Genes & Genomics

Hypoxia-induced PLOD2 regulates invasion and epithelial-mesenchymal transition in endometrial carcinoma cells

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was induced in hypoxia and participated in cancer development. However, the role of PLOD2 in endometrial carcinoma remains unclear. To explore the influences and regulation mechanism of PLOD2 in endometrial carcinoma under hypoxic condition. The small interfering RNA (siRNA) targeting to PLOD2 and pcDNA3.1-PLPD2 were transfected to endometrial carcinoma cells to alter PLOD2 expression. Cell proliferation ability was determined by colony formation assay. Wound healing assay used to detect cell migration ability. Transwell invasion assay was used to detect cell invasion ability. PLOD2 and Hypoxia-inducible factor-1α (HIF-1α) were induced by hypoxia. Down-regulation of PLOD2 did not affect endometrial carcinoma cell proliferation ability, while inhibited cell migration, invasion under hypoxic condition. Besides, down-regulation of PLOD2 increased the levels of γ-catenin and E-cadherin and decreased levels of Fibronectin and Snail under hypoxic condition. Down-regulation of PLOD2 also inactivated Src and phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) signaling under hypoxic condition. The promoting effects of PLOD2 overexpression on migration, invasion and epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells were reversed by Akt inhibitor (MK2206) under hypoxic condition. PLOD2 expression was increased in endometrial carcinoma cells under hypoxic condition. PLOD2 modulated migration, invasion, and EMT of endometrial carcinoma cells via PI3K/Akt signaling. PLOD2 may be a potential therapeutic target for endometrial carcinoma.

LncRNA LINC01123 promotes malignancy of ovarian cancer by targeting hsa-miR-516b-5p/VEGFA

Long non-coding RNAs (lncRNAs) play a critical role in the development of ovarian cancer (OC). The study aimed to determine the role of LncRNA LINC01123 in OC bio-progression, which is upregulated in OC tissues during OC progression. Bioinformatics methods, GEPIA, and qRT-PCR were used to reveal the level and correlation of LINC01123, hsa-miR-516b-5p, and VEGFA, in OC cell lines. MTT, EdU, TUNEL, and Transwell assays were performed to assess the bioactivity of OC cell. Target sites of LINC01123 and hsa-miR-516b-5p were predicted using Starbase, and the potential linkage points of VEGFA and hsa-miR-516b-5p were predicted using TargetScan. These sites and linkage points were confirmed by double luciferase reporter assay. LINC01123 was upregulated in OC cell lines and LINC01123 silencing suppressed the proliferation and metastasis of OC cells, but promoted cell apoptosis. hsa-miR-516b-5p was linked to LINC01123 and. VEGFA was downstream of hsa-miR-516b-5p. Importantly, silencing of hsa-miR-516b-5p reversed the inhibitory impact of si-LINC01123. The result of hsa-miR-516b-5p inhibitor + si-LINC01123 co-transfection were rescued by si-VEGFA. LINC01123 promotes OC development by dampening miR-516b-5p function, and may be a novel target for treating OC.

MiR-873, as a suppressor in cervical cancer, inhibits cells proliferation, invasion and migration via negatively regulating ULBP2

Cervical cancer (CC) remains a large burden in the developing countries. The tumor inhibitory role of miR-873 has been verified in a variety of cancers, however, whether miR-873 has a suppressive effect on CC remains unclear. The purpose of this study was to investigate the functional role of miR-873 in CC, as well as explore the underlying molecular mechanism. The prognostic values of miR-873 were assessed by Kaplan-Meier methods and cox regression models using the data which were downloaded from TCGA database. The expression of miR-873 was measured by RT-qPCR. Cell counting Kit-8, clone formation, and Transwell assays were used to assess the cell viability and metastasis, appropriately. The targeting relationship between miR-873 and ULBP2 was predicted by biological software and confirmed by dual luciferase reporter assay. Rescue assays were conducted to investigate whether miR-873 affects the phenotype of CC cells via regulating ULBP2. We observed that miR-873 was low-expressed in CC. Up-regulation of miR-873 notably restrained the proliferation, invasion and migration of C33a cells. Meanwhile, down-regulation of miR-873 in SiHa cells presented the opposite outcomes. ULBP2 was forecasted and certified as a target of miR-873. The results of rescue assays showed that overexpression of ULBP2 could restore the proliferation and motility of CC cells that inhibited by miR-873. MiR-873 suppressed the CC cells proliferation, invasion and migration via negatively regulating ULBP2, suggesting that miR-873 could serve as a valuable therapeutic target for CC therapy.

