The Investigation On The Bidirectional Mechanism And Clinical Significance Of PAX9 In Ovarian Cancer Platinum Resistance

Ovarian cancer remains the leading cause of death in women with gynecological malignancies,and platinum-based chemotherapy has been regarded as the first-line chemotherapy.Most of the advanced ovarian cancer patients experience recurrence and become gradually resistant to the first-line chemotherapy.It is thus imperative to elucidate the underlying mechanism of ovarian cancer chemoresistance.The project contains three parts:(1)Gene screening and verification of the chemoresistance related genes in ovarian cancer cells;(2)Exploration of bidirectional effect and mechanism of PAX9 on the regulation of ovarian cancer chemoresistance;(3)Clinical research of PAX9 involved in the chemoresistance and prognosis of ovarian cancer patients.Objective 1.Screen the critical genes involved in cisplatin resistance of epithelial ovarian cancer cells;2.Explore the mechanism of PAX9 on the regulation of ovarian cancer chemoresistance;3.Investigate the correlation between PAX9 expression and progression,pathological characteristics,chemoresistance outcome and prognosis of ovarian cancer patients.Methods Gene screening and analysis was conducted in parent ovarian cancer A2780 cells and cisplatin resistant A2780 DR cells by using human whole genome expression microarray,and the candidate chemoresistant related genes were found out.The difference of the above candidate gene expression level was verified in cisplatin-sensitive and-resistant ovarian cancer cells by PCR.The ovarian cancer cell proliferation was further tested by MTS assay in control or siRNA knockdown group individually.The critical chemoresistant genes were further examined ovarian cancer cell lines by Western blot.PAX9 expression of A2780 DR cells were stably downregulated,then PCR and WB were used to examine the alteration of the chemoresistant related molecules;Series of ovarian cancer cells lines including A2780,ES2,HO8910 and SKOV3 were transfected with Lentivirus plasmid to stably upregulate PAX9 expression,then PCR and WB was used to examine the alteration of the chemoresistant related molecules;Gene microarray was used to find out the PAX9-regulated downstream transcription factor;ChIP-seq was applied to examine the direct binding of PAX9 to the promoter of downstream transcription factor;The regulation of PAX9 and p53 on the sensitivity of various ovarian cancer cells to cisplatin were detected by gene regulation and MTS assay;The regulation of PAX9 on the cell cycle of A2780 DR cells was detected by flow cytometry;The influence of PAX9 silence on the cisplatin-induced apoptosis of A2780 DR cells was evaluated by flow cytometry and Western blot;The expression of p53 and p21 was detected in 5 ovarian cancer cell lines by Western blot.Immunohistochemistry was applied to detect the expression of PAX9 in 40 cisplatin-sensitive and 40 cisplatin–resistant EOC patients,also the clinical feature and pathological characteristics were retrospectively analyzed.ELISA was used to examine the PAX9 expression in preoperative plasma and cancer tissues in 72 EOC patients followed by correlation analysis to evaluate the relationship between PAX9 expression and clinical pathological characteristics.Results 15 chemoresistant related genes(candidate genes) were screened out by gene microarray and analysis.Downregulation of PAX9 and ARNTL2 enhanced the sensitivity of chemoresistant ovarian cancer cells to cisplatin.The chemoresistant ovarian cancer A2780 DR cells showed higher expression of PAX9 protein.Down-or upregulation of PAX9 could alter the expression of most of the classic chemoresistant related molecules including p53,Bax,Bcl-2,ABCB1 and ABCC2;ChIP-seq indicated p53 was directly involved in the PAX9-mediated chemoresistance in ovarian cancer cells;PAX9 upregulation increased IC50 value in A2780 and HO8910 cells,but decreased it in SKOV3 and ES2 cells;PAX9 or p53 downregulation significantly decreased IC50 value in A2780 DR cells;PAX9 silence significantly decreased the proportion of A2780 DR cells in G1 phase and increased the proportion of apoptotic cells as well as the apoptosis related proteins including cleaved caspase-3 and PARP;p53 expression level varied in various ovarian cancer cell lines other than SKOV3,neither ES2 nor SKOV3 cells showed p21 expression.Immunohistochemistry and ELISA results indicated: the chemoresistant ovarian cancer patients showed significant higher expression of PAX9 compared to the chemosensitive patients(P<0.05).The expression of PAX9 expression significantly elevated along with the increased clinical stage(P<0.05).The expression of PAX9 in cancer tissue was negatively correlated with the progression-free survival(P<0.05).Conclusions The chemoresistant ovarian cancer cells and tissues showed higher expression level of PAX9 compared to parent ones.Downregulation of PAX9 could increase the sensitivity of chemoresistant ovarian cancer cells to cisplatin.Multiple chemoresistant related molecules were regulated by PAX9,which could direct binding to the promoter of p53;PAX9 exerted bidirectional regulation on the chemoresistance of ovarian cancer cells depending on the expression of p53: It could promote chemoresistance through cell cycle arrest in G1 phase and increase sensitivity of ovarian cancer cells to cisplatin by upregulating Bax expression upon the presence or absence of functional expression of p53,respectively.The expression of PAX9 was closely correlated with the progression,chemoresistance outcome and survival of epithelial ovarian cancer patients.