| Research background:Breast cancer is one of the most common malignancies in women,ranking first in cancer incidence and second in mortality among women in 2018.Breast cancer is highly heterogeneous.Clinically,breast cancer can be divided into multiple subtypes according to the characteristics of molecular markers on the cell surface.Among them,triple negative breast cancer is the breast cancer whose estrogen receptor,progesterone receptorand human epidermal growth factor receptor 2 are all negatively expressed.TNBC is the most dangerous one of all breast cancer subtypes.Chemotherapy is the main treatment method for TNBC.TNBC lack of clear therapeutic targets,clinical guidelines recommend the use of cytotoxic drugs such as Epirubicin（Epi）or Paclitaxel（Pac）for TNBC treatment.The treatment side effect is big,adverse reaction is much,but the effect is unsatisfactory,the main reason is that the occurrence of acquired drug resistance lead to secondary recurrence or metastasis of the tumor.TNBC has a relatively high proportion of cancer stem cells.Cancer stem cells（CSCs）are a small proportion of cells with stem cell traits in tumor.It is reported that CSCs are associated with drug resistance in a variety of tumors.In breast cancer,CD44+/CD24-ALDH1+cells are generally considered as breast cancer stem cells（BCSCs）.Have been reported in many literature,after chemotherapy stimulating,the majority of cell dies,the residual tumor cells have phenotypic alterations,the most common changes include the epithelial to mesenchymal transition（EMT）and"stemness"phenotype.Cancer stem cells is one of the key factors for a wide variety of tumor chemotherapy resistance.This process involves changes in a variety of signaling pathways,among which TGF-βsignaling pathway has attracted much attention:TGF-βis a multi-functional cytokine that can activate downstream signaling pathways through Smad-dependent or non-Smad dependent ways.TGF-βplays an important role in the generation and formation of tissues and organs,immune response and other biological processes.In recent years,TGF-βsignaling pathway has been considered to play an important regulatory role in the self-renewal and differentiation of CSCs,especially BCSCs,but its precise molecular regulation mechanism in BCSCs and chemotherapy resistance is still unclear.In our previous study,we found that Runx2,a transcription factor,may be associated with this process.Runx2,also known as core binding factor（cbf-α）,is highly expressed in bone and breast tissues,and is a highly active molecule during bone tissue development and differentiation as well as in the development and progression of various tumors,especially in breast cancer bone metastasis.Previous results have shown that Runx2 is highly expressed in TNBC cell lines and tissues,and that high expression is associated with poor outcomes in patients.Knockdown of Runx2 expression in MB-231 cells with high Runx2 expression resulted in down-regulation of the proportion of CD44+/CD24-/lowBCSCs,while overexpression of Runx2 in MCF-7 cells with low Runx2 expression directly up-regulated the proportion of CD44+/CD24-/low/low BCSCs.Western Blot results were consistent.This suggests that Runx2 has a regulatory effect on BCSCs.It is speculated that Runx2 may play a role in the occurrence of TNBC chemotherapy resistance by regulating BCSCs.Research Objective:Based on previous studies,we observed whether epirubicin stimulation resulted in up-regulation of TGF-β,Runx2 and stem cell markers in TNBC.Whether the upregulation is related to the resistance of chemotherapy;Whether Runx2 can regulate the proportion of breast cancer stem cells involved in chemotherapy resistance in TNBC by regulating the expression of TGF-βprompting EMT;Whether Runx2 has other resistance regulatory mechanisms and whether TGF-βalso influences Runx2 expression.The exploration of these molecular mechanisms of drug resistance provides a theoretical basis for the use of Runx2-TGF-βas a target to reverse chemotherapy resistance and improve survival in patients with TNBC.Research content:1.Expression changes of Runx2,TGF-βand stem cell markers were observed in TNBC cell lines MB-231 and SUM-149 treated with Epi,a commonly used TNBC chemotherapy drug.To determine the upregulation and activation of Runx2,TGF-β and breast cancer stem cells by epirubicin in triple-negative breast cancer chemotherapy.2.A stable drug-resistant TNBC cell line MB-231/Epi was induced by chronic low concentration epirubicin gradual dosing for 6-12 months.MTT drug toxicity experiment,plate cloning and drug resistance experiment were used to detect drug resistance of drug-resistant cells,and the drug resistance index RI was calculated. MTT