Mir-326 potentiates radiosensitivity of cervical squamous cell carcinoma through downregulating SMO expression in the Hedgehog signaling pathway

Radiotherapy resistance affects the therapeutic effect of cervical squamous cell carcinoma (CSCC). Smoothened (Smo) is an anticancer target of the Hedgehog (Hh) pathway and its mutation is related to drug resistance. To explore the roles of miR-326 and Smoothened (SMO) on radiation resistance in patients with cervical carcinoma. Expression of miR-326 and SMO in cervical cancer tissue and radioresistant cell lines were analyzed. The radiation response with the expression of miR-326 was evaluated in tissue and cells. Bioinformatics analysis and literature review were performed to explore the target of miR-326. The regulation of miR-326 to SMO mRNA was verified through the dual-luciferase reporter assay. Patients with poor radiation response have lower miR-326 and higher SMO expression. Upregulation of miR-326 decreased SMO expression and its downstream proteins but does not affect the proliferation of CSCC cells. The upregulation of miR-326 increased radiation sensitivity of the CSCC cell through downregulating SMO and its downstream proteins in the Hedgehog (Hh) signaling pathway. miR-326 may predict the treatment response to radiation, and upregulating miR-326 may improve the treatment response to radiation.

ANXA3, associated with YAP1 regulation, participates in the proliferation and chemoresistance of cervical cancer cells

Cervical cancer, as one of the most common cancers in women, remains a major health threat worldwide. Annexin A3 (ANXA3), a component of the annexin family, is upregulated in numerous cancers, with no explicit role in cervical cancer. This study aims to investigate the function of ANXA3 in cervical cancer. Differential expression genes between the cervical cancer tissues of patients and the controls were analyzed in The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) database. Using transfection approaches to either upregulate or downregulate ANXA3, its role in cell proliferation and chemosensitivity of human cervical cancer cell lines (HeLa and C33A) was evaluated. Furthermore, the binding activity between YAP1 and ANXA3 was also explored. Genomics analysis indicated that differential genes were mostly associated with cell cycle progression and DNA replication. ANXA3 was highly expressed in the cervical cancer tissues and closely linked to malignancy degree. Knockdown of ANXA3 in cervical cancer cells inhibited cell cycle progression. A similar result was observed in the reduction of cyclin D, CDK4, cyclin E, and CDK2 in cervical cancer cells with ANXA3 silencing. Cervical cancer cells obtained high sensitivity to cisplatin (DDP) when ANXA3 was downregulated. Conversely, these capabilities were the opposite in cervical cancer cells overexpressing ANXA3. Furthermore, the expression levels of ANXA3 and YAP1 were positively correlated. YAP1 upregulation was positively connected with malignant behaviors, which were reversed by ANXA3 downregulation. In light of our findings, targeting ANXA3 expressed in cervical cancer might contribute to more potential therapeutic strategies.

Inhibition of FOSL2 aggravates the apoptosis of ovarian cancer cells by promoting the formation of inflammasomes

Abstract Background Ovarian cancer is a common gynecological malignancy among female patients and poses a serious threat to women’s health. Although it has been established that Fos-like antigen 2 (FOSL2) is linked to ovarian cancer (OC), its exact role in the development of OC remains unknown. Objective This article aims to investigate the role of FOSL2 in ovarian cancer development. Methods FOSL2 expression in ovarian carcinoma and adjacent tissues was assessed using real-time fluorescent quantitative PCR and western blot. We constructed OE/sh-FOSL2 plasmids and Caspase-1 specific inhibitors (Yvad-CMK) and transfected A 2780 cells with them to identify the relevant cell functions. Furthermore, we used western blot assay to determine the changes in expression of apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific proteasezymogen procaspase 1 (pro-caspase-1), cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1β precursor (pro-IL-1β), interleukin-1β (IL-1β), interleukin-18 precursor (pro-IL-18), and interleukin-18 (IL-18). In addition, we measured the concentration of IL-1β and IL-18 using an enzyme-linked immunosorbent assay (ELISA). Moreover, Tthe level of lactate dehydrogenase (LDH) in the cell supernatant was measured by LDH release assay kit. Results The expression of FOSL2 was significantly higher compared with the surrounding tissues. The proliferation, migration, and invasion of A2780 cells were enhanced after transfection with OE-FOSL2 plasmids; however, the cell apoptosis was significantly decreased. When FOSL2 was overexpressed, the inflammasome-associated proteins such as ASC, caspase-1, IL-1β, and IL-18 were downregulated. Furthermore, FOSL2 induced apoptosis and activated the production of inflammasomes in A2780 cells. Co-therapy with Yvad-CMK and substantially inhibited apoptosis and activation of inflammasomes. Conclusions Inhibition of FOSL2 promotes the apoptosis of OC cells by mediating the formation of an inflammasome.

