Silencing The Expression And Function Of Breast Cancer Resistance Protein In MCF7/MX100Cells By RNA Interference

A sequence was found, which can generate the best interference effect on the human breast cancer resistance protein(ABCG2/BCRP) through shRNA expressing plasmids and synthetized siRNA and used to establish the MCF7/MX100cell line stably expressing siRNA targeted to human ABCG2/BCRP by lentivector-mediated gene transfer system.This cell line can effectively restore the chemosensitivity to miotxantrone by suppressing the BCRP/ABCG2mRNA and protein expression level. Furthermore, it could be used in screening active ingredients so as to make sure whether they are BCRP substrates or inhibitors, and it might be a good cell model in drug development and rational use.1. Construction of shRNA expressing plasmids and their interference to BCRPMCF7/MX100cell lines resistant to mitoxantrone were established through "two phases screening"-increasing dose mitoxantrone and inducing time which were confirmed by western blot and MTT, with a significant increase in BCRP expression level and an increasing IC50from1.014±0.11μmol/L in MCF7parent cells to4.064±0.38μmol/L (P<0.001,***).Firstly, shRNA expressing plasmids were constructed based on pSilencer4.1plasmid and identified the interference effect by RT-PCR. Although several shRNA expressing plasmids were constructed successfully, the interference effect was very weak compared to negative control, mainly because of imperfected design of siRNA.2. Synthetized siRNAs and their interference to BCRP Three synthetized siRNAs (si-BCRP1, si-BCRP2, and si-BCRP3) were transfected into MCF7/MX100cells by liposome. The BCRP expression of MCF7/MX100cell was measured by real-time RT-PCR and western blot methods. Flow cytometry assay was applied to measure the change of chemosensitivity to miotxantrone. The results from RT-PCR and western blot demonstrated that BCRP expression was significantly decreased by transfecting si-BCRP2and si-BCRP3, the mRNA inhibition percentages of si-BCRP2, si-BCRP3was97%and95%, the protein inhibition percentages was76%and71%. The results from flow cytometry assay showed that the chemosensitivity to mitoxantrone was improved, especially by si-BCRP2and si-BCRP3(P<0.05).RT-PCR and western blotting demonstrated that BCRP expression was significantly decreased by transfecting si-BCRP2and si-BCRP3, the mRNA inhibition of si-BCRP2, si-BCRP3was97%and95%, the protein inhibition was76%and71%. Flow cytometry assay demonstrated the chemosensitivity to mitoxantrone was improved, especially by si-BCRP2and si-BCRP3(P<0.05).3. Establishment of the MCF7/MX100cell line stably expressing siRNA targeted to human ABCG2/BCRP by lentivector-mediated gene transfer systemFirstly, RNA interference sequence was inserted into lentiviral expression plasmid pTRIPZ after digested by restricted enzymes, then packaged the lentivirus in lenti293t cells by transfection reagent Roche X-tremeGENE for48h. The MCF7/MX100cell line was infected by lentivirus with polybrene(8μg/mL). Finaly, the interference effects were identified after screening with puromycin(1μg/mL) and inducing with doxycycline(1μg/mL). After infecting lentivirus into MCF7/MX100cell line, the mRNA inhibition percentages by Lenti-BCRP2, Lenti-BCRP3was72%and56%, and the protein inhibition percentages was70%and53%. Meanwhile, the chemosensitivity to mitoxantrone was significantly improved in Lenti-BCRP3(P<0.05).4. The application of the stably transfected MCF7/MX100cell lineThe MCF7/MX100cell line stably expressing siRNA targeted to human ABCG2/BCRP were used in screening active ingredients in TCMs(Traditional Chinese medicines), such as dihydromyricetin(30μmol/L), icariin(10μmol/L), isorhamnetin(25μmol/L). For accumulation study, the incubation time was45min, while for interaction study, mitoxantrone would incubated for30min after preincubation in other compounds for15min. The samples were analysed by UPLC-MS/MS methods. Then calculated the concentration of ingredients in cells, compared the accumulation per minute by certain protein. The results showed that accumulation of isorhamnetin was significantly increased after inducing with doxycycline, meanwhile, the accumulation of mitoxantrone were significantly increased after preincubation with isorhamnetin. So isorhamnetin might be both a BCRP substrate and inhibitor.