Development Of Cervical Squamous Cell Carcinoma Research Articles

N6-methyladenosine methylation on FSCN1 mediated by METTL14/IGF2BP3 contributes to human papillomavirus type 16-infected cervical squamous cell carcinoma.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.

Impact of CD40 gene polymorphisms on the risk of cervical squamous cell carcinoma: a case-control study

BackgroundCervical cancer is the fourth most common cancer among women worldwide. Genome-wide association studies have revealed multiple susceptible genes and their polymorphisms for cervical cancer risk. Therefore, we aimed to investigate the correlation between single nucleotide polymorphisms (SNPs) of the CD40 gene and susceptibility to cervical squamous cell carcinoma (CSCC) in a population from the northeastern Han Chinese population.MethodsThe three SNPs (rs1800686, rs3765459, and rs4810485) of the CD40 gene were analyzed by multiplex polymerase chain reaction (PCR) combined with next-generation sequencing methods in 421 patients with CSCC, 594 patients with high-grade squamous intraepithelial lesions (HSIL), and 504 healthy females. Multivariate logistic regression analysis was used to analyze the potential relationship between CD40 gene polymorphisms and CSCC, or HSIL.ResultsOur research results showed the AA genotype of rs1800686 had a protective effect on CSCC in comparison to the GG genotype and AG+GG genotypes (AA vs. GG: p = 0.0389 and AA vs. AG+GG: p = 0.0280, respectively). After FDR correction, the results were still statistically significant (p = 0.0389 and p = 0.0389, respectively). Similarly, rs3765459 showed a reduced risk association for CSCC in the codominant and recessive models (AA vs. GG: p = 0.0286 and AA vs. AG+GG: p = 0.0222, respectively). Significant differences remained after FDR correction (p = 0.0286 and p = 0.0286, respectively). However, these differences were no longer significant after the Bonferroni correction. In addition, the genotypes for the rs4810485 polymorphisms were associated with parity of the patients with CSCC. The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC. The 3 SNPs genotypes of the CD40 gene were closely related to the squamous cell carcinoma antigen (SCC) of the patients with HSIL.ConclusionsThe CD40 gene may play a role in the occurrence and development of CSCC.

Correlation and clinical significance of VISTA expression and cervical squamous cell carcinoma

Objective: To explore the relationship between the expression of the T-cell activation suppressor-immunoglobulin variable region (VISTA) and the development of cervical squamous cell carcinoma (CSCC), and the impact on the prognosis of CSCC patients. Methods: Cervical tissue samples from 116 CSCC, including 23 cervical intraepithelial neoplasia (CIN) grade I, 23 CIN grade Ⅱ-Ⅲ, and 23 chronic cervicitis patients, were collected from the First Hospital of Soochow University between March 2014 and April 2019. The expression of VISTA in each group was detected by immunohistochemistry (IHC). Survival data of CSCC patients were obtained by follow-up. The survival analysis was performed by Kaplan-Meier method, and survival differences between groups were compared by Log rank test. Prognostic impact factors were analyzed using a multifactorial Cox proportional hazards model. Results: The positive rate of VISTA expression in CSCC group was 32.8% (38/116), and which of grade Ⅱ-Ⅲ was 17.4% (4/23). VISTA expression results showed no positive expression patients in the cervical intraepithelial neoplasia grade I and chronic cervicitis groups. The differences between the CSCC group and other groups were statistically significant (P<0.01). In 116 CSCC patients, VISTA expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis (P<0.01). The mean survival time of patients in the VISTA positive expression group was 30.7 months, and the 3-year survival rate was 44.7% (17/38). However, the mean survival time of the patients in the VISTA negative expression group was 49.1 months, and the 3-year survival rate was 87.2% (68/78). The Cox regression model found that VISTA expression positivity (P=0.001) and FIGO stage (P=0.047) were prognostic factors for CSCC, and patients with VISTA-positive CSCC had a 4.130-fold risk of death higher than those with VISTA-negative expression. Conclusions: The VISTA protein is highly expressed in CSCC tissues, and its expression level is closely related to the occurrence and development of CSCC. The expression of VISTA can be used as an independent predictor of CSCC prognosis and can provide a strong basis for the treatment of CSCC with immune checkpoint inhibitors.

