ovarian cancer is barely known. This study was carried out to investigate the role of PRNCR1 and the underlying mechanisms in OC. The expression of PRNCR1 and miR-653-5p in OC cell lines and tissues were detected by qRT-PCR. The expression of ELF2 protein was evaluated by Western blot analysis. Cell proliferation was measured by colony formation and MTT assay. Cell invasion and migration were evaluated by Transwell and wound healing assay. Luciferase reporter assay and RNA-binding protein immunoprecipitation assay were performed to determine the interaction between miR-653-5p and PRNCR1, as well as between miR-653-5p and ELF2. In vivo tumor xenograft model was established to evaluate the role of PRNCR1 in tumor growth. Our results demonstrated that PRNCR1 was significantly upregulated in both OC cell lines and tissues, and high expression of PRNCR1 was correlated with poor survival of OC patients. Overexpression of PRNCR1 accelerated OC cell invasion, migration and proliferation. Besides, the expression of PRNCR1 was negatively correlated with the expression of miR-653-5p, while positively correlated with the expression of E74-like factor 2 in OC tissues. Importantly, ELF2 could target miR-653-5p, and PRNCR1 increased the expression levels of ELF2 by sponging miR-653-5p in OC cells. Furthermore, the miR-145-5p/ELF2 axis was involved in the regulation of PRNCR1 in OC progression in vivo. PRNCR1 promotes OC tumor progress via the miR-653-5p/ELF2 axis and might be a potential therapeutic target for OC.

To investigate the important roles of the cancer-promoting long non-coding RNAs (lncRNAs) in cervical cancer, the up-regulated lncRNAs and prognostic analysis were identified through Lnc2Cancer and Lncar. LncRNA-regulated miRNA and miRNA-target mRNA were analyzed based on starBase v2.0 and miTarbase to predict the lncRNA-miRNA-mRNA ceRNA network. Based on the above findings, the abnormally expressed histocompatibility leukocyte antigen complex P5 (HCP5) was identified in 31 cervical cancer patients through RT-qPCR. The stable cell lines were constructed to explore the effect of HCP5 on the promotion of cervical cancer and the regulatory role on the expression of miR-216a-5p and CDC42. Cell Counting Kit-8 (CCK8) assay, cell clone formation, and transwell assay were used to examine proliferation and migration ability of cervical cancer cells. The results displayed that the overexpression of HCP5 promoted cervical cancer cell proliferation and migration in vitro, and the elevated HCP5 can also promote tumor growth in vivo. Besides, RT-qPCR and western blot assay revealed that elevated HCP5 suppressed miR-216a-5p expression and then up-regulated the expression of CDC42. In contrast, knocking down HCP5 resulted in increased expression of miR-216a-5p and then downregulated the expression of CDC42. Rescue experiments also demonstrated that miR-216a-5p could in part intercept in promotion impact caused by HCP5 on cervical cancer cells. Above all, HCP5, as an oncogene, can promote proliferation and migration ability of cervical cancer via the regulation of the miR-216a-5p/CDC42 axis.

Human growth hormone (hGH) is widely used to treat several diseases for decades. However, the current treatment regime requires frequent injections via subcutaneous route due to short in vivo half-life, which leads to pain and poor patient compliance. In this study, a novel transdermal (non-invasive) hGH loaded niosomes gel was prepared to reduce the frequency of subcutaneous injections and to improve the patient compliance. Niosomes were prepared by film hydration technique at three levels of cholesterol. The particle size and entrapment efficiency increases with an increase in the level of cholesterol. Transmission electron microscopy images confirmed the spherical shape of niosomes without aggregation. Texture profiles analysis indicates that the niosomal gel has the required mechanical properties for transdermal application. The ex vivo permeation profile showed sustain hGH release for 4 days from the niosomal gel compared to 24 h from the control gel without niosomes. A rabbit skin irritation study showed no sign of irritation after application of niosomal gel. The pharmacokinetic parameters in the rat model showed 7.22-fold higher bioavailability with niosomal gel compared to control gel. In conclusion, the study demonstrated the potential of niosomal gel as an effective long-term sustained release strategy for hGH delivery to replace traditional subcutaneous injections.

