| Background: Breast cancer is one of the most common and the highest mortality female tumors in the world.The United States expects 276480 newly diagnosed cases of breast cancer in 2020,accounting for 30% of the newly diagnosed cases of cancer in women,seriously affecting the physical and mental health of female patients.Due to the significant progress of treatment level,breast cancer related deaths have been reduced.Surgical treatment,radiotherapy and chemotherapy,hormone treatment and targeted treatment are mature strategies for cancer treatment at present.Therefore,it is necessary to elucidate the molecular mechanism of tumorigenesis and development,identify new therapeutic targets and prognostic biomarkers.STAT1,located on human chromosome 2q32.2,is composed of 750 amino acid residues with a molecular weight of 91 kDa.It is the first family of signal transducer and activator of transcription,STAT1,a member of STAT),is often considered to have the function of inhibiting tumor.Its main function is to induce cell cycle arrest,promote cell apoptosis and inhibit tumor cell metastasis.We found that STAT1 was highly expressed in breast cancer tissue in the early experiment of our group,and by constructing MDA-MB-231,a three negative breast cancer cell line with low expression of STAT1 gene,we confirmed that STAT1 gene played an anti-cancer role in MDA-MB-231 cell line.Objective: In this study,we constructed MCF7 cell line with STAT1 gene silencing,and observed its effect on the biological function of MCF7 cell line,preliminarily explored the mechanism of this gene in the development of breast cancer,and sought for a new target for clinical treatment of breast cancer.Method: 1.Guangzhou Funeng gene Co.,Ltd.was responsible for the construction of STAT1 gene silencing plasmids.The STAT1 gene silencing plasmids psi-LVRU6MP-STAT1 and packaging plasmids PMD2.G、PRSV-Rev、PMDLg/PRRE were used to co transfect 293 T cells to obtain psi-LVRU6MP-STAT1-shRNA1、2、3、4 and lentivirus venom of negative control group.2.Breast cancer cells MCF7 were infected with lentivirus venom of psi-LVRU6MP-STAT1-shRNA1 、 2 、 3 、 4 and negative control group respectively,and breast cancer cells MCF7 successfully infected were screened with puromycin.Four groups of breast cancer cell lines silenced by STAT1 gene were obtained,which were STAT1-shRNA1、STAT1-shRNA2、STAT1-shRNA3、STAT1-shRNA4 and breast cancer cell lines negative control group infected with empty body lentivirus respectively Cell line MCF7.STAT1-shRNA1 、STAT1-shRNA2、STAT1-shRNA3、STAT1-shRNA4,and STAT1 gene in MCF7 cells of negative control group infected with lentivirus were detected by quantitative real-time polymerase chain reaction(qRT PCR).3.MCF7 cells were used as the blank control group(Normal,N),MCF7 cells transfected with empty vector lentivirus were used as the negative control group(Negative control,NC),STAT1-shRNA3 cells with the best silencing effect were used as the positive control group.Cell proliferation rate was detected by cell counting kit-8(CCK-8),The changes of cell migration and invasion ability were detected by scratch test,Transwell cell migration and invasion test,cell cycle and apoptosis rate were detected by flow cytometry.4.The original data were processed and analyzed by spss24.0,and the measurement data conforming to normal distribution was expressed by mean ± standard deviation(x ± s).T-test was used to compare the mean of two samples,and one-way ANOVA was used to compare the mean of multiple samples(three groups and more than three groups).The test level was 0.05,P < 0.05,indicating that the difference was statistically significant.Result: 1.The breast cancer cell lines STAT1-shRNA1、STAT1-shRNA2、STAT1-shRNA3、STAT1-shRNA4,which silenced STAT1 gene,and the breast cancer cell MCF7 of negative control and NC group which were infected with empty body lentivirus were successfully constructed.The expression of STAT1 mRNA in MCF7 was detected by qRT-PCR.The results showed that STAT1 mRNA in MCF7 negative control group,STAT1-shRNA1、STAT1-shRNA2、STAT1-shRNA3 and STAT1-shRNA4 was detected by qRT-PCR The relative expression of mRNA was 1,1.76,0.99,0.11 and 0.80 respectively.It was found that STAT1-shRNA3 had the best silencing effect and the silencing efficiency was 89%.Therefore,MCF7 cells of STAT1-shRNA3 were selected as the positive control group for subsequent cell function experiment.2.The results of cell proliferation experiment showed that the odvalue of MCF7 cells in STAT1-shRNA3 group was significantly higher than that in the negative control group at 12 hours,24 hours,36 hours and 48 hours,and the difference was statistically significant(P < 0.05).The proliferation rate of STAT1-shRNA3 cells in the silence group was higher than that of MCF7 cells in the negative control group and the blank control group at 12 hours,24 hours,36 hours and 48 hours,the difference was statistically significant(P < 0.01),indicating that the silence of STAT1 gene promoted the proliferation of MCF7 cells.The results of scratch test showed that the migration rates of STAT1-shRNA3 cells in silence group,negative control group and blank control group were 45.46% ± 14.91%,19.27%± 9.49% and 23.63% ± 5.51%,respectively.There was a significant statistical difference(P < 0.05),indicating that STAT1 gene silencing promoted the migration of MCF7 cells.The results of Transwell migration experiment showed that: the number of MCF7 cells passing through Transwell cells in the silence group,negative control group and blank control group were 47 ± 18.67,17.11 ±14.97 and 11 ± 7.98,respectively,which showed a significant difference(P <0.05),indicating that the silence of STAT1 gene promoted the migration of MCF7 cells.The results of Transwell invasion showed that the number of MCF7 cells passing through Transwell cells in silence group,negative control group and blank control group were 53.4 ± 20.54,18 ± 3.93 and 14 ± 6.16,respectively,which showed a significant difference(P < 0.05),indicating that silence of STAT1 gene promoted the invasion of MCF7 cells.The results showed that the apoptosis rate of STAT1-shRNA3 was 16.54% ± 1.39% in silence group,20.32% ± 2.26% in blank control group and 22.18% ± 0.35% in negative control group.Compared with the blank control group and the negative control group,the apoptosis rate of breast cancer cell MCF7 in STAT1-shRNA3 group was lower,the results showed that there was statistical significance(P < 0.05),indicating that the silencing of STAT1 gene inhibited the apoptosis of breast cancer cell MCF7.The results of the cycle experiment showed that the percentage of S-phase content of STAT1-shRNA3 in breast cancer cells of the silence group was 21.50% ± 3.25%,18.70% ± 0.62% and 20.33% ± 4.15%compared with that of the blank control group and the negative control group,respectively.The difference was not statistically significant(P > 0.05),indicating that the silence of STAT1 gene had no effect on the cycle of breast cancer cell MCF7.Conclusion: This study confirmed that in MCF7,STAT1 gene silencing improved the proliferation,migration and invasion of breast cancer cells,and inhibited the apoptosis of breast cancer cells,but had no effect on the cycle of breast cancer cells.Therefore,STAT1 gene plays an anti-cancer role in breast cancer cell MCF7,and STAT1 is expected to be a potential therapeutic target for breast cancer. |
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