The Effects Of Hi-Lnc70 On The Differentiation Efficiency Of HESCs Into IPCs And Its Regulatory Mechanism

Diabetes is divided into insulin-dependent type 1 Diabetes,T1D(T1D),and non-insulin-dependent type 2 Diabetes(T2D).Human embryonic stem cells(hESCs)are the cells that have the potential of continuous renewal and multi-directional differentiation.How to use embryonic stem cells to obtain enough insulin-secreting cells through in vitro differentiation or stem cell transplantation has been the focus of the treatment diabetes by stem cells.Long non-coding RNAs(lncRNAs)are non-coding RNAs that are greater than 200 nucleotides in length and cannot independently encode proteins.In this study,a pancreas-specific lnc RNA that accumulates in the nucleus named Hi-Lnc70 is choosen.Through construction and transfection of Hi-Lnc70 overexpression and suppression vectors,the Hi-Lnc70 expression during the differentiation of hESCs into insulin producing cells(IPCs)was altered and the effects of Hi-Lnc70 was investageted by gene and protein expression of pancreatic related factors.When fluorescent protein gene was introduced into HiLnc70 promoter by CRISPR/Cas9,Hi-Lnc70 promoter can successfully switch on the expression of fluorescent protein.When Hi-Lnc70 was inhibited during the differentiation,Ngn3 and Pdx1 expression were increased at the middle stage,the expression of Pax6 and Maf A was decreased at the middle and late stages,and the expression of Foxa2 and Cxcr4 was decreased at the late stage of differentiation,.Inhibition of HiLnc70 promoted the protein expression of PDX1 and INSULIN.Finally,transcriptome sequencing was perfomed on the high Hi-Lnc70 expression and its inhibitory samples of the middle stage of differentiation.The results shown that the differential genes are enriched in the cell functions,such as the signals mediated by the second messenger and the regulation of neurotransmitter.In the enrichment of KEGG cell pathways,the differential genes are mainly enriched in neural active ligand-receptor pathways,calcium signaling pathways and other pathways.Finally,a protein interaction network of differential genes was constructed,which showed that Hi-Lnc70 is related to intracellular signal transmission and nervous system development.This study laid the foundation for elucidating the role of Hi-Lnc70 in the transcriptional regulatory network during the pancreatic differentiation of hESCs.