Experimental Biology and Medicine

Identification of two molecular subtypes of dysregulated immune lncRNAs in ovarian cancer

Long non-coding RNA (lncRNA) has increasingly been identified as a key regulator in pathologies such as cancer. Multiple platforms were used for comprehensive analysis of ovarian cancer to identify molecular subgroups. However, lncRNA and its role in mapping the ovarian cancer subpopulation are still largely unknown. RNA-sequencing and clinical characteristics of ovarian cancer were acquired from The Cancer Genome Atlas database (TCGA). A total of 52 lncRNAs were identified as aberrant immune lncRNAs specific to ovarian cancer. We redefined two different molecular subtypes, C1(188) and C2(184 samples), in “iClusterPlus” R package, among which C2 grouped ovarian cancer samples have higher survival probability and longer median survival time ( P <0.05) with activated IFN-gamma response, Wound Healing and Cytotoxic lymphocytes signal; 456 differentially expressed genes were acquired in C1 and C2 subtypes using limma (3.40.6) package, among which 419 were up-regulated and 37 were down-regulated, in TCGA dataset. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis revealed that these genes were actively involved in ECM-receptor interaction, PI3K-Akt signaling pathway interaction KEGG pathway. Compared with the existing immune subtype, the Cluster2 sample showed a substantial increase in the proportion of the existing C2 immune subtype, accounting for 81.37%, which was associated with good prognosis. Our C1 subtype contains only 56.49% of the existing immune C1 and C4, which also explains the poor prognosis of C1. Furthermore, 52 immune-related lncRNAs were used to divide the TCGA-endometrial cancer and cervical cancer samples into two categories, and C2 had a good prognosis. The differentially expressed genes were highly correlated with immune-cell-related pathways. Based on lncRNA, two molecular subtypes of ovarian cancer were identified and had significant prognostic differences and immunological characteristics.

Clinicopathological significance and underlying molecular mechanism of downregulation of basonuclin 1 expression in ovarian carcinoma

In this study, we aim to identify the clinical significance of basonuclin 1 ( BNC1) expression in ovarian carcinoma (OV) and to explore its latent mechanisms. Via integrating in-house tissue microarrays, gene chips, and RNA-sequencing data, we explored the expression and clinical value of BNC1 in OV. Immunohistochemical staining was utilized to confirm the protein expression status of BNC1. A combined SMD of –2.339 (95% CI: –3.649 to –1.028, P <  0.001) identified that BNC1 was downregulated based on 1346 samples, and the sROC (AUC = 0.93) showed a favorable discriminatory ability of BNC1 in OV patients. We used univariate and multivariate Cox regulation to evaluate the prognostic role of BNC1 for OV patients, and a combined hazard ratio of 0.717 (95% CI: 0.445–0.989, P <  0.001) revealed that BNC1 was a protective factor for OV. Furthermore, the fraction of infiltrating naive B cells, memory B cells, and other immune cells showed statistical differences between the high- and low- BNC1 expression groups through cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm. Enrichment analysis showed that BNC1 may have a relationship with immune-related items in OV. By predicting the potential regulatory transcription factors (TFs) of BNC1, friend leukemia virus integration 1 ( FLI1) may be a potential upstream TF of BNC1. Corporately, a decreasing trend of BNC1 may serve as a tumor suppressor and prognostic biomarker in OV patients. Moreover, BNC1 may take part in immune-related pathways and influence the fraction of tumor-infiltrating immune cells.

Adipose-derived stem cells in ovarian cancer progression, metastasis, and chemoresistance

Ovarian cancer is the deadliest gynecological malignancy due to its symptomless early stage, metastasis, and high recurrence rate. The tumor microenvironment contributes to the ovarian cancer progression, metastasis, and chemoresistance. Adipose-derived stem cell in the tumor microenvironment of ovarian cancer, as a key player, interacts with ovarian cancer cells to form the cancer-associated fibroblasts and cancer-associated adipocytes, and secretes soluble factors to activate tumor cell signaling, which can promote ovarian cancer metastasis and chemoresistance. We summarize in this review the recent progress in the studies of interactions between adipose-derived stem cell and ovarian cancer, thus, to provide some insight for ovarian cancer therapy through targeting adipose-derived stem cell.

