Functional & Integrative Genomics

Genetic variants of LncRNA associated with splicing regulation and their impact on ovarian cancer development

Ovarian cancer (OC) is a highly lethal gynecologic malignancy that lacks reliable early biomarkers. Numerous long non-coding RNAs (lncRNAs) have been found to play critical regulatory roles in OC, yet the mechanisms underlying most of them remain unclear. Recently, lncRNAs have emerged as key regulators of RNA splicing, while splicing dysregulation is widespread and plays critical roles in cancer. In addition, genetic variants of splicing regulators have been shown to contribute to disease etiology. In this study, we comprehensively analyzed 202 °C samples and characterized 21,129 lncRNA splicing quantitative trait loci (sQTLs) involving both event-level and transcript-level. We found that lncRNA sQTLs differ significantly from lncRNA expression quantitative trait loci (eQTLs), and genes regulated by lncRNA sQTLs are involved in cancer hallmark pathways and associated with immune cell infiltration and drug sensitivity. Additionally, these lncRNA sQTLs are significantly enriched in histone markers, transcription factor (TF) binding sites, and RNA-binding protein (RBP) binding sites, including several critical splicing factors (SF) in OC. Based on SF binding and partial correlation analysis, a potential lncRNA-SF-mRNA regulatory network was constructed. Finally, by integrating GWAS data, we elucidated that a specific lncRNA sQTL rs1549334 generates different isoforms by regulating the splicing of the HOXD3 gene and thus impacting OC risk. Our study provides new insights into the mechanisms of splicing regulation in OC involving lncRNA sQTL and reveals potential biomarkers for early detection and clinical treatment of OC.

FAM72A promotes cervical cancer progression by regulating the PI3K pathway

Cervical cancer (CC) is a major malignancy and a serious threat to women's health worldwide. The role of FAM72A in CC remains poorly defined. This study aimed to investigate its function in CC progression and its impact on the PI3K/AKT/mTOR pathway. FAM72A expression in CC was examined using TCGA-CESC and GEO (GSE63514) datasets, and then validated in CC tissues and cell lines. Functional assays assessed cell proliferation (CCK-8, EdU), invasion (Transwell), apoptosis (flow cytometry), and epithelial-mesenchymal transition (EMT; Western blot for E-cadherin/N-cadherin). The role of FAM72A in the PI3K/AKT/mTOR pathway was further evaluated by Western blot and pharmacological modulation with the activator 740 Y-P and the inhibitor LY294002. In vivo, a xenograft model with BALB/c nude mice was used to assess tumor growth, apoptosis (TUNEL staining), proliferation (Ki-67 IHC), and pathway activation (p-PI3K, p-AKT, and p-mTOR). FAM72A expression was upregulated in CC tissues and correlated with poor survival. Subgroup analysis showed that high FAM72A expression was associated with advanced FIGO stage, lymph node metastasis, and deep stromal invasion, indicating a link with aggressive clinical features. FAM72A silencing suppressed proliferation and invasion but promoted apoptosis, mainly through inhibition of the PI3K/AKT/mTOR pathway. Conversely, FAM72A overexpression enhanced these malignant traits. In vivo, FAM72A knockdown reduced tumor burden and altered EMT markers and PI3K/AKT/mTOR pathway activity. FAM72A promotes CC progression, at least in part, through activation of the PI3K/AKT/mTOR pathway, supporting its value as a potential therapeutic target.

Single-cell RNA sequencing revealed changes in the tumor microenvironment induced by radiotherapy for cervical cancer and the molecular mechanism of mast cells in immunosuppression

Radiotherapy (RT) is an important treatment for cervical cancer (CC), effectively controlling tumor growth and improving survival rates. However, radiotherapy-induced cell heterogeneity and its underlying mechanisms remain unclear, which may potentially impact treatment efficacy. This study aims to investigate tumor microenvironment changes following radiotherapy for CC, hoping to provide evidence to improve the therapeutic effects of radiotherapy. For the first time, we applied single-cell RNA sequencing (scRNA-seq) to analyze tissue samples from three CC patients pre- and post-radiotherapy. We obtained gene expression data from 52,506 cells to identify the cellular changes and molecular mechanisms induced by radiotherapy. Radiotherapy significantly alters cellular composition and gene expression within the tumor microenvironment (TME), notably upregulating mast cell expression. Mast cells are involved in multiple cell axes in the CC ecosystem after radiotherapy, and play a pivotal role in tumor immunosuppression and matrix remodeling. scRNA-seq revealed gene expression variations among cell types after radiotherapy, underscoring the importance of specific cell types in modulating the TME post-treatment. This study revealed the molecular mechanism of radiotherapy for CC and the role of mast cells, providing a foundation for optimizing the personalized treatment of CC.