EGR1 promotes stemness and predicts a poor outcome of uterine cervical cancer by inducing SOX9 expression

Early growth response-1 (EGR1) is a transcription factor involved in the progression of several cancer types. However, the expression and clinical significance of EGR1 in uterine cervical cancer (CC) have not been elucidated. To investigate the expression, clinical significance and prognostic value of EGR1 in CC. The expression of EGR1 was detected in 13 CCs and paired adjacent tissues with qRT-PCR and in 144 CC tissues with immunohistochemistry (IHC). The IHC scores were used to divide the patients into subsets with low and high EGR1 expression. The correlations between the EGR1 expression and clinicopathological factors were analyzed with the chi-square test, and the prognostic significance of EGR1 expression was evaluated with univariate and multivariate analyses. The functions of EGR1 in the proliferation, invasion and stemness of CC cells were investigated, and the molecular mechanism was assessed by in vitro experiments. High expression of EGR1 was significantly associated with low survival rates of CC. EGR1 is an independent prognostic biomarker of CC, and its high expression predicted a poor outcome. EGR1 facilitated stemness and thus promoted proliferation and invasion of CC cells. SOX9 played an essential role in the EGR1-induced progression of CC cells. EGR1 is an independent prognostic biomarker of CC. High EGR1 expression promoted proliferation, invasion and stemness by increasing SOX9 expression in CC cells. Our results suggested that the EGR1-SOX9 axis may be a potential drug target and that blocking the EGR1-SOX9 axis may be a possible approach to treating CC.

Iron metabolism protein transferrin receptor 1 involves in cervical cancer progression by affecting gene expression and alternative splicing in HeLa cells

Transferrin receptor 1 (TfR1), encoded by TFRC, is a key regulator of iron homeostasis and plays important roles in many diseases, including cancers. To decipher the underlying molecular functions of TfR1 based on its influence on transcriptome profile in cancer cells. In this study, we first identified the expression pattern and prognostic influence of TFRC in cervical cancer patients from TCGA database. To explore the regulatory outcomes of TfR1 from the view of whole transcriptome profile, we generated TFRC knockdown (TFRC-KD) HeLa cells and negative control (NC) cells using short hairpin RNA (shRNA) method. Unbiased transcriptome sequencing (RNA-seq) experiment was used to analyze the global expression level and alternative splicing (AS) changes between TFRC-KD and NC cells. We found TFRC was consistently elevated in cervical cancer samples and tightly associated with prognosis of patients. Differential expression analysis revealed that 629 differentially expressed genes (DEGs) were identified between TFRC-KD and NC. Functional enrichment analysis of these DEGs revealed that TFRC-KD extensively disturbed cell physiology related pathways, including immunity, cell metabolism and gene expression. Moreover, dysregulated AS profile also indicated that TfR1 has important roles in the AS regulation. Hundreds of TfR1-regulated AS genes were involved in DNA repair, cell death, transcription and viral reproduction pathways, which were tightly associated with cancer cell progression. In summary, we for the first time explored the molecular functions of TfR1 at transcriptional and post-transcriptional levels. These results demonstrate TfR1 participates in the progression of cervical cancer by affecting the expression and AS levels of genes in cancer associated pathways, which greatly extends our understanding of TfR1 functions besides iron homeostasis and provide novel options in cancer treatment by targeting TfR1.

Effects of Zonula occludens-1 (ZO-1) tight junction protein on tumor characteristics in human ovarian cancer cells

Ovarian cancer is among the most lethal malignancies affecting women, largely due to its asymptomatic progression in early stages, rapid advancement, and high mortality rate. Tight junction protein 1 (TJP1), also known as Zonula occludens-1 (ZO-1), plays a critical role in epithelial and endothelial cell integrity by regulating paracellular permeability. Additionally, ZO-1 is involved in cell-cell communication networks, influencing cellular proliferation, differentiation, and metastasis. While previous studies have demonstrated the significance of ZO-1 in tumorigenesis and cancer progression, its precise mechanistic role remains to be fully elucidated. This study aims to investigate the functional role of ZO-1 in human ovarian cancer cells to provide a molecular perspective on its impact on tumor progression. Human ovarian cancer cell lines SNU119 and SKOV3 were utilized. ZO-1 knockout (KO) was achieved using the Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system in combination with single-guide RNA (sgRNA) targeting ZO-1. Hygromycin B selection was employed to establish stable ZO-1 KO SNU119 and ZO-1 KO SKOV3 cell lines. The successful knockout of ZO-1 was confirmed at both the transcript and protein levels via real-time quantitative PCR (RT-qPCR) and Western blotting. Functional assays, including cell proliferation, migration, and invasion assays, were conducted to assess the effects of ZO-1 KO on key tumor-associated characteristics. CRISPR-Cas9-mediated ZO-1 KO in SNU119 and SKOV3 ovarian cancer cell lines resulted in a significant reduction of ZO-1 expression at both the transcript and protein levels. The loss of ZO-1 led to a disruption of cell-cell junctions. Functionally, ZO-1 KO cells exhibited reduced proliferation, whereas cell migration and invasion were significantly enhanced, suggesting a shift toward a more aggressive phenotype. The findings indicate that ZO-1 KO in ovarian cancer cells suppresses cell proliferation while promoting migratory and invasive properties, hallmarks of tumor progression. These results underscore the complex role of ZO-1 in ovarian cancer and highlight the need for further investigation into its broader regulatory impact on oncogenic pathways.

