Roles Of P300 And PAX6 In Human Breast Cancer

PARTⅠChromatin immunoprecipitation?sequencing predicts p300 binding sites in human breast cancer cell line MCF7Objective: This study aimed to reveal the distribution of p300 binding sites in human breast cancer cell line MCF7 before and after estrogen(E2) stimulation, and identify the regulation mechanisms of P300 in breast cancer and its relations with estrogen receptor signaling pathway.Methods: Based on the Ch IP-seq data of P300 in human breast cancer cell line MCF7 before and after E2 stimulation(GSE39623), the distribution of p300 binding sites in chromosome and combined signal intensity were firstly analyzed. Then the surrounded target genes in P300 binding site were identified and functional enrichment analysis was carried out by GO database. Besides, specific target genes with significantly enhanced combined signal intensity were also identified, and then eveluated by GO functional enrichment analysis. Finally, Motif findings analysis was performed on pecific binding sites of P300, and newly emerged transcription factors co-located with P300 were further analyzed by GO functional enrichment analysis.Results: The binding sites of p300 in human breast cancer cell line MCF7 were mainly located in 5’ UTR, 3’ UTR, transcriptional start site(TSS) and transcriptional termination site, and E2 stimulation elevated the density of P300 binding sites in TSS. A total of 24899 differential binding sites of p300 was revealed in MCF7 after E2 stimulation, and the combined signal intensity of 39% sites was significanly increased. Before E2 stimulation, target genes of P300 were mainly enriched in biological processes of neural cell differentiation, while increased target genes enriched in the processes of cell-cell junction organization and regulation of cell adhesion were emerged by E2 stimulation. Besides, target genes with significant enhanced signal intensity also enriched in the process of neural differentiation, while increased target genes enriched in the processes of cell locomotion were revealed by E2 stimulation. Finally, a total of 225 and 176 transcription factors was found around P300 binding sites in breast cancer cell line MCF7 before and after E2 stimulation. GO functional enrichment showed these transcription factors were mainly enriched in the process of regulation of cell proliferation, negative regulation of cell differentiation and cell migration. Besides, estrogen receptor signaling pathway related factors ESR1 and BRCA1 were all found to be co-located with P300.Conclusion: P300 exhibited extensive transcriptional regulatory effects on breast cancer, which was closely related with estrogen receptor signaling pathway.PARTⅡThe expression of PAX6 and P300 in human breast cancer cell lines and breast cancer tissuesObjective: This study aimed to reveal the expression level of PAX6 and P300 in breast cancer cell lines and breast cancer tissues.Methods: Real time PCR was performed to detect the expression of PAX6 and P300 in breast cancer cell lines BT-474, MDA-MB-231 and MCF-7, as well as normal breast cell line HBL-100 in m RNA level. Meanwhile, the expression levels of PAX6 and P300 in breast cancer tissues in patients with(n=30) or without(n=30) lymphatic metastasis, and their corresponding non-tumor tissues next to carcinoma(control) were also detected.Results: Compared with the normal breast cell line HBL-100, the expression of PAX6 was increased in breast cancer cell lines BT-474(P < 0.01), but not in MDA-MB-231 and MCF-7. P300 was higher in the three breast cancer cell lines(P < 0.01). In all of the 60 patients, breast cancer tissues exhibited a higher expression level of PAX6 and P300 than non-tumor tissues(P < 0.05). In addition, breast cancer tissues in metastatic patients exhibited a higher expression level of PAX6 and P300 than those in the non-metastatic patients(P < 0.05).Conclusion: Both PAX6 and P300 were highly expressed in breast cancer, and the expression level was closely realted with the metastasis potential of human breast cancer.PART Ⅲ The effects of P300 silence on the the ability of proliferation, apoptosis, invasion and migration in breast cancer cell MCF-7Objective: This study aimed to reveal the effects of P300 on the ability of proliferation, apoptosis, invasion and migration in breast cancer cell MCF-7.Methods: A total of 3 si RNA fragments(si P300-1, si P300-2, si P300-3) were constructed by chemical synthesis to inhibit the expression of P300 in breast cancer cell MCF-7. After transfection, Real-time PCR was firstly performed to detect the silencing efficiency of these 3 si RNAs, and the expression of tumor invasion and metastasis related genes such as MMP9, VEGF and u PA was also detected. Then Western blot was performed to identified the inhibition effects of si P300-1 on the expression of P300 and PAX6. The effects of si P300-1, si P300-2 and si P300-3 on the proliferation and apoptosis of breast cancer cell MCF-7 were detected by CCK-8 and Flow cytometry. Finally, cell wound scratch assay and transwell invasion assay were performed to detect the inhibition effects of si P300-1 on the ability of migration and invasion in breast cancer cell MCF-7.Results: The expression of P300 in breast cancer cell MCF-7 could be obviously inhibited by either si P300-1, si P300-2 or si P300-3, while the inhibition efficiency was different in some degree. After transfection for 3 days, the proliferation of breast cancer cell MCF-7 was significantly inhibited by either si P300-1, si P300-2 or si P300-3(P < 0.05). After transfection for 2 days, the apoptosis rate of NC, si P300-1, si P300-2 and si P300-3 group was 18.22%, 18.60%, 21.94% and 17.94%, respectively, and no significantly differences were revealed. The expressions of MMP9, VEGF and u PA were also significantly inhibited by silence of P300(P < 0.05). Meanwhile, significantly slowed healing and reduced invasive cells further indicated the ability of migration and invasion in breast cancer cell MCF-7 was obviously inhibited by si P300-1. Furthermore, the expression of PAX6 was also revealed to be inhibited by si P300-1.Conclusion: The ability of proliferation, invasion and migration of breast cancer cell MCF-7 was all inhibited by silence of P300, while the apoptosis of MCF-7 was not significantly changed.