CLDN6 Down-regulates Cyclin D1 Via ERK-Sp1 Signaling Pathway To Inhibit Breast Cancer Cells Proliferation

The proliferation process of breast cancer is regulated by tight junctions(Tight Junctions,TJs)between cells.TJs regulate epithelial proliferation through different molecular mechanisms,which generally inhibit proliferation.Claudins(CLDNs)are the major components of TJs,with four transmembrane domains,and a carboxy terminus containing a PDZ domain.The domain can bind to a variety of signal proteins which also contain a PDZ domain.In this way,CLDNs participate in signaling pathways such as ERK signaling pathway.However,the specific mechanism by which CLDNs target downstream proteins through signaling pathways to regulate cell proliferation remains unclear.The preliminary work of our study showed that CLDN6 expression was down-regulated in breast cancer cells,and overexpression of CLDN6 significantly inhibited the proliferation of breast cancer cells.According to the results of gene sequencing,the expression of cyclin D1 was down-regulated by overexpression of CLDN6.The pre-experimental results showed that overexpression of CLDN6 and down-regulation of cyclin D1 expression in breast cancer cells were consistent with sequencing results.It’s suggested that CLDN6 may inhibit the proliferation of breast cancer cells by inhibiting the expression of cyclin D1.Cyclin D1 is a type of cyclin,which the promoter region contains a variety of transcription factor binding sites,including the transcription factor Sp1.When Sp1 binds to the binding site of the cyclin D1 promoter region,it can promote the transcription level of cyclin D1 and regulate cell proliferation.Sp1 is a transcriptional regulator widely present in human cells and belongs to the Sp/KLF zinc finger family.As a major GC box transcription factor,it is involved in the regulation of target gene expression.Sp1 targets a variety of proteins and is regulated by many proteins and signaling pathways.Among them,the ERK-Sp1 signaling pathway plays a role in the development of many cancers.The extracellular signal-regulated kinase(ERK)pathway is a highly conserved signaling cascade that regulates the proliferation of a variety of cancers.In cancer,Sp1 expression is up-regulated after activation of the ERK signaling pathway.In the previous work,it was found that in cervical cancer cells,CLDN6 can bind to AF6,inhibit the binding of AF6 to Ras,competitively inhibit the binding of Ras and Raf,then inhibit the activation of ERK signaling pathway.We hypothesized that CLDN6 may down-regulate cyclin D1 expression via ERK-Sp1 signaling pathway to inhibit the proliferation of breast cancer cells.Objective:To explore the mechanism of CLDN6 down-regulating the expression of cyclin D1 by ERK-Sp1 signaling pathway to inhibit the proliferation of breast cancer cells,and provide experimental evidence for the treatment of breast cancer targeting CLDN6.Method:Human breast cancer cell lines MCF-7 and MDA-MB-231(clone group)stably overexpressing CLDN6 gene obtained in previous work;MCF-7 and MDA-MB-231 transfected with empty pc DNA3.1 plasmid Cells(empty group)were the subjects of the study: the expression of CLDN6 was identified by RT-PCR and Western-Blot assay.1.Overexpression of CLDN6 inhibits the proliferation of breast cancer cellsThe effect of CLDN6 on cell proliferation was detected by CCK8 assay and colony formation assay.2.The role of Sp1 in the inhibition of cyclin D1 expression by CLDN6The expression of Sp1 and cyclin D1 was detected by Western-Blot assay.The expression of Sp1 vector was constructed in overexpressing CLDN6 cells and the expression was identified by RT-PCR and Western-Blot.Cyclin D1 protein expression was detected by Western-Blot after transfection Sp1.3.Role of ERK in down-regulating cyclin D1 inhibition of breast cancer cell proliferation by CLDN6Western-Blot detected the phosphorylation level of ERK in overexpressing CLDN6 cells;Western-Blot detected the optimal concentration of ERK agonist PMA,and the protein expression level of Sp1 and cyclin D1 after treatment;CCK8 method,clone formation experiment examined proliferation ability of cells after PMA treatment.CCK8 method was used to detect the IC50 value of Sp1 inhibitor MTM;Western-Blot method was used to detect the optimal concentration and time of MTM;Western-Blot method was used to detect the expression of cyclin D1 after inhibiting Sp1 and activating ERK signaling pathway;CCK8 method and colony formation experiment were detected to the effect of activating ERK signaling pathway and inhibiting Sp1 on cell proliferation.Result:1.Overexpression of CLDN6 inhibits the proliferation of breast cancer cellsCompared with the empty group,the m RNA and protein expression levels of CLDN6 in the overexpressed CLDN6 group were significantly increased;CLDN6 decreased cell viability and inhibited cell proliferation;CLDN6 down-regulated cyclin D1 expression.2.The role of Sp1 in the inhibition of cyclin D1 expression by CLDN6Compared with the empty group,the expression of Sp1 was decreased in overexpressing CLDN6 cells;the expression of cyclin D1 was up-regulated after transfection with Sp1.3.Role of ERK in down-regulating cyclin D1 inhibition of breast cancer cell proliferation by CLDN6CLDN6 inhibits the phosphorylation level of ERK;the optimal concentration of ERK agonist PMA is 15 n M,and the expression of Sp1 and cyclin D1 is up-regulated after PMA treatment,which promotes cell proliferation.The IC50 value of Sp1 inhibitor MTM was 503.4n M,the optimal concentration of MTM was 100 n M,and the treatment time was 24 h.The expression of cyclin D1 was decreased after the inhibition of Sp1 and activiting ERK signaling pathway,while the cell proliferation ability was weakened.Conclusion:CLDN6 inhibits the proliferation of breast cancer cells by inhibiting ERK-Sp1 signaling pathway and down-regulating cyclin D1 expression.