LncRNA HOXD-AS1 affects proliferation and apoptosis of cervical cancer cells by promoting FRRS1 expression via transcription factor ELF1

ABSTRACT To investigate the function of lncRNA HOXD-AS1 in cervical squamous cell carcinoma (CESC) and the underlying mechanism. The expressions of HOXD-AS1 and FRRS1 were analyzed on the online software GEPIA based on CESC-related information in The Cancer Genome Atlas (TCGA). Cervical cancer cells (SiHa and Hela) were accordingly transfected with pCDNA3.1-HOXD-AS1, sh-HOXD-AS1, sh-FRRS1 or pCDNA3.1-ELF1. After cell transfection, CCK-8, EDU and flow cytometry were applied for measurement of cell vitality, quantity and apoptosis, respectively. The relationship between HOXD-AS1 and FRRS1 was predicted on the online software LncMap and further verified by RNA binding protein immunoprecipitation. Nude mice were injected with stabilized SiHa cells transfected with sh-HOXD-AS1 to assess the tumorigenic ability of HOXD-AS1 in vivo. Immunohistochemistry detected the expression of the proliferation marker Ki-67. The levels of HOXD-AS1, ELF1 and FRRS1 were measured in vivo and in vitro. HOXD-AS1 and FRRS1 were overexpressed in CESC. After transfection of sh-HOXD-AS1, sh-ELF1 or sh-FRRS1, the proliferation of SiHa and Hela cells was inhibited and their apoptosis was promoted; while HOXD-AS1 overexpression had opposite effects on CESC development. Co-transfection of sh-FRRS1 and pCDNA3.1-HOXD-AS1 could abolish the tumor suppressive effect of FRRS1 knockdown. HOXD-AS1 elevated the level of FRRS1 by binding ELF1. Furthermore, HOXD-AS1 contributed to the CESC growth in mouse models. HOXD-AS1 promotes CESC by up-regulating FRRS1 via ELF1.

Detection of DKK-1 gene methylation in exfoliated cells of cervical squamous cell carcinoma and its relationship with high risk HPV infection.

To detect the methylation of Dickkopf-associated protein 1 (DKK-1) gene promoter in cervical exfoliated cells and to study its clinical significance in cervical squamous cell carcinoma (CSCC) and its relationship with high-risk HPV infection. Methylation-specific PCR (MSP) was utilized to detect the methylation of DKK-1 gene promoter in cervical exfoliated cells from 40 patients with CSCC and 40 patients with chronic cervicitis in the Affiliated Hospital of Inner Mongolia Medical University. The methylation rate of DKK-1 gene promoter in different clinicopathological factors and its relationship with high-risk HPV infection was compared, and different detection methods were compared. The degree of methylation of DKK-1 gene promoter in CSCC group was significantly higher than that in cervicitis group (P < 0.05). In CSCC group, the degree of methylation was significantly different in high-risk HPV infection, histological differentiation, tumor size, lymph node metastasis and the International Federation of Gynecology and Obstetrics (FIGO) staging (all P < 0.05). The degree of methylation is not related to the type of high-risk HPV infection (P > 0.05). The one-year survival rate of CSCC patients with high-risk HPV positive and DKK-1 gene promoter methylation is relatively low, only 74.1%. The sensitivity, specificity and accuracy of DKK-1 gene methylation combined with high-risk HPV detection were 96.7%, 78.0% and 85.0%, respectively. Methylation of DKK-1 gene promoter in cervical exfoliated cells of patients with CSCC is related to high-risk HPV infection and different clinicopathological factors, but the degree of methylation of DKK-1 gene is not related to the type of high-risk HPV infection. It may become an indicator different from HPV typing detection, which may play a shunt role in suggesting whether further invasive cervical examination is needed and reduce cervical invasive examination and overtreatment. It may be related to the survival rate of patients, which can be used to estimate the development and prognosis of CSCC and may play a good role in early warning in follow-up monitoring of CSCC after treatment. DKK1 gene methylation combined with HPV detection can improve the sensitivity, specificity and accuracy of diagnosis, which may improve the detection rate of early CSCC and make up for the deficiency of HPV and TCT detection. That may become a non-invasive screening method for CSCC.

Building a circular RNA centered gene regulation network associated with cervical squamous cell carcinoma.

