Use of Nomogram to Predict the Risk of Lymph Node Metastasis among Patients with Cervical Adenocarcinoma.

Background: The objective of this study was to develop a nomogram that can predict lymph node metastasis (LNM) in patients with cervical adenocarcinoma (cervical AC). Methods: A total of 219 patients with cervical AC who had undergone radical hysterectomy and lymphadenopathy between 2005 and 2021 were selected for this study. Both univariate and multivariate logistic regression analyses were performed to analyze the selected key clinicopathologic features and develop a nomogram and underwent internal validation to predict the probability of LNM. Results: Lymphovascular invasion (LVI), tumor size ≥ 4 cm, and depth of cervical stromal infiltration were independent predictors of LNM in cervical AC. However, the Silva pattern was not found to be a significant predictor in the multivariate model. The Silva pattern was still included in the model based on the improved predictive performance of the model observed in the previous studies. The concordance index (C-index) of the model increased from 0.786 to 0.794 after the inclusion of the Silva pattern. The Silva pattern was found to be the strongest predictor of LNM among all the pathological factors investigated, with an OR of 4.37 in the nomogram model. The nomogram developed by incorporation of these four predictors performed well in terms of discrimination and calibration capabilities (C - index = 0.794; 95% confidence interval (CI), 0.727-0.862; Brier score = 0.127). Decision curve analysis demonstrated that the nomogram was clinically effective in the prediction of LNM. Conclusion: In this study, a nomogram was developed based on the pathologic features, which helped to screen individuals with a higher risk of occult LNM. As a result, this tool may be specifically useful in the management of individuals with cervical AC and help gynecologists to guide clinical individualized treatment plan.

Down-regulation of ZNF252P-AS1 alleviates ovarian cancer progression by binding miR-324-3p to downregulate LY6K.

BACKGROUND: Ovarian cancer is a common gynecological malignant disease in women. Our work aimed to study the specific functions of ZNF252P antisense RNA 1 (ZNF252P-AS1) in ovarian cancer. METHODS: ZNF252P-AS1, miR-324-3p, and lymphocyte antigen 6 family member K (LY6K) expression were analyzed by bioinformatics tools in ovarian cancer tissues and was quantified by qRT-PCR in ovarian cancer cells. The effect of ZNF252P-AS1 knockdown, miR-324-3p suppression, and LY6K over-expression on apoptosis, cell viability, invasion, migration, and epithelial to mesenchymal transition (EMT) was determined in vitro by using colony formation and EdU assays, flow cytometry, transwell assay, and Western blot. The interactions between ZNF252P-AS1 and miR-324-3p and between miR-324-3p and LY6K were validated by luciferase assays. The effects of restraining ZNF252P-AS1 in vivo were studied using BALB/c male nude mice. RESULTS: ZNF252P-AS1 and LY6K levels were up-regulated, while miR-324-3p was declined in ovarian cancer tissues and cells. ZNF252P-AS1 knockdown reduced ovarian cancer cell proliferation, invasion, migration, and EMT, whereas promoted its apoptosis. Besides, ZNF252P-AS1 interacted with miR-324-3p and reversely regulated its level, and miR-324-3p was directly bound to LY6K and negatively regulated its expression. Moreover, ZNF252P-AS1 knockdown reversed the effect of miR-324-3p on cancer cell apoptosis, growth, migration, invasion, and EMT. Similar results were discovered in the rescue experiments between miR-324-3p and LY6K. Additionally, mouse models in vivo experiments further validated that ZNF252P-AS1 knockdown distinctly inhibited tumor growth. CONCLUSION: ZNF252P-AS1 mediated miR-324-3p/LY6K signaling to facilitate progression of ovarian cancer.

Long non-coding RNA TDRG1 promotes hypoxia-induced glycolysis by targeting the miR-214-5p/SEMA4C axis in cervical cancer cells.

