| Backround:Breast cancer is one of the most common malignant tumor in Chinese women,which seriously threatens the health of chinese women.according to statistics,the number of new cases of breast cancer in in China was about 304,000 in 2015,with an incidence rate of 45.29/100,000,ranking first among female tumors in China.in terms of regions,compared with rural areas,the incidence of breast cancer in urban areas was higher,about 54.32 per 100,000,while the incidence in rural areas was only 33.64 per 100,000.in patients with estrogen receptor(ER)positive and progesterone receptor(PR)positive breast cancer,hormone level is closely related to the occurrence and development of breast cancer.Tamoxifen(TAM),an estrogen receptor(ER)modulator,is widely used in endocrine therapy of breast cancer.whether the status of progesterone receptor(PR)affects the therapeutic effect of tamoxifen in breast cancer is controversial.what kind of role it plays in endocrine therapy of breast cancer needs further research and discussion.based on whether PR status affects the therapeutic effect of tamoxifen in endocrine therapy of breast cancer,this study knockdown the expression of PR gene in MCF-7 breast cancer cells by lentivirus transfection to obtain ER+PR-MCF-7 breast cancer cells,and made breast cancer xenograft tumor model to observe the therapeutic effect of tamoxifen in the xenograft tumor of each nude mouse.the clonal formation ability of cells was detected by plate cloning assay and the proliferation ability of cells in each group was evaluated.by prote in immunoblot technique(Western Blotting,WB)detection of progesterone receptor and epidermal growth factor receptor(epidermal growth factor receptor,EGFR),rapamycin target protein(mammalian target of rapamycin,mTOR).Objective:Investigate the effects of TAM on the growth of ER+PR-cell and ER+PR-xenograft tumors after PR gene knockdown in breast cancer cell and the changes of protein expression levels of EGFR and mTOR after PR knockdown.Methods:1.A short hairpin RNA(shRNA)and a negative control sequence were designed to knockdown PR gene expression and constructed into lentiviral vectors respectively for virus packaging in vitro,MCF-7 breast cancer cell as the target,infecting MCF-7 cell with successfully packaged virus particles.The low expression of PR gene in MCF-7 cells was detected by qPCR and WB.2.Clonal formation ability of cells in sh-PR TAM group,sh-PR control group,vector TAM group and vector control group was detected by plate cloning experiment,and then the proliferation ability of cells in each group was evaluated.3.Build the ER(+)PR(+)HER2(-)and ER(+)PR(-)HER2(-)subcutaneous xenograft tumor model of nude mice in vivo experiment:ER+PR+MCF-7 breast cancer cells(vector group cells)and ER+PR-MCF-7 breast cancer cells(sh-PR group cells)were injected into the right armpit of nude mice to establish an ectopic model of breast cancer in nude mice,and the effect of PR on TAM treatment was studied in vivo.Mice were randomly divided into 2 groups(10 in each group),sh-PR group and Vector group.After tumor formation,the two groups were divided into 2 groups,namely sh-PR TAM group,sh-PR control group,vector TAM group and vector control group.during the two weeks of medication,the volume of the xenograft tumor and the body weight of the nude mice were measured.after 2 weeks of medication,the nude mice were killed,the weight of the tumor in each group was weighed and evaluating the tumor inhibition rate.4.The expression of mTOR and EGFR protein in PR-shRNA group and Vector group was detected by Western blot.5.Statistical methods GraphPadPrism8.2,ImageJ and SPSS22.0 statistical software were used for plotting and data analysis.measurement samples were represented as mean±standard deviation(x±s).statistical methods:T Test of two independent samples,one way Analysis of Variance(ANOVA),Least Significant Difference T Test,and P value<0.05 was considered statistically significant.Results:1.The results of plate cloning experiment showed that the number of colony formation in sh-PR TAM group,sh-PR control group,vector TAM group and vector control group were 378.00±10.37,432.33±8.99,728.33±6.90,1141.67±8.12.compared with the other three groups,the vector control group has more colony-forming ability.after knockdown PR gene expression in MCF-7 breast cancer cell,the colony-forming number of cells in sh-PR TAM group and sh-PR control group decreased,and knockdown PR gene inhibited the colony-forming ability of cells.compared with untreated sh-PR control group,there was no significant difference in cell cloning ability(P>0.05).after TAM treatment,the number of cell clones in vector TAM group decreased compared with vector control group(P<0.05),and the proliferation ability in sh-PR TAM group was weaker than that in vector TAM group(P<0.05).2.In vivo experiments in nude mice showed that on the 15th day after administration,the average tumor volume of sh-PR TAM group and sh-PR control group were(71.96±4.30)mm3/(64.63±8.56)mm3 respectively,and the average tumor volume of vector TAM group and vector control group was(107.42±10.60)mm3/(205.942±25.63)mm3 respectively,compared with sh-PR control group,the xenograft tumor volume in sh-PR control group has no significant difference(P>0.05).the xenograft tumor volume in sh-PR control group is smaller than that in vector control group and sh-PR TAM group is smaller than that in vector TAM group.3.The weight of xenograft tumor was(0.03 ±0.007)g and(0.03 ±0.005)g in sh-PR TAM group and sh-PR control group,(0.11 ±0.012)g and(0.06±0.003)g in vector control group and vector TAM group respectively.the tumor weight of sh-PR control group is smaller than that of vector control group,which is larger than that of vector TAM group.there is no significant difference between sh-PR control group and sh-PR TAM group,but the tumor weight of sh-PR TAM group is smaller than that of vector TAM group.the tumor inhibition rates of sh-PR TAM group was 72%,the tumor inhibition rates of sh-PR control group was 72%,and vector TAM group was 45%.4.The results of western blot showed that the expression levels of EGFR and mTOR protein in shRNA group were higher than those in vector group,and the difference was statistically significant(P<0.05).5.After transfection of lentivirus into MCF-7 breast cancer cells,the results of qPCR and western blot showed that the relative expression of PR gene in positive sequence shRNA group(PR-shRNA group)was significantly lower than that in blank group(control group)and negative sequence shRNA group(vector group),and there was a statistical difference between them(p<0.05)Conclusion(s):1.In vitro and in vivo experiments have shown that knockdown of progesterone receptor gene in MCF-7 cells lead to slower cell proliferation in vitro and in vivo.2.Knockdown of PR gene expression in MCF-7 breast cancer cell reduced the therapeutic effect of tamoxifen on ER+PR-MCF-7 cells,that is,progesterone receptor affect the therapeutic effect of TAM on ER+PR-cell and xenograft tumor in nude mice.3.The expression levels of EGFR and mTOR in ER+PR-MCF-7 cells were increased after the knockdown of PR gene expression,and PR was negatively correlated with EGFR and mTOR.4.The low expression of PR gene in MCF-7 breast cancer cells was achieved by successfully transfecting the PR-shRNA positive sequence into MCF-7 breast cancer cells by lentivirus transfection technique. |
PDF Full Text Request