proliferation assay,transwell,flow cytometry and mammospheres assay were used to detect the phenotypic differences between drug-resistant cells and parental cells.Western blot analysis of TGF-β,BCSC markers and some proteins related to TGF-βregulation.Immunohistochemistry was used to detect the pathological sections of TNBC and non-TNBC patients,and kaplan-meier was used to draw the survival curve of TNBC patients with high or low TGF-βexpression after chemotherapy.Through the above experiments,the drug resistance characteristics of TNBC resistant cells and the role of TGF-βin the epirubicin chemoresistance of TNBC were determined.3.Knockdown of Runx2 expression in drug-resistant cell line and elevation of Runx2 expression in normal TNBC cell line SUM-149 using lentivirus infection method, and stable transgenic strains were selected by puromycin.Drug resistance was detected by MTT toxicity assay,expression of TGF-β,EMT and stem cell markers was detected by Western Blot,and stemness was detected by flow cytometry and mammospheres assay.Using transwell assay to detect their invasion and migration ability.Through the above experiments,we can determine the role of Runx2 in the regulation of chemotherapy-resistance and stemness,TGF-βsignal activation.4.Western Blot and RT-PCR were used to detect the changes in the expression of markers related to the mechanism of drug resistance,such as TGF-β,p-Smad2/3, MDR1,BCRP,P53 and so on,so as to explore the exact molecular mechanism of Runx2 in the regulation of epirubicin resistance and stemness in TNBC.5.The gradient doses of TGF-βcytokine were used to induce MB-231 cells at different times to explore the regulation role of TGF-βon Runx2 expression and its effect on chemoresistance.Research result:1.After acute epirubicin stimulation,the expression of TGF-β,BCSCs marker CD44+ /CD24-and Runx2 in TNBC cells showed a dose-dependent increasing trend by Western Blot.2.Chronic induction successfully established a TNBC epirubicin resistant cell line MB-231/Epi;Using lentivirus infection,Epi-Runx2i（Epi-KD）cell line with knocking down Runx2 expression and its negative control cell line Epi-NC were established;SUM-Runx2（SUM-OE）cell line with elevating Runx2 expression and its negative control cell line SUM-NC were established.3.Western blot results showed increased expression of Runx2,TGF-βand BCSCs markers in drug-resistant cells.The results of MTT drug toxicity experiment,plate cloning and drug resistance experiment,MTT proliferation experiment,transwell experiment,flow cytometry,mammospheres assay and other experiments showed that drug resistance of MB-231/Epi increased,and RI was 6.747 times of that of MB-231,MB-231/Epi has reduced proliferation ability,enhanced EMT related invasion and migration ability,and enhanced stemness.4.Western blot,MTT drug toxicity assay,plate cloning and its resistance assay, transwell assay,flow cytometry,mammospheres assay and other experimental results showed that knockdown of Runx2 expression in TNBC drug-resistant MB-231/Epi could reverse drug resistance,down regulate stemness,expression of TGF-β,EMT related invasion and migration ability and corresponding molecular markers expression.However,overexpression of Runx2 in SUM-149 had contrariwise results.5.RT-PCR and Western blot were used to detect the expression of downstream molecular markers of the TGF-βsignaling pathway and some drug-resistant related markers such as Hif-a,MDR1,BCRP,P53 and so on.The results showed that Runx2 knockdown could reverse the chemotherapy resistance of TNBC,which may because Runx2 regulate the activation of the TGF-βclassical Smad pathway lead to EMT and the expression of multi-drug resistant protein MDR1.6.Western Blot analysis of Runx2 expression in TNBC cells under TGF-βcytokine gradient doses and different time induction,we found that TGF-βhas a feedback regulation on Runx2 expression and the degree of up-regulation of Runx2 is positively correlated with the degree of drug resistance induced.Conclusion:1.Both acute and chronic induction of epirubicin upregulates TNBC cells TGF-β,Runx2,and BCSC stem cell markers expression.2.Runx2 regulates the proportion of CD44+/CD24-breast cancer stem cells by regulating the expression of TGF-βleading to EMT,thereby prompting the chemoresistance of TNBC.3.Knockdown of Runx2 reverses the drug resistance of TNBC drug-resistant cell line which is related not only to the regulation of the classical Smads pathway of TGF-β,but also to the regulation of the expression of MDR1（multi-drug resistant protein1）.4.TGF-βalso has feedback regulation on the expression of Runx2,and its induced expression level of Runx2 is positively correlated with its induced drug resistance level. |
