| Objective To investigate the effect of Sphingosine kinase 1(SphK1)gene silencing on the sensitivity of cisplatin(DDP)in human colon cancer RKO cells and its potential mechanisms.Methods The pLenti6.3-shRNA-SphK1 lentiviral vector targeting Sph K1 gene was designed by recombinant DNA technique,and the p Lenti6.3-EGFP lentiviral vector was used as a negative control(NC)to transfect human colon cancer RKO cells.A monoclonal RKO cell line with stable low expression of SphK1 was obtained by screening with blasticidin(BSD).Then cells were classified into three groups: Sph K1 low-expression group(shSphK1 group),negative control group(NC group)and wild-type RKO cells without transfected as blank control group(Control group).To detect cell transfection efficiency,the expression of fluorescent protein in transfected cells was observed under inverted fluorescence microscope,Real-time quantitative PCR(RT-qPCR)was used to detect the mRNA level of SphK1 and the expression of SphK1 protein level was detected by Western blot.The experiment will be divided into non-DDP group and DDP group according to with or without DDP treatment.The non-DDP group includes shSphK1 group,Control group and NC group;theDDP group includes shSphK1+ group,Control+ group and NC+ group.Cells of these groups were incubated with or without(0,2,4,8,16,32)μg / mL DDP for0,24,48 and 72 hours.After treatment,cell viability was detected by MTT assay.Apoptosis was evaluated by TdT-mediated dUTP-biotin nick end labeling(TUNEL).Western blot was uesed to detect protein level of Ki-67(proliferation related marker),apoptosis-related proteins Bcl-2,Caspase-9 and Caspase-3.Results A large number of green fluorescent protein expression was observed in the shSphK1 and NC groups under inverted fluorescence microscope.RT-q PCR and Western blot showed that SphK1 expression of shSphK1 group was significantly lower than Control group and NC group(P<0.05).MTT assay indicated that 24 h cell viability of shSphK1 group,Control group and NC group were(1.1063 ± 0.0316),(1.2821 ± 0.0463)and(1.2508 ±0.0365);48 h cell viability of three groups were(1.1937 ± 0.0347),(1.5733 ±0.0554)and(1.5314 ± 0.0572);72 h cell viability of three groups were(1.2288± 0.0495),(1.6907 ± 0.0785)and(1.6667 ± 0.0658).Cell proliferation of shSphK1 group was significantly reduced when compared with Control group and NC group(P<0.05),indicating that down-regulation of SphK1 can inhibit RKO cells proliferation.MTT assay also showed that after treated with 2 μg / ml DDP,24 h cell viability of shSphK1+ group,Control+ group and NC+ group were(0.7587 ± 0.0422),(0.9379 ± 0.0535)and(0.9189 ± 0.0512);48 h cell viability of three groups were(0.6675 ± 0.0429),(0.8151 ± 0.0704)and(0.7868± 0.0893);72 h cell viability of three groups were(0.5224 ± 0.0535),(0.7510 ±0.0632)and(0.7456 ± 0.0605).When treated with 4 μg / ml DDP,24 h cell viability of three groups were(0.6332 ± 0.0303),(0.8561 ± 0.0697)and(0.8364 ± 0.0714);48 h cell viability of three groups were(0.5236 ± 0.0219),(0.7121 ± 0.0253)and(0.6971 ± 0.0536);72 h cell viability of three groupswere(0.4035 ± 0.0709),(0.6495 ± 0.0375)and(0.6002 ± 0.0218).Then treated with 8 μg / ml DDP,24 h cell viability of three groups were(0.5447 ± 0.0399),(0.7683 ± 0.0402)and(0.7409 ± 0.0622);48 h cell viability of three groups were(0.4343 ± 0.0529),(0.5938 ± 0.0701)and(0.5576 ± 0.0365);72 h cell viability of three groups were(0.2781 ± 0.0195),(0.4725 ± 0.0427)and(0.4543 ± 0.0594).When treated with 16 μg / ml DDP,24 h cell