Long non-coding RNA (lncRNA) has been demonstrated as vital regulator in human cancer. However, the precise role of lnc-TDRG1 in cervical cancer (CC) remains unclear, so this study was aimed to clarify the role and underlying molecular mechanism of lnc-TDRG1 in CC. The real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to assess the expression levels of lnc-TDRG1, miR-214-5p and Semaphorin 4C (SEMA4C). Under hypoxia condition, the biological behaviors of CC cell, including invasion and glycolysis were determined by transwell assay and Glucose Assay Kit and Lactate Assay Kit, respectively. The Western blot assay was employed to test the expression level of SEMA4C and hexokinase 2 (HK2) expression. The interaction relationship between miR-214-5p and lnc-TDRG1 or SEMA4C was analyzed bioinformatics database and confirmed by dual-luciferase reporter assay, respectively. A xenograft experiment in nude mice was established to clarify the functional role of lnc-TDRG1 in vivo. We found Lnc-TDRG1 was highly expressed in CC tissues and cells and it was upregulated in response to hypoxia. Loss-of-functional experiment suggested that knockdown of lnc-TDRG1 impede invasion, hypoxia-induced glycolysis in vitro and tumor growth in vivo, which was abolished by knockdown of miR-214-5p or overexpression of SEMA4C. Moreover, we confirmed that miR-214-5p specifically bound to SEMA4C and negatively correlated with SEMA4C expression. Collectively, lnc-TDRG1 regulated SEMA4C expression by sponging miR-214-5p in CC. Collectively, mechanistically, lnc-TDRG1 could act as a sponge of miR-214-5p to regulate the expression of SEMA4C, and further regulate invasion and hypoxia-glycolysis in CC cells.

Characterization of DNA hydroxymethylation profile in cervical cancer.

Cervical cancer is one of the most common gynecological tumors in females. DNA methylation alteration is a type of epigenetic decoration that controls the gene transcriptional regulation and is essential for the pathological progression of cervical cancer to reflect the prognosis and therapeutic sensitivity in clinical practice. Beyond DNA methylation, DNA hydroxymethylation considered as a more stable biomarker draws the outline of the reversible cycle from DNA methylation and demethylation. However, the landscape of 5-hydroxymethylcytosine (5hmC) distributed in the genome is never characterized in cervical cancer. In this study, we presented the whole 5-methylcytosine (5mC) and 5hmC profile in cervical cancer of I-IIa and IIb to IV stages and cervicitis tissues as control by dot plot assay and immunohistochemistry. We observed that the total 5mC was up-regulated while 5hmC was down-regulated in cervical cancer group compared to the control group. Furthermore, we investigated the distribution of 5mC and 5hmC on genomic DNA by MeDIP- and hMeDIP-Seq. 53 differential methylation/hydromethylation regions (DMRs/DHMRs) displayed a continuously increasing or decreasing trend of 5mC or 5hmC from cervicitis to I-IIa and from I-IIa to IIb-IV stages of cervical cancer. Thirty-seven DMRs and DHMRs have a similar variation trend while the other 8 have the opposite trend compared between CSCC and cervicitis. Moreover, the DMR/DHMR associated genes were closely related to Wnt, MAPK, Rap1 and other important signaling pathways. Finally, 5hmC beyond 5mC at the genes such as ACTG1, SALL3, DNAJA3, SERPINB6, CDC14B and CALN1 were considered as the putative novel hallmarks for cervical cancer diagnosis and prognosis. Altogether, this study first describes the DNA hydroxymethylation atlas of cervical cancer and shows a list of novel genes transcriptionally regulated by DNA methylation and hydroxymethylation.

An integrated analysis reveals the oncogenic function of lncRNA LINC00511 in human ovarian cancer.

Ovarian cancer is one of the most common female reproductive system malignancies worldwide. Recently, the aberrant long noncoding RNAs (lncRNAs) expression has been identified in multiple cancers. Emerging evidence has highlighted the critical roles of lncRNAs in carcinogenesis and tumor progression, including ovarian cancer. The objective of this study is to comprehensively analyze lncRNAs expression pattern, and explore their clinical significance and underlying mechanism in human ovarian cancer. In this study, we found hundreds of dysregulated lncRNAs in ovarian cancer by performing genome-wide analysis using RNA sequencing data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) project, and three microarray datasets from Gene Expression Omnibus (GEO). Moreover, our results revealed that up- or down-regulation of some lncRNAs expression in ovarian cancer is accompanied by their genomic loci copy number amplification or deletion. Importantly, some lncRNAs expression levels are significantly associated with ovarian cancer patients' poor prognosis. Further experimental validation and mechanistic investigation indicate that LINC00511 exerts oncogenic function in ovarian cancer cells through interacting with EZH2 and repressing P21 expression. Taken together, the findings in the current study may provide a useful resource of novel ovarian cancer associated lncRNAs and potential diagnostic biomarker and therapeutic targets for ovarian cancer.

Changes of Th17/Tc17 and Th17/Treg cells in endometrial carcinoma.

