Study Of The Secreted Factors Of HUC-MSCs Inducing MCF7 Cells Pyroptosis

Breast cancer is a kind of malignant tumor that occurs in the glandular epithelial tissue of breast.According to the statistics of 2015,about 2.1 million women suffered from breast cancer and 533600 people died of it.Although China is not a high-incidence country,the growth rate has been higher than that in other high-risk countries in recent years.At present,the main methods to treat breast cancer are surgery,chemotherapy,radiotherapy and targeted drug therapy with Chinese medicine assistance.However,breast cancer is classified by different classification systems and different type has different prognosis and treatment response.In addition,radiation therapy and chemotherapy can not only kill cancer cells but also kill normal cells.Therefore,it is necessary to find new and more effective targeted therapy.Human umbilical cord mesenchymal stem cells(hUC-MSCs)derived from umbilical cord Wharton's jelly.They are more primitive than marrow-derived MSCs and are stem cells between adult stem cells and embryonic stem cells.hUC-MSCs have all characteristics of MSCs and are superior to MSCs from other sources:the content of hUC-MSCs is higher,the immunogenicity is lower,and in vitro culture,the doubling time is shorter,the telomere is longer and more stable,and hUC-MSCs will not spontaneously transform into teratoma.It does not involve any ethical issues and does not cause trauma to donors when materials are taken.Therefore,hUC-MSCs are more suitable for clinical research and application.In our previous studies,we found the secreted factors of hUC-MSCs can inhibit some cancer cells,including breast cancer cells.In view of the current situation that new treatment methods for breast cancer need to be developed and the good prospect of hUC-MSCs in anti-cancer,our project focused on hUC-MSCs and breast cancer cell line--MCF7 cells to do the follow studies:1.Spindle cells were successfully isolated from human umbilical cord Wharton's jelly by the tissue explant technique.The identity of hUC-MSCs was confirmed by cell surface proteins,which provided cell materials for subsequent experiments.2.hUC-MSCs conditioned medium(hUC-MSCs CM)was prepared with hUC-MSCs cultured medium.MCF7 cells were cultured in hUC-MSCs CM and cell morphological changes were observed.It was found that MCF7 cells showed the morphological characteristics of pyroptosis.MTS assay showed that the viability of MCF7 cells continued to decrease with the increase of treatment time.Flow cytometry showed no significant changes in cell cycle of MCF7 cells.Annexin V/PI analysis showed that the number of PI+/FITC-cells increased significantly,which is also the characteristic of pyroptosis.Furthermore,TUNEL assay,quantification of secreted IL-1? assay and western-blot were used to verify that MCF7 cells were indeed pyroptosis.3.RNA-seq and bioinformatics analysis were carried out on MCF7 cells which were pyroptosis induced by hUC-MSCs CM.Finally,1822 up-regulated genes and 775 down-regulated genes were obtained.Among significantly up-regulated genes,pyroptosis related genes caspase-4 and NLRP1 were found,suggesting that caspase-4 and NLRP1 played an important role in this process.Therefore,these two genes were selected for subsequent experiments.GO enrichment analysis of the DEGs revealed that the main biological reactions were the response to external stimuli,cell signal transduction,cell structure changes and immune system processes;the main changes of cell components happened to cell surface,cell membrane and extracellular part;the main molecular function change was related to ion binding.KEGG analysis of the DEGs revealed 14 pathways were significant difference,which were mainly related to immune system,endocrine system,nervous system and diseases related to these systems.4.Caspase-4 or NLRP1 genes were successfully inhibited in MCF7 cells at the levels of both transcriptional and protein expression.After inhibited the caspase-4 or NLRP1 gene,it was found that the pyroptosis of MCF7 cells induced by the secreted factors of hUC-MSCs were mediated by both classical and non-classical pathways.When one of them was inhibited,another would be enhanced.Therefore,MCF7 cells would eventually be pyroptosis.To sum up,our project targeted hUC-MSCs,which are ideal seed cells for clinical stem cell therapy,and studied their effect on MCF7 cells.Transcriptome sequencing was performed on the MCF7 cells which were pyroptosis induced by hUC-MSCs CM by RNA-seq technique.The shRNA vectors of caspase-4 or NLRP1 were constructed to study the role of these two genes in the process of MCF7 pyroptosis.We found hUC-MSCs can promote MCF7 cells pyroptosis,the mechanism of which is closely related to caspase-4 or NLRP1 gene-mediated pyroptosis pathway.These results provide a theoretical reference for the further application of hUC-MSCs in the clinical treatment of breast cancer.At the same time,the research on the mechanism of pyroptosis and the important genes related to pyroptosis provide useful information for finding new targets to treat pyroptosis-related diseases.