| Background and ObjectiveMethodsUsing high dose doxorubicn(DOX)combined with time-increasing to establishment of human bladder cancer doxorubicin-resistant cell line T24/DOX;Observing the morphological changes of T24 and T24/DOX cell lines of different parental strains under inverted microscope;CCK-8 was used to detect IC50 of T24 and T24/DOX at different stages,calculate the resistance index(RI).The growth curves of T24 and T24/DOX were plotted and calculate the multiplication time,the T24/DOX resistance was initially measured.Plate colony formation to Detect Single Cell Clone Formation Ability of T24 and T24/DOX;Rhodamine 123(Rh123)accumulation assay was used to detect the amount of rhodamine accumulation in the T24 and T24/DOX and to detect the activity of drug-resistance-associated protein P-gp;PI/Hoechst33342 double staining method was used to detect the apoptosis rate of T24 and T24/DOX under different drug concentrations;Western Blot was used to detect the expression of drug-resistance-associated proteins in the T24 and T24/DOX,preliminary explored the mechanism of T24/DOX resistance;Using subcutaneous tumor transplantation to establish a drug-resistance model of transplanted tumor in nude mice with T24 and T24/DOX.and detect T24/DOX and its mechanism were verified in vivo.High-throughput sequencing was used to screen Inc RNAs differentially expressed in T24 and T24/DOX.Results1.Establishment of T24/DOX-resistant cell line:Successfully establish human bladder cancer T24/DOX-resistant cell line(the drug-resistant cell is homologous to the parental cell).The IC50 of the T24 and the T24/DOX were 247.6 ng/ml,3221 ng/ml,and RI 13.01.2.Verification of drug resistance:In the inverted microscope,it was observed that T24/DOX had a significant change in morphology compared to the T24,and the proportion of deformed cells is increased;The cell growth curve showed that the doubling times of T24 and T24/DOX cells were(53.41 ± 8.24)h and(44.36 ± 6.64)h respectively,compared with the T24,T24/DOX extend doubling time(P<0.05);The colony formation rates of T24 and T24/DOX cells were(18.4± 1.83)%and(44.93±3.03)%,respectively,and the T24/DOX colony formation rate was significantly increased compared with T24 cells(P<0.01);Rhl23 accumulation experiment results showed that compared with T24 cells(6.43±0.75%),the fluorescence intensity in T24/DOX-resistant cells was significantly reduced,and the positive cell rate was also significantly reduced(2.15±0.81)%(P<0.01).The results of PI/Hoechst33342 showed that T24/DOX red fluorescence was decreased compared with T24 cells at different drug concentrations(DOX 2.5,1.25,and 0.625 ?g/ml)(P<0.05).The results of PI/Hoechst33342 showed that T24/DOX red fluorescence was decreased compared with T24 cells at different drug concentrations(DOX 2.5,1.25,and 0.625 ?g/ml)(P<0.05);Successfully established a drug-resistant model of transplanted tumor in nude mice,and the tumor formation rate was 100%.The tumor inhibition rate of transplanted tumors in T24 nude mice was 76.18%,and the tumor inhibition rate of T24/DOX in nude mice was 32.23%(P<0.05).In vivo experiments showed that T24/DOX had obvious drug resistance.3.Expression of drug-resistant proteins in the T24 and T24/DOX:Compared with T24 cells,the expression of Bcl-2 was increased in the T24/DOX-resistant cell line,the ratio of Bax/Bcl-2 was significantly decreased(P<0.05),and the expression of P-gp protein was increased(P<0.01).The expression of caspase-3 decreased(P<0.05);The expression of protein in nude mice transplanted tumor tissue was higher in the T24/DOX group than in the T24 nude mice.The expression of Bax was decreased and the ratio of Bax/Bcl-2 was decreased in the T24/DOX group(P<0.01).).Caspase-3 expression decreased(P<0.01);Compared with DOX T24 in nude mice,the expression of Bcl-2 was increased,the expression of Bax was decreased,the ratio of Bax/Bcl-2 was decreased(P<0.05),and the expression of Caspase-3 was decreased(P<0.05),the expression of the protein in the tumor tissue of nude mice was consistent with the expression in the cells.4.High-throughput sequencing results:High-throughput sequencing was performed on three T24.T24/DOX(RI= 6.12 and 13.01,respectively)find 292 new Inc RNAs.Compared with T24 cells,there were 11 differentially expressed Inc RNAs in T24/DOX(RI = 6.12 and 13.01,respectively).There were 5 Inc RNAs shared by the two resistant cells,respectively MSTRG.14409(FC<-1.5),MSTRG.16907(FC<0),MSTRG.121(FC<0),MSTRG.17(FC<0),MSTRG.16359(FC>1.5).Conclusions1.Establishment of stable human bladder cancer doxorubicin-resistant cell line T24/DOX2.The mechanism of drug resistance of T24/DOX was related to inhibition of apoptosis and increased expression of P-gp protein;3.High-throughput sequencing results showed that there were abnormally expressed Inc RNAs in the resistant strain T24/DOX and the parent strain T24. |
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