| Objective:Observe the changes in autophagy activity of triple negative breast cancer MDA-MB-231 cells after BECN1 gene silencing,and explore the effect of autophagy on the sensitivity of triple negative breast cancer docetaxel.Methods:1.According to the c DNA sequence of BECN1 provided by Genbank,three si RNAs for BECN1 gene are designed and synthesized and packaged into sh RNA slow virus(LV-sh RNA-BECN1)and negative control slow virus(LV-sh RNA-NC);2.Establishment of LV-sh RNA-BECN1 MDA-MB-231 cell line;3.Use real-time fluorescence quantitative PCR to detect the expression of each group of cells BECN1 gene;4.The application of protein immuno-printing reaction to detect the expression changes of BECN1 protein and autophagic marker protein LC3B in each group of cells;5.Using a transmission electron microscope to observe the autophagy in each group of MDA-MB-231 cells;6.CCK-8 method was used to detect the IC50 value of docetaxel in each group of cells before and after transfection;7.Flow cytometry was used to detect the apoptosis rate of each group of cells after 48 hours of intervention with docetaxel;8.Using nude mice tumorigenesis experiment,observe the change of tumorigenic ability of MDA-MB-231 cells after down-regulation of autophagy.Results:1.After sequencing identification,the successful synthesis of three si RNA for BECN1 and packaged into a slow virus,slow virus titer are up to 1×10~8TU/ml;2.Successful use of slow viral infection MDA-MB-231 cells,under the microscope can be observed to have transfection efficiency of more than 95%;3.The results of real-time quantitative PCR showed that the relative expression levels of BECN1 m RNA in the blank group,negative control group,interference group(841),interference group(1056),and interference group(1245)were(1±0)and(0.98±0.03),(0.34±0.01),(0.11±0.01),(0.66±0.03),there was no difference in the relative expression of BECN1 m RNA between the blank group and the negative control group(P>0.05);compared with the blank group and the negative control group,The relative expression of BECN1 m RNA in cells of each interference group was significantly reduced,the difference was statistically significant(P<0.05),and the relative expression of BECN1 m RNA in the lentiviral interference group(1056)was the lowest;4.The results of Western blot analysis showed that the relative expression levels of BECN1 protein in blank group,negative control group,interference group(841),interference group(1056),and interference group(1245)were(1±0)and(1.03±0.07),(0.78±0.05),(0.11±0.00),(0.51±0.01),there was no significant difference between the blank group and the negative control group(P>0.05);each interference group was compared with the blank group and the negative control group,The expression of BECN1 protein was significantly reduced,the difference was statistically significant(P<0.05),among which the relative expression of BECN1 protein was the lowest in the lentiviral interference group(1056);the blank group,negative control group,interference group(841),The relative expression levels of autophagy marker protein LC3B in the interference group(1056)and interference group(1245)were(1±0),(0.98±0.01),(0.81±0.02),(0.56±0.03),(0.89±0.00),compared with the blank control group and the negative control group,the expression of LC3B protein in each interference group was significantly reduced(P<0.05);5.The results of the transmission electron microscopy(TEM)experiment showed that the number of autophagy vesicles in the mice in the slow virus interference group was significantly reduced compared with the blank group and the negative control group.6.CCK-8 experimental results show that after 48 hours of intervention,the blank group,the negative control group and the interfering group cell IC50 values were(0.09±0.00),(0.09±0.01),(0.05±0.01),blank group compared with the negative control group,the difference was not statistically significant(P﹥0.05).Compared with blank group and negative control group,the interference group cell IC50 decreased significantly,the difference was statistically significant(P﹤0.05),and the inhibition rate of cell proliferation in each group increased with the increase of docetaxel intervention concentration,showing dose dependence;7.The results of flow cytometry showthat that the apoptosis rate of blank group,negative control group and interference group was:(35.42±3.90)μg/ml,(35.39±3.40)μg/ml and(19.24±4.86)μg/ml,compared with the blank group and negative control group,the apoptosis rate of the interference group decreased significantly,the difference was statistically significant;8.The results of tumor formation experiments in nude mice showed that after inoculation of tumor cells in nude mice,the nude mice in the interference group began to have visible tumors under the arm on the 5th day after inoculation.Tumor body.By the 7th day,tumors were formed under the skin of nude mice in each group.Compared with the blank group and the negative control group,the tumor speed of growth of the interference group increased significantly(P<0.05);31days after tumor cell inoculation,the tumor volume of the blank group,negative control group,and interference group were:(24.49±5.91)mm3,(23.40±12.7)mm3,(236.02±68.37)mm3,tumor weights are:(41.52±13.24)mg,(42.00±12.79)mg,(200.22±50.70)mg.Compared with the blank group and the negative control group,the tumor volume and weight of the interference group were larger than that of the blank group and the negative control,the difference was statistically significant(P<0.05)Conclusions:1.The recombinant BECN1 interference lentivirus(LV-sh RNA-BECN1)constructed in this research can specifically silence the expression of BECN1 gene in MDA-MB-231 cells and inhibit the autophagic activity of MDA-MB-23 cells;2.Inhibiting the autophagy activity of cells can effectively increase the proliferation inhibitory effect of docetaxel on MDA-MB-231 cells,reduce the effect of docetaxel-induced apoptosis,can improve the tumorigenic ability of MDA-MB-231 cells in nude mice and promotes the growth of transplanted tumors in nude mice. |
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