Aim: The roles of circular RNA (circRNA) in tumor development are not fully understood. This study aims to explore the possible role of circRNAs as drug targets for the treatment of cervical squamous cell carcinoma (CSCC). Materials & methods: Through the bioinformatics approach to integrating multiple types of gene expression profiles, a circRNA-centered gene regulatory network was generated to explore the role of circRNAs in CSCC. Results: The expression levels of hsa_circ_0043280 and hsa_circ_0027821 were significantly different in tissues and might function as competing endogenous RNAs and play vital roles in CSCC development. Conclusion: We constructed a regulatory network consisting of two circRNAs, two miRNAs, 22 mRNAs and 19 drugs/chemicals and provided new insight into the prognosis and therapy of CSCC.

Polymorphisms and haplotypes of TLR4, TLR9 and CYP1A1 genes possibly interfere with high-risk human papillomavirus infection and cervical cancer susceptibility in Jharkhand, India

BackgroundExpression and single nucleotide polymorphisms (SNPs) of TLR4/9 and CYP1A1 genes are vital for cervical squamous cell carcinoma (CSCC) but considerably vary in different populations. MethodsA total of 255-subjects from Jharkhand (130-cases, 125-controls) were utilized to obtain the expression/SNP status of TLR4/9, CYP1A1, and E6 (HPV16/18) by RT-PCR, WB, and allele-specific-PCR followed by sequencing. ResultsOver-expression of TLR4/9 and high infection of HPV16/18(78.5%) were found to be associated with CSCC. Among the seven SNPs(p1-p7) tested, the CT-genotype (p3:rs1927911; OR = 2.142; p = 0.004) and ‘T’-allele (p3; OR = 1.694; p = 0.0061) of TLR4; CC-genotype (p4:rs5743836; OR = 3.307; p = 0.0018), ‘C’-allele (p4; OR = 1.895; p = 0.0009), GA-genotype (p5:rs352140; OR = 2.064; p = 0.0172), AA-genotype (p5; OR = 2.602; p = 0.0021) and ‘A’-allele (p5; OR = 1.939; p = 0.0002) of TLR9; and the TC-genotype (p6:rs4646903; OR = 1.967; p = 0.0452) and GG-genotype (p7:rs1048943; OR = 2.336; p = 0.0287) and ‘G’-allele (p7; OR = 1.685; p = 0.0082) of CYP1A1 were associated with an increased-risk of CSCC. Similarly, the p3:CT-genotype (OR = 1.993; p = 0.0134); p4:CC-genotype (OR = 3.071; p = 0.0057) and ‘C’-allele (OR = 1.838; p = 0.0029); p5:AA-genotype (OR = 2.231; p = 0.0147) and ‘A’-allele (OR = 1.756; p = 0.0032); p6:TC-genotype (OR = 2.370; p = 0.02); and the p7:GG-genotype (OR = 2.255; p = 0.0488) and ‘G’-allele (OR = 1.691; p = 0.0118) showed an association with HPV16/18 infection. Conversely, TLR4 (p1-p2-p3:A-G-T; OR = 3.361; p = 0.029), TLR9 (p4-p5:C-A; OR = 1.786; p = 0.032) and CYP1A1 (p6-p7:C-G; OR = 1.783; p = 0.033) haplotypes with CSCC susceptibility was observed, whereas the TLR4 (p1-p2-p3:A-C-C; OR = 0.4675; p = 8.E-3) and TLR9 (p4-p5:T-G; OR = 0.3937; p = 0.00) haplotypes showed protection against the development of CSCC. Further, though p1:rs10759931 and p2:rs11536889 were found to be insignificant, the p3:CT-genotype, p5:GA/AA-genotype, and p7:GG-genotype were associated with elevated protein; the p4:CC-genotype and p6:TC-genotype were associated with increased mRNA compared to their respective-wild-type-groups. ConclusionThe present study revealed an association between TLR4/9 and CYP1A1 polymorphisms with increased HPV16/18 infection susceptibility and CSCC risk among the women of Jharkhand state.

CircRNA circ_POLA2 Promotes Cervical Squamous Cell Carcinoma Progression via Regulating miR-326/GNB1.