Overexpression of TRIM44 mediates the NF-κB pathway to promote the progression of ovarian cancer

Ovarian cancer (OC) is the second most commonly seen cancer in the US, and patients with OC are commonly diagnosed in the advanced stage. Research into the molecular mechanisms and potential therapeutic targets of OC is becoming increasingly urgent. In our study, we worked to discover the role of TRIM44 in OC development. This study explored whether the overexpression of TRIM44 mediates the NF-kB pathway to promote the progression of OC. A TRIM44 overexpression model was constructed in SKOV3 cells, and the proliferation ability of the cells was detected using the CCK-8 assay. The migration healing ability of cells was detected using cell scratch assay. Cell migration and invasion were detected using Transwell nesting. TUNEL was applied to detect apoptosis, and ELISA and western blot were used to detect the expression of NF-κB signaling pathway proteins. The pathological changes of the tumor tissues were observed using HE staining in a mouse ovarian cancer xenograft model. Immunofluorescence double staining, RT-PCR, and western blot were used to determine the expression of relevant factors in tumour tissues. TRIM44 overexpression promoted the proliferation, migration, and invasion of SKOV3 cells in vitro and inhibited apoptosis while enhancing the growth of tumours in vivo. TRIM44 regulated the NF-κB signaling pathway. TRIM44 overexpression can regulate the NF-κB signaling pathway to promote the progression of OC, and TRIM44 may be a potential therapeutic target for OC.

Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells

Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

Effects of thymosin β4-derived peptides on migration and invasion of ovarian cancer cells

Thymosin β4 (Tβ4) is a highly conserved actin binding protein associated with the metastatic potential of tumor cells by stimulating cell migration. The role of Tβ4 and its derived fragment peptides in migration of ovarian cancer cells has not been studied. To analyze the effects of Tβ4 and its derived fragment peptides on ovarian cancer cell migration and invasion, we applied Tβ4 and three Tβ4-derived synthetic peptides to SKOV3 ovarian cancer cells. The migration and invasion of SKOV3 cells treated with Tβ4(1-43), Tβ4(1-15), Tβ4(12-26), Tβ4(23-), and untreated control were analyzed by in vitro migration and invasion assay with transwell plate. Cell proliferation assay was conducted to identify the effect of Tβ4 and its derived peptide on SKOV3 cell proliferation. The expression of Tβ4 related proteins related with cell proliferation was analyzed by Western blot after treatment with Tβ4 and its derived peptides. Cell migration and invasion were significantly increased in Tβ4 peptide-treated SKOV3 cells compared with untreated control. All three Tβ4-derived fragment peptides including those without an actin binding site significantly stimulated migration and invasion of SKOV3 cells. Tβ4 and its derived peptide significantly stimulated SKOV3 cell proliferation and up-regulated the expression of RACK-1 protein. The Tβ4 peptide and all of its derived fragment peptides including those without an actin binding motif stimulate migration and invasion of SKOV3 ovarian cancer cells. All peptides significantly increased RACK-1 expression and cell proliferation of SKOV3 cells. These results suggest that Tβ4 stimulates migration and invasion of SKOV3 cells by stimulation of cell proliferation through up-regulation of RACK-1 protein.

Effect of human endogenous retrovirus-K env gene knockout on proliferation of ovarian cancer cells

Among various human endogenous retroviruses (HERVs), the HERV-K (HML-2) group has been reported to be highly related to cancer. In pancreatic cancer cells, shRNA-mediated downregulation of HERV-K env RNA decreases cell proliferation and tumor growth through the RAS-ERK-RSK pathway; in colorectal cancer, CRISPR-Cas9 knockout (KO) of the HERV-K env gene affects tumorigenic characteristics through the nupr-1 gene. The effect of HERV-K env KO has not been studied in ovarian cancer cell lines. In this study, we analyzed the tumorigenic characteristics of ovarian cancer cell lines, including cell proliferation, migration, and invasion, and the expression patterns of related proteins after CRISPR-Cas9 KO of the HERV-K env gene. The HERV-K env gene KO was achieved using the CRISPR-Cas9 system in ovarian cancer cell lines SKOV3 and OVCAR3. Tumorigenic characteristics including cell proliferation, migration, and invasion were analyzed, and related protein expression was investigated by western blot analysis. The expression of the HERV-K env gene in KO cells was significantly reduced at RNA and protein levels, and tumorigenic characteristics including cell proliferation, migration, and invasion were significantly reduced. In HERV-K env KO SKOV3 cells, the expression of the RB protein was significantly up-regulated and the cyclin B1 protein level was significantly reduced. In contrast, in HERV-K env KO OVCAR3 cells, the level of phospho-RB protein was significantly reduced, but other protein levels were not changed. The results of this study showed that HERV-K env gene KO affects cell proliferation, invasion, and migration of ovarian cells through RB and Cyclin B1 proteins, but the specific regulation pattern can differ by cell line.

