Investigator
Kosin University
Loss of Zonula Occludens-1 (ZO-1) Enhances Angiogenic Signaling in Ovarian Cancer Cells
Zonula occludens-1 (ZO-1), encoded by the TJP1 gene, is a crucial scaffolding protein within tight junctions that maintains epithelial and endothelial barrier integrity. In addition to its structural role, ZO-1 participates in signal transduction pathways that influence various cellular processes such as proliferation, differentiation, and apoptosis. Increasing evidence suggests that tight junction proteins, including ZO-1, play important regulatory roles in tumor progression, particularly by modulating metastasis, cell polarity, and vascular remodeling. Ovarian cancer, the most lethal gynecologic malignancy, is characterized by rapid growth, peritoneal dissemination, and a strong reliance on tumor angiogenesis. However, the specific role of ZO-1 in regulating angiogenesis within ovarian cancer remains poorly defined. In this study, we used CRISPR-Cas9-mediated gene editing to generate TJP1 knockout (KO) ovarian cancer cell lines and investigated the impact of ZO-1 loss on the expression of angiogenesis-related genes. Transcriptomic and qRT-PCR analyses revealed upregulation of KLF5 and IL-8, both of which are well-established pro-angiogenic factors. Furthermore, functional assessment using a Matrigel™ tube formation assay demonstrated that conditioned media from ZO-1-deficient cells significantly enhanced endothelial tube formation. These findings indicate that ZO-1 loss promotes a pro-angiogenic tumor microenvironment, likely through modulation of key signaling molecules such as KLF5 and IL-8. Therefore, ZO-1 may serve as a potential suppressor of angiogenesis and a therapeutic target in ovarian cancer.
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.
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.
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.
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.
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.
