The Action And Mechanism Research Of Long Non-Coding RNA Opbr6 During Cell Reprogramming

Background:Stem cell has the ability of extensive self-renewaling and differentiating offspring,which is the focus of regenerative medicine.Stem cell technology is a major hope for many diseases that are currently difficult to cure,and it has also promoted the development of new cancer stem cell therapies.Terminal-differentiated cells can be reprogrammed to a pluripotent state induced by factors such as pluripotency-related transcription factors,small chemical complexes,and nuclear transfer.Somatic cell reprogramming is the process by which we induce the reverse differentiation of somatic cells into pluripotent embryonic stem cells.However,this reprogramming process is time consuming and relatively inefficient,limiting the clinical application of induced pluripotent stem cells as a regenerative medicine method.The successful preparation of artificially induced pluripotent stem cells has given scientists new hope.Artificially induced pluripotent stem cells were obtained by Japanese scientist Yamanaka in 2006.Four pluripotency-related transcription factors Oct3/4,Sox2,Klf4,and c-Myc were transferred into somatic cells for the first time.Artificially induced pluripotent stem cells do not involve ethical or operational problems associated with human embryonic stem cells and are the most promising methods for regenerative medical cell therapy.At present,the molecular mechanism of cell reprogramming and maintenance of stem cell pluripotency are not completely clear.The mechanism of maintaining pluripotency can not only promote the development of tissue engineering,embryonic development and tumor research,but also promote the development of induced pluripotent stem cells technology.We now have made it clear that to successfully reprogram cells and achieve pluripotency,we need to cross a powerful epigenetic barrier.In the previous experiments,we used the modified Crispr Cas9 chromatin immunoprecipitation technique to capture lnc RNAs that bind to the Oct4 and Sox2 promoters,revealing the DNA-lnc RNA binding network of the pluripotent gene Sox2 and Oct4 promoters in cell reprogramming.In the sequencing results,we predict lnc RNA Opbr6 is closely related to cell reprogramming and maintenance of stem cell pluripotency.Objective:This study aimed to investigate the role of Opbr6 in cell reprogramming and stem cell pluripotency maintenance and its regulatory mechanisms.Methods:Real-time quantitative PCR was used to detect the expression of Opbr6 and three pluripotent reprogramming factors(Oct4,Sox2,Nanog)in various stages of cell reprogramming,embryonic stem cell differentiation and mouse tissues.RNA FISH was used to describe Opbr6 localization in cells.The function of Opbr6 was confirmed by Opbr6 knockdown or overexpression.The function of Opbr6 was verified by Opbr6-induced secondary reprogramming.The dual luciferase reporter detection system was used to determine the binding of Opbr6 to the dry gene promoter.The binding of Opbr6 to DNA was clarified by reverse transcription correlation capture technique.The binding of Opbr6 to pluripotency protein was determined by RNA-protein immunoprecipitation technique.Results:1.The expression of lnc RNA Opbr6 was positively correlated with cellular pluripotency,silenced in FBC,expressed very little in URC,highly expressed in ESC and i PS.The expression of lnc RNA Opbr6 down-regulated during embryonic body differentiation,and the trend of down-regulation was parallel with Oct4 and Sox2.Opbr6 was also expressed in differentiated mouse tissues.2.After the Opbr6 overexpression treatment in FBC,the expression of Opbr6 and three pluripotent reprogramming factors(Oct4,Sox2,Nanog)increased significantly,the cells obtained partial pluripotency,and the morphology developed to stem cells.After Opbr6 knockdown treatment in ESC,the expression of Opbr6 and three pluripotent reprogramming factors(Oct4,Sox2,Nanog)decreased significantly,the cells lost pluripotency,the morphology became flat and slender,and the cells adhered firmer.The results of immunofluorescence showed that pluripotency molecular markers AP and Sox2 were down-regulated in the Opbr6 knockdown cells.Opbr6 overexpression treatment increased the efficiency of secondary MEF reprogramming.3.The luciferase reporter system test proved that Opbr6 activated the promoter of Oct4 and Sox2 genes.RAT experiments showed that Opbr6 binded to the promoter of Oct gene,distal enhancer,and proximal enhancer.Opbr6 formed specific chromatin conformation around Oct gene.It also showed that Opbr6 combined with other pluripotency regulators,which could form a regulatory network.RIP confirmed that Opbr6 binded to SMC1,thereby recruiting SMC1 to Oct4 promoter and promoting its transcription.Conclusions:1.The expression of lnc RNA Opbr6 in nucleus is closely related to the state of cell reprogramming.And it has the function of promoting cell reprogramming and maintaining stem cell pluripotency.2.The mechanism of how Opbr6 promotes stem cell pluripotency is that Opbr6 activates the promoters of pluripotency genes Oct4 and promote the expression of Oct4.In addition,Opbr6 forms a complex with SMC1.This complex promotes the formation of the inner loop of chromatin by binding to the distal enhancer and promoter of Oct4 gene and pulling the distal enhancer to the promoter.And it maintains a specific spatial conformation of chromatin and the pluripotent epigenetic state of the cell.