Circular RNAs (circRNAs) are a group of non-coding RNAs that have an essential function in the development and progression of various cancers. The expression pattern and function of circRNA in cervical squamous cell carcinoma (CESC) are not fully understood. In the present study, we aimed to investigate the expression profiles and regulation mechanism of circRNA circ_POLA2 in CESC. Circ_POLA2 was highly expressed in CESC tissues and positively correlated with poor prognosis in CESC patients. Knockdown of circ_POLA2 using shRNA inhibited cervical cancer cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, circ_POLA2 could sponge endogenous microRNA-326 (miR-326) and inhibit its expression. Furthermore, miR-326 negatively regulated G protein subunit beta 1 (GNB1) by targeting its 3′-UTR. Intriguingly, we found that GNB1 was overexpressed and associated with poor prognosis in CESC patients. Overexpression of GNB1 could antagonize the inhibitory effect of miR-326 on cervical cancer cell proliferation, migration, and invasion. In addition, we demonstrated that circ_POLA2/miR-326/GNB1 axis regulated ERK signaling. In conclusion, circ_POLA2 promotes cervical squamous cell carcinoma development and progression via regulating the miR-326/GNB1 axis, which might serve as a novel therapeutic target for CESC patients.

Methylation-driven genes and their prognostic value in cervical squamous cell carcinoma.

Abnormal gene methylation is crucial for tumor progression. This study explored a cluster of methylation-driven genes involved in cervical squamous cell carcinoma (CESC). The data on RNA expression, methylation and clinical outcomes of CESC patients were downloaded from The Cancer Genome Atlas (TCGA) database. Protein-protein interaction (PPI) network was constructed. Gene Ontology (GO) and KEGG analyses were performed to identify the biological functions of methylation-driven genes, and univariable and multivariate Cox analyses to screen out the key prognostic genes. A risk signature was established and its predictive value was evaluated with Kaplan-Meier and ROC curves. The key genes were further investigated by Cox regression analyses, gene set enrichment analysis (GSEA), and methylation site analysis. Additionally, "rms" package was used for establishing nomogram and calibrate curve. We found 144 differentially expressed methylation-driven genes. A risk model was constructed with three key prognostic genes (ITGA5, HHEX and S1PR4). The risk score was an independent risk factor for CESC prognosis. Lowly-expressed and hypermethylated ITGA5, highly-expressed and hypomethylated HHEX and S1PR4 were associated with better CESC prognosis. The methylation sites and biological functions enriched in ITGA5, HHEX and S1PR4 were uncovered. Additionally, the nomogram also validated the performance of risk model. Methylation-driven ITGA5, HHEX and S1PR4 are associated with CESC development. The three genes might serve as potential targets in the treatment of CESC.

Abstract B56: YAP1 inhibits host cell immunity and increases human papillomavirus (HPV) infection to drive cervical tumorigenesis

Abstract Although human papillomavirus (HPV) is believed to be the major causative agent for cervical cancer, accumulating evidence indicates that HPV alone is insufficient to induce cervical cancer. The molecular mechanisms underlying cervical cancer development remain unclear. In the present study, we provide evidence showing that the Hippo/YAP pathway plays a critical role in cervical carcinogenesis. We found that genetic alteration of the Hippo/YAP pathway frequently occurred in human cervical cancer patients and was associated with poor prognosis of these patients. Keratin 14 promoter-driven expression of constitutively active YAP1 (YAP S127A) in mouse cervical epithelial cells induced invasive cervical squamous cell carcinoma (CVSCC), suggesting that hyperactivation of YAP1 is sufficient to induce cervical cancer. Importantly, hyperactivation of YAP1 in cervical epithelial cells not only increased the putative HPV receptor molecules and cellular susceptibility to HPV infection, but also disrupted the host cell innate immune system, resulting in failure of HPV viral recognition, suppression of type I IFN production, inhibition of the IFNRs/JAKs/STATs pathway, and blockage of production of antiviral interferon-stimulated genes. Cervical epithelial-specific YAP1 and HPV double knockin mouse models showed that YAP1 synergizes with HPV to promote the initiation and progression of CVSCC. The present study demonstrates that the Hippo/YAP pathway plays a central role in the development of CVSCC and the synergism between hyperactivated YAP1 and high-risk HPV may be the key driver of cervical cancer development. The Hippo/YAP signaling pathway may represent a promising target for developing novel strategies to prevent and treat cervical cancer. Citation Format: Xiangmin Lv, Chunbo He, Cong Huang, Hongbo Wang, Li Chen, Peichao Chen, John S. DavisS, Bo R. Rueda, Cheng Wang. YAP1 inhibits host cell immunity and increases human papillomavirus (HPV) infection to drive cervical tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B56.

