| Objective:Breast cancer is the most common malignant tumor in women worldwide.PTX is a commonly used chemotherapy drug for breast cancer,and it has a good effect in the treatment of breast cancer.However,with the continuous use of drugs,drug resistance seriously restricts its clinical efficacy.UA is a pentacyclic triterpenoid widely distributed in plants such as Hawthorn,Bearberry,Hedyotis diffusa,etc.It has anti-tumor,anti-oxidation and other effects.Our previous study found that UA can reverse breast cancer paclitaxel MCF-7.Cell resistance,and can promote the expression level of miR-149,reduce the expression level of MyD88,and further explore the role of UA in reversing paclitaxel resistance of breast cancer and its molecular mechanism through in vivo and in vitro experiments,which is UA combined with PTX.The clinical application provides a theoretical basis.Methods:1.CCK-8 method was used to detect the proliferation inhibition rate of 231 and231/PTX cells treated with different concentrations of UA and PTX,and the safe drug concentration was screened;the apoptosis of PTX was detected by flow cytometry;q RT-PCR The expression levels of Bax and Bcl-2 genes were detected after PTX treatment.The protein expression levels of Bax and Bcl-2 were detected by Western Blot,and the drug resistance of 231/PTX resistant strains was identified.2.231/PTX cells were used as experimental subjects and divided into 6 groups: blank control group,UA20?M treatment group,PTX30?M treatment group,PTX60?M treatment group,PTX30?M combined with UA20?M group,PTX60?M combined with UA20?M group,and apoptosis was detected by flow cytometry.q RT-PCR was used to detect the gene expression levels of miR-149-5p,MyD88,Bax and Bcl-2,Western Blot was used to detect the protein expression levels of MyD88,Bax and Bcl-2,and UA was used to reverse the drug resistance of paclitaxel in breast cancer.The effects of apoptosis.3.Through the lentiviral transfection,construct a 231/PTX cell line that overexpresses miR-149-5p,interferes with miR-149-5p,overexpresses MyD88,interferes with MyD88,and interferes with miR-149-5p,q RT-The m RNA expression levels of miR-149-5p and MyD88 were detected by PCR;the effect of drugs on cell proliferation was detected by CCK-8 method;the expression of miR-149-5p was overexpressed by Western Blot,and 231/PTX cells MyD88 and Bax were interfered with MyD88.Protein expression levels of Bcl-2,PI3 K,Akt,and P-Akt.Detect the effect of genetic alterations on signaling pathways.4.According to the software prediction results,the p GL3-MyD88 3'UTR luciferase reporter vector was constructed to transfect 231/PTX cells,and the transcriptional activity of MyD88 was observed to detect the regulation of miR-149-5p on MyD88.5.A nude mouse model of breast cancer 231/PTX cells was established,and the model was divided into 5 groups,namely,control group,UA10mg/kg medication group,PTX10mg/kg medication group,PTX20mg/kg medication group,UA10mg/kg combined with PTX10mg/kg.The drug group was used to record the change of tumor growth.The tumor weight and volume were detected after tumor removal.The protein expression levels of Bax and Bcl-2 were detected by Western Blot.The reversal effect of UA on paclitaxel resistance in nude mice was detected.Results:1.The safe drug concentration of UA was selected to be 0-40 ?M.The inhibitory effect of PTX on the proliferation of 231/PTX cells was significantly higher than that of231 cells,and the pro-apoptotic effect on 231/PTX cells was significantly lower than that of 231 cells.The expression level of Bax gene and protein in 231/PTX cells was lower than that of 231 cells,and the expression level of Bcl-2 gene and protein in231/PTX cells was higher than that of 231 cells.2.UA combined with PTX can promote the proliferation inhibition and apoptosis-promoting effect of PTX on 231/PTX cells,promote the expression of miR-149-5p gene,Bax gene and protein,and reduce the genes of Bcl-2 and MyD88.And protein expression levels.3.UA promoted the expression of miR-149-5p in 231/PTX cells and decreased the expression of MyD88.4.Overexpression of miR-149-5p and interference with MyD88 increased the sensitivity of 231/PTX cells to PTX.After interference with miR-149-5p,the sensitivityof 231 cells to PTX decreased.After interfering with miR-149-5p and overexpressing MyD88,UA reversed the effect of paclitaxel resistance in 231/PTX cells.Overexpression of miR-149-5p and interference with MyD88 decreased the expression levels of PI3 K and P-Akt.5.Luciferase reports show that miR-149-5p directly targets the 3'-UTR of MyD88.6.UA reversed the effect of paclitaxel resistance in breast cancer in nude mice.Conclusion:1.UA reverses PTX resistance in breast cancer 231/PTX cells.2.miR-149-5p can directly target the MyD88 gene in 231/PTX cells.3.MyD88 mediates breast cancer resistance to PTX via the PI3K/Akt pathway.4.UA reverses the resistance of PTX in breast cancer by promoting the expression of miR-149-5p and decreasing the expression of MyD88. |
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