| Platinum chemotherapeutic agent cisplatin is still the first-line drug in the treatment of non-small cell lung cancer(NSCLC),which mainly causes DNA double-strand breaks.Increased repair capacity of double-stranded DNA is associated with innate or acquired cisplatin resistance.Although Kinesin family member 4A(KIF4A)has been reported to regulate a variety of DSB repair related proteins,such as breast protein 2(breast cancer 2,BRCA2)and poly(ADP ribose)polymerase-1,PARP 1),the relationship between KIF4 A and cisplatin sensitivity in human NSCLC cells remains unclear,and little is known regarding the effect of targeting KIF4 A on the function of DSB repair-related proteins in these cells.Therefore,we have carried out the following research.1.Objective:By targeting the expression of KIF4 A molecule,the effect of KIF4 A protein expression on the sensitivity of NSCLC to cisplatin was studied,and the effect of silencing KIF4 A protein on the function of DSB repair-related proteins in NSCLC was explored,which provided a novel thread for the treatment of NSCLC with chemotherapy resistance.2.Methods:(1)In order to study whether cisplatin affects the expression of KIF4 A in human NSCLC cells.H1299 cells were treated with or without 10 ?mol/L of cisplatin for different times including 0h,12 h,24h,48 h and72h.Western-blot assay was used to detect the expression of KIF4 A protein.(2)In order to explore the effect of silencing KIF4 on the function and cisplatin sensitivity of NSCLC cells,H1299 cells were infected with multiple lentiviral vectors shRNA-KIF4 A,targeting human KIF4 A gene by shrna-kif4 a vector(shrna-nc)as control.Then H1299 cells were selected by puromycin,and stable KIF4 A knockdown H1299 cell lines shRNA-KIF4A-(3)and shRNA-KIF4A-(4)were established.The stable knockdown KIF4 A H1299 cell line was treated with different concentrations of cisplatin,and the effect of silencing KIF4 A on the cisplatin sensitivity of NSCLC cells was observed by clonal formation assay and apoptosis assay(Annexin V and Propidium iodide(PI)double staining).(3)Given that DNA damage induced by cisplatin can lead to cell cycle arrest in S and G2 phases,cell cycle plays an important role in the selection of DSB repair pathway.The stable knockdown KIF4 A H1299 cell line was treated with different concentrations of cisplatin,and the cell cycle changes were detected by PI staining to observe the effect of knockdown of KIF4 A on cisplatin induced cell cycle arrest.(4)To investigate the possible mechanism of enhancement cisplatin induced DNA damage by inhibition of KIF4 A in H1299 cells,with shRNA-NC KIF4 A H1299 cells as contrast,the stable knockdown KIF4 A H1299 cell line was treated with cisplatin after different time,with detected the focus of BRCA2 and RAD51(focus)form in the nuclei with immunofluorescence,using Western blot to detect BRCA2,RAD51 and PARP-1 protein expression and activity changes involved in DSB repair.3.Results:(1)Cisplatin treatment stimulates the expression of KIF4 A protein in human H1299 NSCLC cells.KIF4A was abundantly expressed in H1299 cells.At 12,24,48,or 72 hr after treatment with cisplatin,KIF4 A expression in these cells was increased by about 69%,168%,130%,and 150%,respectively.These results indicate that cisplatin treatment enhances the expression of KIF4 A protein in human NSCLC cells.(2)KIF4A knockdown enhances cisplatin sensitivity of human H1299 NSCLC cells.Lentiviruses were used to infect H1299 cells and establish stable silenced KIF4 A cells.Two groups of cells were treated with cisplatin.After exposure to 1 and 2 ?mol/L of cisplatin,about 54 and 26 clones of H1299 cells infected stably with control shRNA were still alive,respectively.However,knockdown of KIF4 A significantly increased cisplatin cytotoxicity in H1299 cells;after infection stably with shRNA-KIF4A-(3)and shRNA-KIF4A-(4),approximate 16 and 43 clones of H1299 cells still survived at 1 ?mol/L of cisplatin,and just about 11 and 18 clones of H1299 cells were left alive at 2 ?mol/L of cisplatin,respectively.These results indicate that KIF4 A silencing potentiates cisplatin-induced cytotoxicity in human H1299 NSCLC cells.In addition,the early