Human papillomavirus (HPV) integration in the human genome is suggested to be an important cause of cervical cancer. With the development of sequencing technologies, an increasing number of integration “hotspots” have been identified. However, this HPV integration information was derived from analysis of whole cervical cancer tissue, and we know very little about the integration in different cancer cell subgroups or individual cancer cells. This study optimized the preparation of probes and provided a dual-color fluorescence in situ hybridization (FISH) method to detect HPV integration sites in paraffin-embedded cervical cancer samples. We used both HPV probes and site-specific probes: 3p14 ( FHIT ), 8q24 ( MYC ), 13q22 ( KLF5 / KLF12 ), 3q28 ( TP63 ), and 5p15 ( TERT ). We detected HPV signals in 75 of the 96 cases of cervical cancer; 62 cases showed punctate signals, and 13 cases showed diffuse punctate signals. We identified 3p14 as a high-frequency HPV integration site in 4 cervical cancer cases. HPV integration at 8p14 occurred in 2 cases of cervical cancer. In the same cervical cancer tissue of sample No.1321, two distinct subgroups of cells were observed based on the HPV probe but showed no difference in cell and nucleus morphology. Our study provides a new method to investigate the frequent HPV integration sites in cervical cancer and reports the heterogeneity within cervical cancer from the perspective of HPV integration.

This study was aimed to analyze the role of hepatocyte nuclear factor -1α (HNF-1α) in regulating migrative and invasive potentials in cervical cancer via the involvement of pyruvate kinase L/R (PKLR). The expression of HNF-1α and PKLR in cervical cancer tissues classified by tumor size and FIGO (Federation International of Gynecology and Obstetrics) stage were detected by qRT-PCR. The expression correlation between HNF-1α and PKLR in cervical cancer tissues was analyzed by Pearson correlation test. After intervening HNF-1α and PKLR levels in SiHa and Hela cells, their migratory and invasive abilities were examined by the Transwell assay. HNF-1α was upregulated in cervical cancer tissues, particularly those with large tumor size or advanced FIGO stage. PKLR was highly expressed in cervical cancer tissues as well, presenting a positive correlation with the HNF-1α level. Knockdown of HNF-1α attenuated migratory and invasive abilities in SiHa cells, whereas overexpression of HNF-1α enhanced migratory and invasive abilities in SiHa cells. PKLR was able to abolish the regulatory effects of HNF-1α on cervical cancer metastasis. HNF-1α and PKLR synergistically promote cervical cancer to migrate and invade.

Integration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.

Objectives This study aimed to assess the effect of neoadjuvant chemotherapy (NACT) on the rate of lymph node metastasis (LNM) in FIGO stage IB1-IIB cervical cancer patients and compare the LNM between NACT plus surgery and surgery only. Methods We identified 34 eligible studies in PubMed, Web of Science, Cochrane Library, and EMBASE from inception to July 27, 2019. Data analyses were performed by Stata (version 13) and Revman (version 5.3). Results In these 34 included studies, the pooled incidence of LNM was estimated as 23% (95% CI, 0.20-0.26; I2 = 79.6%, P<0.001). In the subgroup analysis, we identified five factors, including study type, year of publication, continents from which patients came, histological type and the FIGO stage. When taking FIGO stage into consideration, the LNM rate was 13% in stage IB (95% CI: 0.10-0.15; I2 = 5.5%, P=0.385), 23% in stage IIA (95% CI: 0.18-0.28; I2 = 0%, P=0.622), and 27% in stage IIB (95% CI: 0.20-0.33; I2 = 0%, P=0.898), respectively. Through the comparison between NACT plus surgery and surgery only based on the six randomized controlled trials, the incidence of positive lymph nodes was lower in patients receiving NACT plus surgery than surgery only (RR=0.57, 95% CI: 0.39-0.83; I2 = 60.5%, P=0.027). The 5-year OS was higher in the NACT + surgery group than surgery-only group (RR=1.13, 95% CI: 1.03-1.23; I2 = 0.0%, P=0.842). Conclusions Among cervical cancer in stage IB1-IIB, the preoperative NACT plus radical surgery resulted in a 23% probability of LNM, which was lower than those receiving radical surgery only. In stage IIA and IIB, the effect of NACT to reduce LNM was more obvious.

