The Expression Of PAX8 Was Inhibited By Trametinib Through ELK1 In Ovarian Cancer

Objective 1.To explore the differential expression of PAX8 between normal ovarian tissue and ovarian cancer tissue.2.Searching for small molecule inhibitors to inhibit PAX8 expression.3.To explore the mechanism of Trametinib inhibiting PAX8 expression.Methods 1.The expression of PAX8 in ovarian cancer was investigated by reading literatures and TCGA database.2.The expression of PAX8 in ovarian cancer tissues of different pathological types was detected by immunohistochemistry.3.Western blotting was used to detect PAX8 expression in ovarian cancer cells treated with different inhibitors.4.The effect of Trametinib on PAX8 expression in KURAMHCHI ovarian cancer cells were detected by Western blotting,immunofluorescence staining and real-time quantitative PCR。 5.Gene enrichment analysis（GSEA）was used to detect the expression differences of related genes in KURAMHCHI cells treated with Trametinib.6.The expression of PAX8 in HEY and KURAMOCHI ovarian cancer cells were detected after the ELK1 gene was down-regulated by si RNA small interference assay.7.CCK8 assay was used to detect the effect of ELK1 interference on the proliferation of HEY and KURAMOCHI cells.Results 1.The expression of PAX8 in ovarian cancer tissues was higher than that in normal ovarian tissues.（1）The high expression of PAX8 in ovarian cancer was found by TCGA database.（2）Immunohistochemical analysis of human ovarian cancer tissue showed that PAX8 was highly expressed in ovarian cancer tissue.2.The expression of PAX8 was inhibited by Trametinib in KRAS mutant ovarian cancer cells（1）The KURAMOCHI ovarian cancer cell line was treated with different inhibitors,and The result of western blotting showed that Trametinib significantly inhibited the expression of PAX8 in KURAMOCHI cells.（2）Eight ovarian cancer cell lines were treated with Trametinib,and the result of western blotting showed that Trametinib inhibited the expression of PAX8 in ovarian cancer cell lines,but the expression was most significantly inhibited in ovarian cancer cell lines containing KRAS mutation.（3）Ovarian cancer cells treated with Trametinib in different times were collected for western blotting and real-time quantitative PCR experiments.The results showed that the PAX8 m RNA level decreased first,and then the PAX8 protein level decreased.It was considered that Trametinib inhibited PAX8 expression in ovarian cancer cells,but it does not directly affect PAX8 protein.3.Trametinib inhibits the proliferation of KRAS mutant ovarian cancer cells Ovarian cancer cell lines with KRAS mutation（KURAMOCHI,HEY,OV56,TOV21G）treated with Trametinib at different concentrations（0,0.03,0.1,0.3,1,3u M）for 72 h were stained with crystal violet.Crystal violet staining showed that Trametinib had the most obvious inhibitory effect on KURAMOCHI cell proliferation.0.1u M may be the minimum concentration at which Trametinib achieves optimal inhibitory effect.4.Trametinib inhibited the expression of ELK1 gene set in KURAMOCHI cells Differential genes of KURAMOCHI cells treated with Trametinib were analyzed by gene enrichment,and GSEA enrichment analysis results showed that the ELK1 target gene set and the gene set positively regulated by KRAS pathway were down-regulated,suggesting that ELK1 gene may be involved in Trametinib inhibiting the proliferation of KRAS mutant ovarian cancer cells.5.Trametinib inhibits PAX8 expression and proliferation of ovarian cancer cells by ELK1 gene（1）Western blotting was performed to detect the protein levels of PAX8 and ELK1 in KURAMOCHI and HEY cells after Trametinib treatment.Western blotting results showed that the protein levels of ELK1 and PAX8 in ovarian cancer cells decreased after Trametinib treatment.（2）The expression of ELK1 in KURAMOCHI and HEY cells was down-regulated by si RNA small interference assay,Western blotting assay showed that interfering ELK1 gene could inhibit the expression of PAX8 gene in KURAMOCHI cells.（3）After the expression of ELK1 in KURAMOCHI and HEY cells was interfered,the result of CCK8 assay showed that the proliferation activity of ovarian cancer cells was reduced with ELK1 gene interfered.Conclusion 1.The expression of PAX8 is different in ovarian cancer.2.Trametinib inhibits PAX8 expression and cell proliferation in KRAS mutant ovarian cancer cells.3.Trametinib inhibits PAX8 expression and cell proliferation in KRAS mutant ovarian cancer cells by ELK1 gene.