Function And Mechanism Of KLF9 In Ovarian Cancer Cells

Ovarian cancer is a malignant neoplastic disease that seriously threatens the safety and quality of life of women.Because of the original diversity of ovarian cancer cells,complex histological types,atypical clinical features,and lack of early symptoms,the early diagnosis of it is still very hard.When patients are diagnosed in an advanced stage,the five-year survival rate is very low.If being diagnosed in an early stage,patients will be very sensitive to platinum-based chemotherapeutics.However,they are very prone to relapse and drug resistance,and are often accompanied by extensive pelvic-abdominal metastasis and distant metastasis with a poor prognosis.A large number of previous studies has shown that Kruppel-like factors(KLFs)not only participate in the regulation of various physiological processes of normal cells,but also play a critical role in cancer cell proliferation,apoptosis,metastasis,and et al.Among them,KLF9 is determined to regulate the tissue development and cell proliferation.In recent years,the roles of KLF9 in the development and progression of a variety of malignant diseases have been reported.However,the study of malignant disease in the female reproductive system is still unknown.In order to clarify the role and mechanism of KLF9 in the development and progression of ovarian cancer,we analyzed the significance of the KLF9 expression level in the prognosis of ovarian cancer patients,and further investigate the biological function and associated mechanism of KLF9 in ovarian cancer cells.Our study here provides a fundamental understanding of KLF9 in the development and progression of ovarian cancer.Objective:To determine the significance of the KLF9 expression level on the correlation of prognosis of ovarian cancer patients;to investigate the effect of KLF9 on proliferation,migration,and apoptosis of ovarian cancer cells;to investigate the effect of KLF9 on the sensitivity of ovarian cancer cells to anoikis;to explore the fundamental mechanism of KLF9 on the regulation of anoikis in ovarian cancer cells.Methods:The KM-plotter database was used to analyze the correlation between the expression level of KLF9 and the prognosis of ovarian cancer patients.The lentiviral delivery system was used to overexpress or knock down KLF9 in SKOV3 and A2780 ovarian cancer cells,respectively.Using qRT-PCR and Western Blot to confirm the expression of KLF9 in stable cell clones;using CCK-8 assay and clone formation assay to detect the effect of KLF9 on proliferation of ovarian cancer cells;using the transwell to detect the effect of KLF9 on cancer migration;using flow cytometry to detect apoptosis of ovarian cancer cells in adherent and suspension culture system,respectively;and using qRT-PCR and Western Blot to explore the fundamental mechanism of KLF9 on the regulation of anoikis sensitivity in ovarian cancer cells.Results:1.Analysis of the correlation between the expression of KLF9 and the prognosis of ovarian cancer patientsIn all analyzed ovarian cancer patients,compared with the low-expression group,the survival rate of the KLF9 high-expression group was significantly shorter.It indicates that the high level of KLF9 expression is related to the poor prognosis in ovarian cancer patients,and KLF9 could be a potential evaluation index for the prognosis of ovarian cancer patients.2.Establishment and identification of stable ovarian cancer cell clones with different expression levels of KLF9Using lentiviral delivery system,we stably overexpressed or silenced KLF9 in two ovarian cancer cell lines,respectively.We performed qRT-PCR and Western Blot to confirm the upregulation or downregulation of KLF9 in stable cell lines.The results suggests that we successfully establish the stable ovarian cancer cell clones with different expression levels of KLF9 and provide potential tools to determine the biological function and fundamental mechanism of KLF9 in ovarian cancer cells.3.The effect of KLF9 on proliferation in ovarian cancer cellsWe used the CCK-8 assay and plate clone formation assay to test the effect of KLF9 on proliferation in SKOV3 and A2780 ovarian cancer cells.The result showed that knockdown of KLF9 in ovarian cancer cells significantly suppresses proliferation in two cancer cell lines.Meanwhile,overexpression of KLF9 in cancer cells significantly promotes proliferation capability of cancer cells.The results suggest that KLF9 plays a critical role in the regulation of ovarian cancer cell proliferation.4.The effect of KLF9 on the capability of migration in ovarian cancer cellsWe used the transwell to test the effect of KLF9 on migration of ovarian cancer cells in vitro.The result showed that knockdown of KLF9 in ovarian cancer cells significantly inhibits migration of cancer cells.Meanwhile,overexpression of KLF9 in cancer cells significantly promotes migration capability of ovarian cancer cells.The results suggest that KLF9 plays a critical role in the regulation of ovarian cancer cell migration in vitro.5.The effect of KLF9 on apoptosis of ovarian cancer cells in the monolayer culture conditionWe cultured ovarian cancer cell lines with different levels of KLF9 in the monolayer culture condition.Then,using the flow cytometry,we performed the Annexin-V staining to test the effect of KLF9 on apoptosis of ovarian cancer cells.The result showed that KLF9 has no obvious effect on apoptosis of ovarian cancer cells in the monolayer culture condition.6.The effect of KLF9 on anoikis of ovarian cancer cellsWe cultured ovarian cancer cell lines with different levels of KLF9 in the suspension cell culture condition.Then,using the flow cytometry,we performed the Annexin-V staining to test the effect of KLF9 on apoptosis of ovarian cancer cells.The result showed that knockdown of KLF9 in ovarian cancer cells significantly promotes anoikis of ovarian cancer cells.Meanwhile,overexpression of KLF9 significantly protects ovarian cancer cells from suspension culture-induced anokis.The results suggest that KLF9 is essential in the regulation of anoikis sensitivity in ovarian cancer cells.7.Fundamental mechanism of KLF9 on the regulation of anoikis phenotype of ovarian cancer cellsROS is an important factor in inducing cell apoptosis,and Glutathione peroxidase 4(GPX4)is an important glutathione peroxidase involved in the regulation of ROS.Based on the results of the above study on anoikis of suspension cultured cells,we further focused on the changes in the expression of GPX4 and explored the mechanism of KLF9 regulating the anoikis sensitivity of ovarian cancer cells.The result of western blot results showed that in overexpression of KLF9 upreuglates the expression the GPX4 in the protein levels,while the result of loss of functional experiment showed that knockdown of KLF9 decreases the expression the GPX4 in the protein levels.Given that KLF9 is a transcriptional factor,we suspected that it may regulate GPX4 in the transcriptional level.However,when we performed the qRT-PCR assay,Strikingly,we did not find that KLF9 has any obvious effect on the regulation of the GPX4 expression in the mRNA level,no matter upregulation or downregulation of KLF9 in ovarian cancer cells.The result suggests that KLF9 regulates the expression of GPX4 in a transcription-independent mechanism.Conclusions:In the present study,we found that the high level of KLF9 expression is related to the poor prognosis in ovarian cancer patients,and KLF9 could be a potential evaluation index for the prognosis of ovarian cancer patients.Our in vitro assay demonstrates that KLF9 plays a critical role in the regulation of ovarian cancer cell proliferation and migration,while has no obvious effect on apoptosis of ovarian cancer cells in the monolayer culture condition.However,it is essential in the regulation of anoikis sensitivity in ovarian cancer cells.And our mechanism indicates that KLF9 regulates the expression of GPX4 in a transcription-independent mechanism.Given the effect of GPX4 is a scavenger of ROS and ROS is a driver promoting anoikis of cells.Fundamentally,we provide that a rational KLF9-GPX4 axis may protect ovarian cancer cells from suspension culture-induced anoikis.And further mechanism study should be deeply explored in the future project.