ISG15 Is Competitively Regulated By KLF1 And KLF12 And Implicated In Maintenance Of Cancer Stem Cell Like Features In Ovarian Cancer

Objective: Ovarian cancer is a kind of malignant tumor which seriously endangers women’s health.Cisplatin is an effective drug in the treatment of ovarian cancer.Cisplatin resistance directly affects the survival rate of patients with ovarian cancer.Therefore,studying the molecular mechanism of cisplatin resistance in ovarian cancer is helpful for clinical treatment of potential targets.The ubiquitin-like protein interferon-stimulated gene 15(ISG15)encodes a 17 k Da precursor,which is proteolytically processed to form a mature form of 15 k Da.It can covalently bind to a substrate to form ISGylated proteins.Except for conjugated form ISG15 also exists in free form,and plays both pro-tumoral and anti-tumoral functions in various tumors.Krüppel-like factors(KLFs)are transcription factors with evolutionarily conserved zinc(Zn)-finger domains in their C-terminal regions.In the present study,we identified that ISG15 was downregulated in cisplatin resistant cells of ovarian cancer.Both ectopic wild type and non ISGylatable mutant ISG15 overexpression inhibited cancer stem cell(CSC)-like phenotypes of cisplatin resistant ovarian cancer cells.In addition,knocking of ISG15 promoted CSC-like features of cisplatin sensitive ovarian cancer cells.The current study demonstrated that KLF1 played a role of activator of transcription in cisplatin-resistive ovarian cancer,and KLF12 played a role of repressor of transcription in cisplatin resistant cells.In conclusion,we continued to explore the specific mechanisms of KLF1,KLF12 and ISG15 and their effects on cisplatin resistant ovarian cancer stem cell like phenotype on the basis of previous experiments,so as to provide new therapeutic targets for the treatment of ovarian cancer.Methods: 1.Cisplatin-sensitive and cisplatin-resistant Human Ovarian Cancer Cell Lines were constructed,and the stable overexpression or knockdown ISG15 cell lines were constructed by lentiviral infection.2.The CCK8 assay was used to detect the proliferation of various target cell lines.3.Western blot was used to analyze the protein expression level of each target gene.4.Dot blot was used to detect the exocytosis of ISG15 in cisplatin resistant and cisplatin sensitive ovarian cancer cells.5.The clonogenic ability of ovarian cancer cells was detected by clonogenic assay.6.Transwell test was used to detect the migration and invasion ability of ovarian cancer cells when ISG15 was over over-expressed or under-expressed.7.QRT PCR was used to detect the expression of ISG15 m RNA in each cell line.8.The kit was used to label and capture the m RNA of new ISG15 in ovarian cancer cells.9.The stability of ISG15 m RNA was detected by treating ovarian cancer cells with actinomycin D according to time gradient.10.Spheroid formation ability of overian cancer cells with ISG15 was detected by balloon formation assay.11.Luciferase reporter gene test was used to detect the active site of the binding of transcription factors KLF1,KLF12 and ISG15.12.Chromatin immunoprecipitation assay was used to detect the recruitment of transcription factors KLF1,KLF5 and KLF12 by ISG15.13.The effect of ISG15 overexpression on the ability of ovarian tumor formation was detected by Nude mice xenograft experiments.14.ANOVA and Dunnett’s t test were used to analyze the statistical significance of different expressions of ISG15 in ovarian cancer patients.Results: 1.ISG15 was down regulated in cisplatin resistant ovarian cancer cells,and over expression of ISG15 inhibited the characteristics of cancer stem cells.2.ISG15 deletion promoted cancer stem cell like features in cisplatin-sensitive ovarian cancer cells.3.Cisplatin resistant ovarian cancer cells overexpressed ISG15 inhibited tumor formation in nude mice.Clinical data analysis showed low expression of ISG15 was associated with poor prognosis of ovarian cancer.4.The activity of ISG15 promoter was inhibited in cisplatin resistant ovarian cancer cells,and the stability of ISG15 m RNA was decreased in cisplatin resistant ovarian cancer cells.The expression of ISG15 was regulated at the level of transcriptional activation and posttranscriptional stability.5.ISG15 was competitively regulated by KLF1 and KLF12 at the transcriptional activation level via the CACCC elements on the ISG15 promoter.Conclusion: The down-regulation of free ISG15 expression in cisplatin-resistant ovarian cancer promotes the phenotype of ovarian cancer tumor stem cells.ISG15 is competitively regulated by KLF1 and KLF12 and implicated in maintenance of cancer stem cell like features in ovarian cancer.