LncRNA MAGI2-AS3 is involved in cervical squamous cell carcinoma development through CDK6 up-regulation

BackgroundIt has been reported that lncRNA MAGI2-AS3 can promote many types of cancer, such as breast cancer and bladder cancer, by regulating cell behaviors, such a proliferation, invasion, and migration. However, its role in cervical squamous cell carcinoma (CSCC) is unclear. This study aims to investigate the role of MAGI2-AS3 in CSCC.MethodsSixty-four CSCC patients (36 to 68 years, 46.3 ± 5.1 years) out of 136 CSCC patients admitted by Shengjing hospital affiliated to China Medical University from June 2010 to October 2013 were included in the present study. Among the 64 enrolled patients, 20 were HPV-16 positive cases, 24 were HPV-18 positive cases and 20 were HPV negative. CSCC and non-tumor biopsies from CSCC patients as well as C-33A cell lines were used. Cells were transfected with MAGI2-AS3 and CDK6 expression vectors as well as with MAGI2-AS3 siRNA to analyze gene interactions. Cell cycle analysis was performed to analyze the effects of transfections on cell cycle progression. qPCR and western blot were applied to analyze gene expression. Paired t-test and ANOVA (one-way) combined with Tukey test were used for data comparisons. Survival analysis was performed by plotting and comparing survival curves.ResultsqRT-PCR results showed that CDK6 and MAGI2-AS3 were both up-regulated in CSCC and positively correlated with each other. MAGI2-AS3 and CDK6 expression was not significantly affected by HPV infections. High levels of MAGI2-AS3 were associated with the poor survival of CSCC patients. In CSCC cells, MAGI2-AS3 over-expression up-regulated CDK6, while MAGI2-AS3 siRNA down-regulated CDK6. In CCK-8 assay, MAGI2-AS3 and CDK6 over-expression led to increased proliferation rate of CSCC cells by reducing CDK6 levels, while MAGI2-AS3 siRNA didn’t. In addition, CDK6 over-expression attenuated the effect of MAGI2-AS3 siRNA silencing.ConclusionsIn conclusion, MAGI2-AS3 promoted CSCC cell proliferation by up-regulating CDK6.

Human papillomavirus 16 infection alters the Toll-like receptors and downstream signaling cascade: A plausible early event in cervical squamous cell carcinoma development

ObjectiveToll-like receptors constitute an important component of innate immune mechanism. HPV is a known etiological factor of cervical cancer and is known to interfere with the expression of TLRs and downstream signaling pathway. It remains poorly understood whether HPV modulates the expression of TLRs. Hence, understanding HPV mediated immune alterations might aid in identifying novel therapeutic targets. The aim was to study the relative gene expression of TLRs & downstream signaling pathway in cervical carcinoma. MethodsCervical squamous cell carcinoma (CSCC) and normal cervical tissues were obtained. Subsequent to HPV genotyping, mRNA expression profiling using PCR Array was performed. Protein expression of relevant genes with western blot was studied. Levels of cytokines in cervicovaginal washes were estimated using a Luminex multiplex platform. ResultsAll cases of cervical cancer were HR-HPV positive and predominant subtype was HPV16 (71.1%). Significant TLR4 upregulation and TLR2,7 downregulation were observed in HR-HPV infected cervix. TLR4,7 demonstrated low expression in CSCC. Molecules from cancer allied pathways; RELA, AKT, CDKN2A, and MDM2 demonstrated upregulation in CSCC. Protein expression data corroborated with gene expression profile. A diminished level of Th1 cytokines TNF-α, IFN-ɣ, IL-17, and IL-12 was observed in CSCC. Significantly increased levels of IL-1β, IL-6 and IL-2 were detected in HR-HPV infected cervix. Kaplan Meier curve demonstrated high TLR4 and low TLR7 expression was associated with poor prognosis. ConclusionThe study demonstrates the HPV mediated dampening of the innate immune response in CSCC and provides support for exploring potential TLR2, 7 agonists as an adjunct therapy in CSCC patients.