Epigenetic regulation of Cereblon in cancer: molecular mechanisms and clinical implications

Transcriptome analysis of the effect of HERV-K env gene knockout in ovarian cancer cell lines

Human endogenous retroviruses (HERVs) have been implicated in the pathogenesis of various diseases, particularly cancers. Previous investigations from our group demonstrated that targeted knockout (KO) of the HERV-K env gene led to a significant reduction in tumorigenic attributes, including proliferation, migration, and invasion of ovarian cancer cells. In this study, we aimed to elucidate the impact of HERV-K env KO on gene expression in ovarian cancer cell lines through comparative RNA sequencing (RNA-Seq) analysis with two distinct HERV-K env KO ovarian cancer cell lines, SKOV3 and OVCAR3. HERV-K env gene KO was achieved in SKOV3 and OVCAR3 ovarian cancer cell lines using the CRISPR-Cas9 system. Next-generation mRNA sequencing was employed to assess the gene expression profiles of both mock and HERV-K env KO ovarian cancer cells. Furthermore, comprehensive analyses involving gene ontology and pathway assessments were conducted. Transcriptome analysis revealed that 23 differentially expressed genes (DEGs) were upregulated and 17 DEGs were downregulated in SKOV3 cells. In OVCAR3 cells, 198 DEGs were upregulated, and 17 DEGs were downregulated. Notably, 53 DEGs exhibited statistically significant differences among the 1,612 DEGs identified. Our findings indicate that HERV-K env gene KO exerts a profound influence on gene expression patterns in OVCAR3 cells, while genetic alterations in expression were relatively modest in SKOV3 cells. Nevertheless, genes ND1, ND2, and CYTB displayed a common increase in expression, while ERRFI1 and NDRG1 exhibited a decrease in expression in both cell lines. Our study demonstrates that KO of the HERV-K env gene in ovarian cancer cell lines has a substantial impact on gene expression patterns and can be used to identify potential therapeutic targets for ovarian cancer and related diseases.

Suppression of synuclein gamma inhibits the movability of endometrial carcinoma cells by PI3K/AKT/ERK signaling pathway

Although overexpression of synuclein gamma (SNCG) has been reported in several cancers, few studies have been performed onSNCG in endometrial carcinomas. This study aimed to investigate the role of SNCG in the progression of endometrial carcinoma. The expression pattern and function ofSNCG gene were analyzed using the Gene Expression Omnibus (GEO) and Gene Set Enrichment Analysis (GSEA) datasets. Two vector types, containing either SNCG or negative control shRNAs, were used to evaluate cell proliferation, apoptosis, and metastasis using Cell Counting Kit 8, colony formation, flow cytometry, wound-healing, transwell, and invasion assays. The relative protein levels of N-cadherin, E-cadherin, vimentin, p-PI3K, PI3K, p-AKT, AKT, p-ERK, and ERK were determined by western bloting. Our results revealed thatSNCG mRNA expression and SNCG protein levels in shRNA-treated SPEC2 cells were lower than in the negative control cells. Furthermore, cell proliferation, migration, and invasion were significantly inhibited in SNCG shRNA-treated cells, but apoptosis was increased. The results of western blot analysis indicated that SNCG silencing reduced the protein levels of N-cadherin, vimentin, p-PI3K, p-AKT, and p-ERK, but not those of total PI3K, AKT, and ERK. Therefore, shRNA-mediated suppression of SNCG inhibited SPEC2 cell proliferation, migration, and invasion, and promoted SPEC2 cell apoptosis, which was presumably accomplished via regulation of the PI3K/AKT/ERK signaling pathway.