N-glycosylation of GSTO1 promotes cervical cancer migration and invasion through JAK/STAT3 pathway activation

Protein glycosylation is strongly associated with tumor progression. Glutathione S-transferase omega 1 (GSTO1) is a member of the glutathione S-transferase family. The significance of GSTO1 N-glycosylation in the progression of cervical cancer (CC) has remained elusive. In this study, we investigated the functional significance of GSTO1 N-glycosylation in CC progression. We employed immunohistochemistry to detect the relative expression of evaluating the link between GSTO1 in CC and benign tissues and the overall survival (OS) and progression-free survival (PFS) in CC patients.In vitro and in vivo experiments to detect CC cell proliferation or metastatic ability after GSTO1 downregulation. NetNGly1.0 Server database predicts potential N-glycosylation modification sites of GSTO1 (Asn55, Asn135, Asn190). Investigating GSTO1 N-glycosylation's function in cellular migration, invasion and epithelial-mesenchymal transition (EMT), we mutated the N-glycosylation sites of GSTO1 through lentivirus-based insertional mutagenesis. Detection of signalling pathways associated with N-glycosylation-modified GSTO1 by enrichment analysis and Western blot. Compared to normal cervical tissue, CC tissue showed significantly higher GSTO1 expression. Further, high GSTO1 levels were a poor predictor of OS and PFS. Both cell and animal experiments suggested that down-regulation of GSTO1 inhibited cell proliferation and metastasis. Glycosylation modification of targeted mutant GSTO1 at positions 55, 135 and 190 significantly inhibits migration and invasion of CC cells. GSTO1 N-glycosylation fixed point mutation inhibits EMT process in CC cells. Mechanistically, N-glycosylated GSTO1 promoted the expression of JAK/STAT3 pathway related markers. GSTO1 N-glycosylation is associated with CC progression and may promote EMT via JAK/STAT3 signaling.

NSUN7 promotes cervical cancer progression through activation of ErbB signaling pathway

We aimed to investigate the role of NSUN7 in the progression of Cervical Cancer through a combination of bioinformatics analysis and cell and animal culture experiments. We comprehensively assessed the expression levels of NSUN7 in the TCGA and CCLE databases, and explored its correlations with clinicopathological features, immune cell infiltration, DNA damage repair gene function, drug sensitivity, and methylation status. The NSUN7 gene was disrupted through lentiviral infection, and the effects on cell proliferation, invasion, and apoptosis were evaluated using CCK-8 assay, Transwell migration assay, and flow cytometry analysis. Gene enrichment analysis wasidentify the biological pathways associated with NSUN7 and cervical cancer development. Additionally, a xenograft model of cervical cancer was established to assess the in vivo inhibitory effect of NSUN7 and its impact on pathway molecules. The results of both in vitro and in vivo experiments confirmed that silencing the NSUN7 gene significantly inhibited the growth, spread, and metastasis of cervical cancer cells, while promoting apoptosis. TUNEL assay and HE staining further verified the apoptotic effect of NSUN7 on tumor tissues, and KEGG enrichment analysis revealed a significant enrichment of NSUN7 in the ErbB pathway. Silencing of NSUN7 resulted in a significant down-regulation of key ErbB pathway proteins (HER2, STAT5, PI3K/p-PI3K) as demonstrated by quantitative real-time PCR and Western blot. These findings suggest that NSUN7 may affect the biological behavior of cervical cancer cells and promote tumor development by activating the ErbB signaling pathway.