Proteomics analysis of proteins interacting with heat shock factor 1 in squamous cell carcinoma of the cervix.

Protein interactions are crucial for maintaining homeostasis. Heat shock factor 1 (HSF1), a transcription factor that interacts with various proteins, is highly expressed in squamous cell carcinoma (SCC) of the cervix. The aim of the present study was to investigate the protein interaction profile of HSF1 in cervical SCC. Proteins interacting with HSF1 in SCC tissue and non-cancerous control (Ctrl) tissue were obtained by immunoprecipitation, separated by SDS-PAGE, identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and analyzed using bioinformatics methods. A total of 220 and 241 proteins were identified by mass spectrometry in the tissues of Ctrl and SCC samples, respectively, among which 172 were detected exclusively in SCC (Pro-S), 151 exclusively in Ctrl (Pro-C) and 69 in both groups (Pro-B). The protein interaction profiles were different in each group; the STRING database identified three proteins that interacted with HSF1 directly, including insulin-like growth factor 1 receptor and small nuclear RNA-activating protein complex subunit 4 in Pro-C and small ubiquitin-related modifier 1 in Pro-S. Functional enrichment analysis of Gene Ontology revealed that the top terms were alternative splicing in Pro-S and polymorphism in Pro-C. In Pro-S, more categories were related to protein modification, such as phosphorylation, ubiquitination and acetylation. Therefore, HSF1 may influence the occurrence and development of cervical SCC by interacting with specific proteins.

Mutational signatures efficiently identify different mutational processes underlying cancers with similar somatic mutation spectra

Compared to analyzing mutations with conventional spectra, deciphering mutational signatures provides much greater resolution of biological processes that generate somatic mutations during cancer development. Previous studies of bladder urothelial cancer (BLCA) and cervical squamous cell carcinoma (CESC) mutational signatures failed to uncover different mutational processes underlying the two cancers, which diminishes the capability of mutational signature to differentiate between the two cancers. In this study, we deciphered and compared the mutational signatures of BLCA and CESC. Four BLCA mutational signatures were deciphered from 37,098 somatic mutations of 130 exomes. Five CESC mutational signatures were deciphered from 44,206 somatic mutations of 194 exomes. Three BLCA mutational signatures were very similar to the three CESC signatures. These mutational signatures exhibited common endogenous mutational processes during BLCA and CESC development. The respective BLCA and CESC mutational signature 4 revealed the role of viral infection in both cancers. Noticeably, CESC mutational signature 4 is a novel one that has not been described in other studies. In summary, we have demonstrated the similarities and differences between BLCA and CESC by deciphering mutational signatures. This study will shed light on the use of mutational signatures to clarify the mechanisms of endogenous and exogenous carcinogens that cause somatic mutations in human cancers.

Characterization of the microRNA profile in early-stage cervical squamous cell carcinoma by next-generation sequencing.

Squamous cell carcinoma (SCC) is histologically the most prominent type of cervical cancer. There is accumulating evidence suggesting that microRNAs (miRNAs) play important regulatory roles in the biological processes of cervical squamous cell carcinoma (CSCC). Deciphering the miRNA regulatory network in CSCC could deepen our understanding at the molecular level of CSCC initiation and progression. In the present study, we performed next‑generation sequencing (NGS) to profile miRNA expression in 3pairs of early-stage CSCC samples. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify primary findings in another 20pairs of CSCC samples. We identified 37known miRNAs that exhibited significant alterations in expression (2-fold change or greater), among which 8miRNAs were upregulated and 29miRNAs were downregulated. Nine of these miRNAs were selected for further qRT-PCR validation. A novel miRNA candidate was also reported for the first time in the present study to be upregulated. The Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that its target genes were involved in MAPK, calcium and adherent junction signaling pathways. The present study systematically characterized the miRNA expression variation in early-stage CSCC and provides novel biomarkers for diagnosis and treatment as well as an opportunity for further investigation of the molecular mechanisms underlying the pathogenesis and development of CSCC.