Enhancing the sensitivity of ovarian cancer cells to olaparib via microRNA-20b-mediated cyclin D1 targeting

We previously reported that cyclin D1 silencing interferes with RAD51 accumulation and increases the sensitivity of BRCA1 wild-type ovarian cancer cells to olaparib. However, the mechanisms associated with cyclin D1 overexpression in ovarian cancer are not fully understood. TargetScan predicted the potential binding sites for microRNA-20b (miR-20b) and the 3′-untranslated region of cyclin D1 mRNA; thus, we used luciferase reporter assay to verify those binding sites. The Kaplan-Meier method and log-rank test were used to examine the relationship between miR-20b and progression-free survival of ovarian cancer patients in The Cancer Genome Atlas ( n = 367) dataset. In vitro experiments were performed to evaluate the effects of miR-20b on cyclin D1 expression, cell cycle and response to olaparib. A peritoneal cavity metastasis model of ovarian cancer was established to determine the effect of miR-20b on the sensitivity of olaparib. Immunohistochemistry was performed to evaluate molecular mechanisms. In this work, we demonstrated that miR-20b down-regulates cyclin D1, increases the sensitivity of ovarian cancer cells to olaparib, reduces the expression of RAD51, and induces cell cycle arrest in G0/G1 phase. Ovarian cancer patients with higher expression of miR-20b had significantly longer progression-free survival. These results indicate that miR-20b may be a potential clinical indicator for the sensitivity of ovarian cancer to olaparib and the survival of ovarian cancer patients. Our findings suggest that miR-20b may have therapeutic value in combination with olaparib treatment for ovarian cancer.

Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker

Circulating cell-free DNA (cfDNA) has attracted attention as a non-invasive biomarker for diagnosing and monitoring various cancers. Given that human papillomavirus (HPV) DNA integration and overexpression of E6/E7 oncogenes are pivotal events for carcinogenesis, we sought to determine if HPV E7 cfDNA could serve as a specific biomarker for cervical cancer detection. We applied droplet digital PCR (ddPCR) to quantify HPV16/18 E7 cfDNA from the serum of patients with cervical cancer, cervical intraepithelial neoplasia, and controls. HPV16/18 E7 cfDNA was highly specific for cervical cancer, displaying 30.77% sensitivity, 100% specificity, and an area under the curve of 0.65. Furthermore, we developed a sensitive isothermal detection of HPV16/18 E7 and the PIK3CA WT reference gene based on recombinase polymerase amplification combined with a lateral flow strip (RPA-LF). The assay took less than 30 min and the detection limit was 5–10 copies. RPA-LF exhibited 100% sensitivity and 88.24% specificity towards HPV16/18 E7 cfDNA in clinical samples. The agreement between RPA-LF and ddPCR was 83.33% ( κ = 0.67) for HPV16 E7 and 100% ( κ = 1.0) for HPV18 E7, indicating a good correlation between both tests. Therefore, we conclude that HPV E7 cfDNA represents a potential tumor marker with excellent specificity and moderate sensitivity for minimally invasive cervical cancer monitoring. Moreover, the RPA-LF assay provides an affordable, rapid, and ultrasensitive tool for detecting HPV cfDNA in resource-limited settings.

Establishment of a mouse model for ovarian cancer–associated venous thromboembolism

Patients with ovarian cancer are at increased risk of venous thromboembolism (VTE), and the cumulative incidence is high, particularly at advanced stages of this disease. Nevertheless, it is challenging to investigate the molecular mechanisms of ovarian cancer–associated VTE (OC-VTE), mainly due to the lack of a well-developed animal model for this disease. We generated a mouse model for developing OC-VTE using ovarian cancer cell injection in combination with the inferior vena cava stenosis method. The rate of thrombosis in the OC-VTE group was 50%, compared with 0 in the control group. Moreover, we conducted a proteomic analysis using platelets from these models and revealed differentially expressed proteins between the OC-VTE and control groups, including upregulated and downregulated proteins. Gene Ontology analysis revealed that these differentially expressed proteins were mostly enriched in the biological process of negative regulation of fibrinolysis and the cellular component of the fibrinogen complex, both of which play key roles in thrombosis. In conclusion, this study lays the foundation for further investigation of the underlying mechanisms of how ovarian cancer promotes VTE formation.