TBP activates DCBLD1 transcription to promote cell cycle progression in cervical cancer

Discoidin, CUB, and LCCL domain-containing (DCBLD) proteins have been associated with poor prognosis of human cancers. This study investigated the function of DCBLD1 in the development of cervical cancer (CC) and explored its associated mechanism. DCBLD1 was identified as a dysregulated gene in CC via bioinformatics analysis. Immunohistochemistry and RT-qPCR assays revealed increased DCBLD1 expression in CC specimens and cells. Artificial DCBLD1 knockdown blocked the proliferation, invasion, and migration of cells, while promoting cell apoptosis and inducing cell cycle arrest in the G1 phase. Following bioinformatic predictions and subsequent chromatin-immunoprecipitation and luciferase reporter assays, TATA-box binding protein (TBP) was found to be a transcription factor that binds to the DCBLD1 promoter region for transcriptional activation. Knockdown of TBP similarly blocked the malignant properties of CC cells and induced cell cycle arrest, but these changes were reversed by further DCBLD1 overexpression. Xenograft mouse tumors were generated for in vivo validation. Consistently, the tumorigenic activity of CC cells in nude mice was suppressed by TBP knockdown, but restored by DCBLD1 overexpression. In conclusion, this study provides novel evidence that TBP-mediated DCBLD1 activation is correlated with cell cycle and CC progression. TBP and DCBLD1 may serve as potential therapeutic targets for CC management.

Low FHL1 expression indicates a good prognosis and drug sensitivity in ovarian cancer

Chemotherapy resistance is the main reason for the poor prognosis of ovarian cancer (OC). FHL1 is an important tumour regulator, but its relationship with the prognosis, drug resistance, and tumour microenvironment of OC is unknown. Immunohistochemistry was used to determine FHL1 expression in OC. Kaplan‒Meier plotter was used for survival analysis. The value of gene expression in predicting drug resistance was estimated using the area under the curve (AUC). Bivariate correlation was used to determine the coexpression of two genes. Functional cluster and pathway enrichment were used to uncover hidden signalling pathways. The relationship between gene levels and the tumour microenvironment was visualised through the ggstatsplot and pheatmap packages. The mRNA and protein levels of FHL1 were downregulated in 426 and 100 OC tissues, respectively. Low FHL1 expression was correlated with good progression-free survival (PFS), postprogression survival, and overall survival (OS) in 1815 OC patients, and was further confirmed to be associated with good OS by immunohistochemistry in 152 OC tissues. Furthermore, FHL1 was downregulated in drug-sensitive tissues, while its high expression predicted drug resistance (AUC > 0.65). Mechanistically, FHL1 was coexpressed with FLNC, CAV1, PPP1R12B, and FLNA at the mRNA and protein levels in 558 and 174 OC tissues, respectively, and their expression was downregulated in OC. Additionally, very strong coexpression of FHL1 with the four genes was identified in at least 23 different tumours. Low expression of the four genes was associated with good PFS, and the combination of FHL1 with the four genes provided better prognostic power. Meanwhile, the expression of all five genes was strongly and positively associated with the abundance of macrophages. Low FHL1 expression acts as a favourable factor in OC, probably via positive coexpression with FLNC, CAV1, PPP1R12B, and FLNA.

Upregulation of hsa-miR-141-3p promotes uterine cervical carcinoma progression via targeting dual-specificity protein phosphatase 1

We aimed to explore the aberrant expression status of hsa-miR-141-3p and dual-specificity protein phosphatase 1 (DUSP1) and their relative mechanisms in uterine cervical carcinoma (UCC).Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was conducted to detect the expression of hsa-miR-141-3p. Immunohistochemical (IHC) staining was performed to examine the expression of DUSP1 in UCC. Gene chips and RNA-seq datasets were also obtained to assess the expression level. Integrated standardized mean difference (SMD) was calculated to evaluate the expression status of hsa-miR-141-3p in UCC tissues comprehensively. DUSP1-overexpression and hsa-miR-141-3p-inhibition HeLa cells were established, and CCK-8, transwell, wound healing, cell cycle, and apoptosis assays were implemented. The targets of hsa-miR-141-3p were obtained with online tools, and the combination of hsa-miR-141-3p and DUSP1 was validated via dual-luciferase reporter assay. Single-cell RNA-seq data were analyzed to explore hsa-miR-141-3p and DUSP1 in different cells. An integrated SMD of 1.41 (95% CI[0.45, 2.38], p = 0.0041) with 558 samples revealed the overexpression of hsa-miR-141-3p in UCC tissues. And the pooled SMD of -1.06 (95% CI[-1.45, -0.66], p < 0.0001) with 1,268 samples indicated the downregulation of DUSP1. Inhibition of hsa-miR-141-3p could upregulate DUSP1 expression and suppress invasiveness and metastasis of HeLa cells. Overexpression of DUSP1 could hamper proliferation, invasion, and migration and boost apoptosis and distribution of G1 phase. The dual-luciferase reporter assay validated the combination of hsa-miR-141-3p and DUSP1. Moreover, the targets of hsa-miR-141-3p were mainly enriched in the MAPK signaling pathway and activated in fibroblasts and endothelial cells. The current study illustrated the upregulation of hsa-miR-141-3p and the downregulation of DUSP1 in UCC tissues. Hsa-miR-141-3p could promote UCC progression by targeting DUSP1.

