| The circadian protein TIMELESS is a member of the core molecular clock.TIMELESS regulates cell proliferation,cell metabolism and the recognition and repair of DNA damage,etc.In recent years,related research has reported that TIMELESS expression is significantly correlated with the initiation and progress of hepatocellular carcinoma,lung cancer and colorectal cancer,etc.Research on TIMELESS regulating breast cancer were not consistent.Some studies have indicated that the protein level of TIMELESS was lower in breast cancer.However,other studies reported that TIMELESS could promote the proliferation of breast cancer by activating MYC or estrogen signaling pathways.Abnormal cell proliferation is one of the most common cytological features of tumors,and the infiltration and mobility of cancer cells is directly related to malignant tumors.Meanwhile,the disrupted circadian rhythm and abnormal activation of EMT both promote the initiation and metastasis of the tumor.However,the specific mechanism on EMT contributing to the transition from epithelial cancer to mesenchymal cancer is still not very clear.Especially,there is a lack of the mechanism on TIMELESS regulating breast cancer metastasis.In this study,the specific mechanism of TIMELESS regulating the proliferation,metastasis and invasion of breast cancer was explored.Therefore,it may provide a new theoretical basis for circadian rhythm regulating the initiation and progress of tumor and open new avenues for the accurate treatment of breast cancer.The main work includes as follows:Analysis of the patient survival with all types of breast cancer indicated that the expression of the circadian gene TIMELESS was negatively correlated with the patient survival.In addition,western blot analysis of endogenous TIMELESS in ZR-75-30,T-47 D and MCF-7 breast cancer cells confirmed that TIMELESS expressed at different levels.1.MTT and colony formation assays showed that TIMELESS overexpression promoted the proliferation of breast cancer cells.Furthermore,Luciferase reporter assay confirmed that TIMELESS overexpression up-regulated ERα-mediated transcriptional activation of ERE.2.The KM analysis of patients with Basal-like breast cancer revealed that TIMELESS expression was positively correlated with the survival.Western blot assay found that the protein level of TIMELESS in ZR-75-30 cells is lower than that in T-47 D and MCF-7 cells.What’s more,scratch wound-healing assay found that TIMELESS overexpression inhibit the migration of breast cancer cells.Conversely,TIMELESS knowndown promote the migration of cells.3.Transwell assay found that TIMELESS overexpression inhibited the invasion and metastasis of breast cancer cells and TIMELESS knockdown promoted these properties of breast cancer cells.In addition,immunofluorescence assay showed that TIMELESS overexpression inhibited the assembly of cytoskeletal actin into F-actin,and conversely,TIMELESS knockdown promoted the process.4.Luciferase reporter and RT-PCR assays indicated that TIMELESS overexpression down-regulated the transcription and protein levels of EMT inducers SNAIL1 and MMP9 and TIMELESS may inhibit the transcriptional activity of MMP9 promoter by the elements or motifs between-556 bp and-293 bp.Moreover,western blot assay of EMT markers showed that TIMELESS overexpression changed the protein levels of EMT pathway-related markers corresponding to the transcriptional repression of SNAIL1,with E-cadherin up-regulated and N-cadherin down-regulated.In addition,Cytokeratin 18 and Vimentin were also up-and down-regulated,respectively.Collectively,differential regulation of TIMELESS was confirmed in this study,that is,TIMELESS can promote the proliferation of breast cancer cells by activating estrogen signaling pathways and block the EMT pathway by down-regulating the transcription and protein levels of SNAIL1 and MMP9,thereby inhibiting the invasion and metastasis of breast cancer cells. |
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