Relationship between excision repair cross-complementation group 1 methylation and sensitivity to cisplatin-based concurrent chemoradiotherapy in cervical squamous cell carcinoma

Objective To investigate the correlation of the excision repair cross-complementation group 1(ERCC1) gene methylation status with sensitivity to cisplatin-based concurrent chemoradiotherapy and clinical pathological characteristics in patients with cervical squamous cell carcinoma (CSCC). Methods Methylation specific polymerase chain reaction was used to determine the ERCC1 gene methylation status in cervical tissue from 20 healthy people and 60 patients with CSCC. All patients with CSCC were treated with cisplatin-based concurrent chemoradiotherapy. The treatment outcomes were evaluated using the Response Evaluation Criteria in Solid Tumors. Based on the treatment outcomes, patients with a complete response were assigned to chemoradiotherapy-sensitive group, and patients with a partial response, stable disease, or progressive disease were assigned to chemoradiotherapy-resistant group. Comparison of the ERCC1 gene methylation status between the two groups was made by χ2 test, and multivariate logistic regression analysis was used to analyze the relationship of the ERCC1 gene methylation status with various clinical pathological characteristics and sensitivity to chemoradiotherapy. Results The ERCC1 gene methylation rate was significantly higher in cervical tissue with CSCC than in normal cervical tissue (60% vs. 0%, P=0.000). The multivariate analysis showed that ERCC1 gene methylation was an independent influencing factor for sensitivity to cisplatin-based concurrent chemoradiotherapy in CSCC (P=0.022); ERCC1 gene methylation was significantly correlated with histological grade of CSCC (P=0.030); there was no significant relationship of ERCC1 gene methylation with clinical pathological characteristics including age, tumor size, lymph node metastasis, FIGO stage, and pretreatment hemoglobin and platelet count (P=0.729, 0.707, 0.340, 0.747, 0.073, 1.000). Conclusions The ERCC1 gene promoter methylation status may be involved in the development and progression of CSCC. It may also influence the sensitivity of patients with CSCC to cisplatin-based concurrent chemoradiotherapy by playing a role in cell differentiation. Key words: Excision repair cross-complementation group 1; Radiosensitivity; Cervical neoplasms/radiotherapy; Cervical neoplasms/chemotherapy

Prognostic significance of histone methyltransferase enhancer of zeste homolog 2 in patients with cervical squamous cell carcinoma.

Histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been reported to be associated with certain malignant phenotypes in cervical cancer. However, clinicopathological parameters and clinical outcomes of EZH2 in cervical cancer, particularly in cervical squamous cell carcinoma (CSCC) remain largely unknown. The retrospective cohort comprising of 117 consecutive patients with CSCC was incorporated into a tissue microarray which also included 23 paired normal tissues. Immunohistochemical analysis was performed to evaluate the correlation between EZH2 expression and clinicopathological implications. Aberrant overexpression of EZH2 was frequently observed in CSCCs as compared with adjacent normal tissues (P=0.0005). Expression of EZH2 is associated with poor tumor differentiation grade (P=0.020) and lymphovascular invasion (P=0.012). Univariate analysis revealed that the patients with CSCC whose tumors exhibited higher EZH2 levels had inferior overall survival (OS) compared to those whose tumors expressed lower EZH2 (log rank P=0.004). In the multivariate analysis, EZH2 expression was an independent predictor of OS (hazard ratio = 1.836, 95% confidence interval: 1.090-2.993, P=0.022). EZH2 overexpression is common in the development of CSCC and is a promising prognostic predictor for patients with CSCC.

Genetic susceptibility to cervical squamous cell carcinoma is associated with HLA-DPB1 polymorphisms in Taiwanese women.

