The Function And Mechanism Of The Network Of CREB1/Lin28/miR-638/VASP In The Migration Of Breast Cancer

Backgroud:Tumor metastasis, especially distant metastasis of tissues and organs is the leading cause of death in patients with breast cancer. In the process of tumor cell migration, the reconstruction of cytoskeleton is the basis of invasive behavior of tumor cells.The dynamic reconstruction of actin fiber provides the necessary power for the migration of tumor cells. The platelet pseudopodia and linear pseudopodia formed by the dynamic reconstruction is also the structural basis of tumor cell movement. VASP is involved in the process of cytoskeleton rearrangement, and plays an important role in the occurrence and progression of malignant tumor. VASP plays an important role in the metastasis of malignant tumors in the literature and the present study. But the current research still cannot fully explain the molecular mechanism of abnormal expression of VASP in breast cancer tissue, and has not yet established a VASP as the core of the regulatory network. The role of VASP in breast cancer cell migration and its regulatory mechanism still need further exploration.Objective:To clarify the role of VASP in the migration of breast cancer cells and the regulation mechanism of abnormal expression, Constructing the regulation network of CREB/Lin28/mir-638 which with VASP as the core. Explain the role and mechanism of VASP and its regulatory network in breast cancer development and progression. Provide a theoretical basis for expanding the clinical treatment of breast cancer.Methods:1. To construct the breast cancer metastatic cell line which VASP-specific knocked out, toexamine the changes of cytoskeleton morphology, cell proliferation and cell migration and verify in tumor nude mouse models to investigatethe effects of VASP on the proliferation and migration of breast cancer cells2. To investigate the mechanism of CREB 1 transcriptional regulation, hsa-miR-638 in the regulation of VASP expression at post-transcriptional level, to construct a regulatory network with VASP as the core, to explain the possible mechanism of VASP aberrant expression in breast cancer;3. To explorethe possible mechanisms of hsa-miR-638 down-regulation in breast cancer cells was from the perspective of the inhibition of hsa-miR-638 maturation by Lin28 protein, and perfect the regulatory network of CREB/Lin28/miR-638 /VASP.Results:1. Found from gene chip data that the expression level of VASP, CREB1 and Lin28 in breast cancer tissues and cells were higher than those in normal tissues and cells.2. The morphological changes of actin filaments in breast cancer cells after VASP knock-down were obvious, The morphological changes of actin filaments in breast cancer cells were significantly after knockdown of VASP, regularity disappeared, disorder curled, assembled and shorted, and the migration ability of breast cancer cells decreased obviously. In the nude mice model with tumors, the ability of breast cancer cell metastasis significantly reduced after knockdowning VASP.3. The expression of CREB1 in breast cancer tissues was significantly higher than corresponding to the adjacent tissue, the expression level in breast cancer cell lines was higher than that in normal breast cells.4. Luciferase Assay and ChIP showed that CREB1 could start up the CRE site of the VASP promoter regionby combining the VASP, activate the expression of VASP at the transcriptional level, VASP protein and mRNA levels were significantly lower after knock downing VASP.5. MiR-638 can inhibit the migration of breast cancer cells and inhibit the expression of VASP protein at the post-transcriptional level bycombining to the seed sequence of the 3'UTR region of VASP.6. CREB1 could activate the expression of Lin28 at the transcriptional level by binding to the CRE site of the Lin28 promoter region. After knocking down CREB1, the mRNA levels of Lin28A and Lin28B were significantly decreased.7. The effect of Lin28 on miR-638 maturation and the expression of miR-638 in mature stromal cells were studied by the combination of the "GGAG" sequence of miR-638 stem-loop region and the up-regulation of VASP protein level and migration of breast cancer cells. Lin28 can down-regulate the level of mature miR-638, thus up-regulating the protein level of VASP and promoting the migration of breast cancer cells, by combining theGGAG sequence of the miR-638 stem cell cycle and affect the process of miR-638 maturation.8. TUT4 may be involved in the Lin28A-mediated degradation of pre-miR-638.Conclusions:In this study, the network of EGF/CREBl/Lin28/miR-638/VASP was found in breast cancer cells. On the one hand, EGF could up-regulate the phosphorylation levels of CREB1 to increase the transcriptional level of VASP and Lin28A and Lin28B. On the other hand, it could promote down-regulating the expression level of miR-638 mature cells, and releasing the inhibition of miR-638 on the post-transcriptional level of VASP, further promoting the expression of VASP protein. Abnormal high expression of VASP will enhance the migration ability of breast cancer cells, thereby enhancing the probability of distal metastasis of breast cancer patients, shorten the survival period of patients. This study attempted to fully explain the development and progression of breast cancer of VASP and its regulatory network, to provide a theoretical basis for expanding the clinical treatment of breast cancer.