The Effects Of SIRT1 Gene On Proliferation, Migration And Inwasion Of Human Breast Cancer MCF-7 Cell Line And Its Initial Exploration Of Mechanism

Objective:The aims of this study are to investigate the expression level of silent information regulator1(SIRT1)in breast cancer and its effect on proliferation,migration and invasion of hunman breast cancer cell line MCF-7,and to explore its possible mechanism,in order to provide new ideas for the diagnosis and treatment of breast cancer.Methods:1.The expression of SIRT1 in breast cancer tissues and adjacent normol tissues was detected by immunohistochemistry.2.To detect the expression level of SIRT1 m RNA and protein in human normal mammary epithelial cell line MCF-10 A,human breast cancer cell line MCF-7,cell line MDA-MB-231 by real-time quantitative PCR(q RT-PCR)and Western-blot.3.The expression of SIRT1 in MCF-7 cells was interfered by lentiviral vector transfection technique,and the transfection efficiency was verified by q RT-PCR and Western-blot techniques to obtain stable cell lines with transfected.4.The proliferation ability of each group of cells after silencing and overexpression of SIRT1 was detected by CCK-8 kit assay.5.The migration abilityand invasion ability of cells in each group after silencing and overexpression of SIRT1 were detected by Transwell migration assay and Transwell invasion assay.6.The effect of SIRT1 silencing and overexpression on the expression levels of p53 protein and p125 protein was detected by Western-blot technique.Results:1.Immunohistochemistry results showed that the expression of SIRT1 in breast cancer tissues was higher than that in adjacent normol tissues.The difference was statistically significant(p<0.05).2.The results of q RT-PCR showed that the relative expression of SIRT1 m RNA in breast cancer cell lines MCF-7(3.27±0.09)and MDA-MB-231(2.65±0.23)was higher than that of normal breast epithelial cells MCF-10A(1.01±0.12).The difference was statistically significant(p < 0.05).Western-blot results showed that the relative expression of SIRT1 protein in breast cancer cell lines MCF-7(1.21±0.06)and MDA-MB-231(1.01±0.03)was higher than that of normal breast epithelial cells MCF-10A(0.71±0.07).The difference was statistically significant(p < 0.05).3.The short hairpin-RNA(sh RNA)-induced RNA interference(RNAi)was used to knock down or overexpress SIRT1 in MCF-7 cells.The recombinant lentivirus vectors includes LV-sh RNA-SIRT1-KD,LV-sh RNA-SIRT1-OE and the corresponding negative control(LV-sh RNA-NC-KD,LV-sh RNA-NC-OE).MCF-7 cells were used to transfected.The cells were divided into five groups including LV-sh RNA-SIRT1-KD transfected cells(KD group),LV-sh RNA-NC-KD transfected cells(NC-KD group),LV-sh RNA-SIRT1-OE transfected cells(OE group),LV-sh RNA-NC-OE transfected cells(NC-OE group)and non-transfected cells(Control group).The results of q RT-PCR and Western-blot showed that the relative expression levels of SIRT1 m RNA and SIRT1 protein in KD group(0.138±0.05?0.25±0.06)was significantly lower than that in NC-KD group(1.08±0.16 ? 1.12±0.07)and Control group(0.99±0.09?1.08±0.06).The relative expression levels of SIRT1 m RNA and SIRT1 protein in OE group(2.26±0.14?2.24±0.13)were higher than those in NC-OE group(1.10±0.21?1.22±0.18)and Control group(0.94±0.12?1.19±0.13).The difference was statistically significant(p<0.05).4.The results of CCK-8 kit assay revealed that the proliferation ability of KD group was significantly lower than that of NC-KD group and Control group(p<0.05),the difference was statistically significant.The proliferation of OE group was significantly higher than that of NC-OE group and Control groups(p<0.05),the difference was statistically significant.5.Transwell migration assay and Transwell invasion assay revealed that cells number passing through the membrane in the KD group(68.40±5.07?37.40±3.64)was significantly less than that in the NC-KD group(141.40±5.17?94.63±3.05)and the Control group(145.71±3.89?98.01±4.52)(p<0.05),and the difference was statistically significant.The OE group(198.81±6.41?133.40±6.18)passed more cells than those in NC-OE group(144.60±5.17 ? 96.42±5.17)and the Control group(147.60±4.66 ?99.25±4.91),the difference was statistically significant(p<0.05).6.Western-blot results showed that the protein expression level of p53 in KD group(0.92±0.08)was higher than that in NC-KD group(0.53±0.03)and Control group(0.52±0.04),and the protein expression level of p125 in KD group(0.23±0.03)was lower than that in NC-KD group(1.12±0.06)and Control group(1.15±0.05),the difference was statistically significant(p<0.05).The protein expression level of p53 in OE group(0.49±0.03)was lower than that in NC-OE group(0.91±0.05)and Control group(0.93±0.06),and the protein expression level of p125 in OE group(1.91±0.11)was higher than that in NC-OE group(1.25±0.08)and Control group(1.29±0.03),the difference was statistically significant(p<0.05).Conclusin:1.SIRT1 is upregulated in human breast cancer tissue and breast cancer cell line MCF-7.2.Silencing of SIRT1 gene expression can inhibit the proliferation,migration and invasion of MCF-7 cells;overexpression of SIRT1 gene can promote the proliferation,migration and invasion of MCF-7 cells.3.The effects of SIRT1 on proliferation,migration and invasion of MCF-7 cells may be related to the regulation of p53 and p125 proteins by SIRT1.