Cervical cancer is a multifactorial disease, and increasing evidence suggests that host immunogenetic background may contribute to its pathogenesis. Genetic variations in human leukocyte antigen (HLA) genes may alter the efficiency of immune response to human papillomavirus (HPV) antigens and have been implicated in the risk of cervical cancer. We investigated whether polymorphisms in the HLA-DPB1 gene were associated with cervical cancer risk in a Taiwanese population. HLA-DPB1 alleles and +550 G/A polymorphism were genotyped in a case-control study of 473 women with cervical squamous cell carcinoma (CSCC) and 676 healthy controls. The presence and genotypes of HPV in CSCC were determined. We found that the DPB1*05:01 and +550 A alleles were associated with decreased and increased risk of CSCC, respectively [odds ratio (OR) = 0.72, Pc = 0.001; OR = 1.25, Pc = 0.03]. In subgroup analysis based on HPV type 16 positivity, significant associations were shown in the DPB1*05:01 and *13:01 alleles (OR = 0.65, Pc = 0.0007; OR = 1.83, Pc = 0.004). Furthermore, the DPB1*05:01-G and *13:01-G haplotypes conferred decreased and increased risk of both CSCC and HPV-16 positive CSCC women, respectively (OR = 0.72, Pc = 0.0009; OR = 0.63, Pc = 0.0004 for DPB1*05:01-G; OR = 1.55, Pc = 0.03; OR = 1.84, Pc = 0.004 for DPB1*13:01-G). A risk haplotype DPB1*02:01-A was also observed in the HPV-16 positive CSCC women (OR = 1.51, Pc = 0.05). These findings suggest that HLA-DPB1 gene is involved in the CSCC development.

The landscape of alternative splicing in cervical squamous cell carcinoma.

BackgroundAlternative splicing (AS) is a key regulatory mechanism in protein synthesis and proteome diversity. In this study, we identified alternative splicing events in four pairs of cervical squamous cell carcinoma (CSCC) and adjacent nontumor tissues using RNA sequencing.MethodsThe transcripts of the four paired samples were thoroughly analyzed by RNA sequencing. SpliceMap software was used to detect the splicing junctions. Kyoto Encyclopedia of Genes and Genomes pathway analysis was conducted to detect the alternative spliced genes-related signal pathways. The alternative spliced genes were validated by reverse transcription-polymerase chain reaction (RT-PCR).ResultsThere were 35 common alternative spliced genes in the four CSCC samples; they were novel and CSCC specific. Sixteen pathways were significantly enriched (P<0.05). One novel 5′AS site in the KLHDC7B gene, encoding kelch domain-containing 7B, and an exon-skipping site in the SYCP2 gene, encoding synaptonemal complex 2, were validated by RT-PCR. The KLHDC7B gene with 5′AS was found in 67.5% (27/40) of CSCC samples and was significantly related with cellular differentiation and tumor size. The exon-skipping site of the SYCP2 gene was found in 35.0% (14/40) of CSCC samples and was significantly related with depth of cervical invasion.ConclusionThe KLHDC7B and the SYCP2 genes with alternative spliced events might be involved in the development and progression of CSCC and could be used as biomarkers in the diagnosis and prognosis of CSCC.

Expressions of Toll-like receptors 3, 4, 7, and 9 in cervical lesions and their correlation with HPV16 infection in Uighur women

Recent findings show that Toll-like receptors (TLRs) expressed in immune cells play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection on tissue injury. Furthermore, expression of TLRs in cancer cells is associated with tumor proliferation and invasion. To explore the role of TLRs expression in cervical Carcinogenesis in Uighur women, we detected the expressions of TLR3, TLR4, TLR7, and TLR9 in 25 normal cervical tissues, 64 cervical intraepithelial neoplasia (CIN) tissues, and 63 cervical squamous cell carcinoma (CSCC) tissues using immunohistochemical staining, as well as human papillomavirus type 16 (HPV16) infection using PCR. All samples used in this study were from Xinjiang Uighur women. We found the expression levels of TLR4, TLR7, and TLR9 were significantly higher in CIN and CSCC than in normal controls (P < 0.05). Up-regulation of TLR4 and TLR7 were correlated with tumor differentiation but not FIGO stage or lymph node metastasis (P > 0.05). Up-regulation of TLR9 was correlated with lymph node metastasis (P < 0.05) but not tumor differentiation or FIGO stage (P > 0.05). We also analyzed the correlation between the expressions of TLRs and HPV16 infection and found that the expressions of TLR4 and TLR9 significantly correlated with HPV16 infection in CIN (r = 7.434, P = 0.006; r = 7.123, P = 0.008) and CSCC (r = 6.423, P = 0.001; r = 8.478, P = 0.004), whereas the expression of TLR3 was not significantly different in any of the three groups and had no significant correlation with HPV16 infection. Our results suggest that high expression of TLR4, TLR7, and TLR9 may play important roles in the development and progression of CIN and CSCC in Uighur women, and the expressions of TLR4 and TLR9 can be up-regulated by HPV16 infection.