| Objective:1.Examining the expression of CXCL1 in human breast cancer tissues.2.Exploring the influence of CXCL1 on the biological behavior of human breast cancer MDA-MB-231 cells and it’s possible preliminary mechanism.Methods:The first part: 1.Detecting the expression of CXCL1 protein in54 cases of breast cancer tissues and 30 cases of benign breast lesions by IHC staining;2.According to two molecular classifications of three-negative and non-three-negative types,54 cases of breast cancer were classified Organize the specimens into groups to dissecting the relationship between CXCL1 and the two groups.The second part: Part 2: Dividing MDA-MB-231 cells into three groups: Control group(normal cell group),NC group(cell group transfected with empty plasmid),CXCL1 group(cell group transfected with CXCL1plasmid)).1.Carry out cell transfection experiments through the technology of full gene automatic synthesis,construct the MDA-MB-231 cell line transfected with empty plasmid and the MDA-MB-231 cell line with the transfected plasmid and stable high expression of CXCL1;2.Choosing Western blot method,using GAPDH as an internal reference to determine the protein content of CXCL1 in the Control group,NC group,and CXCL1 group;3.MTT method was used to detect the absorbance value(OD)of cells in each group at 0h,24 h,48h,and 72 h.The purpose is to evaluate the proliferation ability of cells;4.Making use of the Transwell method to measure the number of MDA-MB-231 cells that penetrate the Transwell membrane into the lower chamber by MDA-MB-231 cells in the upper chamber,and evaluating the migration and invasion ability;5.Choosing flow Cytometry was used to determine the changes in survival and apoptosis of MDA-MB-231 cells at24h;6.Western blot was used again to determine the protein content of NF-KB in the three groups of cells.Results:1.The positive rate of CXCL1 expression in breast cancer tissues is 70.4%(38/54),while the positive rate of expression in benign breast lesions is 30.0%(9/30).The comparison between the two is statistically significant(P<0.001);2.The positive expression rate of CXCL1 in the triple-negative group was 87.5%(21/24),and the positive expression rate of CXCL1 in the non-triple-negative group was 56.7%(17/30).Compared the two,the statistical difference indicates significance(P=0.014);3.The relative expression levels of CXCL1 protein(0.650±0.012)in the CXCL1 transfected group measured by Western blot was higher than those of the other two groups,and it was statistically significance(P <0.05),but the CXCL1 protein and the NF-kB protein is no significant difference between the Control group and the NC group;4.The OD value of the CXCL1 group measured by the MTT method is higher than that of the other two groups from 24h(1.142±0.045),and showsing significant difference from 48h(1.550±0.035)(P<0.05);5.The cell migration number(155.667±7.506)and invasion number(110.000±16.703)of the CXCL1 transfected group measured by Transwell method were higher than the other two groups of MDA-MB-231 cells(P<0.05);6.Flow cytometry The apoptosis rate(5.497±0.469%)of the transfected CXCL1 group was lower than the other two groups of MDA-MB-231 cells(P<0.05);7.The NF-kB protein(0.601±0.021)of CXCL1 group was higher than that of Control group(0.364±0.039)and NC group(0.408±0.032)by Western blot.Conclusion:1.The expression of CXCL1 in breast cancer tissues is higher than the benign breast lesions,and it is highly expressed in triple-negative breast cancer tissues;2.The up-regulation of CXCL1 expression makes the ability of breast cancer MDA-MB-231 cells enhancing with proliferation,migration and invasion,and the ability of apoptosis is weakened.The mechanism may be related to the up-regulation of NF-kB expression. |
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