| Cisplatin has been widely used in treatment of a various types of cancer including triple negative breast cancer(TNBC)by damaging DNA and inducing apoptosis.However,its anti-cancer effects are often limited due to chemo resistance,which is one of main reasons causing cancer relapse and metastasis.To overcome resistance,cisplatin is often used in combination with other drugs or molecules.Herein,we detected a much higher expression of miR-221/222 in the cisplatin-resistant TNBC cells,as well as in cisplatin-resistant breast cancer patients.Targeted inhibition of miR-221/222 in TNBC MDA-MB-231 cells promoted cisplatin-induced cell apoptosis,and increased the cell sensitivity to cisplatin in vitro.The combination chemotherapy of cisplatin with anti-miR-221/222 showed much higher efficiency in suppressing tumor growth in the mice model carrying TNBC tumors in vivo.In addition,miR-221 and miR-222 showed synergetic effects on regulating sensitivity to cisplatin in MDA-MB-231 cells.In mechanism,suppression of socs1-stat3-bcl2 pathway and activation of p53-Pten signaling both contribute toanti-miR-221/222-induced sensitivity to cisplatin in MDA-MB-231 cells.Thesefindings demonstrated a novel approach for combination chemotherapy of cisplatinwith small non-coding RNAs in treatment of human TNBC.Materials and Methods1.Animal studies were approved by the Institutional Animal Care and Use Committee of the Tongji University Shanghai East Hospital.6-week-old nude female mice were provided by the Silaike Animal Company(Shanghai,China).2.Human breast cancer cell lines MDA-MB-231 were originally purchased from ATCC(Manassas,VA,US)and maintained in our laboratory.The cisplatin-resistant MDA-MB-231 cells were a gift fromDr.Hongfeng Chen at Shanghai University LonghuaHospital.doxorubicin-resistant MDA-MB-231 cells were collected from the survival cells after treatment with increasing gradient concentrations of doxorubicin.3.anti-miR-221,anti-miR-222 and negative control oligos were designed following LNA Oligo Tools and Design Guidelines of Exiqon(Vedbaek,Denmark),and synthesized by GenScript(Nanjing,China).The HiPerFect transfection reagent from Qiagen(Venlo,The Netherland)was used for cell transfection following the manufacturer's instructions.Final concentration of 50nM of the RNA oligo was used for all in vitro assays.4.Total RNA was extracted with Trizol reagent(Invitrogen,US).First-strand complementary DNA of miRNAs was prepared using the M&G miRNA Reverse Transcription kit(miRGenes,Shanghai,China)following 6 the manufacturer's instruction.Forward primer sequences for real-time PCR of miRNAs were:miR-16,5'-agcagcacgtaaatattggc-3';miR-221,5'-agctacattgtctgct-3';miR-222,5'-agctacatctggctac t-3';5s ribosomal RNA,5'-agtacttggatgggagaccg-3'.Allprimers were synthesized and purified by GenScript(Nanjing,China).The SYBRGreenMaster Mix was ABI product(Applied Biosystem,Life Technologies).TheABI7900 HT Sequence Detection System(Applied Biosystem,Life Technologies)was used for quantitative real time PCR assay.5s ribosomal RNA was used fornormalization.5.Cell proliferation assay(CCK8)assay:96-well plates were prepared,and 2*103cells were respectively planted in the plates.After a period of culture,the cells were stained for 3 hours under the conditions of cell culture according to the CCK8 kit,and OD values were detected by the labelling apparatus at the wavelength of 450nm.6.Apoptosis assay:cells were cultured for 24-48h at the presence of cisplatin,followed by Annexin V staining.Flow cytometric analysis was performed using Annexin v-fitc/IP kit(Bestbio,China)as per manufacturer's instructions.7.Cell lysates(50mg)were prepared from cells after 48h transfection with anti-miRNA or control,and separated by 10%SDS/PAGE.The proteins were transferred to nitrocellulose membrane.After being blocked in 5%milk(w/v)at room temperature for 1 hour,the membranes were incubated at 4°C overnight with primary antibodies(1:1000).Following 1XTBST washing,the membranes were incubated with secondary antibodies(1:3000)at room temperature for 1 hour followed by ECL staining.The following antibodies were purchased from Santa Cruz Biotechnology for western blot:anti-p27(sc-776),socs-1(sc-9021),c-myc(s-40),Pten(sc-7974),p53(sc-6243),Stat3(4904s),BCL-2(sc-492)and?