Advances in application of circulating tumor DNA in ovarian cancer

Ovarian cancer is the third most common gynecologic cancer worldwide and has the highest mortality rate among gynecologic cancers. Identifying timely and effective biomarkers at different stages of the disease is the key to improve the prognosis of ovarian cancer patients. Circulating tumor DNA (ctDNA) is a fragment of free DNA produced by tumor cells in the blood. Current techniques for detecting ctDNA mainly include quantitative polymerase chain reaction (PCR), targeted next-generation sequencing (NGS), and non-targeted NGS (such as whole exon or whole genome sequencing). As a non-invasive liquid biopsy technique, ctDNA has a good application prospect in the ovarian cancer diagnosis, monitoring of treatment response and efficacy evaluation, detection of reverse mutation and related medication guidance, and prognosis evaluation. This article reviews the advances in application of ctDNA in ovarian cancer.

SORL1 stabilizes ABCB1 to promote cisplatin resistance in ovarian cancer

Ovarian cancer (OC) has the worst prognosis among gynecological malignancies. Cisplatin (CDDP) is one of the most commonly used treatments for OC, but recurrence and metastasis are common due to endogenous or acquired resistance. High expression of ATP-binding cassette (ABC) transporters is an important mechanism of resistance to OC chemotherapy, but targeting ABC transporters in OC therapy remains a challenge. The expression of sortilin-related receptor 1 (SORL1; SorLA) in the response of OC to CDDP was determined by analysis of TCGA and GEO public datasets. Immunohistochemistry and western blotting were utilized to evaluate the expression levels of SORL1 in OC tissues and cells that were sensitive or resistant to CDDP treatment. The in vitro effect of SORL1 on OC cisplatin resistance was proven by CCK-8 and cell apoptosis assays. The subcutaneous xenotransplantation model verified the in vivo significance of SORL1 in OC. Finally, the molecular mechanism by which SORL1 regulates OC cisplatin resistance was revealed by coimmunoprecipitation, gene set enrichment analysis and immunofluorescence analysis. This study demonstrated that SORL1 is closely related to CDDP resistance and predicts a poor prognosis in OC. In vivo xenograft experiments showed that SORL1 knockdown significantly enhanced the effect of CDDP on CDDP-resistant OC cells. Mechanistically, silencing of SORL1 inhibits the early endosomal antigen 1 (EEA1) pathway, which impedes the stability of ATP-binding cassette B subfamily member 1 (ABCB1), sensitizing CDDP-resistant OC cells to CDDP. The findings of this study suggest that targeting SORL1 may represent a promising therapeutic approach for overcoming CDDP resistance in OC.

