The Mechanism Study Of SIPA1 Regulating EMT And Autophagy In Breast Cancer Cells And Breast Cancer Cells AFAM Imaging

Tumor metastasis remains one of the major causes of death in breast cancer patients.It has been found that the gene expression diversities of E-cadherin and vimentin can cause the transformation between epithelial cell and interstitial cell,which is well-known as EMT,and induce the cell migration.However,the regulation mechanism of EMT related protein expression is still unknown during the tumor metastasis.SIPA1 molecule(signal-induced proliferation-associated protein 1)is one member of Rap GAP protein family.It has been found that SIPA1 molecule is able to regulate tumor cell adhesion,infiltration and metastasis in breast cancer,prostate cancer,colorectal cancer,oral squamous cell carcinoma,and bladder cancer.In the previous study,it is investigated the methylation level of Cp G island-rich region of Sipa1 gene in several tumor cells is negatively related to the transcription and translation levels of Sipa1 gene.Whether the methylation level of Sipa1 gene regulates the migration of cancer cells or not? How does SIPA1 help breast cancer cell gain the properties of migration?This study firstly found when treated MCF7 cells with 5-Aza-Cd R,the methylation status of Cp G islands region of Sipa1 gene was tested at a low level and the SIPA1 expression was upregulated by Western Blotting test.And further the expression of EMT related molecules of E-cadherin was downregulated,and of vimentin was upregulated in a concentration-dependent way.Knockdown SIPA1 in BT549 cells could downregulate vimentin and upregulate E-cadherin and further inhibits its EMT and migration.In addition,the q RT-PCR assay indicated that SIPA1 protein could promote breast cancer cell EMT and migration by activating the transcription of TGFB1 and ZEB1.In order to investigate more mechanisms of SIPA1 molecule regulating breast cancer cell migration,the expression of total MAP1LC3 B in four cancer cells were tested and found that the expression of SIPA1 protein was positively related to the expression of total MAP1LC3 B.When downregulated SIPA1 in MDA-MB-231,the expression of total MAP1LC3 B was reduced and when rescued SIPA1 expression in MDA-MB-231/sh SIPA1,the expression of total MAP1LC3 B was upregulated.On the other hand,when upregulated SIPA1 expression in MCF7 cells,the expression of total MAP1LC3 B was enhanced.Then,the aggregation of MAP1LC3 B and the formation of autophagosome were further detected by confocal microscopy and discovered that SIPA1 could promote the formation of autophagosome.By CHIP and dual-luciferase report assay in MDA-MB-231 cells,the binding of SIPA1 with the region of MAP1LC3 B promoter were confirmed and found that SIPA1 knockdown could inhibit the activity of MAP1LC3 B promoter.When treated with CQ(Chloroquine)inhibiting the fusion of autophagosome and lysosome,the autophagy of MDA-MB-231 cells was found lower and the migration capability of MDA-MB-231 cells was decreased significantly in vitro and in vivo.The morphorlogy of cancer cell will change during migration.In this thesis,the morphorlogy and subcellular structure were imaged and analyzed between MDA-MB-231 and MCF7 cells by an atomic force acoustic microscopy(AFAM)technique.After optimizing the preparing process,the problem of crystallization on the cell surface was solved and the high-quality imagies for MDA-MB-231 and MCF7 cells were obtained by optimizing the scanning frequency,ultrasonic amplitude and ultrasonic frequency of AFAM and their three-dimensional ultrasonic images were accquired via CSPM Imager.The morphology differences on sags and crests at surface between MDA-MB-231 and MCF7 cells were found.Taken together,the thesis aimed for studying the breast cancer cell properties including the external cell morphology,subcellular structure and the internal molecular mechanisms that could regulate the migration of breast cancer cell.Our study confirmed that the low methylation status of Cp G islands in the promoter of Sipa1 gene could upregulate the expression of SIPA1 protein and further promote breast cancer cell EMT and migration.It also found that SIPA1 could bind to the region of MAP1LC3 B promoter,activate its promoter activity and upregulate MAP1LC3 B transcription and expression,which could promote the migration of breast cancer cells.Furthermore,the morphology differences between MDA-MB-231 and MCF7 cells were observed by AFAM image.These results uncovered in deep the migration mechanism for breast cancer cells and provided the theoretical support for research and development of the effective anti-tumor drugs and also provided new methods for imaging the tumor cells.