Role And Mechanism Of Circular RNA Circ PLCE1 In Differentiation Of HiPS Cells Into Urothelial Cells

Objective: To study the role of circPLCE1 in the differentiation of human induced pluripotent stem cells(hiPSCs)into urothelial cells(UCs),and explore the molecular mechanism of circPLCE1,in order to provide intervention strategies for resolving the differentiation of target cells during the repair process of urinary system tissue engineering.Methods:(1)The expression of the pluripotent markers NANOG and OCT4 of hiPSCs was identified by flow cytometry and immunofluorescence assay.According to the "three step method",hiPSCs were first differentiated into endodermal cells,followed by a stage of hindgut cells,and finally differentiated into UCs;western blot and quantitative real-time polymerase chain reaction(q RT-PCR)were used to detect the expression of endodermal cell markers SOX17,CDX2,hindgut cell markers HOXA13,HOXD13,and differentiated UCs markers UPKI,UPKIII,CK13,CK20,and E-Cadherin.(2)High throughput sequencing was performed on differentiated UCs and hiPSCs.The differences in the sequencing results were analyzed using bioinformatics to screen for high expression abundance and differentiation related circular RNAs,which the sequencing results were validated by q RT-PCR experiments.(3)Using the ring forming experiment to test whether circPLCE1 could form a ring correctly.The differentially expressed hiPSCs were constructed using circPLCE1 small interfering RNA and an overexpression virus vector,then differentiated into UCs.Western blot and q RT-PCR experiments were used to detect the expression of markers at various stages of differentiation;(4)The localization of circPLCE1 in cells was determined by nucleocytoplasmic separation experiments.The bioinformatics technology was used to predict the mi RNA binding to circPLCE1,which was verified by RNA pull down experiment and dual luciferase experiment.hiPSCs cell lines overexpressing mi R-1184 constructed by lentivirus vectors differentiated into UCs.Western blot and q RT-PCR experiments were used to detect the expression of markers at various stages.Result:(1)The results of flow cytometry and immunofluorescence assay showed that the multipotent marker proteins NANOG and OCT4 were expressed in hiPSCs.hiPSCs were successfully induced to differentiate into UCs after 18 days.During the differentiation process,the expression of endodermal cell markers SOX17 and CDX2 were significantly increased after 3 days of differentiation(P<0.05),and the expression of hindgut cell markers HOXA13 and HOXD13 were significantly increased after 7 days of differentiation(P<0.05),and the expression of UCs markers UPKI,UPKIII,CK13,CK20,and E-Cadherin were significantly increased after 18 days of differentiation(P<0.05).(2)High throughput sequencing results showed significant differential expression of circRNA during differentiation.According to expression abundance,bioinformatics and q RT-PCR experiments,circPLCE1 might promote the differentiation of hiPSCs into UCs.(3)The ring formation experiment showed that circPLCE1 could be looped correctly.The results of q RT-PCR and western blot experiments showed that overexpression of circPLCE1 significantly promoted the differentiation of hiPSCs into UCs,and the lower expression of circPLCE1 inhibited the expression of UCs markers.(4)The results of nucleocytoplasmic separation experiments showed that circPLCE1 mainly exists in the cytoplasm of hiPSCs.Prediction results from CRI,mir CRC,and mi RNADa databases suggested that mi R-1184,mi R-127-5p,and mi R-663 b might bound to circPLCE1.RNA pull down and dual luciferase experiments showed that circPLCE1 and mi R-1184 could bind directly.Western blot and q RT-PCR results showed that overexpression of mi R-1184 inhibited the differentiation of hiPSCs into UCs,and decreased the differentiation promoting effect of circPLCE1.Conclusion:(1)hiPSCs can be induced in vitro to differentiate into UCs through the endoderm and hindgut stages.(2)Overexpression of circPLCE1 promotes the differentiation of hiPSCs into UCs,while low-expression of circPLCE1 inhibits the differentiation of hiPSCs into UCs.(3)CircPLCE1 targeted binding to mi R-1184 plays a role in promoting differentiation.Meaning: This study provides a theoretical basis for clarifying the mechanism of circPLCE1 promoting the differentiation of hiPSCs into UCs,provides a new idea for solving the problem of difficult access to seed cells in urinary system tissue engineering,and brings new hope for the defect repair and functional reconstruction of urinary system diseases.