ER? Is Required For Suppressing OCT4-induced Proliferation Of Breast Cancer Cells Via DNMT1/ISL1/ERK Axis

Objective:To investigate the mechanism of OCT4-dependent ER? on the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis,and to provide experimental basis for targeted OCT4 therapy in different subtypes of breast cancer.Methods and Results:1.Expression of OCT4 in breast cancer tissues and breast cancer cells(1)The expression of OCT4 in breast cancer tissues was analyzed by Oncomine database and immunohistochemistry assay.The results showed that the expression of OCT4 in breast cancer tissues was significantly lower than that in breast epithelial tissues.(2)The expression of OCT4 in breast cancer cells was analyzed by Expression Atlas database,RT-PCR and Western blot assays.The results showed that the expression of OCT4 in breast cancer cells MDA-MB-231 and MCF-7 was significantly lower than that in breast epithelial cells HBL-100.These results indicate that the expression of OCT4 is low in breast cancer tissues and cells.2.Establishment of OCT4 gene overexpression in breast cancer cellsBreast cancer cells MCF-7 and MDA-MB-231 were overexpressed by lentivirus transfection,and the expression of OCT4 was identified by RT-PCR,Western blot and immunofluorescence assays.The results showed that the expression of OCT4 in MCF-7-OCT4 and MDA-MB-231-OCT4 cells was significantly higher than that in MCF-7-p EF and MDA-MB-231-p EF cells,and OCT4 expression was mainly located in the nucleus.3.Effect of OCT4 on proliferation of breast cancer cells(1)ICELLigence,plate colony formation and soft agarose colony formation assays were used to detect the effect of OCT4 on the proliferation of breast cancer cells.The results showed that the slope of proliferation curve in MCF-7-OCT4 group was lower than that in MCF-7-p EF group,and the number of colony formation in MCF-7-OCT4 group was significantly lower than that in MCF-7-p EF group.The slope of proliferation curve in MDA-MB-231-OCT4 group was higher than that in MDA-MB-231-p EF group,and the number of colony formation in MDA-MB-231-OCT4 group was significantly higher than that in MDA-MB-231-p EF group.(2)The expression of Ki67,a proliferation marker,was detected by immunofluorescence assay.The expression of Ki67 in MCF-7-OCT4 cells was significantly lower than that in MCF-7-p EF cells,and mainly expressed in the nucleus.The expression of Ki67 in MDA-MB-231-OCT4 cells was significantly higher than that in MDA-MB-231-p EF cells,and mainly expressed in the nucleus.The results demonstrate that OCT4 inhibits the proliferation of MCF-7 cells and promotes the proliferation of MDA-MB-231 cells.4.The mechanism of OCT4 regulating the proliferation of breast cancer cells(1)Regulation of DNMT1/ISL1,Ras/Raf1/ERK signaling pathway and ER? by OCT4DNMT1,ISL1,Ras/Raf1/ERK signalings and ER? were detected by Western blot.The results showed that OCT4 up-regulated ISL1 and ER? expression,down-regulated the expression of DNMT1,Ras,Raf1 and p-ERK in MCF-7 cells.OCT4 down-regulated ISL1 expression,up-regulated the expression of DNMT1,Ras,Raf1 and p-ERK in MDA-MB-231 cells.However,OCT4 did not regulate ER? expression in MDA-MB-231 cells,and ER? was not expressed in MDA-MB-231 cells.(2)Mechanism of OCT4 inhibiting proliferation of MCF-7 cells1)The relationship between OCT4 and ER? was detected by Co IP assay.The results showed that OCT4 binds to ER? and up-regulates the expression of ER? in MCF-7 cells.The relationship between ER? and DNMT1 was detected by Ch IP assay.The results showed that ER? binds to the promoter region of DNMT1 and inhibits its transcription in MCF-7-OCT4 cells.Co IP assay was used to analyze the relationship between ISL1 and Ras in MCF-7-OCT4 cells.The results showed that ISL1 interacts with Ras in MCF-7-OCT4 cells.2)Western blot assay was used to detect the effect of ER? inhibitor AZD9496 on the Ras/Raf1/ERK signaling pathway in MCF-7-OCT4 cells.The results showed that the expression of DNMT1,Ras,Raf1,p-ERK was up-regulated and the expression of ISL1 was down-regulated in AZD9496 treated cells compared with DMSO group.3)The effects of ER? inhibitor AZD9496 on the proliferation of MCF-7-OCT4 cells were detected by plate colony formation assay and soft agarose colony formation assay.The results showed that the number of colony formation in AZD9496 group was significantly higher than that in DMSO group.(3)Mechanism of OCT4 promoting proliferation of MDA-MB-231 cells1)The relationship between OCT4 and DNMT1 was detected by Ch IP assay.The results showed that OCT4 binds to the promoter region of DNMT1 in MDA-MB-231-OCT4 cells and promotes its transcription.2)Western blot,i CELLigence,plate colony formation assay and soft agarose colony formation assay were used to detect the effects of DNMT1 inhibitors 5-aza-d C and zebularine on the proliferation and Ras/Raf1/p-ERK expression in MDA-MB-231-OCT4 cells.The results showed that compared with DMSO group,the expression of DNMT1,Ras,Raf1,p-ERK was down-regulated and the expression of ISL1 was up-regulated in MDA-MB-231-OCT4 cells treated with 5-aza-d C and zebularine,and the slope of cell proliferation curve in 5-aza-d C group and zebularine group was lower than that in DMSO group.The number of colony formation in 5-aza-d C group and zebularine group was significantly lower than that in DMSO group.These results demonstrate that OCT4 affects the proliferation of breast cancer cells by regulating DNMT1/ISL1 and Ras/Raf1/ERK pathway.5.Effect of OCT4 on proliferation of breast cancer cells in vivoThe effect of OCT4 on the proliferation of breast cancer cells was verified by tumor formation assay in nude mice.The results showed that the tumor volume of MCF-7-OCT4 group was significantly smaller than that of MCF-7-p EF group.The tumor volume of MDA-MB-231-OCT4 group was significantly larger than that of MDA-MB-231-p EF group.Conclusion:1.OCT4 inhibits proliferation of breast cancer cells MCF-7 with positive expression of ER?,and promotes proliferation of breast cancer cells MDA-MB-231 with negative expression of ER?,indicating that the different effects of OCT4 on breast cancer cells may be related to the expression of intrinsic ER?.2.In ER?-positive breast cancer cells MCF-7,OCT4 upregulates the expression of ER? which inhibits DNMT1 transcription and suppresses cell proliferation through DNMT1/ISL1/ERK axis.3.In ER?-negative breast cancer cells MDA-MB-231,OCT4 directly promotes the transcription of DNMT1,down-regulates the expression of ISL1,and promotes cell proliferation through Ras/Raf1/ERK signaling pathway.