Glycolysis Induced by METTL14 Is Essential for Macrophage Phagocytosis and Phenotype in Cervical Cancer.

N 6-methyladenosine (m6A) is the most abundant mRNA modification in mammals and it plays a vital role in various biological processes. However, the roles of m6A on cervical cancer tumorigenesis, especially macrophages infiltrated in the tumor microenvironment of cervical cancer, are still unclear. We analyzed the abnormal m6A methylation in cervical cancer, using CaSki and THP-1 cell lines, that might influence macrophage polarization and/or function in the tumor microenvironment. In addition, C57BL/6J and BALB/c nude mice were used for validation in vivo. In this study, m6A methylated RNA immunoprecipitation sequencing analysis revealed the m6A profiles in cervical cancer. Then, we discovered that the high expression of METTL14 (methyltransferase 14, N6-adenosine-methyltransferase subunit) in cervical cancer tissues can promote the proportion of programmed cell death protein 1 (PD-1)-positive tumor-associated macrophages, which have an obstacle to devour tumor cells. Functionally, changes of METTL14 in cervical cancer inhibit the recognition and phagocytosis of macrophages to tumor cells. Mechanistically, the abnormality of METTL14 could target the glycolysis of tumors in vivo and vitro. Moreover, lactate acid produced by tumor glycolysis has an important role in the PD-1 expression of tumor-associated macrophages as a proinflammatory and immunosuppressive mediator. In this study, we revealed the effect of glycolysis regulated by METTL14 on the expression of PD-1 and phagocytosis of macrophages, which showed that METTL14 was a potential therapeutic target for treating advanced human cancers.

APLN promotes the proliferation, migration, and glycolysis of cervical cancer through the PI3K/AKT/mTOR pathway.

Apelin (APLN) is an endogenous ligand of the G protein-coupled receptor APJ (APLNR). APLN has been implicated in the development of multiple tumours. Herein, we determined the effect of APLN on the biological behaviour and underlying mechanisms of cervical cancer. The expression and survival curves of APLN were determined using Gene Expression Profiling Interactive Analysis. The cellular functions of APLN were detected using CCK-8, clone formation, EdU, Transwell assays, flow cytometry, and seahorse metabolic analysis. The underlying mechanisms were elucidated using gene set enrichment analysis and Western blotting. APLN was upregulated in the samples of patients with cervical cancer and is associated with poor prognosis. APLN knockdown decreased the proliferation, migration, and glycolysis of cervical cancer cells. The opposite results were observed when APLN was overexpressed. Mechanistically, we determined that APLN was critical for activating the PI3K/AKT/mTOR pathway via APLNR. APLN receptor inhibitor ML221 reversed the effect of APLN overexpression on cervical cancer cells. Treatment with LY294002, the PI3K inhibitor, drastically reversed the oncological behaviour of APLN-overexpressing C-33A cells. APLN promoted the proliferation, migration, and glycolysis of cervical cancer cells via the PI3K/AKT/mTOR pathway.

hsa_circ_0000745 promotes cervical cancer by increasing cell proliferation, migration, and invasion.

Circular RNAs (circRNAs) participate in gene regulation and malignant tumor progression, including uterine cervical cancer (CC). In this study, the expression profile of circRNAs in CC was detected using circRNA microarrays. Then, we selected hsa_circ_0000745 for further examination from the significantly dysregulated circRNAs. Proliferation assays, Transwell assays, quantitative reverse transcription polymerase chain reaction, western blot analysis and tumorigenesis tests in vivo were used to validate the role of hsa_circ_0000745 in CC. hsa_circ_0000745 was upregulated in CC, and its level positively correlated with the level of its linear messenger RNA isoform. Patients with poorly differentiated tumors or vascular/lymphatic invasion presented higher expression of hsa_circ_0000745. The role of hsa_circ_0000745 was illuminated by knocking down hsa_circ_0000745 in CC cells, and the results revealed that reducing hsa_circ_0000745 inhibited cell proliferation, migration, and invasion in CC by upregulating E-cadherin (E-cad) expression. In summary, as a tumor promoter in CC, hsa_circ_0000745 enhances the cell's ability to proliferate, migrate, and invade by reducing the expression of E-cad. hsa_circ_0000745 is a candidate target for the treatment of CC in the clinic.

Targeting tumor-associated macrophage-derived CD74 improves efficacy of neoadjuvant chemotherapy in combination with PD-1 blockade for cervical cancer.

