The Role Of Long Noncoding RNA UCA1 In The Differentiation Of Human Umbilical Cord Mesenchymal Stem Cell Into Hepatocyte

Background: End-stage liver disease often lead to liver failure which brings about tremendous harm to human health.And it is one of the main causes of death in the world.Liver and hepatocyte transplantation have been regarded as important methods to treat end-stage liver disease.However,organ shortage remains a major limitation step of this procedure.Human umbilical cord mesenchymal stem cells(UC-MSCs)could be induced into hapatocyte through adding growth factors,which makes it a reliable resource of hepatocyte.But the low function of these hepatocytes could not meet the clinical demands.It has been reported that long noncoding RNA(lncRNA)plays an important part in stem cell differentiation.Our research focus on the lncRNA UCA1 in the differentiation of UC-MSCs to hepatocytes and wanna to gain a more functional hepatocyte in the end.Objective: To explore the role of UCA1 in the differentiation of UC-MSCs to hepatocyes and how UCA1 influences the differentiation.Materials and methods: At the first part of experiments,we isolated primary UC-MSCs from umbilical cord and after 3 passages,flow cytometry was used to examine the cellular phenotype.Moreover,UC-MSCs were induced into osteocytes,chondrocytes and adipocytes.At the second part of experiments,UC-MSCs were induced into hepatocytes through adding cytokines and growth factors in vitro.To observe the morphological change,inverted microscope was used.We also used immunofluorescence technique to examine liver specific proteins after differentiation.At the third part in vitro experiments,UC-MSCs were induced to differentiate into hepatocytes and were compared with bone marrow mesenchymal stem cells(BM-MSCs)for their hepatic differentiation potential.To compare the proliferative ability of UC-MSCs and BM-MSCs,mean values of cell counts were calculated.The phenotype of UC-MSCs and BM-MSCs were evaluated by flow cytometry using a flow cytometer.They were also induced into osteocytes,chondrocytes and adipocytes to compare their differentiation potential.Immunofluorescence and Western-Blot techniques were used to compare their expression of liver specific proteins after hepatic differentiation.Liver specific gene expression were compared by reverse transcription polymerase chain reaction(RT-PCR)after hepatic differentiation of UC-MSCs and BM-MSCs.Cell culture supernatants of UC-MSCs and BM-MSCs were collected and examined for the level of secreted ALB and BUN using ELISA kit.At the fourth part in vitro experiments,the expression of UCA1 was measured by RT-PCR.Lentivirus transfection method was used to achieve UCA1 overexpression.The hepatocyte related proteins and mRNAs was then examined by immunofluoresence analysis,ELISA and RT-PCR.To examine the expression of liver enriched factors after overexpression of UCA1,RT-PCR technique was used.We also focused on variation of Wnt/β-catenin signaling by immunofluorescence,RT-PCR and TOP/FOP assay.Results: We could acquire UC-MSCs successfully by primary cell isolation tech nique.UC-MSCs could be induced into hepatocytes through adding growth factors and cytokines.UC-MSCs had greater proliferative ability than BM-MSCs.RT-PCR analysis revealed a higher expression of liver specific genes in differentiated UC-MSCs when compared with differentiated BM-MSCs.It was shown in our research that UCA1 was upregulated during hepatic differentiation of UC-MSCs.Upregulation of UCA1 could improve hepatic differentiation of UC-MSCs including hepatocyte related genes and proteins.In addition,we also found liver-enriched transcription factors were upregulated after overexpression of UCA1.Moreover,there was a correlation between Wnt/β-catenin signaling and UCA1.Conclusions: UC-MSCs were ideal source for hepatic differentiation.Overexpression of UCA1 could improve the hepatic differentiation of UC-MSCs and the mechanism was associated with liver-enriched transcription factors and Wnt/β-catenin signaling.