Study On The Molecular Mechanism Of The Interaction Between PGRN And ECM1 Mediating Breast Cancer Proliferation And Metastasis

Progranulin(PGRN)is a kind of secreted cell growth factor having multi-functions and involved in many physiological process,such as cell proliferation,injury repair,nervous system development,angiogenesis etc.In addition,PGRN also participated in several pathological process,such as tumor development,inflammation,and neurodegenerative disease etc.High expression levels of PGRN were detected in malignant tumors including breast cancer,ovarian cancer and cervical cancer etc.However,the specific mechanism of PGRN acting as an important tumor related molecular is still unclear.By using yeast two hybrid system(Y2H)to screen the molecules interacting with PGRN,extracellular matrix protein 1(ECM1)was found.ECM1 is a kind of secreted glycoprotein and its molecular weight is 85 kDa.It has been reported to be closely related with tumor development,cartilage formation,angiogenesis and epidermal differentiation.High expression levels of ECM1 have been found in malignant epithelial tumors,including invasive ductal carcinoma,esophageal squamous cell carcinoma,liver-cancer,gastric and colorectal cancer,which were positively related to the malignancy grade and lymph node metastasis.Considering the important roles of ECM1 and PGRN in the process of tumor development,the project was focused on the signaling pathways involved in tumor development.This study would lay the foundation for clarifying the biological functions mediated by the interaction of ECM1 and PGRN.The project was carried from 5 aspects and the results were shown as follow:1.The preparation,identification and function study of monoclonal antibodies targeting ECM1.Six strains of monoclonal antibodies targeting ECM1(1C4,1G4,2E3,2G3,2A3 and 4C2)were obtained by monoclonal antibody preparation techniques.The WB results indicated that all antibodies could react with prokaryotic and eukaryotic ECM1.All antibodies could recognize natural structure of ECM1 and be used for immunohistochemical staining except for 4C2.The WB results showed that 1C4 and 1G4 recognized ECM1 N-termianl,2E3 recognized ECM1 Repeat ?,2G3 recognized ECM1 Repeat ? and 2A3 and 4C2 recognized ECM1 C-termianl.Although 1C4 and 1G4 recognized ECM1 N-termianl and 2A3 and 4C2 recognized ECM1 C-termianl,the results of competitive ELISA assay suggested that 1C4 and 1G4 recognozed different epitopes,and the similar conculsion was obtained with 2A3 and 4C2.Furthermore,the antibodies 1C4,2E3,2G3 and 2A3 could be used for IP and IF assays,and 1C4 could be used for IHC assay.The results indicated that 1C4,2E3 and 2A3 could inhibit the proliferation,migration and invasion of MDA-MB-231 cells.In conclusion,monoclonal antibodies targeting ECM1 provide a valuable tool for the function study of ECM1.2.Establishment of sandwich ELISA kit for detecting ECM1.Based on the antibodies targeting four domains of ECM1,the sandwich ELISA kit for detecting ECM1 was developed and the standard curve was y=0.17397x+0.06148(R=0.998),the detection range is from 575.0 pg/mL to 9.2 ng/mL.The serum levels of ECM1 of tumor patients and normal persons were detected by this kit.The results showed the serum levels of ECM1 of tumor patients,especially breast and ovarian cancer patients,were significantly higher than those of normal people.Based on the sandwich ELISA kit,the luciferase-luciferin system was introduced to further improve the sensitivity and the standard curve was y=458.54x+194.25(R=0.992),the detection range is from 287.5 pg/mL to 9.2 ng/mL.The sensitivity has been successfully improved.3.The validation of the interaction about ECM1 and PGRN and the identification of the regions present in each molecule responsible for the interation of two molecules.The interaction of PGRN and ECM1 in vivo and in vtro were confirmed by Y2H,Co-IP and GST pull-down.The IHC result showed PGRN and ECM1 were colocalizated in malignant breast cancer.By Y2H,B domain of PGRN was found to be responsible for interacting with ECM1,and ECM1 Repeat ?,Repeat ? and C-terminal domains were found to be responsible for interacting with PGRN.Because ECM1 Repeat ?,Repeat ? and C-termianl domains had