Inferences of carboplatin response-related signature by integrating multiomics data in ovarian serous cystadenocarcinoma

Platinum-based chemotherapy, especially carboplatin, is the primary measure to treat patients with ovarian cancer (OC). However, OC patients still have an adverse prognosis due to emergency of chemotherapy resistance. Ovarian serous cystadenocarcinoma (OSC) is the most common histological subtype of OC. Therefore, identifying the key factors that affect chemotherapy resistance and searching novel treatments had become a top priority. In this study, we analyzed carboplatin response-related mRNA, miRNA, DNA methylation, and alternative splicing (AS) and established a drug-resistant signature for carboplatin in OSC. This drug-resistant signature was obviously higher in resistant group than in non-resistant group and had accuracy predictive performance, which demonstrated that this signature could be considered as a superior indicator for OSC patients with carboplatin resistance. Furthermore, we selected three potential small molecule drugs including liranaftate, siguazodan, and tramiprostate to inhibit carboplatin resistance of OSC. In addition, we also identified ZINC00000205417, ZINC00000140928, and ZINC00021908260 were potential small molecule compounds for SLC17A7 based on Molecular Operating Environment (MOE) virtual screening. Finally, we confirmed the drug-like properties of these small molecule drugs via evaluating absorption, distribution, metabolism, elimination, and toxicity (ADMET) property. In summary, the signature could be used as biomarker for carboplatin resistance and small molecule drugs targeting these genes could improve clinical treatment for OSC in the future.

SNHG1 represses the anti-cancer roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin signaling

As a flavonoid, baicalein exhibits remarkable anti-cancer roles in several cancers. However, the factors regulating the antitumorigenic roles of baicalein in cervical cancer remain undefined. Here, we revealed that long noncoding RNA SNHG1 is implicated in the tumor-suppressive roles of baicalein. Functional assays demonstrated that ectopic expression of SNHG1 attenuates the roles of baicalein in repressing cervical cancer cell viability, inducing apoptosis, and repressing migration. SNHG1 silencing promotes the tumor-suppressive roles of baicalein in cervical cancer cell viability, apoptosis, and migration. Xenograft assays showed that SNHG1 reverses the tumor-suppressive roles of baicalein in repressing cervical cancer growth in vivo. Mechanistic investigations revealed that SNHG1 directly binds miR-3127-5p and up-regulates FZD4, a target of miR-3127-5p. Via regulating miR-3127-5p/FZD4, SNHG1 activates Wnt/β-catenin signaling. Moreover, SNHG1 reverses the repressive role of baicalein on Wnt/β-catenin signaling. The effect of SNHG1 on the antitumorigenic process of baicalein was abolished by Wnt/β-catenin signaling inhibitor ICG-001. Together, our observations demonstrated that SNHG1 represses the tumor-suppressive roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin axis, and suggested that targeting SNHG1 represents a potential strategy to enhance the tumor-suppressive roles of baicalein in cervical cancer. Impact statement Baicalein exhibits anti-cancer roles in several cancers. However, the factors influencing the antitumorigenic efficiencies of baicalein in CC remain largely unclear. Here, we provide convincing evidences that lncRNA SNHG1 attenuates the tumor-suppressive roles of baicalein in CC cell viability, apoptosis, migration, and CC tumor growth. This study further demonstrates that the influences of SNHG1 in the antitumorigenic process of baicalein are achieved through modulating the miR-3127-5p/FZD4Wnt/β-catenin axis. SNHG1 attenuates the repressive role of baicalein on Wnt/β-catenin. Therefore, SNHG1 is a novel modulator of the tumor-suppressive roles of baicalein and SNHG1 represents a therapeutic intervention target to reinforce the tumor-suppressive roles of baicalein in CC.

Identifying a cervical cancer survival signature based on mRNA expression and genome-wide copy number variations

Cervical cancer mortality is the second highest in gynecological cancers. This study developed a new model based on copy number variation data and mRNA data for overall survival prediction of cervical cancer. Differentially expressed genes from The Cancer Genome Atlas dataset detected by univariate Cox regression analysis were further simplified to six by least absolute shrinkage and selection operator (Lasso) and stepwise Akaike information criterion (stepAIC). The study developed a six-gene signature, which was further verified in independent dataset. Association between immune infiltration and risk score was investigated by immune score. The relation between the signature and functional pathways was examined by gene set enrichment analysis. Ninety-nine differentially expressed genes were detected, and C11orf80, FOXP3, GSN, HCCS, PGAM5, and RIBC2 were identified as key genes to construct a six-gene signature. The prognostic signature showed a significant correlation with overall survival (hazard ratio, HR = 3.45, 95% confidence interval (CI) = 2.08–5.72, p <  0.00001). Immune score showed a negative correlation with the risk score calculated by the signature ( p <  0.05). Four immune-related pathways were closely associated with risk score ( p <  0.0001). The six-gene prognostic signature was an effective tool to predict overall survival of cervical cancer. In conclusion, the newly identified six genes may be considered as new drug targets for cervical cancer treatment.

