Abstract:
Background Breast cancer has become the malignant tumor with the highest incidence rate in women, and therefore, it is of great importance to find effective therapeutic targets to control the development and progression of breast cancer.
Objective To construct the eukaryotic expression vector of Sp1 with green fluorescent protein reporter (pEGFP-C1), and to validate the function of pEGFP-Sp1 protein.
Methods With the human mammary library as the template, the CDS sequence of the Sp1 gene amplified by polymerase chain reaction was inserted into pEGFP-C1 vector; after enzyme digestion and sequence confirmation, the recombinant plasmid was transfected into human embryonic kidney 293T cells, and Western blotting was used to measure the expression level of pEGFP-Sp1 protein. After pEGFP-C1 and pEGFP-Sp1 were transfected into human breast cancer ZR75-1 cells, fluorescence microscopy was used to observe the localization of Sp1 protein in cells, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the transcriptional levels of cell cycle genes.
Results Double-enzyme digestion and sequencing confirmed that the pEGFP-Sp1 plasmid was successfully constructed, and Western blotting showed the successful expression of Sp1 protein. Fluorescence microscopy showed that Sp1 protein was mainly localized in the nucleus of human breast cancer ZR75-1 cells, and RT-PCR showed that Sp1 overexpression changed the transcriptional level of cell cycle-related genes.
Conclusion The eukaryotic expression vector pEGFP-Sp1 is successfully constructed, and it is confirmed that Sp1 protein is expressed in human breast cancer ZR75-1 cells, mainly in the nucleus. In addition, Sp1 can downregulate the mRNA expression levels of Cyclin D2 and CDK6 and upregulate the mRNA expression levels of Cyclin G2 and p18. In summary, the results of this study show that Sp1 plays an important role in regulating the cell cycle pathway, which provides a basis for further in-depth research on the function of Sp1 transcription factor.
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