Regulation Mechanism Of Breast Carcinoma Metastasis By Peptidylprolyl Isomerase(Cyclophilin)-like 2(Ppil2)

Breast cancer is a heterogeneous malignancy with the highest disease incidence in women.Cancer metastasis and tumor drug resistance are the major treatment problems of breast cancer.Therefore,it will be of great clinical significance to study new anti-metastatic and sensitive drugs.Studying the anti-metastatic effect and molecular mechanism of clinical drugs is the most rapid and effective way to develop new drugs.Cyclosporine A(CsA)is an immunosuppressant commonly used to suppress rejection after organ transplantation.Rencently,CsA has been found to inhibit proliferation and metastasis in breast cancer cells.However,some studies showed that the incidence of breast cancer in people treated with CsA was higher than that in the general population.Therefore,further study on the role and mechanism of CsA in breast cancer metastasis is helpful to extend its application.CsA exerts the immunosuppressive function through the intracellular Peptidylprolyl isomerase family,namely the cyclophilin family.Recent omics analysis found that Peptidylprolyl isomerase(cyclophilin)-like 2(PPIL2),a member of the cyclophilin family,regulates the distribution of cytoskeletal protein F-actin.It is also the downstream target gene of small RNA miR-31 which related with tumor metastasis.These results indicated that PPIL2 might be involved in breast cancer metastasis.Therefore,this article aims to study the function and mechanism of PPIL2 in breast cancer metastasis,to explore the impact and mechanism of CsA in breast cancer metastasis.It might provide the possibility to expand the application of CsA.The main work of this thesis is as follows:1.The human breast cancer cell lines MCF-7,T47D,ZR-75-30 cells and breast cells MCF-10A were used as experimental materials.We chose phase contrast microscopy imaging,Western blot and immunofluorescence for study.The results showed that the expression of PPIL2 was correlated with breast cancer cell morphology and F-actin distribution.Cell scratch,Transwell invasion chamber and mice lung metastasis model was used to evaluate the function of PPIL2 on breast cancer cell metastasis.2.The results of Western blot,RT-PCR and gene report showed that PPIL2 regulated the expression of Epithelial-mesenchymal transition(EMT)markers.Furthermore,PPIL2 reduced the binding of SNAI1 on the promoter of E-cadherin by downregulating its concentration.PPIL2 is thus determined to be a negative regulator of EMT.3.The human breast cancer cell lines MCF-7 and ZR-75-30 were used as experimental materials.The interaction between PPIL2 and SNAI1 was confirmed by co-immunoprecipitation,mammalian two-hybrid and GST-pulldown experiments.The plasmids of truncated PPIL2 and SNAI1 were constructed.The result of co-immunoprecipitation showed that the U-box domain of PPIL2 mediated its binding to SNAI1,while the N-terminal end of SNAI1 mediated its binding to PPIL2.PPIL2 and SNAI1 co-localized in the nucleus of breast cancer cells according to the result of immunofluorescence.4.By using human breast cancer cell lines MCF-7 and ZR-75-30 as experimental materials,experiments of immunoprecipitation,immunoprecipitation and nucleus/cytoplasm separation were chosed.The results showed that PPIL2 prompted K48 type ubiquitination of SNAI1 in the nucleus to downregulate SNAI1 protein level,thereby inhibiting the EMT process.5.The human breast cancer cell line ZR-75-30 cells were used as experimental material to test the effect of CsA.The results of immunoprecipitation,immunofluorescence,cell scratch,Transwell invasion chamber and mouse lung metastasis model of breast cancer showed that CsA inhibite breast cancer cell invasion and metastasis depends on PPIL2.6.Using human breast cancer tissue samples as experimental materials,the expression of PPIL2 and SNAI1 in breast cancer tissues was examined by immunochemistry.The results showed that the expression of PPIL2 and SNAI1 was negatively correlated,and there was a significant correlation between the level of PPIL2 and SNAI1 with the infiltration and metastatic status of breast cancer tissues.In summary,this paper reveals the molecular mechanism by which PPIL2 inhibits EMT and metastasis in breast cancer.PPIL2 changes F-actin distribution and promotes SNAI1 ubiquitination to degradation.This is the mechanism by which CsA inhibits breast cancer metastasis.The results help us to understand the function of CsA and cyclophilin family in breast cancer metastasis more deeply,and provide a new orientation for developing cyclosporine as a novel anti-cancer drug.