Background: Persistent infection with the high-risk of human papillomavirus (HR-HPVs) is the primary etiological factor of cervical cancer; HR-HPVs express oncoproteins E6 and E7, both of which play key roles in the progression of cervical carcinogenesis. Zinc Finger Nucleases (ZFNs) targeting HPV E7 induce specific shear of the E7 gene, weakening the malignant biological effects, hence showing great potential for clinical transformation. Objective: Our aim was to develop a new comprehensive therapy for better clinical application of ZFNs. We here explored the anti-cancer efficiency of HPV targeted ZFNs combined with a platinum-based antineoplastic drug Cisplatin (DDP) and an HDAC inhibitor Trichostatin A (TSA). Methods: SiHa and HeLa cells were exposed to different concentrations of DDP and TSA; the appropriate concentrations for the following experiments were screened according to cell apoptosis. Then cells were grouped for combined or separate treatments; apoptosis, cell viability and proliferation ability were measured by flow cytometry detection, CCK-8 assays and colony formation assays. The xenograft experiments were also performed to determine the anti-cancer effects of the combined therapy. In addition, the HPV E7 and RB1 expressions were measured by western blot analysis. Results: Results showed that the combined therapy induced about two times more apoptosis than that of ZFNs alone in SiHa and HeLa cells, and much more inhibition of cell viability than either of the separate treatment. The colony formation ability was inhibited more than 80% by the co-treatment, the protein expression of HPV16/18E7 was down regulated and that of RB1 was elevated. In addition, the xenografts experiment showed a synergistic effect between DDP and TSA together with ZFNs. Conclusion: Our results demonstrated that ZFNs combined with DDP or TSA functioned effectively in cervical cancer cells, and it provided novel ideas for the prevention and treatment of HPV-related cervical malignancies.

Background : The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is becoming a promising gene therapy method. Herein, we evaluated the therapeutic effect of CRISPR/Cas9 system in cervical carcinogenesis, especially cervical precancerous lesions. Methods : In cervical cancer/pre-cancer cell lines, we transfected the CRISPR/Cas9, transcription activator–like effector nuclease (TALEN), and zinc finger nuclease (ZFN) plasmids, respectively. We used the cell apoptosis, cell viability, and colony formation assays to examine the efficiency and specificity of inhibition of cell apoptosis and growth between the different gene editing tools. Western blotting was used to estimate the related protein expression. We used xenograft formation assays to examine the ability of inhibition of cell growth in vivo. In the K14-HPV16 transgenic mice model of HPV-driven cervical carcinogenesis, we investigated the therapeutic effect by vaginal administration. Results : Compared to ZFN and TALEN, CRISPR/Cas9 has shown comparable efficiency and specificity of inhibition of cell apoptosis and growth in cervical cancer cell lines, which seem to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. In xenograft formation assays, CRISPR/Cas9 could inhibit tumor formation in vivo and affects the expression of the corresponding protein. In the K14-HPV16 transgenic mice, CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. Conclusion : In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively reduce the expression of E7 protein in vitro. Additionally, it could revert the HPV-related cervical carcinogenesis in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment.

Integration of human papillomavirus (HPV) DNA into the human genome is a reputed key driver of cervical cancer. However, the effects of HPV integration on chromatin structural organization and gene expression are largely unknown. We studied a cohort of 61 samples and identified an integration hot spot in the CCDC106 gene on chromosome 19. We then selected fresh cancer tissue that contained the unique integration loci at CCDC106 with no HPV episomal DNA and performed whole-genome, RNA, chromatin immunoprecipitation and high-throughput chromosome conformation capture (Hi-C) sequencing to identify the mechanisms of HPV integration in cervical carcinogenesis. Molecular analyses indicated that chromosome 19 exhibited significant genomic variation and differential expression densities, with correlation found between three-dimensional (3D) structural change and gene expression. Importantly, HPV integration divided one topologically associated domain (TAD) into two smaller TADs and hijacked an enhancer from PEG3 to CCDC106, with a decrease in PEG3 expression and an increase in CCDC106 expression. This expression dysregulation was further confirmed using 10 samples from our cohort, which exhibited the same HPV-CCDC106 integration. In summary, we found that HPV-CCDC106 integration altered local chromosome architecture and hijacked an enhancer via 3D genome structure remodeling. Thus, this study provides insight into the 3D structural mechanism underlying HPV integration in cervical carcinogenesis.