-actin(sc-47778).8.Experiments on animals:1×10~6 MDA-MB-231 cells were mixed with matrigel,and injected intothe fat pad of the fourth mammary gland of nude mice(n=30).The mice were separated randomly into three groups(n=10 for each group).Blank control group:mice were intraperitoneally injected with 0.9%Nacl(5mg/kg body weight/dose),once a week for three weeks,a total of three times.Tumor local injection 0.9%Nacl(5ug in 10ul:0.5ug/ul):injected every 72 hours,a total of four times;The methods of cisplatin group and combination group were the same as above:cisplatin group:mice were intraperitoneally injected with cisplatin(5mg/kg body weight/dose),and local injection of tumor anti-miRNA-NC.(5ug in 10ul:0.5ug/ul).Cisplatin+anti-miR group:mice were intraperitoneally injected with cisplatin(5mg/kg body weight/dose),and local injection of tumor anti-miR-221/222(5ug in 10ul:0.5ug/ul).The volume of the tumors was measured every other day,and the growth curve of tumors was plotted.After 3-weeks experiments,all mice were sacrificed at day 23 after cell transplantation.All tumors were weighted,and aplied to isolate RNA and proteins for further analysis.9.Statistical analysis:Data are presented as meanąSEM unless stated otherwise.The standard two-tailed student's t-test was used for statistical analysis,in which p<0.05was considered significant.Results1.MiR-221/222 was specifically upregulated in cisplatin/carboplatin resistant breast cancerIn view of the recurrence and metastasis of chemotherapy resistance in breast cancer,miRNA expression screening of MDA-MB-231-R cell was performed to determine the key genes regulating cisplatin resistance in TNBC.In MDA-MB-231-R cell,including miR-221/222,a miRNA subgroup with differential expression was found.In addition to cisplatin,miR-221/222 expression was up-regulated in MDA-MB-231cells.The comparative analysis of miRNA expression between chemotherapy-resistant breast cancer and chemotherapy-sensitive breast cancer in TCGA database showed that the levels of miR-221 and miR-222 in cisplatin/carboplatin resistant breast cancer patients were significantly higher than those in chemotherapy-sensitive breast cancer patients.However,there was no difference in the expression of miR-221 and miR-222in other drugs(such as tamoxifen resistant and sensitive breast cancer patients),suggesting that miR-221/222 was specifically upregulated in cisplatin/carboplatin resistant breast cancer.2.Knockdown of miR-221/222 by anti-miR-221 and anti-miR-222 inhibitors significantly increased the sensitivity of cells to cisplatinIn view of the upregulation of miR-221/222 in cisplatin resistant breast cancer/carboplatin resistant breast cancer,the regulatory function of miR-221/222 was further determined by targeting and knocking down the expression of miR-221/222 in MDA-MB-231-R cells,followed by cisplatin treatment and cell viability detection.It was shown that knockdown of miR-221/222 with anti-miR-221 and anti-miR-222inhibitors significantly increased the sensitivity of cells to cisplatin,and annexin V staining and flow cytometry were used to detect the apoptotic cells treated with and without cisplatin before and after transfection of miR-221/222 with MDA-MB-231 cells.In the absence of cisplatin stimulation,anti-miR-221/222 did not affect apoptosis compared with anti-nc.However,in the presence of cisplatin at 20uM,anti-miR-221/222 significantly