LncRNA SLC25A21-AS1 increases the chemosensitivity and inhibits the progression of ovarian cancer by upregulating the expression of KCNK4

Owing to high mortality rate, ovarian cancer seriously threatens women's health. Extensive abdominal metastasis and chemoresistance are the leading causes of ovarian cancer deaths. Through lncRNA sequencing, our previous study identified lncRNA SLC25A21-AS1, which was significantly downregulated in chemoresistant ovarian cancer cells. In this study, we aimed to evaluate the role and mechanism of SLC25A21-AS1 in ovarian cancer. The expression of SLC25A21-AS1 was analyzed by qRT-PCR and online database GEPIA. The biological functions of SLC25A21-AS1 and KCNK4 were analyzed by CCK-8, transwell, and flow cytometry. The specific mechanism was analyzed by RNA-sequencing, RNA binding protein immunoprecipitation, rescue experiments, and bioinformatic analysis. SLC25A21-AS1 was decreased in ovarian cancer tissues and cell lines. Overexpression of SLC25A21-AS1 enhanced the sensitivity of ovarian cancer cells to paclitaxel and cisplatin, and inhibited cell proliferation, invasion, and migration, while SLC25A21-AS1-silencing showed the opposite effect. Potassium channel subfamily K member 4 (KCNK4) was significantly up-regulated upon enforced expression of SLC25A21-AS1. Overexpression of KCNK4 inhibited cell proliferation, invasion, migration ability, and enhanced the sensitivity of ovarian cancer cells to paclitaxel and cisplatin. Meanwhile, KNCK4-overexpression rescued the promotive effect of SLC25A21-AS1-silencing on cell proliferation, invasion and migration. In addition, SLC25A21-AS1 could interact with the transcription factor Enhancer of Zeste Homolog 2 (EZH2), while EZH2 knockdown increased the expression of KCNK4 in some of the ovarian cancer cell lines. SLC25A21-AS1 enhanced the chemosensitivity and inhibited the proliferation, migration, and invasion ability of ovarian cancer cells at least partially by blocking EZH2-mediated silencing of KCNK4.

Regulation of m6A (N6-Methyladenosine) methylation modifiers in solid cancers

High expression of formin-2 can promote ovarian cancer chemoresistance via immunosuppressive macrophages

Ovarian cancer (OC) remains a major threat to women's health, with chemoresistance driven by the immunosuppressive tumor microenvironment. Formin-2 (FMN2), a cytoskeletal regulator, was investigated for its role in OC chemoresistance and macrophage polarization. Bioinformatics analysis identified high FMN2 expression in chemotherapy-resistant OC cell lines, validated experimentally. Stable FMN2 knockdown cell lines were generated via lentiviral transfection. Functional assays revealed that FMN2 overexpression conferred chemoresistance in vitro and in vivo and promoted M2 macrophage polarization via the CCL2/JAK2/STAT3 pathway. Co-culture with M2 macrophages enhanced cisplatin (DDP) resistance in OC cells, mediated by CXCL1 secretion, which activated the epithelial-mesenchymal transition (EMT) pathway. Clinically, FMN2 levels correlated with CCL2 and CD206 (M2 marker) in platinum-resistant patients, and high FMN2, CCL2, or CD206 expression predicted poorer overall and disease-free survival. This study identifies FMN2 as a key mediator of chemoresistance and immune evasion in OC, proposing FMN2-CCL2-CD206 signaling and macrophage-derived CXCL1 as therapeutic targets and prognostic markers for chemotherapy response.

Application of single cell sequencing technology in ovarian cancer research (review)

Ovarian cancer is a malignant tumor of ovary. It has the characteristics of difficult early diagnosis, poor late curative effect and high recurrence rate. It is the biggest disease that seriously threatens women's health. Single cell sequencing technology refers to sequencing the genetic information carried by it at the single cell level to obtain the gene sequence, transcript, protein and epigenetic expression profile information of a certain cell type and conduct integrated analysis. It has unique advantages in the study of tumor occurrence and evolution, and can provide new methods for the study of ovarian cancer. This paper reviews the single cell sequencing technology and its application in ovarian cancer.

CRISPR-Cas9-mediated deletion enhancer of MECOM play a tumor suppressor role in ovarian cancer

MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion.