Insulin-like growth factor 2 mRNA-binding protein 2 is a therapeutic target in ovarian cancer

Ovarian cancer (OC) is a fatal gynecologic disease. The most common treatment for OC patients is surgery combined with chemotherapy but most patients at advanced stages eventually develop relapse due to chemoresistance. This study examined the role and function of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) in OC. We observed that the expression of IGF2BP2 mRNA and protein was up-regulated in OC cells and tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. An increase in IGF2BP2 expression at mRNA and protein levels was verified by the analyses of The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC), respectively. Gene Expression Omnibus (GEO) and Cancer Cell Line Encyclopedia (CCLE) databases were applied to analyze the expression and clinical value of IGF2BP2. Gene set enrichment analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) analyses explored biological functions and the involvement of IGF2BP2 in cell growth. Indeed, the knockdown of IGF2BP2 resulted in the inhibition of OC cell proliferation evaluated by the Cell Counting Kit-8 assay. Genomic amplification of IGF2BP2 partly accounted for its overexpression. High expression of IGF2BP2 was associated with signal transducer and activator of transcription 1 (STAT1) and drug sensitivity and was correlated with an unfavorable survival outcome in OC patients. Furthermore, the responsiveness of chemotherapy and immunotherapy were analyzed using the “pRRophetic” R package and The Cancer Immune Atlas (TCIA) database, respectively. The low expression of IGF2BP2 was associated with chemoresistance but with high tumor microenvironment scores and tumor-infiltrating immune cells, suggesting that immunotherapy may apply in chemoresistant patients. The alteration of IGF2BP2 expression may respond to chemotherapy and immunotherapy. Thus, IGF2BP2 shows potential as a therapeutic target and diagnostic biomarker for OC.

Identifying immune subtypes of uterine corpus endometrial carcinoma and a four-paired-lncRNA signature with immune-related lncRNAs

Uterine corpus endometrial carcinoma (UCEC) is the third most frequent gynecological malignancies in the female reproductive system. Long non-coding RNAs (lncRNAs) are closely involved in tumor progression. This study aimed to develop an immune subtyping system and a prognostic model based on lncRNAs for UCEC. Paired lncRNAs and non-negative matrix factorization were applied to identify immune subtypes. Enrichment analysis was conducted to assess functional pathways, immune-related genes, and cells. Univariate and multivariate Cox regression analysis were performed to analyze the relation between lncRNAs and overall survival (OS). A prognostic model was constructed and optimized by least absolute shrinkage and selection operator (LASSO) and Akaike information criterion (AIC). Two immune subtypes (C1 and C2) and four paired-prognostic lncRNAs closely associated with overall survival were identified. Some immune features, sensitivity of chemotherapy and immunotherapy, and the relation with immune escape showed variations between two subtypes. A nomogram established based on prognostic model and clinical features was effective in OS prediction. The immune subtyping system based on lncRNAs and the four-paired-lncRNA signature was predictive of UCEC prognosis and can facilitate personalized therapies such as immunotherapy or RNA-based therapy for UCEC patients.

Megestrol acetate drives endometrial carcinoma cell senescence via interacting with progesterone receptor B/FOXO1 axis

Megestrol acetate is a common and efficient anticancer progesterone. To explore the activity and the therapeutic mechanisms of megestrol acetate in endometrial cancer, human endometrial cancer cell lines Ishikawa and HHUA overexpressing progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were treated with megestrol acetate. Cell viability, apoptosis, cycle arrest, and senescence, as well as the expressions of p21 and p16, two hallmarks of cellular senescence, were evaluated. Compared with the control, >10 nmol/L megestrol acetate treatment could significantly reduce endometrial cancer cell growth, and induce the irreversible G1 arrest and cell senescence. The expression of cyclin D1 in megestrol acetate treated cells was downregulated, while the expressions of p21 and p16 were upregulated via PR-B isoform. FOXO1 inhibitor AS1842856 could significantly abrogate megestrol acetate-induced cell senescence, suggesting that FOXO1 was involved in megestrol acetate/PR-B axis. These findings may provide a new understanding for the treatment of human endometrial cancer.