increased the apoptosis rate of MDA-MB-231cells from~12%to~22%,further indicating that the application of anti-miR-221/222 promoted the sensitivity of MDA-MB-231cells to cisplatin.3.The synergistic effect of anti-miR-221 and anti-miR-222 was further verified by flow cytometry analysis after annexin V stainingSince miR-221 and miR-222 have the same seed sequence and may have the same target gene,it is necessary to determine the compensatory and/or synergistic effects of miR-221 and miR-222 in the regulation of drug sensitivity in TNBC cells.Only a few anti-miR-221 or anti-miR-222 cells affected cisplatin sensitivity,while the combination of anti-miR-221 and anti-miR-222 significantly increased the sensitivity ofMDA-MB-231cells to cisplatin.Therefore,the synergistic effect of anti-miR-221 and anti-miR-222 on promoting the sensitivity of TNBC cells to cisplatin was further confirmed by flow cytometry analysis after annexin V staining.4.In vivo experiments on mice carrying TNBC tumors,the results showed that anti-miR-221/222 combined with cisplatin had a significant effect in inhibiting tumor growth.In order to further determine the sensitivity of anti-miR-221/222 to cisplatin in TNBC cells,MDA-MB-231 cells were inoculated in the fourth pair of breast fat pads of nude mice,and then intraperitoneally injected with cisplatin for treatment,and anti-miR-221/222 was given locally in the tumor.Mice were sacrificed 23 days after MDA-MB-231 cells were inoculated,and the tumors were removed and weighed.The results showed that the average tumor size and tumor weight of cisplatin+anti-miR group were smaller than those of cisplatin group and control group.In vivo experiments on mice carrying TNBC tumors,the results showed that anti-miR-221/222 combined with cisplatin had a significant effect in inhibiting tumor growth.5.Inhibition of socs1-stat3-bcl2 pathway and activation of p53-pten signaling pathway can induce anti-miR-221/222 sensitivity of MDA-MB-231 cells to cisplatinTo determine the mechanism by which miR-221/222 regulates cisplatin sensitivity in TNBC,p27 and socs1,we selected two target genes previously identified by us in miR-221/222 in TNBC and analyzed the mRNA and protein levels in MDA-MB-231cells before and after transfection of anti-miR-221/222.The up-regulation of p27 and socs1 was related to the knockdown of miR-221/222.Further analysis of Stat3 as a widely confirmed target gene downstream of socs1 showed that anti-miR-221/222inhibited its expression,and Stat3 transcriptional regulation regulated the expression of a group of genes including c-myc and bcl2,and regulated tumor occurrence and drug resistance.Analysis of Stat3 downstream genes showed that anti-miR-221/222down-regulated c-myc and bcl2,which was consistent with the up-regulation of socs1and down-regulation of Stat3.Further analysis of key regulatory genes for apoptosis showed that miR-221/222 targeted to down-regulate the expression of p53 and pten in MDA-MB-231cells.These results indicated that inhibition of socs1-stat3-bcl2 pathway and activation of p53-pten signaling pathway could induce anti-miR-221/222 sensitivity of MDA-MB-231cells to cisplatin.Conclusions1.miR-221/222 was highly expressed in drug-resistant TNBC cells.2.The inhibition of miR-221/222 promoted the sensitivity of TNBC cells tocisplatin.3.anti-miR-221 and anti-miR-222 synergistically promoted the sensitivity of TNBC cells to cisplatin.4.anti-miR-221 and anti-miR-222 combined with cisplatin can further improve the sensitivity of TNBC cells to cisplatin in the tumorigenesis experiment of mice carrying TNBC tumor.5.The mechanism by which anti-miR-221/222 regulates TNBC's sensitivity to cisplatin is mediated by inhibiting the socs1-stat3-bcl2 pathway and activating the p53-Pten signaling pathway. |
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