The Role And Mechanism Of AND-1 In Tumor Cisplatin Resistance

Tumors,especially malignant tumors,are one of the major diseases that currently seriously affect human health and threaten human life,and their morbidity and mortality are increasing year by year.In modern medicine,the development of a variety of tumors is controlled or treated by surgical resection of a combination of chemotherapy drugs.As the most commonly used chemotherapy drug in clinical practice,cisplatin has a good antitumor effect.However,the resistance of tumor cells to cisplatin seriously affects the chemotherapy effect of cisplatin.Cisplatin resistance includes primary and acquired resistance,and acquired resistance in most cancer patients during chemotherapy is the main cause of clinical failure.Cisplatin resistance is a multi-factor,multi-gene complex process in which cisplatin can cause DNA strands or interchain damage,preventing DNA replication leading to tumor cell apoptosis,and most of the DNA cross-linkage damage can be nuclear.The nucleoside excision repair(NER)system recognizes and repairs tumor cells against cisplatin by inhibiting apoptosis.AND-1 is an acidic nuclear DNA-binding protein that forms part of a DNA replicator and regulates genome stability through a variety of mechanisms.AND-1 is highly expressed in various tumor cells such as lung cancer and esophageal cancer,and expression studies in osteosarcoma and ovarian cancer have not been reported.Previous studies have found that the knockdown of AND-1 affects the sensitivity of tumor cells to cisplatin,and then constructs ovarian cancer cisplatin-resistant A2780/CDDP,and detects the expression levels of AND-1 and NER-related proteins in A2780/CDDP cells.AND-1 regulates the molecular mechanism of ovarian cancer cell resistance to cisplatin.Objective:1.To investigate whether the expression of AND-1 in tumor cells affects its resistance to cisplatin.2.To analyze the expression of AND-1 in ovarian cancer resistant cells and to elucidate the molecular mechanism of AND-1 regulating cisplatin resistance.Methods: 1.AND-1 affects the sensitivity of tumor cells to cisplatin: an AND-1 stable knockout osteosarcoma cell line(U2OS)and ovarian cancer cell line(A2780)were established by shRNA interference technology;Cell morphology and cell counting experiments were used to investigate the changes in morphology and growth rate of U2 OS cells and A2780 cells after AND-1 knockout;It was tested by CCK-8 assay and cell monoclonal formation assay whether the knockdown of AND-1 affects the sensitivity of U2 OS cells and A2780 cells to cisplatin;The expression of apoptosis-related proteins in U2 OS cells and A2780 cells and their AND-1 knockout cell lines was detected by Western blotting.2.Expression of AND-1 in ovarian cancer cell line A2780 and ovarian cancer drug resistant cell A2780/CDDP: The ovarian cancer resistant cell line was constructed by in vitro induction of small dose and cisplatin concentration and intermittent action;The morphology and growth rate of A2780 cells and A2780/CDDP cells were studied by cell morphology observation and cell counting experiments;The resistance index of A2780/CDDP cells to cisplatin was detected by CCK-8 assay;The resistance of A2780 cells and A2780/CDDP cells to cisplatin was investigated by cell monoclonal formation assay;Flow cytometry was used to study the apoptosis rate of A2780 cells and A2780/CDDP cells induced by different concentrations of CDDP;The expression of apoptosis-related proteins in A2780 cells and A2780/CDDP cells treated with different concentrations of CDDP was detected by Western blotting.3.Regulation of cisplatin resistance in ovarian cancer: Western blotting and Q-PCR were used to detect the expression level of AND-1 in ovarian cancer cell lines with different concentrations of CDDP;The expression of AND-1 and NER-related proteins in A2780/CDDP cells was detected by Western blotting;It was investigated by immunoprecipitation experiments whether AND-1 interacts with NER related proteins ERCC1,XPA and XPF;Whether the knockdown of AND-1 in A2780/CDDP cells affects the resistance of A2780/CDDP cells to cisplatin by CCK-8 assay;Detection of AND-1 and NER-related protein expression in A2780/CDDP cells after AND-1 knockout by Western blotting.Results:1.Successfully established an AND-1 stable knockout osteosarcoma cell line and ovarian cancer cell line;The volume of the AND-1 knockout osteosarcoma cell line was increased compared with the unknocked AND-1 tumor cell,and the AND-1 knockout ovarian cancer cell line surrounded by cell lengths,AND-1 knock The growth rate of the tumor cell lines was significantly slower(P<0.05);CCK-8 assay and cell clone formation experiments showed that knockdown of AND-1 significantly increased the sensitivity of U2 OS cells and A2780 cells to cisplatin(P<0.05);Western blotting results showed that the knockdown of AND-1 increased the expression of cisplatin-related apoptosis proteins in tumor cells.2.A2780/CDDP cisplatin-resistant ovarian cancer cells with a concentration of 8.5 ?g/ml were successfully constructed.The A2780 cells were small in size,regular in shape and fast in growth,and the volume of A2780/CDDP cells increased and the growth rate was slow;According to the Snow standard,the CCK-8 experiment showed that the resistance index of A2780/CDDP cells was 2.67,which was low-resistance cells;Western blotting showed that Bak,Bax,cleaved-Caspase 3 and cleaved-Parp significantly downregulated Bcl-2 protein levels in A2780 cells,and the level of related apoptotic proteins in A2780/CDDP cells did not change significantly.CDDP can induce apoptosis in A2780 cells,but has less apoptosis in A2780/CDDP cells.3.Western blotting and Q-PCR results showed that the expression level of AND-1 in ovarian cancer drug-resistant cells with different drug resistance concentrations was significantly up-regulated;Western blotting results showed that the expressions of AND-1,ERCC1,XPA and XPF were up-regulated;The CCK-8 results showed that the resistance of A2780/CDDP cells to CDDP after AND-1 knockdown decreased;Western blotting showed that the expression of AND-1,ERCC1,XPA and XPF in A2780/CDDP cells was down-regulated.Conclusion: 1.The knockdown of AND-1 in tumor cells increases the sensitivity of tumor cells to cisplatin.2.The expression level of AND-1 in A2780/CDDP cells was significantly higher than that in A2780 cells.3.AND-1 can regulate the resistance of ovarian cancer resistant cells to cisplatin.