viability of three groups were(0.4575 ± 0.0622),(0.6387 ± 0.0899)and(0.6193 ± 0.0323);48 h cell viability of three groups were(0.2519 ± 0.0353),(0.4163 ± 0.0532)and(0.3952 ± 0.0413);72 h cell viability of three groups were(0.1472 ±0.0477),(0.3061 ± 0.0319)and(0.2707 ± 0.0242).While treated with 32 μg / ml DDP,24 h cell viability of three groups were(0.2867 ± 0.0746),(0.4519 ±0.0698)and(0.4298 ± 0.0706);48 h cell viability of three groups were(0.1120± 0.0493),(0.2194 ± 0.0389)and(0.1888 ± 0.0439);72 h cell viability of three groups were(0.0478 ± 0.0073),(0.1399 ± 0.0231)and(0.1040 ± 0.0365).The above results revealed that DDP inhibit cell proliferation in a dose and time-dependent manner.When incubated with same concentration of DDP for equal time,cell proliferation of shSph K1+ group was obviously decreased compared with Control+ group and NC+ group(P < 0.05).The results of TUNEL showed that the apoptotic rate of shSphK1 group(13.54 ± 2.19)% was significantly higher than that of Control group(5.04 ± 1.05)% and NC group(6.15 ± 1.56)%(P < 0.05).Then treated with 8 μg / ml DDP for 24 h,cell apoptotic rate of shSphK1+ group,Control + group and NC+ group were(25.91± 3.01)%,(18.68 ± 3.16)% and(19.28 ± 2.97)%.When treated with 16 μg / ml DDP for 24 h,cell apoptotic rate of three groups were(55.26 ± 4.71)%,(35.82 ±3.97)% and(37.52 ± 3.51)%.When treated with 32 μg / ml DDP for 24 h,cell apoptotic rate of three groups were(76.04 ± 4.52)%,(59.60 ± 4.87)% and(62.28± 3.11)%.These results indicated that DDP induced RKO cells apoptosis in a concentration-dependent form,and the apoptosis rate of shSphK1+ group was obviously higher than that of Control+ group and NC+ group(P <0.05).Western blott showed after SphK1 gene silencing,the protein expression of Ki-67 in shSphK1 group,Control group and NC group were(0.9410 ± 0.0437),(1.1592 ±0.0753)and(1.1543 ± 0.0981);the protein expression of Bcl-2 in three groups were(0.7317 ± 0.0374),(0.9180 ± 0.0633)and(0.9205 ± 0.0495);the protein expression of Caspase-9 in three groups were(0.4367 ± 0.0757),(0.2317 ±0.0593)and(0.2495 ± 0.0435);the protein expression of Caspase-3 in three groups were(0.6479 ± 0.0762),(0.3989 ± 0.0499)and(0.3942 ± 0.0614).After treated with 16 μg / ml DDP for 24 h,protein level of Ki-67 in shSph K1+ group,Control+ group and NC+ group were(0.3105 ± 0.0367),(0.8049 ± 0.0684)and(0.8138 ± 0.0596);protein level of Bcl-2 in three groups were(0.2329 ± 0.0389),(0.5203 ± 0.0312)and(0.5270 ± 0.0497);protein level of Caspase-9 in three groups were(1.8941 ± 0.1025),(0.8627 ± 0.0624)and(0.8785 ± 0.0858);protein level of Caspase-3 in three groups were(2.5849 ± 0.1586),(0.8955 ±0.0735)and(0.8872 ± 0.0942).These results indicated that with SphK1 silenced,the expression of Ki-67 and Bcl-2 were reduced;the expression of Caspase-9and Caspase-3 were enhanced.After treated with DDP,the protein level of Ki-67 and Bcl-2 in shSphK1+ group was significantly lower than that in Control+ group and NC+ group(P <0.05),while the expression of Caspase-9and Caspase-3 protein in shSphK1+ group was significantly higher than that in Control+ group and NC+ group(P <0.05).Conclusion SphK1 gene silencing can inhibit the proliferation of colon cancer RKO cells.Down-regulation of SphK1 gene may promote apoptosis of RKO cells by inhibiting the level of Bcl-2 and activating Caspase 9/3 expression.SphK1 gene silencing may enhance the chemosensitivity of colon cancer RKO cells to DDP by activating the Caspase-9/3 dependent apoptotic signaling pathway. |