MATN2 overexpression suppresses tumor growth in ovarian cancer via PTEN/PI3K/AKT pathway

The incidence rate of developing ovarian cancer decreases over the years; however, mortality ranks top among malignancies of women, mainly metastasis through local invasion. Matrilin-2 (MATN2) is a member of the matrilin family that plays an important role in many cancers. However, its relationship with ovarian cancer remains unknown. Our study aimed to explore the function and possible mechanism of MATN2 in ovarian cancer. Human ovarian cancer tissue microarrays were used to detect the MATN2 expression in different types of ovarian cancer using immunohistochemistry (IHC). CCK-8, wound scratch healing assay, transwell assay, and flow cytometry were used to detect cell mobility. Gene and protein expression were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. MATN2 interacts with phosphatase, and the tensin homolog (PTEN) deleted on chromosome 10 was analyzed using TCGA database and co-immunoprecipitation (Co-IP). In vivo experiments were conducted using BALB/c nude mice, and tumor volume and weight were recorded. Tumor growth was determined using hematoxylin and eosin (H&E) and IHC staining. MATN2 was significantly downregulated in ovarian cancer cells. The SKOV3 and A2780 cell mobility was significantly inhibited by MATN2 overexpression, while the cell apoptosis rate was significantly increased. MATN2 overexpression decreased transplanted tumor size in vivo. These results were reversed by inhibiting MATN2. Furthermore, we found that PTEN closely interacted with MATN2 using bioinformatics and Co-IP. MATN2 overexpression significantly inhibited the PI3K/AKT pathway, however, PTEN suppression reversed this effect of MATN2 overexpression. These results indicated that MATN2 may play a critical role in ovarian cancer development by inhibiting cells proliferation and migration. The mechanism was related to interacting with PTEN, thus inhibiting downstream effectors in the PI3K/AKT pathway, which may be a novel target for treating ovarian cancer.

CRISPR du-HITI an attractive approach to targeting Long Noncoding RNA HCP5 as inhibitory factor for proliferation of ovarian cancer cell

This research provides a glimmer of hope that the knockout of HCP5 leads to a therapy response to considerably prolong the life of patients with OC. RT-PCR evaluated the expression of lncRNA HCP5 in the ovarian cancer OVCAR-3 cell line. CRISPR knockout cell lines validated by western blot. Small genomic deletions at the targeted locus were induced. CCK-8 colony formation assays were used to analyze the effect of HCP5 knockout on the proliferation capacity of OVCAR-3 cells. Transwell migration and invasion assayed. Furthermore, the Sphere-formation assay isolated the most aggressive population of cancer stem cells. Bioinformatic analysis showed a significant correlation between lncRNA HCP5 up-regulation and OVCAR-3 cell proliferation. The ChIP technique assesses specific sites of interaction between transcription factors and DNA. Real-time PCR assays explored the relationship between HCP5, Hsa-miR-9-5p, CXCR4, CDH1, caspase-3, p53, bcl2 and survivin. PCR carried out amplification of the 448-bp band for sgRNA1 and sgRNA2 after the use of particular primers for HCP5. the number of breast cancer cells that moved to the bottom chamber reduced considerably after transfection with PX461-sgRNA1/2 vectors compared to the Blank control groups (P < 0.05). MTT assay designated growth curves that showed the rate of OVCAR-3 growth was significantly repressed (***P < 0.001) when compared with control OVCAR-3 cells after HCP5 knockdown. Also, the survival results of W.T cells in 24, 48 and 72 h showed 92%, 87% and 85%, respectively. This is while the cells of the CRISPR/Cas9 group in which LncRNA HCP5 was knocked out had 42% (*P < 0.05), 23%(**P < 0.01) and 14% (**P < 0.01) survival, respectively. The expression levels of caspase-3, Hsa-miR-9-5p, P53 genes in the HCP5 deletion of CRISPR/Cas9 group significantly increased than the W.T. control group; the deletion group showed a considerable reduction in HCP5 expression compared to the blank control group (3.6-fold, p < 0.01). Whereas BCL2, SURVIVIN, CXCR4, CDH1 genes expression markedly increased than in HCP5 knockout cells (5.8-fold, p < 0.05). These results indicate that CRISPR/Cas9-mediated HCP5 disruption on OVCAR-3 cell lines promotes anti-tumor biomarkers, suppressing ovarian cancer progression. Consistent with these results, HCP5 is one of the most critical lnc for the efficient proliferation and migration of OVCAR-3 cell lines.

