| Objective:Based on RNA sequencing(RNA-seq)technique,the target and mechanism of compound GA5 in overcoming the drug resistance of human breast cancer multidrug resistant cell line MCF-7/Adr(Muti-drug Resistance,MDR)were studied.Methods:Human breast cancer cell line MCF-7/Adr with multidrug resistance induced by adriamycin(DOX)was used as a model.1.Using MTT assay GA5 alone and in combination with a chemotherapeutic agent effect GA5,chemosensitivity of cells resistant MCF-7/Adr;2.Screening genes by RNA-Seq:(1)screening differentially transcribed genes of MCF-7/Adr by RNA-Seq technique with MCF-7 sensitive cell lines as reference,(2)screening differential transcriptional genes of MCF-7/Adr cells treated with compound GA5 by RNA-Seq technique,comparing with MCF-7/Adr cells,and screening differentially transcribed genes of MCF-7/Adr cells by using RNA-Seq technique to find possible genes related to drug resistance,(3)from the differentially transcribed genes screened from MCF-7/Adr and MCF-7,and the differentially transcribed genes screened from MCF-7/Adr and MCF-7/Adr treated by GA5,the common differentially transcribed genes were found in the two groups of screened differentially transcribed genes,so as to find the possible intervention targets for GA5 to overcome drug resistance.3.qRT-PCR was used to verify the gene transcription level,(1)the transcription level of ABCB1,BCL2A1,CYP1A2,ERCC1,GSTP1 in MCF-7 and MCF-7/Adr,(2)the changes of differential transcriptional genes in TENM4,PCDHA12,PLPPR4,HIST1H1B,HFM1,BMP6,ANGPTL4,TLE3,NDRG1,GPR78 and CAVIN-2 between MCF-7 and MCF-7/Adr.(3)the changes of TENM4,PCDHA12,PLPPR4,HIST1H1B,HFM1,BMP6,ANGPTL4,TLE3,NDRG1,GPR78 and CAVIN-2 differentially transcribed genes in MCF-7/Adr and MCF-7/Adr after GAS treatment.4.Western Blot was used to verify the changes of BMP6,TLE3 and NDRG1 protein expression in MCF-7 and MCF-7/Adr cells and the effect of GA5 on BMP6,TLE3 and NDRG1 protein expression in MCF-7/Adr cells.Results:1.The results of MTT detection showed that the 50%inhibitory concentration(IC50)of doxorubicin(Doxorubicin,DOX),paclitaxel(Paclitaxel,PTX),cisplatin(Cisplatin,DDP)and etoposide(Etoposide,VP-16)on MCF-7 sensitive cells were 1.47±0.08,2.89±0.50,16.15±0.32,92.85±3.90 ?m,respectively.The other three chemotherapeutic agents showed significant inhibitory activity on MCF-7 cells.Under the same experimental conditions,that is,MCF-7/Adr cells were resistant not only to DOX,but also to PTX,DDP and VP-16.The RI of each chemotherapeutic drug was 159.23,1.1,1.2,5.0 respectively.The above results show that MCF-7/Adr cells are highly resistant to DOX,obviously resistant to VP-16(but MCF-7 is not sensitive to VP-16),and resistant to PTX and DDP,so MCF-7/Adr shows the characteristics of multidrug resistant cells,and this model meets the requirements of this study.2.The IC50 of compound GA5 to MCF-7 and MCF-7/Adr cells was 54.94±0.99,13.55±0.28 ?M.Compared with the above chemotherapeutic drugs,GA5 had stronger activity against MCF-7/Adr,and the effect of GA5 on multidrug resistant cell line MCF-7/Adr was superior to that of sensitive cell line MCF-7/Adr,which was similar to the previous results.The effect of GA5 on MCF-7/Adr in combination with different chemotherapeutic drugs:(1)the effect of GA5 combined with DOX on drug resistance of MCF-7/Adr cells.The results showed that the IC50 of MCF-7/Adr cells treated with DOX alone and in combination with different concentrations of compound GA5 were 234.07 ± 13.55,188.43±57.93,175.90±35.70,142.94 ±30.21 ?M,and Rf were 1.24,1.33,1.64 times.It is suggested that compound GA5 can increase the sensitivity of drug-resistant cell line MCF-7/Adr to chemotherapeutic drug DOX.