BACKGROUND: Cervical cancer has the second-highest mortality rate among malignant tumors of the female reproductive system. Immune checkpoint inhibitors such as programmed cell death protein 1 (PD-1) blockade are promising therapeutic agents, but their efficacy when combined with neoadjuvant chemotherapy (NACT) has not been fully tested, and how they alter the tumor microenvironment has not been comprehensively elucidated. METHODS: In this study, we conducted single-cell RNA sequencing using 46,950 cells from nine human cervical cancer tissues representing sequential different stages of NACT and PD-1 blockade combination therapy. We delineated the trajectory of cervical epithelial cells and identified the crucial factors involved in combination therapy. Cell-cell communication analysis was performed between tumor and immune cells. In addition, THP-1-derived and primary monocyte-derived macrophages were cocultured with cervical cancer cells and phagocytosis was detected by flow cytometry. The antitumor activity of blocking CD74 was validated in vivo using a CD74 humanized subcutaneous tumor model. RESULTS: Pathway enrichment analysis indicated that NACT activated cytokine and complement-related immune responses. Cell-cell communication analysis revealed that after NACT therapy, interaction strength between T cells and cancer cells decreased, but intensified between macrophages and cancer cells. We verified that macrophages were necessary for the PD-1 blockade to exert antitumor effects in vitro. Additionally, CD74-positive macrophages frequently interacted with the most immunoreactive epithelial subgroup 3 (Epi3) cancer subgroup during combination NACT. We found that CD74 upregulation limited phagocytosis and stimulated M2 polarization, whereas CD74 blockade enhanced macrophage phagocytosis, decreasing cervical cancer cell viability in vitro and in vivo. CONCLUSIONS: Our study reveals the dynamic cell-cell interaction network in the cervical cancer microenvironment influenced by combining NACT and PD-1 blockade. Furthermore, blocking tumor-associated macrophage-derived CD74 could augment neoadjuvant therapeutic efficacy.

The Imbalance Expression of DLX3 May Perform Critical Function in the Occurrence and Progression of Preeclampsia.

BACKGROUND: The present research focuses on preeclampsia (PE), a clinically relevant pregnancy disease. To date, the majority of research on PE was centered on placental insufficiency. However, the genes that regulate these processes, and the exact molecular mechanisms modulating these processes, are still unclear. METHODS: We obtained placentae from a clinically well-specified group of patients with preeclampsia and gestationally matched control pregnancies in order to evaluate the expression of homeobox gene DLX3 by immunohistochemical staining, real-time PCR, and Western immunoblotting and determine the function of DLX3 utilizing lentivirus transfection in HTR-8/SVneo cells. RESULTS: In the present study, we detected DLX3 expression in a clinically well defined cohort of preeclampsia-affected and gestation-matched control pregnancies. As opposed to the controls, DLX3 was overexpressed in preeclampsia-affected placentae. Moreover, we found that the in vitro cell growth and invasive ability of HTR8/SVneo cells was enhanced by the exogenous overexpression of DLX3 (P < 0.05). It can be seen that DLX3 influences the cell cycle of HTR-8/SVneo cells in vitro. CONCLUSIONS: DLX3 has been shown to be strongly related to normal placental growth as well as the pathophysiology of preeclampsia. The imbalanced expression of DLX3 may perform an integral function in the occurrence and progression of preeclampsia.

Development of a Novel Deep Learning-Based Prediction Model for the Prognosis of Operable Cervical Cancer.

Background: Cervical cancer ranks as the 4th most common female cancer worldwide. Early stage cervical cancer patients can be treated with operation, but clinical staging system is not a good predictor of patients' survival. We aimed to develop a novel prognostic model to predict the prognosis for operable cervical cancer patients with better accuracy than clinical staging system. Methods: A total of 13,952 operable cervical cancer patients were retrospectively enrolled in this study. The whole dataset was randomly split into a training set (n = 9,068, 65%), validation set (n = 2,442, 17.5%), and testing set (n = 2,442, 17.5%). Cox proportional hazard (CPH) model and random survival forest (RSF) model were used as baseline models for the prediction of overall survival (OS). Then, a deep survival learning model (DSLM) was developed for OS prediction. Finally, a novel prognostic model was explored based on this DSLM. Results: The C-indexes for the CPH and RSF model were 0.731 and 0.753, respectively. DSLM, which had four layers that had 50 neurons in each layer, achieved a C-index of 0.782 in the validation set and a C-index of 0.758 in the testing set. The novel prognostic model based on DSLM showed better performances than the conventional clinical staging system (area under receiver operating curves were 0.826 and 0.689, respectively). Personalized survival curves for individual patient using this novel model also showed notably different survival slopes. Conclusions: Our study developed a novel, practical, personalized prognostic model for operable cervical cancer patients. This novel prognostic model may have the potential to provide a more prognostic information to oncologists.

The Regulatory Role of circRNA_101308 in Cervical Cancer and the Prediction of Its Mechanism.