the typical structure of CC-(X7-10)C forming the double loop structure,they have similar function for binding to PGRN.4.The study of mechanisms involving tumor metastasis mediated by the interaction of PGRN and ECM1.The MDA-MB-231 ECM1-/-and MDA-MB-231 PGRN-/-cell lines had been constructed successfully by CRISPR/Cas9 system.The proliferation rate of PGRN gene knockout cells was decreased significantly and that of ECM1 gene knockout cells was slightly inhibited compared with wild type.The results of Transwell assays indicated that ECM1 promoted invasion and migration of breast cancer cells,while PGRN inhibited invasion and migration of breast cancer cells.The mRNA expression levels of molecules promoting Epithelial-Mesenchymal Transition(EMT)processes(N-cadherin and SNAIL)and metastasis(MMP9,MMP14 and ICAM1),the protein expression levels of ?-catenin,ICAM1 and the phosphorylation levels of ERK1/2 were decreased in ECM1 knockout cells.But the mRNA expression levels of N-cadherin,SNAIL,MMP9,MMP14,ICAM1and?-catenin,the protein expression of ?-catenin,ICAM1 and phosphorylation levels of ERK1/2 were increased in PGRN knockout cells.The results indicated that PGRN could inhibit the ?-catenin and ERK1/2 signaling pathways.The in vivo studies showed the tumor from PGRN knockout cells showed slowest growth in nude mice,and the ECM1 knockout tumor cells showed slower growth than that from wild type group.The tumor sizes were positively related to the survival time of nude mice.These results indicated PGRN interacted with ECM1,and the two molecules together regulated the expression of important molecules related to metatasis of breast cancer cells by an antagonistic manner.5.The study of mechanism involving the balance of proliferation and metastasis of cancer cells by the interaction of PGRN with EGFR or ECM1.The interaction between PGRN and EGFR was identified by Co-IP and solid binding assay in vivo and in vitro.By solid competition assay,we found that ECM1 could compete with EGFR resulting the disassociated of PGRN and EGFR.Furthermore,EGFR could compete with ECM1,which resulted the disassociated of ECM1 and PGRN.The results indicated EGFR could competitively bind to PGRN with ECM1.The results of Y2H showed PGRN could not directly bind with EGFR,and the interation between the two molecules was mediated by other unknown molecules.The EGFR knock down(KD)MDA-MB-231 cell lines was constructed by CRISPR/Cas9 technique.In terms of proliferation,the proliferation rate of the EGFR KD cells was significantly slower than that of the PGRN knockout cells or wild type.The Transwell results showed that the invasion and migration of EGFR KD cells were promoted.But the mRNA expression levels of N-cadherin,SNAIL,ICAM1 and?-catenin,the protein expression of ?-catenin,ICAM1 and phosphorylation level of ERK1/2 were increased in EGFR KD cells.These results indicated that PGRN and EGFR could promote proliferation by inhibiting its invasion and migration.The proliferation and metastasis of breast cancer cells were balanced by PGRN interacting with EGFR or ECM1.In conclusion,ECM1 was found as a new molecule interacting with PGRN by Y2H.Six strains of monoclonal antibodies targeting ECM1 were obtained by monoclonal antibody preparation techniques and they provides a valuable tool for the function study of ECM1.Based on the antibodies obtained,the sandwich ELISA kit for detecting ECM1 was developed and the luciferase-luciferin system was introduced to further improve the sensitivity.The interaction of PGRN and ECM1 in vivo and in vitro were confirmed by Y2H,Co-IP,GST pull-down and IHC,and the signaling pathways involved in the process were deeply investigated.This study illustrated the biological functions mediated by the interaction between PGRN and ECM1 involving breast cancer metastasis.Moreover,EGFR as the receptor of PGRN was for the first time identified.The balance of proliferation and metastasis of breast cancer cells was mediated by the interaction of PGRN with EGFR or ECM1.These results laid an important foundation for further clarifying the biological functions of PGRN and the mechanism of breast cancer development.It will provide a new insight for the discovery of new targets for tumor treatment.