Gene therapy targeted human papillomavirus (HPV) is a promising treatment for cervical cancer, and the key for clinical application depends on an effective gene delivery method. Our aim was to formulate a new pharmaceutical formula for appropriate gene delivery intravaginally. For the first time, we here developed a new polyethylenimine (PEI) based vaginal suppository. The sectional immunofluorescence results confirmed the delivery efficacy both in vivo and in vitro. The quenching fluorescence and decreased gene expression in topical epithelium of green fluorescence protein (GFP) transgenic mice demonstrated the efficient targeting potential of the suppository. The other aim of this study was to evaluate the biocompatibility of the PEI based transfer. To our knowledge, this was also the first study to explore the toxicity in vivo systematically and comprehensively. Our study provided novel ideas for the translational application of PEI based suppository to the prevention and treatment of cervical cancer.

Cervical cancer is a serious threat to women’s health and is the third most common malignancy in women worldwide. Recent studies indicate that the long non-coding RNA CCAT1 plays a role in the malignant behavior of many tumors. However, the role of CCAT1 in cervical cancer is still unknown. Our aim is to evaluate the expression and investigate the regulatory role and potential mechanism of CCAT1 in cervical cancer. CCAT1 expression was measured by qRT-PCR. In addition, CCK-8 assays, colony formation assays, qRT-PCR assays, Transwell assays and xenograft experiments were performed to determine the role of CCAT1 in the proliferation and invasion in cervical cancer cells. The expression of CCAT1 in the cervical cancer tissues was higher than in the adjacent normal tissues. Overexpressing CCAT1 promoted cervical cancer cell proliferation, colony formation, and invasion in vitro. Elevated CCAT1 suppressed miR-181a expression, which was accompanied by an increased expression of MMP14 and HB-EGF. In contrast, knocking down CCAT1 resulted in increased expression of miR-181a, along with decreased expression of MMP14 and HB-EGF. Thus, CCAT1 is a key oncogenic lncRNA associated with cervical cancer and plays a role in promoting cervical cancer cell proliferation and invasion by regulating the miR-181a-5p/MMP14 axis.

Background: Integration of human papillomavirus (HPV) DNA into human genome is the reputed key driver of cervical cancer. However, the effects of HPV integration on chromatin structural organization and gene expression are largely unknown in such cancer. Methods: We investigated a cohort of 61 samples for HPV integration analysis. And selected a fresh cancer tissue from the set of clinical samples whose tissue only contained unique integration loci at CCDC106 and contained no HPV episomal DNA. A combination of WGS, RNA-seq, ChIP-seq and Hi-C data analysis were applied to identify the mechanisms of HPV integration in cervical carcinogenesis. IHC staining was used to validate the expression change after HPV-CCDC106 integration. Results: Based on the cohort of 61 samples, we identified a hot integrated site in the CCDC106 gene on chromosome 19. Additional analysis showed that this chromosome was enriched with genome variation and differential expression densities in the same HPV-CCDC106 integrated carcinoma sample. More importantly, we identified that HPV divided one topologically associated domain (TAD) into two TADs and hijacked an enhancer from PEG3 to CCDC106, thus leading to a decrease in PEG3 expression and high CCDC106 expression. This expression dysregulation was further confirmed by 10 samples exhibiting the same HPV-CCDC106 integration from our cohort. Interpretation: We found that HPV-CCDC106 integration tended to alter the architecture of the local chromosome and hijacked an enhancer via 3D genome structure remodeling, thus providing insight into the 3D structural mechanism underlying HPV integration in cervical carcinogenesis. Funding Statement: This work was supported by Natural Science Foundation of China (81630060 to P. W., 81830074 and 81772786 to H. W., 81572569 to G. C., 31771402 to G. L. and 81772775 to J. W.), National Science and Technology Major Project (2018ZX10301402-002 to Q. G.), and the research-oriented clinician funding program of Tongji Medical College, Huazhong University of Science and Technology for P.W.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: All tumor specimens, ThinPrep cytologic test (TCT) samples, and normal cervical tissues were obtained from the Department of Gynecological Oncology at Tongji Hospital under ethics board approval (TJ-IRB20180611) and with documented informed consent from all patients.

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Viral infections cause at least 10%–15% of all human carcinomas. Over the last century, the elucidation of viral oncogenic roles in many cancer types has provided fundamental knowledge on carcinogenetic mechanisms and established a basis for the early intervention of virus-related cancers. Meanwhile, rapidly evolving genome-editing techniques targeting viral DNA/RNA have emerged as novel therapeutic strategies for treating virus-related carcinogenesis and have begun showing promising results. This review discusses the recent advances of genome-editing tools for treating tumorigenic viruses and their corresponding cancers, the challenges that must be overcome before clinically applying such genome-editing technologies, and more importantly, the potential solutions to these challenges.