PURPOSE: Accumulating evidence indicates that circular RNAs (circRNAs) are closely involved in canceration and cancer progression. However, the role of circRNAs in cervical cancer (CC) is largely unknown. Here, we characterized the role of circRNA_101308 in CC. MATERIALS AND METHODS: The expression of circRNA_101308 in CC tissues was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Then, circRNA_101308 was overexpressed in CC cells to detect its function by proliferation and apoptosis assays, Transwell assays and animal experiments. The potential mechanism of circRNA_101308 in CC was explored by RNA pull-down, Gene Ontology (GO) and pathway analyses. RESULTS: CircRNA_101308 was significantly downregulated in CC tissues. The level of circRNA_101308 was much lower in CC patients with lymph node metastasis or deep myometrial invasion compared to those patients without lymph node metastasis and superficial myometrial invasion. CircRNA_101308 overexpression inhibited CC cell proliferation, invasion and migration. MiR-26a-5p, miR-196a-5p, miR-196b-5p, miR-335-3p, and miR-1307-3p were found to be sponged by circRNA_101308 in CC cells. Further, GO and pathway analyses predicted the potential functional processes and pathways of circRNA_101308 in CC. CONCLUSION: CircRNA_101308 is downregulated and acts as a tumor suppressor in CC. CircRNA_101308 can participate in many different processes by sponging different miRNAs in CC cells. This exploration of circRNA_101308 provides new directions for research on cancer development and the clinical treatment of CC.

Development and Validation of a Personalized Survival Prediction Model for Uterine Adenosarcoma: A Population-Based Deep Learning Study.

BACKGROUND: The aim was to develop a personalized survival prediction deep learning model for adenosarcoma patients using the surveillance, epidemiology and end results (SEER) database. METHODS: A total of 797 uterine adenosarcoma patients were enrolled in this study. Duplicated and useless variables were excluded, and 15 variables were selected for further analyses, including age, grade, positive lymph nodes or not, marital status, race, tumor extension, stage, and surgery or not. We created our deep survival learning (DSL) model to manipulate the data, which was randomly split into a training set (n = 519, 65%), validation set (n = 143, 18%) and testing set (n = 143, 18%). The Cox proportional hazard (CPH) model was also included comparatively. Finally, personalized survival curves were plotted for randomly selected patients. RESULTS: The c-index for the CPH model was 0.726, and the Brier score was 0.17. For our deep survival learning model, we achieved a c-index of 0.774 and a Brier score of 0.14 in the external testing set. In addition, the limitations of the traditional staging system were revealed, and a personalized survival prediction system based on our risk scoring grouping was developed. CONCLUSIONS: Our study developed a deep neural network model for adenosarcoma. The performance of this model was superior to that of the traditional Cox proportional hazard model. In addition, a personalized survival prediction system was developed based on our deep survival learning model, which provided more accurate prognostic information for adenosarcoma patients.

Upregulated MELK Leads to Doxorubicin Chemoresistance and M2 Macrophage Polarization via the miR-34a/JAK2/STAT3 Pathway in Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (ULMS) is the most lethal gynecologic malignancy with few therapeutic options. Chemoresistance prevails as a major hurdle in treating this malignancy, yet the mechanism of chemoresistance remains largely unclear. In this study, we certified MELK as a poor prognostic marker through bioinformatic analysis of the GEO database. Cellular experiments in vitro revealed that MELK played an essential role in ULMS cells' chemoresistance and that a high expression of MELK could lead to doxorubicin resistance. mRNA profiling uncovered the pathways that MELK was involved in which led to doxorubicin resistance. MELK was found to affect ULMS cells' chemoresistance through an anti-apoptotic mechanism via the JAK2/STAT3 pathway. miRNA profiling also revealed that upregulated MELK could induce the decrease of miRNA-34a (regulated by JAK2/STAT3 pathway). We detected that MELK overexpression could induce M2 macrophage polarization via the miR-34a/JAK2/STAT3 pathway, contributing to doxorubicin chemoresistance in the tumor microenvironment. OTSSP167, a MELK inhibitor, may increase ULMS sensitivity to doxorubicin. Our investigation could propose novel targets for early diagnosis and precision therapy in ULMS patients.

Promoter methylation of SEPT9 as a potential biomarker for early detection of cervical cancer and its overexpression predicts radioresistance.