Transcription factor Dp-1 knockdown downregulates thymidine kinase 1 expression to protect against proliferation and epithelial-mesenchymal transition in cervical cancer

Thymidine kinase 1 (TK1) level is an independent survival prognostic factor for both premalignant and malignant cervical pathologies. Herein, this study sought to probe the impacts of TK1 on cervical cancer (CC) progression and its underlying mechanism. Transcription factor Dp-1 (TFDP1) and TK1 expression was assessed using qRT-PCR in CC cell lines. After ectopic expression and knockdown experiments, cell counting kit-8 and colony formation assays were adopted to measure cell proliferation, western blot to examine the expression of epithelial-mesenchymal transition (EMT)-related proteins, and Transwell assays to assess cell invasion and migration. The binding of TFDP1 to TK1 was predicted by bioinformatic sites and verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Tumor xenograft experiments in nude mice were performed to validate the influence of TFDP1/TK1 on CC progression in vivo. CC cells had high TK1 and TFDP1 expression. TFDP1 or TK1 knockdown restrained CC cell EMT, invasion, migration, and proliferation. TFDP1 facilitated TK1 expression in CC via transcription. Overexpression of TK1 counteracted the suppressive impacts of TFDP1 knockdown on CC cell malignant behaviors. Moreover, TFDP1 knockdown depressed CC growth in vivo by downregulating TK1. TFDP1 knockdown restricted proliferation and EMT in CC by downregulating TK1 expression.

Identification of seven hypoxia-related genes signature and risk score models for predicting prognosis for ovarian cancer

Abstract Ovarian cancer (OC) is the most common malignant cancer in the female reproductive system. Hypoxia is an important part of tumor immune microenvironment (TIME), which is closely related to cancer progression and could significantly affect cancer metastasis and prognosis. However, the relationship between hypoxia and OC remained unclear. OCs were molecularly subtyped by consensus clustering analysis based on the expression characteristics of hypoxia-related genes. Kaplan–Meier (KM) survival was used to determine survival characteristics across subtypes. Immune infiltration analysis was performed by using Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) and microenvironment cell populations-counter (MCP-Counter). Differential expression analysis was performed by using limma package. Next, univariate Cox and least absolute shrinkage and selection operator (LASSO) regression analyses were used to build a hypoxia-related risk score model (HYRS). Mutational analysis was applied to determine genomic variation across the HYRS groups. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to compare the effectiveness of HYRS in immunotherapy prediction. We divided OC samples into two molecular subtypes (C1 and C2 subtypes) based on the expression signature of hypoxia genes. Compared with C1 subtype, there was a larger proportion of poor prognosis genotypes in the C2 subtype. And most immune cells scored higher in the C2 subtype. Next, we obtained a HYRS based on 7 genes. High HYRS group had a higher gene mutation rate, such as TP53. Moreover, HYRS performed better than TIDE in predicting immunotherapy effect. Combined with clinicopathological features, the nomogram showed that HYRS had the greatest impact on survival prediction and a strong robustness.

Transcriptome sequencing profiles of cervical cancer tissues and SiHa cells

High-risk human papillomavirus (HPV) is a causal factor for cervical cancer, of which HPV16 is the predominant genotype, but the detailed mechanism remains to be elucidated. In this study, we performed transcriptome sequencing in cervical cancer tissues with HPV16-positive and normal tissues with HPV16-negative, and SiHa cells with or without HPV16 E6/E7 knockdown, and identified 140 differential expressed genes (DEGs) in two data sets. We carried out a series of bioinformatic analyses to learn more about the 140 DEGs, and found that 140 DEGs were mostly enriched in cell cycle and DNA repair through Kyoto Encyclopedia of Genes and Genomes pathway enrichment, Gene Ontology annotation, and gene set enrichment analysis. A total of 20 genes including RMI1, MKI67, FANCB, KIF14, CENPI, RACGAP1, EXO1, KIF4A, FOXM1, C19orf57, PSRC1, NUSAP1, CIT, NDC80, MCM7, GINS2, MCM6, ORC1, TLX2, and UHRF1 were screened by co-expression analysis; of those, the expressions of 6 (CENPI, FANCB, KIF14, ORC1, RACGAP1, and RMI1) were verified by qRT-PCR. Further, we found that E2F family, NF-Y, AhR:Arnt, and KROX family may be involved in modulating DEGs by TransFind prediction. TF2DNA database and co-expression analysis suggested that 12 TFs (ZNF367, TLX2, DEPDC1B, E2F8, ZNF541, EGR2, ZMAT3, HES6, CEBPA, MYBL2, FOXM1, and RAD51) were upstream modulators of DEGs. Our findings may provide a new understanding for effects of HPV oncogenes in the maintenance of cancerous state at the transcriptional level.