and late apoptotic rates of shRNA-NC H1299 cells treated with 10?mol/L cisplatin in the control group were 7.8% and 11.4% respectively,but the apoptotic rates of KIF4A-silenced cells increased significantly.The early and late apoptotic rates of stably KIF4 A knockdown H1299 cell line shRNA-KIF4A-(3)were 20.0% and 20.1%,respectively.The early and late apoptotic rates of stably KIF4 A knockdown H1299 cell line shRNA-KIF4A-(4)were 15.6% and 17.8%,respectively.These results suggest that inhibition of KIF4 A gene can significantly increase the early and late apoptosis of H1299 cells induced by cisplatin,and increase the sensitivity of cells to cisplatin.(3)Knockdown of KIF4 A enhances cisplatin-induced cell cycle arrest in human H1299 NSCLC cellsCompared with control shRNA-NC H1299 cells,higher concentration(10 ?mol/L)of cisplatin induced cell cycle arrest in both S and G2/M phases.However,when cisplatin was dropped to 5 ?mol/L,cell cycle G2/M arrest persisted while S arrest decreased.Notably,KIF4 A knockdown significantly enhanced cell cycle G2/M arrest induced by cisplatin at a lower concentration(5 ?mol/L)and cell cycle S arrest induced by cisplatin at a higher concentration(10 ?mol/L).Collectively,these data suggest that KIF4 A inhibition augments cell cycle S and G2/M arrest induced by cisplatin in human NSCLC cells.(4)Knockdown of KIF4 A inhibits DNA damage repair ability of human H1299 NSCLC cells.When cells were treated with 10 ?mol/L of cisplatin,nearly 70% and 60% of BRCA2 and RAD51 foci were induced in H1299 cells infected with lentiviral control shRNA,respectively.However,in KIF4 A shRNA-targeted cells,BRCA2 and RAD51 foci were significantly reduced,with only approximately 40% of cells being BRCA2 or RAD51 foci positive.These results suggest that silencing KIF4 A can inhibit the focal formation of BRCA2 and RAD51 in the nucleus of human NSCLC induced by cisplatin.After treated with 10 ?mol/L cisplatin for 0,12,24,48 and 72 hours,the expression of BRCA2 and RAD51 protein in shRNA-NC H1299 cells increased significantly and reached the highest value at 24 h after treatment.However,Although KIF4 A knockdown did not significantly affect the expression of BRCA2 and RAD51 proteins in H1299 cells,KIF4 A knockdown appreciably inhibited the stimulatory effect of cisplatin on the expression of BRCA2 and RAD51,although there was no significant change in the expression of these proteins in H1299 cells infected with lentivirus shRNA KIF4 A alone compared with control lentivirus shRNA-NC.Taken together,these results suggest that KIF4 A significantly contributes to cisplatin-induced formation of BRCA2 and RAD51 foci in human NSCLC cells.In addition,we found that silencing KIF4 A reduced the expression of PARP-1 by WB assay.Therefore,we want to further observe whether it affects the activity of PARP-1.The activity of PARP-1 reacts by detecting PAR,the active product of PARP-1.Infection with lentiviral KIF4 A shRNA alone increased the PARP-1 activity in these cells,which is consistent with previous reports.Similarly,treatment with 10 ?mol/L of cisplatin gradually increased the activity of PARP-1 in H1299 cells in a time-dependent manner.However,knockdown of KIF4 A decreased the expression of PARP-1,thereby leading to the inability of cisplatin treatment to further increase the PARP-1 activity in H1299 cells infected with lentiviral KIF4 A shRNA.In aggregate,these results suggest that KIF4 A knockdown limits the further increase in PARP-1 activity induced by cisplatin treatment in human NSCLC cells.4.Conclusion Cisplatin treatment stimulates the expression of KIF4 A protein in human H1299 NSCLC cells.KIF4 A knockdown inhibits cisplatin-induced formation of BRCA2 and RAD51 foci,and limits the further increase in PARP-1 activity induced by cisplatin treatment in human NSCLC cells,thereby enhances cisplatin-induced cell cycle arrest and potentiates cisplatin-induced cytotoxicity.These studies thus identify the chromokinesin KIF4 A as a novel modulator of cisplatin sensitivity that is significantly enhanced by the chromokinesin in human NSCLC cells via multiple mechanisms. |
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