| BackgroundBreast cancer is the most common malignant tumor of women in China.It is also one of the most common malignant tumors of women.Estrogen plays an important role in the development of breast cancer.Estrogen receptor(ER)mediates the biological effects of estrogen and participates in the development and progression of breast cancer.Estrogen receptor-positive patients account for 60%-70%of all types of breast cancer.The classic drug for the treatment of estrogen receptor-positive breast cancer is Tamoxifen,but recent studies have shown that long-term use of tamoxifen induces resistance to endocrine therapy in breast cancer,which is an urgent clinical problem needed to be solved.Signal transducers and activators of transcription(STAT)are the important cytoplasmic transcription factors that regulate cell growth,cell survival,cell differentiation and cell apoptosis.STAT1 is a key member of the STAT family and is involved in the regulation of immunity,inflammation,and neoplastic disease.STAT1 is involved in the formation of a variety of tumors,but the mechanism of which was how to act on the ERa pathway remains unclear in breast cancer.Our preliminary findings suggest that STAT]is involved in the regulation of the ERa signaling pathway and promotes breast cancer cell proliferation.In the present study,we aim to study the molecular mechanism of STAT1 regulation and cell proliferation by using real-time quantitative PCR,Western blotting,Chromatin immunoprecipitation asssay,luciferase assay and RNA sequence analysis,which helps to provide new ideas for improving the clinical treatment and reversing clinical endocrine therapy resistance of the breast cancer.ObjectiveThrough molecular biology research on breast cancer cells,we aim to explore the mechanism of STAT1 regulating on estrogen signaling pathway and its effect on cell proliferation,and reveal a feasible approach for clinically reversing the endocrine therapy resistance of breast cancer.Methods1.In order to confirm the tamoxifen resistance of LCC2 cells,the IC50 of LCC2 cells was examined compared to MCF-7 cell line;Unbiased transcriptomic-based RNA sequence analysis of LCC2 and MCF-7 cell lines was performed to determine the signal pathway for higher expression in LCC2 cell lines.2.The differential expressions of STAT1 and IRF9 on mRNA and protein level were examined by RT-qPCR and Western Blot in the MCF-7 and LCC2 cells.The expression of ERa protein in MCF-7 cells was detected by Western Blotting after depleting STAT1 and IRF9 genes.RT-qPCR was used to detect the expression of ERa protein in MCF-7 cells after over expression of STAT1 gene.3.Western Blotting was used to detect the expression of ERa protein levels with depleting of STAT1 gene in LCC2,MCF-7 and T47D cell lines under vehicle/E2 treatment conditions.4.RT-qPCR was used to compare the expression of ERa downstream target genes in LCC2 and MCF-7 cells and to detect the different expression of ERa downstream target genes in LCC2、MCF-7 and T47D cell lines after depleting STAT1 gene under vehicle/E2 or vehicle/Tamoxifen treatment conditions.5.Cell proliferation assay was used to determine the effect of STAT1 on the proliferation of LCC2 and MCF-7 cell lines.6.Luciferase reporter gene assay was used to verify the regulation of ERa by STAT1 in LCC2,MCF-7 and T47D cell lines.7.To elucidate the regulation mechanism of STAT1 on ERa signaling pathway,ChIP experiments were used to explore whether STAT1 and ERa bind to each other,and further explore the specific binding sites of STAT1 and ERa.Result1.Unbiased transcriptomic RNA sequence analysis was performed to determine differentially expressed genes between LCC2 and MCF-7 cells.The entire transcriptomic data showed a significant up-regulation of the JAK-STAT pathway,particularly the ISGF3 complex.As a key effector of JAK-STAT pathway,STAT1 and IRF9 were up-regulated 5-fold and 20-fold respectively in LCC2 cells.2.Results of Western Blotting showed that the expression of ERa protein in MCF-7 cells decreased after depleting STAT1,but the expression of ERa protein did not change after depleting IRF9.RT-qPCR confirmed that after over expression plasmids of STAT1 was transfected into MCF-7 cells,the expression level of ERa was up-regulated by 2.5 fold.3.Under the treatment condition of vehicle or estradiol(E2),the LCC2,MCF-7 and T47D cell lines were compared to confirm the expression of ERa protein after depleting STAT1 by Western Blotting.4.Under the treatment condition of vehicle or estradiol(or tamoxifen),the RT-qPCR,Western Blot,Luciferase reporter gene assay was further used to demonstrated that siSTAT significantly inhibited the expression ERa downstream target genes in the LCC2,MCF-7 and T47D cell lines.5.Cell proliferation experiments showed that the depleting of STAT1 significantly inhibited the proliferation of LCC2 and MCF-7 cells under the treatment condition of vehicle or tamoxifen.6.Chromatin immunoprecipitation(ChIP)assay showed that ERa transcription.vas associated with the recruitment of STAT1 to the ERa promoter region and showed STAT1 binds to the E2 region of ERa promoter but not to promoter A,which suggests that transcriptional regulation is one possible mechanism of STAT1 regulates estrogen signaling and cell proliferation in breast cancer cells.ConclusionSTAT1 regulates the expression of ERa at the transcriptional level by STAT1 binding to the E2 region of ERa promoter.This may be one of the potential mechanism by which STAT1 modulates the ERa signaling pathway.Targeting of STAT1 is a potential treatment strategy for ER-positive breast cancer. |