BRN3A, a transcription factor, regulates the expression of genes involved in biological processes shaping the HPV induced cervical cancer

Cervical cancer is the fourth most common cancer worldwide in females. This occurs primarily due to the infection of high-risk Human Papilloma Virus (HPV), although in advanced stages it requires support from host cellular factors. BRN3A is one such host cellular factors, whose expression remains high in cervical cancers and upregulates tumorigenic HPV gene expression. The effect of BRN3A on HPV-mediated cervical cancer and the underlying mechanism remains obscure. To investigates the effect of BRN3A on cancer-promoting biological processes in HPV-positive uterine cervix cancer cells. We have altered the expression of BRN3A through over-expression (OE) and knock-down (KD) constructs in cervical cancer cell line, SiHa, and did transcriptome profiling through next-generation RNA-sequencing, validation through RT-PCR and BRN3A binding study with in silico promoter study and ChIP PCR methods. This study revealed a substantial change in the expression of several genes associated with cancer-promoting biological processes including viral processes, immune response, cell-death, cell-proliferation, different signaling pathways, etc. Additionally, promoter analysis through in silico mode revealed that a total of 32.7% of genes possess BRN3A binding sites at their promoters. Physical interaction of BRN3A with IFITM1, OAS3, ISG15, BCL2L1 and HSP90AB1 genes was also confirmed. The present study identified molecular targets of BRN3A and provided new insight into the pathogenesis of cervical cancer. According to our knowledge, this is the first report on the effect on eukaryotic transcriptomes after over-expression and knocking down BRN3A.

Single-cell RNA sequencing reveals a pro-metastatic subpopulation and the driver transcription factor NFE2L1 in ovarian cancer cells

Although cytoreductive surgery followed by adjuvant chemotherapy is effective as a standard treatment for early-stage ovarian cancer, the majority of ovarian cancer cases are diagnosed at the advanced stages with dissemination to the peritoneal cavity, leading to a poor prognosis. Therefore, it is crucial to understand the cellular and molecular mechanisms underlying metastasis and identify novel therapeutic targets. In this study, we aimed to elucidate the mechanisms underlying gene expression alterations during the acquisition of metastatic potential and characterize the metastatic subpopulations within ovarian cancer cells. We conducted single-cell RNA sequencing of two human ovarian cancer cell lines: SKOV-3 and SKOV-3-13, a highly metastatic subclone of SKOV-3. Suppression of NFE2L1 expression was performed through siRNA-mediated knockdown and CRISPR-Cas9-mediated knockout. Clustering and pseudotime trajectory analysis revealed pro-metastatic subpopulation within these cells. Furthermore, gene set enrichment analysis and prognosis analysis indicated that NFE2L1 could be a key transcription factor in the acquisition of metastasis potential. Inhibition of NFE2L1 significantly reduced migration and viability of both cells. In addition, NFE2L1 knockout cells exhibited significantly reduced tumor growth in a mouse xenograft model, recapitulating in silico and in vitro results. The results presented in this study deepen our understanding of the molecular pathogenesis of ovarian cancer metastasis with the ultimate goal of developing treatments targeting pro-metastatic subclones prior to metastasis.

LncRNA SNHG20 promotes cell proliferation and invasion by suppressing miR-217 in ovarian cancer

Abstract Background Ovarian cancer is the most common female gynecological malignancy. SNHG20, as a long non-coding RNA, has been proven to be an important regulator in the occurrence and development of various tumors. However, the potential mechanism of SNHG20 in ovarian cancer is unclear. Objective The present study was aimed to investigate the functions and mechanisms of SNHG20 in ovarian cancer. Methods The expression of SNHG20 and miR-217 in ovarian cancer tissues and cell lines was detected by qRT-PCR. CCK-8 assay was used to measure cell proliferation in transfected cells. The transwell assay was used to detect the relative invasion rate of transfected cells. The putative binding sites between SNHG20 and miR-217 were predicted by software LncBase v.2, and the interaction between SNHG20 and miR-217 was confirmed by dual-luciferase reporter assays and RIP assay. The rescue experiments were used to illustrate potential mechanisms. Results SNHG20 was upregulated in ovarian cancer tissues and cell lines. Overexpression of SNHG20 promoted ovarian cancer cell proliferation and invasion. MiR-217 was downregulated in ovarian cancer tissues and cells, and was negatively regulated by SNHG20. Moreover, miR-217 overexpression inhibited ovarian cancer cell proliferation and invasion. Furthermore, miR-217 mimic reversed the inhibitory effect of SNHG20 overexpression on the biological behavior of ovarian cancer cells. Conclusions SNHG20 promoted cell proliferation and invasion by sponging miR-217 in ovarian cancer. These results suggested that SNHG20 and miR-217 might provide new targets for therapeutic application in ovarian cancer.

