The Role And Molecular Mechanism Of MCM6 On Cell Proliferation In Er+ Breast Cancer

BackgroundBreast cancer has become the largest cancer in the world,and its death toll is also in the first place of female cancer deaths.Therefore,although the comprehensive treatment of breast cancer has been developed and improved to a great extent,the recurrence,metastasis and drug resistance of breast cancer are still the great killers of global female health.Estrogen receptor positive breast cancer accounts for about 70% of the total number of breast cancer.The main obstacle to the treatment of estrogen receptor positive breast cancer is the resistance of endocrine therapy.This problem has always existed and needs to be solved urgently.MCM6 is one of the important members of MCM family,which is essential for DNA replication in cells and plays a cancer promoting role in multiple cancers.However,there are few reports about MCM6 in breast cancer.The role of MCM6 in breast cancer and its specific mechanism need to be clarified.ObjectiveTo explore the effect of MCM6 on the proliferation of estrogen receptor positive breast cancer cells,and to further study the specific mechanism of MCM6 in this type of breast cancer.Methods(1)Through the bioinformatics analysis method of WGCNA and literature reading,the differentially expressed gene MCM6 was selected as the research target.The difference of MCM6 expression in breast cancer and adjacent tissues,and the effect of MCM6 on survival in different molecular types of breast cancer was analyzed by online database Oncomine and Ualcan.Chi square test was used to analyze the correlation between MCM6 and clinicopathological features of breast cancer in TCGA database.(2)si RNA was used to transfect estrogen receptor positive breast cancer cells MCF7 and T47 D to knock down the expression of MCM6.Western blot and q RT-PCR were used to verify the knockdown effect at the translation and transcription levels.The optimal sequence was selected to construct lentiviral sh RNA,and then lentiviral transfection was used to generate cell lines stably overexpressing MCM6.CCK8 cell proliferation assay,clone formation assay,Edu assay and xenograft tumor formation assay were used to detect the difference of proliferation ability between MCM6 knockdown group and control group in vitro and in vivo.(3)The effect of E2/ER on MCM6 m RNA level was analyzed using GEO database,and the change of MCM6 protein level was detected by Western blot.(4)PROMO and JASPAR data websites was used to predict the transcription factor of MCM6 promoter sequence and the transcription binding site of ER on the promoter.And then we detected the binding of ER and MCM6 promoter by CHIP experiment.(5)Transcriptome sequencing was performed to explore the main signal pathway of MCM6 in the control and MCM6 knockdown cell lines,and Western blot was used to detect the proteins level of the pathway.The ER+ breast cancer cell line MCF7 was used to perform mass spectrometry analysis of proteome after ER antibody IP,and Western blot was applied to validate the proteins in the results.Results(1)The expression of MCM6 in breast cancer tissues was higher than that in adjacent tissues,and its expression level was related to ER/PR/HER2 status and prognosis of breast cancer.There were differences in prognosis among different molecular types of breast cancer,especially in ER+ breast cancer.(2)Knockdown of MCM6 in estrogen receptor positive breast cancer cell line significantly reduced the proliferation of cancer cells.(3)When estrogen is used as a ligand to activate ER,ER can bind to the promoter of MCM6 gene and play its role as a transcription factor,thus promoting the transcription of MCM6.(4)MCM6 plays an important role in PI3K/AKT and its downstream signaling pathway.Knockdown of MCM6 can inhibit this pathway.MCM6 can combine with many kinds of proteins including GINS3 to form a complex and play its biological function.ConclusionThis study is the first to explore the regulation and mechanism of MCM6 on cell proliferation in ER+ breast cancer.We found that estrogen receptor can act as a transcription factor to promote the transcription of MCM6.After the transcriptional expression of MCM6 increased,it combined with GINS3 and other related proteins to form a complex to play its important role.And then it transmitted more activation signals to the downstream,resulting in the increase of PI3 K and the activation of this pathway,thus promoting the proliferation of cancer cells.Given the regulatory relationships described above,we suggest that MCM6 could perhaps be a novel target for another effective combination therapy after endocrine therapy resistance in ER+ breast cancer,offering new possibilities for future therapeutic modalities for breast cancer patients.