NEDD4 facilitates the progression of endometrial carcinoma by enhancing PAMR1 protein degradation through ubiquitination

Endometrial carcinoma (EC) is an epithelial malignant neoplasm that frequently appears in postmenopausal and perimenopausal women. PAMR1 is related to the prognosis of EC. Here, we probed into the significance of PAMR1 in EC progression and its acting mechanism. In silico analysis was conducted to identify the differentially expressed gene PAMR1 and its upstream gene NEDD4 in EC, followed by the determination of their expression in EC tissues and cells. The gene expression, cell proliferation, angiogenesis, migration, invasion, and apoptosis were examined after ectopic expression or knockdown experiments. The interaction between NEDD4 and PAMR1 and the level of PAMR1 ubiquitination were examined. The injection of Ishikawa cell suspensions into nude mice was carried out to establish a tumor xenograft model, validating the roles of PAMR1 and NEDD4 in EC. EC cells exhibited high NEDD4 expression and low PAMR1 expression. NEDD4 knockdown or PAMR1 overexpression suppressed the invasive, migrating, angiogenic, and proliferative properties of EC cells while promoting apoptosis. NEDD4 facilitated PAMR1 protein degradation through ubiquitination. Deletion of PAMR1 abolished the inhibitory effects of NEDD4 knockdown on the malignant behaviors of EC cells. Furthermore, NEDD4 knockdown restrained EC growth in nude mice by increasing PAMR1 protein expression. NEDD4 facilitated EC progression by enhancing PAMR1 protein degradation through ubiquitination.

HNRNPA2B1-mediated m6A modification of FOXM1 promotes drug resistance and inhibits ferroptosis in endometrial cancer via regulation of LCN2

N6-methyladenosine (m6A) methylation is an extensive posttranscriptional RNA modification, and it is associated with various cellular responses, especially in tumor progression. An m6A "reader"-HNRNPA2B1 has been found oncogenic in multiple malignancies. As a key proliferation-related transcription factor, forkhead box protein M1 (FOXM1) is involved in tumorigenesis. Here, we elucidated the underlying mechanism by which HNRNPA2B1-mediated modification of FOXM1 promotes endometrial cancer (EC). The GSE115810 dataset was used to analyze the upregulated gene mRNA in late-stage EC tissues. The expression levels of HNRNPA2B1, FOXM1, and LCN2 in EC samples were shown by western blotting and qPCR. The interaction among HNRNPA2B1, FOXM1, and LCN2 in EC cells was detected using bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down, RNA decay analysis, and luciferase reporter experiments. Cisplatin (DDP)-resistant EC cells were constructed using HEC-1-A and HEC-1-B cells, named HEC-1-A/DDP and HEC-1-B/DDP, respectively. Proliferation, migration, and invasiveness in treated HEC-1-A/DDP and HEC-1-B/DDP cells were detected by EdU, wound healing, and transwell assays. Ferroptosis-resistant gene expression, MDA level, and ROS level were measured. The m6A modification level in EC tissues was elevated. HNRNPA2B1 and FOXM1 levels were upregulated in EC. HNRNPA2B1 expression was positively related to FOXM1 expression in EC samples, and HNRNPA2B1 bound to the 3'UTR of FOXM1 and stabilized FOXM1 mRNA via m6A modification. FOXM1 positively regulated LCN2 expression in EC cells by binding to the LCN2 promotor. Knockdown of FOXM1 downregulated ferroptosis-resistant gene expression and increased MDA and ROS levels in DDP-resistant EC cells. Rescue assays revealed that LCN2 overexpression eliminated the effects mediated by FOXM1 knockdown on the proliferation, migration, invasiveness, and ferroptosis in DDP-resistant EC cells. In conclusion, HNRNPA2B1-mediated mA modification of FOXM1 facilitates drug resistance and inhibits ferroptosis in EC cells by upregulating LCN2 expression.