Single-cell RNA sequencing reveals a putative lncRNA-associated ceRNA network in high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive ovarian cancer subtype, characterized by high recurrence, chemoresistance, and poor prognosis. Although competing endogenous RNA (ceRNA) networks involving long non-coding RNAs (lncRNAs) have been proposed as key regulators of tumor progression, their cell type-specific roles in HGSOC remain unclear. We aimed to identify lncRNA-associated ceRNA networks active within the HGSOC tumor microenvironment. We performed an integrative analysis combining single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq datasets. Cancer cells were isolated, and coexpression analyses were conducted using high-dimensional weighted gene coexpression network analysis. The resulting ceRNA modules were validated and functionally annotated using The Cancer Genome Atlas and pathway enrichment analysis. Our analysis identified a cancer cell-specific ceRNA network involving MIR100HG, mir-224-5p, and EYA4. Interaction prediction and expression correlation analyses indicated that MIR100HG may function as a molecular sponge for mir-224-5p, thereby alleviating its suppression of EYA4. The presence of a 7mer-m8 seed match between mir-224-5p and EYA4 supported this interaction. Pathway analysis suggested a link between the identified ceRNA network and the Wnt signaling pathway, a key driver of tumor initiation and metastasis. The MIR100HG-mir-224-5p-EYA4 ceRNA network may promote tumor progression by modulating Wnt signaling. These findings offer insights into a potential posttranscriptional regulatory mechanism in tumor development and therapeutic targeting in HGSOC.

Analysis of KAP1 expression patterns and human endogenous retrovirus Env proteins in ovarian cancer

Human endogenous retroviruses (HERVs) constitute around 8% of the human genome and have important roles in human health and disease, including cancers. Previous studies showed that HERV envelope (Env) proteins are highly expressed in cancer tissues and co-related with cancer progression. KAP1 has been reported to play a key role in regulating retrotransposons, including HERV-K, through epigenetic silencing. The relationship between KAP-1 and HERV Envs expressions was analyzed only in tumor cell lines and has not yet been studied in cancer tissues. In this study, we analyzed the expression patterns and relationship between KAP1 and HERV Env proteins in ovarian cancer tissues. The expression patterns of KAP-1 and HERV Env proteins, including HERV-K and HERV-R, were analyzed in ovarian cancer tissue microarrays that contained 80 surgical specimens, including normal ovary and malignant ovarian cancers. The expression of HERV-R Env and KAP1 proteins is significantly higher in ovarian cancer compared with normal ovary tissues. However, the expression of HERV-K Env did not change significantly in cancer tissues. The expression patterns of HERV-K Env and HERV-R Env significantly increased in early stages of cancer and KAP1 expression was higher in certain stage and types of cancers. However, the expression of HERV-K Env, HERV-R Env, and KAP1 did not change in different age groups. The correlation between the expression of KAP1 and HERV-Env, including HERV-K and HERV-R, was not significantly correlated. The results of this study showed that there was no significant correlation between the expression of KAP1 and HERV Env proteins in ovarian cancer tissues, unlike studies with cell lines in vitro. These results suggest that the actual expression of HERV Env proteins in ovarian cancer tissues may be regulated through various complex factors as well as KAP1.

Rab11a promotes the malignant progression of ovarian cancer by inducing autophagy

Rab11a is a novel identified tumorigenic factor involved in different cancers. This study aimed to assess the biological function of Rab11a in ovarian cancer (OC). GEPIA database and real-time PCR were used to determine Rab11a expression in OC tissues and normal ovarian tissues. CCK-8, cell cycle, wound healing, transwell, and enzyme linked immunosorbent assay were used to detect the effects of Rab11a knockdown or overexpression on the proliferation, migration, and invasion of OC cells. Western blot analysis of autophagy-related markers and immunofluorescence staining of LC3 were performed to determine autophagy induction in Rab11a-silenced or overexpressed OC cells. Moreover, autophagy inhibitor 3-MA was employed to clarify the effects of Rab11a-regulated autophagy on the malignant phenotypes of OC cells. The mRNA level of Rab11a was increased in tumor tissues from OC patients as compared to the normal ovarian tissues. Knockdown of Rab11a in OVCAR-3 cells inhibited the growth of OC cells and led to cell cycle arrest, accompanied by reduced expression of PCNA and Cyclin D1. Rab11a deficiency suppressed migration and invasion of OC cells, accompanied by decreased secretion of MMP-2 and MMP-9. Silence of Rab11a impeded autophagy induction, as evidenced by decreased LC3 puncta formation, reduced abundance of LC3II and Beclin1, and increased p62 protein expression. In contrast, the ectopic expression of Rab11a in A2780 cells exerted opposite effects. Interestingly, autophagy inhibitor 3-MA abolished the effects of Rab11a overexpression on autophagy, proliferation, migration, and invasion. Rab11a promotes the malignant phenotypes of OC cells by inducing autophagy.

