Study On The Mechanism Of Interfering NEK2 To Inhibit Carboplatin Resistance In Ovarian Cancer

Background:Ovarian cancer is the most aggressive cancer of the female reproductive system,and a leading cause of cancer-related mortality worldwide every year.Thus,ovarian cancer is greatly contributed to cancer-related death in women.Although patients usually respond to initial therapy by adjuvant chemotherapy with platinum and paclitaxel,most of those with advanced-stage ovarian cancer experience recurrence.In many of these cases,the disease is incurable mainly due to the development of drug resistance.Therefore,there is an ongoing and urgent need to better understand the mechanisms of drug resistance,and that would probably lead to improved treatment strategies and perhaps the better survival.Objectives:NEK2[NIMA（never in mitosis gene A）-related expressed kinase 2],a cell cycle-related protein,is up-regulated in many solid tumors and implicated in regulation of cancer development.However,the studies of the protein with ovarian cancer were limited,and the research on drug resistance in ovarian cancer was rare.On the basis of big data screening and comprehensive bioinformatics analyses,our team previously reported that upregulation of NEK2 was associated with drug resistance in ovarian cancer.By means of research on cell function and molecular changes,this thesis aims to further illustrate the relationships and mechanisms of NEK2 with carboplatin resistance in ovarian cancer,and provide a theoretical basis for its clinical application.Methods:The human ovarian cancer cell line H and carboplatin-resistant cell line H-CBP were maintained in our laboratory;the stable RNA interfering of NEK2 cell line were established by lentivirus transfection;the half maximal inhibitory concentration of carboplatin(IC₅₀)was determined using MTS assay;the colony formation ability was measured by plate colony formation assay;the changes of cell cycle and apoptosis was determined by flow cytometry;the differential expressed genes in different cells was identified using transcriptome sequencing,and gene ontology and pathway enrichment of those genes was subsequently performed;m RNA and protein expression was measured by quantitative reverse transcription polymerase chain reaction（RT-q PCR）and Western blot.Results:We found that the NEK2 m RNA and protein was significantly up-regulated in carboplatin-resistant ovarian cancer cell line H-CBP compared with the parental cell line H,as measured using RT-q PCR and Western blot.RNA interfering of NEK2 in H and H-CBP cells was performed through lentivirus transfection.NEK2 m RNA and protein was significantly down-regulated in NEK2 interfering cells H-sh NEK2 and H-CBP-sh NEK2compared with the expression in scramble cells H-scr and H-CBP-scr.MTS assay revealed that RNA interfering of NEK2 didn’t affect the growth of H-CBP-sh NEK2 compared with H-CBP-scr,but RNA interfering of NEK2could significantly decrease the carboplatin resistance of ovarian cancer cells（especially the carboplatin-resistant cells）.Plate colony formation assay showed that RNA interfering of NEK2 inhibited the colony formation ability of ovarian cancer cells with carboplatin treatment.Flow cytometry analysis indicated that under carboplatin treatment,RNA interfering of NEK2 significantly arrested the cell cycle of carboplatin-resistant ovarian cancer cells,and increased the apoptotic and death rate of ovarian cancer cells.Consistent with the finding,transcriptome sequencing indicated that NEK2 decrease significantly correlated with several signaling pathways including cell cycle,apoptosis and p53signaling when the cells were treated with carboplatin.Besides,Western blot revealed that with or without carboplatin treatment,the expression levels of several key proteins in cell cycle,such as Cyclin D3,Cyclin E1 and p27 Kip1,were variedly suppressed by RNA interfering of NEK2,and the expression level of MDR1 was significantly inhibited by RNA interfering of NEK2.Furthermore,based on the results of transcriptome sequencing,signaling pathway changes in H-sh NEK2 and H-CBP-sh NEK2 cells treated with 125μM and 250μM carboplatin were dynamicly analyzed,and the results indicated that NEK2decrease might participate in regulation of carboplatin resistance via several pathways independent of carboplatin,which including NFKB1 signaling and platinum drug resistance signaling.Conclusion:RNA interfering of NEK2 could significantly increase the sensitivity of ovarian cancer cells to caboplatin,probably via interactions with several biological processes such as cell cycle,apoptosis and drug transport,and via interactions with several pathways such as p53,NFKB1 and platinum drug resistance signaling.The results will set the stage for further application of this gene in treatment of drug-resistant ovarian cancer.