Effects Of Co-culture Of Bone Marrow Mesenchymal Stem Cells And Endometrial Cancer Cells On Malignant Phenotype And Gene Expression Differences

Objective: To establish the co-culture system of human Bone derived mesenchymal stem cells(h BMSCs)and JEC cells of endometrial cancer.Investigate the effects of h BMSCs on JEC cell proliferation,migration and apoptosis.High-throughput sequencing and bioinformatics methods were used to analyze and screen differentially expressed mi RNA and m RNA,to predict the possible targeting relationships and to verify their expressions.Methods: The h BMSCs and JEC cells were cultured separately.JEC cells were infected by lentivirus Lenti-GFP,and the positive cells were screened by blasticidin(BSD).The infected positive JEC cells were labeled as JEC-GFP cells.The experiment had two groups: the JEC-GFP group was cultured with JEC-GFP cells alone.In the JEC-GFP-BM group,JEC-GFP cells were mixed with h BMSCs in 1:3 ratios for co-culture.Three repetition groups were set up for each group.After three days,JEC-GFP cells were isolated by differential digestion.The effects of h BMSCs on JEC cell proliferation,migration and apoptosis were verified by CCK8,transwell and apoptosis experiments,respectively.Flow sorting and high-throughput sequencing were performed for the two groups of cells.Differentially expressed mi RNA and m RNA molecules before and after co-culture were screened by bioinformatics method and analyzed.The predicted targeted relationship was verified by double luciferase assay and its expression was verified by q PCR and WB.Results:(1)HBMSCs can inhibit the proliferation and migration of JEC cells and promote their apoptosis(P < 0.05).(2)A total of 397 differential mi RNAs and 7289 differential m RNAs were screened through sequencing analysis,among which 151 mi RNAs 4177 m RNAs were up-regulated and 246 mi RNA 3112 m RNAs were down-regulated in JEC-BM group compared with JEC group.(3)It was predicted that mi R-125a-5p may have a targeting relationship with m RNA ETS1.(4)QPCR and WB verified the expression of mi R-125a-5p and ETS1,and the variation trend was consistent with the sequencing.(5)Double luciferase reporting experiments showed that mi R-125a-5p had a targeted relationship with ETS1,and mi R-125a-5p had a negative regulatory effect on the expression of ETS1.Conclusions: The co-culture system of h BMSCs and JEC cells was established successfully.Through high-throughput sequencing technology,it was found that RNA changes in a large number of JEC cells after co-culture with h BMSCs predicted that mi R-125a-5p may have a targeting relationship with ETS1.The targeting relationship between mi R-125a-5p and ETS1 was predicted and verified.It will provide experimental basis for further research on mi RNA and m RNA associated with endometrial cancer.