MUC16 facilitates cervical cancer progression via JAK2/STAT3 phosphorylation-mediated cyclooxygenase-2 expression

MUC16 (mucin 16, also known as CA-125, cancer antigen 125, carcinoma antigen 125, or carbohydrate antigen 125) has been predicted as tumor biomarker for therapy. We determined to investigate effects and regulatory mechanism of MUC16 on cervical tumorigenesis. Expression levels of MUC16 in cervical cancer cell lines was analyzed via qRT-PCR (quantitative real-time polymerase chain reaction). Knockdown of MUC16 was conducted via shRNA (Short hairpin RNA) transfection. MTT and colony formation assays were used to investigate effect of MUC16 on cell proliferation. Wound healing assay was utilized to detect migration and transwell assay to detect invasion. The underlying mechanism was demonstrated via western blot analysis. MUC16 was elevated in cervical cancer cell lines. MUC16 knockdown inhibited cell proliferation, invasion and migration. Gain- and loss-of functional assays revealed that over-expression of MUC16 activated Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) via phosphorylation, thus facilitating cyclooxygenase-2 (COX-2) expression, while knockdown of MUC16 demonstrated the reverse effect on JAK2/STAT3 activation and COX-2 expression. Moreover, inhibition of JAK2/STAT3 attenuated the regulation of MUC16 on COX-2. MUC16 enhanced proliferation and invasion of cervical cancer cells via JAK2/STAT3 phosphorylation-mediated cyclooxygenase-2 expression, suggesting the potential therapeutic target ability of MUC16 to treat cervical cancer.

Mechanism of E2F1 in the proliferation, migration, and invasion of endometrial carcinoma cells via the regulation of BMI1 transcription

Endometrial carcinoma (EC) is the most prevalent gynecological cancer. Transcription factor (TF) regulates a large number of downstream target genes and is a key determinant of all physiological activities, including cell proliferation, differentiation, apoptosis, and cell cycle. The transcription factor E2F1 shows prominent roles in EC. BMI1 is a member of Polycomb suppressor Complex 1 (PRC1) and has been shown to be associated with EC invasiveness. It is currently unclear whether E2F1 can participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription. We investigated whether E2F1 could participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription, in order to further clarify the pathogenesis and etiology of EC, and provide reference for identifying potential therapeutic targets and developing effective prevention and treatment strategies for this disease. Human endometrial epithelial cells (hEECs) and human EC cell lines were selected. E2F1 expression was assessed by Western blot. E2F1 was silenced in AN3CA or overexpressed in HEC-1 by transfections, or E2F1 was silenced and BMI1 was overexpressed in AN3CA by cotransfection. Cell proliferation, migration, and invasion were detected by MTT, wound healing, and Transwell assays. The binding sites between E2F1 and BMI1 promoters were predicted through JASPAR website, and the targeted binding was verified by dual-luciferase report and ChIP assays. E2F1 was up-regulated in human EC cell lines, with its expression highest in AN3CA, and lowest in HEC-1. AN3CA invasion, migration, and proliferation were repressed by E2F1 knockdown, while those of HEC-1 cells were promoted by E2F1 overexpression. E2F1 overexpression increased the activity of wild type BMI1 reporter vector promoter, while this promotion was weakened after mutation of the predicted binding site in the BMI1 promoter. In the precipitated E2F1, BMI1 promoter site level was higher than that of IgG immunoprecipitant. BMI1 silencing suppressed AN3CA cell growth. BMI1 overexpression partially abrogated E2F1 silencing-inhibited EC cell growth. E2F1 promoted EC cell proliferation, invasion, and migration by promoting the transcription of BMI1.

B3GNT3 acts as a carcinogenic factor in endometrial cancer via facilitating cell growth, invasion and migration through regulating RhoA/RAC1 pathway-associated markers

Aberrant expression of beta-1,3-N-acetylglucosaminyltransferase-3 (B3GNT3) has been frequently clarified in various cancers, however, its role in endometrial cancer (EC) has not been assessed in detail. This study aimed to investigate the biological role of B3GNT3 in EC and simply explored the detailed mechanism. The EC RNA-Seq dataset from TCGA database was applied to evaluate the expression of B3GNT3 and assess its role on prognostic value. HEC-1-A and KLE cell lines of EC were used to perform loss- and gain-of-function B3GNT3 assays respectively. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure the mRNA and protein levels of indicated molecules respectively. Cell counting kit-8, clone formation tests, and Transwell assay served to determine the changes of proliferative, invasive and migratory abilities of EC cells after altering the expression of B3GNT3. B3GNT3 was found to be highly expressed in EC tissues compared to normal tissues according to the online public databases, which confirmed by the following qRT-PCR in 3 EC cell lines. Besides, high B3GNT3 expression presented a worse overall survival in EC patients as compared with low B3GNT3 expression group. Furthermore, functional experiments in vitro indicated that B3GNT3 could facilitate the cell growth, invasion and migration. Moreover, we found that downregulation of B3GNT3 significantly reduced the expression level of GTP-RhoA and GTP-RAC1, whereas upregulation of B3GNT3 presented the opposite results. The results of current study demonstrate that B3GNT3 acts as an oncogene that promotes EC cells growth, invasion and migration possibly through regulating the RhoA/RAC1 signaling pathway-related markers, suggesting that B3GNT3 may be a candidate biomarker for EC therapeutic intervention.