OBJECTIVES: T helper 17 (Th17), T cytotoxic 17 (Tc17) and regulatory T (Treg) cells are important factors in the pathogenesis of inflammatory and autoimmune diseases. However, information concerning the roles of these cells in antitumor immunity or endometrial tumorigenesis remains limited. In this study, we aimed to describe the distribution of Th17, Tc17 and Treg cells in endometrial carcinoma patients, and elucidate the probable role of these effector T cells. METHODS: We assessed the expression of interleukin (IL)-17 and Foxp3 in the peripheral blood of endometrial carcinoma patients and healthy controls by flow cytometry to determine the relative numbers of Th17, Tc17 and Treg cells. Th17 cells and Tc17 cells were counted as percentages of the total number of CD3(+) T cells; Treg cells were counted as a percentage of the total number of CD4(+) T cells. We also evaluated Th17 and Tc17 cells in tumor tissue by immunohistochemical staining. IL-17 and IL-10, dominant products of these three cell types, were detected by using enzyme-linked immunosorbent assays. RESULTS: The frequencies of Th17, Tc17 and Treg cells, as well as the serum level of IL-10, were significantly elevated in endometrial carcinoma patients compared to healthy controls. The Th17/Tc17 and Th17/Treg ratios were also observed to change significantly. However, there was no significant difference on the IL-17 levels in the serum. Additionally, immunohistochemistry performed on tumor tissues indicated that the amounts of Th17 and Tc17 increased in the cancer patients. CONCLUSIONS: Our data suggests a probable involvement of Th17, Tc17 and Treg cells in the pathogenesis of endometrial carcinoma. Restoring the balance of these cells may help with the research and development of immunotherapies for endometrial carcinoma.

IL-21 and IL-12 inhibit differentiation of Treg and TH17 cells and enhance cytotoxicity of peripheral blood mononuclear cells in patients with cervical cancer.

OBJECTIVES: Interleukin 21 (IL-21) and IL-12 have been known to be effective antitumor agents. In this study, we evaluated whether IL-21 in combination with IL-12 could enhance the cytotoxicity of peripheral blood mononuclear cells (PBMCs) in patients with cervical intraepithelial neoplasia III and cervical cancer. METHODS: Peripheral blood mononuclear cells were isolated from peripheral blood of cervical intraepithelial neoplasia III patients (n = 17) and cervical cancer patients (n = 18). Peripheral blood mononuclear cells were cultured with IL-2 in low concentration as control group. Interleukin 2-stimulated PBMCs were cocultured with anti-human IL-21 neutralizing antibody, IL-21 alone, IL-12 alone, and IL-21 plus IL-12, respectively, as test groups. The cytotoxicity of PBMCs against SiHa tumor cells was examined by lactate dehydrogenase released assay. CD4CD25FOXP3 T regulatory (Treg) cells and CD4IL-17A T helper 17 (TH17) cells were analyzed by flow cytometry. Proliferation and apoptosis were detected by CCK-8 (cell counting kit 8) assay and flow cytometry, respectively. RESULTS: Compared with controls, IL-21 and IL-12 significantly elevated PBMC cytotoxicity against SiHa cells. Moreover, IL-21 plus IL-12 significantly elevated PBMC cytotoxicity in comparison to IL-21 alone and IL-12 alone. We also found that IL-21 plus IL-12 significantly decreased Treg and TH17 cell proportion in comparison to controls. Notably, IL-21 plus IL-12 significantly decreased TH17 cell proportion in comparison to IL-21 alone. Both IL-21 and IL-12 significantly decreased the apoptosis rate of PBMCs, whereas neither IL-21 nor IL-12 had significant effect on PBMC proliferation. CONCLUSIONS: The combination of IL-21 and IL-12 could efficiently stimulate PBMCs with cytotoxicity against SiHa cells, and the possible mechanisms may be due to down-regulated Treg and TH17 cell differentiation.

[Effect of interleukin 21 and/or interleukin 12 on the antitumor activity of peripheral blood mononuclear cells in patients with endometrial cancer].