Silencing of FAM111B inhibits tumor growth and promotes apoptosis by decreasing AKT activity in ovarian cancer

Ovarian cancer is the most lethal gynecological tumor in women worldwide. FAM111B (family with sequence similarity 111 member B) is an oncoprotein associated with multiple cancers, but its biological functions in ovarian cancer remain elusive. In this study, FAM111B was overexpressed in ovarian cancer tissues and cell lines. Functional studies in vitro revealed that silencing of FAM111B inhibited ovarian cancer cell proliferation, invasion, and migration, as well as increased cell apoptosis. Furthermore, FAM111B silencing arrested the ovarian cancer cell cycle at the G1/S phase. Furthermore, western blot assays demonstrated that silencing of FAM111B resulted in downregulation of phospho-AKT (p-AKT) protein expression, as well as upregulation of p53 and caspase-1 protein expression. The xenograft animal model of ovarian cancer demonstrated that FAM111B silencing inhibited tumor growth, enhanced cell apoptosis, and inhibited Ki-67 and proliferating cell nuclear antigen (PCNA) protein expression in vivo. Conversely, the overexpression of FAM111B exhibited opposite effects on the ovarian cancer xenograft. It was previously established that inactivating AKT inhibited ovarian cancer progression. This study found that silencing of FAM111B inhibits tumor growth and promotes apoptosis by decreasing AKT activity in ovarian cancer. Caspase-1 and p53 signaling also influenced the function of FAM111B in SKOV3 cells. Collectively, our results demonstrate that silencing of FAM111B is a potential therapeutic strategy against ovarian cancer.

Role of autophagy and its regulation by noncoding RNAs in ovarian cancer

Autophagy is a self-digestion process by which misfolded proteins and damaged organelles in eukaryotic cells are degraded to maintain cellular homeostasis. This process is involved in the tumorigenesis, metastasis, and chemoresistance of various tumors such as ovarian cancer (OC). Noncoding RNAs (ncRNAs), mainly including microRNAs, long noncoding RNAs, and circular RNAs, have been extensively investigated in cancer research for their roles in the regulation of autophagy. Recent studies have shown that in OC cells, ncRNAs can modulate the formation of autophagosomes, which affect tumor progression and chemoresistance. An understanding of the role of autophagy in OC progression, treatment, and prognosis is important, and the identification of the regulatory roles of ncRNAs in autophagy leads to intervention strategies for OC therapy. This review summarizes the role of autophagy in OC and discusses the role of ncRNA-mediated autophagy in OC, as an understanding of these roles may contribute to the development of potential therapeutic strategies for this disease.

Whole-genome analysis and evolutionary characterization of cervical and oral human papillomavirus 16

High-throughput genome-wide sequencing has revealed high genomic variability of HPV16 in different geographic regions which is the most predominant genotype in human papillomavirus (HPV)–associated malignancies. Analysis of the HPV16 by whole-genome sequence (WGS) is an advanced method for the identification of mutations in the genome. There is limited information about HPV16 diversity in Pakistan, especially at the genomic level. Till now, WGS for HPV16 has not been previously reported in Pakistan. The current study has sequenced three HPV16 viral genomes, from two cervical and one oral cavity positive sample of women presented with general gynecological problems without any evidence of precancerous or cancerous lesions using an ion ampliseq customized panel. Sequencing analysis detected 38 variations, including single-nucleotide polymorphisms (SNPs) and two Indels, across three samples with the highest number of SNPs present in E1, E2, and L2, respectively. A total of 20 non-synonymous and 11 synonymous mutations with amino acid substitutions (T1421C, G1515A, T2223C, T1389C, G1483A, and T2191C) were identified. The phylogenetic analysis revealed the genomes of HPV16 are closely associated with those reported from Thailand and the United States. These are the first HPV16 WGS from Pakistan. However, more research is needed with a large sample size from diversified areas to assess the carcinogenic consequences and impact of HPV vaccinations.

RETRACTED: Smad4 induces cell death in HO-8910 and SKOV3 ovarian carcinoma cell lines via PI3K-mTOR involvement

This study investigated the effect and mechanism of Smad4 in ovarian carcinoma (OC) cell viability and demonstrated that Smad4 acted as a tumor suppressor in OC, which may contribute to the understanding of molecular mechanisms underlying OC occurrence and progression. Smad4 expression was decreased in the OC specimens, but Smad4 recovery in the OC cell lines impaired the survival and viability of OC cells by increasing autophagy and apoptosis. Further investigation showed that Smad4 interacted with the P85 subunit of PI3K and caused deactivation of the PI3K/mTOR pathway. Therefore, Smad4 could be considered as a target in cancer therapy due to its regulatory effect in OC carcinogenesis.