(2)the effect of GA5 combined with PTX on drug resistance of MCF-7/Adr cells.The results showed that the IC50 of MCF-7/Adr cells treated with PTX alone and in combination with different concentrations of compound GA5 were 2.88±0.88,1.67±0.15,1.99±0.47,2.44±0.25 ?M,Rf 1,72,1.44,1.18 times,indicating that Low concentration GA5 showed a certain reversal effect on PTX resistance in MCF-7/Adr cells,but did not show a dose-effect relationship.(3)the effect of GA5 combined with DDP on drug resistance of MCF-7/Adr cells.The results showed that the IC50 of MCF-7/Adr cells treated with DDP alone and in combination with different concentrations of compound GA5 were 18.71 ± 1.99,16.23 ±4.78,20.47 ±6.54,19.25±4.03 ?M,indicating that GA5 did not reverse DDP resistance in MCF-7/Adr cells.(4)the effect of GA5 combined with VP-16 on drug resistance of MCF-7/Adr cells.The results showed that the IC50 of MCF-7/Adr cells treated with VP-16 alone and in combination with different concentrations of compound GA5 were 460.7±58.55,279.23 ± 100.00,328.00±124.00 and 271.25 ±21.00 ?M,respectively,and Rf were 1.65,1.40,1.70 times,respectively,indicating that GA5 could reverse the drug resistance of VP-16 to a certain extent.3.RNA-Seq results showed that:(1)compared with MCF-7 cell line,the multidrug resistance genes ABCB1,BCL2A1,ERCC1,GSTP1 were significantly up-regulated in MCF-7/Adr,and the related genes TENM4,PCDHA12,PLPPR4,GPR78,HIST1H1B,HFM1,CAVIN-2;significantly down-regulated in MCF-7/Adr treated with BMP6,ANGPTL4,TLE3,NDRG1;(2)compared with MCF-7 cells,TENM4,PCDHA12,PLPPR4,GPR78,HIST1H1B,HFM1 and CAVIN-2 genes in MCF-7/Adr treated with GA5 were significantly down-regulated,while BMP6,ANGPTL4,TLE3 and NDRG1 genes were significantly up-regulated.4.The results of qRT-PCR showed that:(1)with MCF-7 as the control group,the ABCB1,BCL2A1,ERCC1,GSTP1 gene transcription level in MCF-7/Adr cells was increased,indicating that MCF-7/Adr cells were a multidrug resistant cell model;(2)MCF-7 was used as a control group,and the transcription level of TENM4,PCDHA12,PLPPR4,HFM1,GPR78,CAVIN-2,ANGPTL4,HIST1H1B in MCF-7/Adr cells was increased gene,the TLE3,BMP6,NDRG1,gene transcription level was down-regulated;(3)with MCF-7/Adr as the control group,after MCF-7/Adr was treated with 8 ?M GA5 for 12,24 and 48 hours,the TENM4,GPR78,HIST1H1B gene transcription level was down-regulated,ANGPTL4,BMP6,NDRG1,gene transcription level is up-regulated,according to the consistency of the above RNA-seq results and PCR results,NDRG1,BMP6 and TLE3 genes were selected for later verification.(3)with MCF-7/Adr as the control group,after 8?M GAS concentration acted on MCF-7/Adr for 12h,24h and 48h,the transcription level of TENM4,PCDHA12,PLPPR4,HFM1,GPR78 gene was down-regulated;CAVIN-2,ANGPTL4,HIST1H1B,TLE3,NDRG1,BMP6 gene transcription level is up-regulated.Based on the consistency of the above RNA-seq results and PCR results,the NDRG1,BMP6,and TLE3 genes were selected for later verification.5.Western Blot results showed that with MCF-7 as the control group,the TLE3 protein level in MCF-7/Adr cells was increased and qRT-PCR results were inconsistent,and the NDRG1 protein level was increased,the difference was not statistically significant.BMP6 protein level Down-regulated;with MCF-7/Adr as the control group,after 8?M GA5 acted on MCF-7/Adr for 12,24 and 48 hours,TLE3 protein levels were down-regulated,and NDRG1 protein levels were down-regulated,BMP6 protein levels were increased.Conclusions:1.Compound GA5 has the effect of overcoming the resistance of breast cancer drug-resistant cells MCF-7/Adr,and its activity on drug-resistant cells is significantly higher than that of sensitive cells MCF-7;2.Under the conditions of this experiment,compound GAS did not show a significant effect of reversing the resistance of other chemotherapeutic drugs in drug-resistant cells MCF-7/Adr;3.The compound GA5 overcoming MCF-7/Adr resistance may be related to its effect on bone morphogenetic protein 6(BMP6),but the specific mechanism of action is unclear. |
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