OBJECTIVE: To observe the role of interleukin (IL) 21 alone, IL12 alone, and IL21 plus IL12 for inducing the antitumor activity of peripheral blood mononuclear cells (PBMCs) in patients with endometrial cancer. METHODS: PBMCs were isolated from peripheral blood in patients with endometrial cancer in vitro, and kept the culture with low-level IL2. IL2-stimulated PBMCs were cocultured under different conditions (with anti-IL21 antibody, IL21 alone, IL12 alone, or IL21 plus IL12) for 72 h. The cytotoxicity of PBMCs was then examined by lactate dehydrogenase(LDH) released assay. CD4(+) CD25(+) FOXP3(+) regulatory (Treg) cell and CD4(+) IL17A(+) T-helper (Th17) cell proportion were determined with flow cytometry. Cell proliferation and apoptosis were measured by cell counting kit-8(CCK-8)assay and flow cytometry, respectively. RESULTS: In comparison to control group, both IL21 and IL12 significantly enhanced the cytotoxicity of PBMCs. The IL21 plus IL12 group had superior effect to IL21 alone and IL12 alone. IL21 and IL12 significantly decreased the percentages of Treg cells and the rate of PBMCs apoptosis. IL21 or IL12 had no significant effect on the differentiation of Th17 cells and the proliferation of PBMCs. CONCLUSIONS: IL21 and IL12 can enhance the cytotoxicity of PBMCs in patients with endometrial cancer, which can be further strengthened with treatment of IL21 plus IL12. Such effects may be achieved by inhibiting the differentiation of Treg cells and the apoptosis of PBMCs, but not by the differentiation of Th17 cell.

Phosphoinositide-3-kinase inhibition enhances radiosensitization of cervical cancer in vivo.

BACKGROUND: Phosphoinositide-3-kinase (PI3K)/Akt pathway is downregulated in several human cancers, and PI3K inhibition can sensitize these cancer cells to radiation. However, no research on cervical cancer in vivo has been reported. The present study further investigated whether PI3K inhibition could sensitize cervical cancer to radiation in vivo. METHODS: HeLa cells with sustained PI3K activity and Akt phosphorylation were injected subcutaneously into BALB/C nude mice to establish tumor cell xenograft, which were randomly assigned to control, PI3K inhibitor LY294002 alone, radiation alone, or combined LY294002 and radiation group. Akt phosphorylation was detected by Western blotting to evaluate the blocking efficiency on PI3K activity. The radiosensitization of PI3K inhibition was measured by clonogenic assays, apoptosis analysis, and tumor regrowth assays. RESULTS: The combination of LY294002 and radiation resulted in significant and synergistic suppression of cervical cancer cells in a dose-dependent manner in clonogenic assays (P < 0.05), higher ratio of apoptosis cells, and more remarkable reduction of Akt phosphorylation. Tumor regrowth delay curve showed the lowest increase of tumor volume in the combined group (37 days in average) (P = 0.003). Besides, LY294002 (100 mg/kg) alone decreased cell survival and produced xenograft regrowth delay. CONCLUSIONS: Phosphoinositide-3-kinase inhibition by LY294002 can synergistically enhance radiation efficacy via dephosphorylation of Akt in cervical cancer, and PI3K inhibition alone can also suppress tumor regrowth. This may provide novel therapeutic opportunities to enhance the effect of radiotherapy against cervical cancer.

The imbalance of Th17/Treg in patients with uterine cervical cancer.

BACKGROUND: Th17/Treg was reported to play critical roles in immunoregulation, and its imbalance may lead to autoimmune diseases and allergic reactions. Information on Th17/Treg in cancer bearing hosts is still limited. METHODS: We examined the expression of IL-17, Foxp3 and IL-10 in uterine cervical cancer (UCC) patients, cervical intraepithelial neoplasia (CIN) patients and healthy controls by flow cytometry and enzyme-linked immunosorbent assay. Interleukin (IL)-17-producing CD4+ cells as Th17 and CD4+CD25+Foxp3+ cells as Treg were expressed as a percentage of the total CD4+ cells. RESULTS: Compared with controls, patients with UCC or CIN had a higher proportion of Th17 cells. UCC patients also revealed a significant increase in Treg number and IL-17 and IL-10 concentrations in plasma, while CIN patients did not. Notably, in UCC patients, the increased Th17 prevalence was associated with clinical stage, lymph node metastases and vasoinvasion, while the increased Treg frequency was associated with tumor differentiation. Remarkably, an attractive imbalance of Th17/Treg was observed in UUC and CIN patients. Furthermore, in UCC patients with lymph node metastases or vasoinvasion, the ratio of Th17/Treg was significantly higher than that in negative patients respectively. CONCLUSIONS: Our results indicated a possible role of Th17 in UCC patients correlated to Treg cells, and the imbalance of Th17/Treg may be involved in the development and progression of UCC.