BACKGROUND: Cervical cancer screening by combined cytology and HPV test has reduced the incidence of cervical cancer, but cytological screening lacks a higher sensitivity while HPV testing possesses a lower specificity. Most patients with invasive cervical cancer are treated with radiotherapy. However, insensitivity to radiotherapy leads to poor efficacy. METHODS: Illumina Methylation EPIC 850k Beadchip was used for genomic screening. We detected methylation of SEPT9 and mRNA expression in different cervical tissues by using methylation-specific PCR and qRT-PCR. Then using CCK8, migration assay, and flow cytometry to detect the biological function and irradiation resistance of SEPT9 in vitro and in vivo. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation (CoIP) were used to find the interacting gene with SEPT9. Immunostaining of CD206 in cervical cancer and polarization of macrophages (M2) were evaluated by immunofluorescence and WB. The Cancer Genome Atlas (TCGA) database was used for screening the potential miRNAs induced by SEPT9. RESULTS: Hyper-methylation of SEPT9 detects cervical cancer and normal tissues, normal+CIN1 and CIN2+CIN3+cancer with high sensitivity and specificity (AUC = 0.854 and 0.797, respectively, P < 0.001). The mRNA and protein expression of SEPT9 was upregulated in cervical cancer tissues when compared to para-carcinoma tissues. SEPT9 promotes proliferation, invasion, migration, and influences the cell cycle of cervical cancer. SEPT9 interacted with HMGB1-RB axis increases irradiation resistance. Furthermore, SEPT9 mediated miR-375 via the tumor-associated macrophages (TAMs) polarization, affecting the resistance to radiotherapy in cervical cancer. CONCLUSIONS: These findings give us the evidence that SEPT9 methylation could be a biomarker for cervical cancer diagnoses. It promotes tumorigenesis and radioresistance of cervical cancer by targeting HMGB1-RB axis and causes polarization of macrophages by mediating miR-375. We suggest SEPT9 could be a potential screening and therapeutic biomarker for cervical cancer.

Increased expression of FHL2 promotes tumorigenesis in cervical cancer and is correlated with poor prognosis.

PURPOSE: Increasing evidence demonstrates that the four and a half LIM domain (FHL) gene and its protein products have different functions in the progression of various malignancies. However, the role of FHL protein 2 (FHL2) in cervical cancer (CC) has not been fully elucidated. In this study, we investigated the prognostic value of FHL2 expression in human CC tissues and the potential molecular mechanisms through which FHL2 modulates CC cell proliferation and apoptosis. MATERIALS AND METHODS: We measured FHL2 expression in CC cell lines and tissues by quantitative real-time polymerase chain reaction and Western blot assays. The effects of FHL2 knockdown on cell proliferation and apoptosis in two CC cell lines were examined using RNA interference, cell counting kit-8, Western blot and flow cytometry assays. Furthermore, we assessed phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) expression in two CC cell lines to determine whether the AKT/mTOR pathway is involved in the effects of FHL2 silencing on cell proliferation and apoptosis. Nude mice tumorigenicity experiments were also performed to evaluate the effects of FHL2 on HeLa cell growth in vivo. RESULTS: We found that FHL2 was significantly upregulated in CC cell lines and tissues. According to survival curves, high FHL2 expression levels in patients were correlated with poor prognosis. Moreover, by decreasing p-AKT and p-mTOR protein levels, silencing FHL2 significantly inhibited cell proliferation and induced apoptosis. FHL2 knockdown also induced apoptosis by increasing the Bax-to-Bcl2 ratio. By contrast, FHL2 overexpression significantly promoted cell proliferation. Finally, decreased tumour growth in an in vivo animal model also demonstrated the tumour-suppressing effects of FHL2 knockdown. CONCLUSION: Our findings indicate that FHL2 is an important prognostic factor in CC and that it plays a crucial oncoprotein role by promoting cell proliferation and inhibiting apoptosis in CC, possibly by targeting the AKT/mTOR pathway.

Aberrant frequency of TNFR2+ Treg and related cytokines in patients with CIN and cervical cancer.

Regulatory T (Treg) cells expressing tumor necrosis factor receptor 2 (TNFR2) are highly suppressive and are associated with immune homeostasis in various diseases. However, the role of TNFR2+Treg subset and relevant cytokines in the development of cervical cancer (CC) remained unclear. In this study, 72 patients with CC, 30 patients with cervical intraepithelial neoplasia (CIN) and 30 healthy volunteers were enrolled. The level of circulating TNFR2+Tregs was investigated through flow cytometry. The plasma concentrations of soluble TNFR1 (s-TNFR1) and soluble TNFR2 (s-TNFR2) were determined by enzyme-linked immunosorbent assay. In addition, the mRNA expression levels of TNF-α, TNFR1, TNFR2, and Foxp3 were measured using real-time polymerase chain reaction. Results showed that both peripheral and tumor infiltrating TNFR2+Tregs significantly increased in patients with CIN and CC and levels of circulating s-TNFR1 and s-TNFR2 increased in patients with CC. Moreover, the percentage of peripheral TNFR2+Tregs was inversely correlated with the clinical stages of CC. Furthermore, the mRNA expression levels of TNF-α, TNFR2, and Foxp3 increased in patients with CIN and CC. Overall, these results indicate that TNFR2+Tregs and relevant cytokines contribute to CC development and are promising targets in future immunotherapeutic approaches.