An In Vitro Study On Long Noncoding RNA-1708 Inhibiting The Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

BackgroundPeriodontitis,malformations,traumas,tumors and other causes often cause severe defects in the maxillofacial bone tissue.Therefore,finding effective methods to treat maxillofacial bone defect has always been the research direction of maxillofacial surgery.Bone tissue engineering has been a research hotspot for its excellent biosafety,biocompatibility,and wide range of sources.Bone tissue engineering includes three factors:plant cells,biomaterial scaffolds,growth and differentiation factors,of which seed cells are the core of tissue engineering.Human bone marrow mesenchymal stem cells(hBMSCs)are ideal seed cells for bone tissue engineering because they are easy to obtain,have strong in vitro proliferation ability,self-renewal ability,and have no immune rejection.Therefore,studying the mechanism of hBMSC osteogenic differentiation is of great significance for exploring the repair methods of maxillofacial bone defects.Long non-coding RNA(lncRNA)is a kind of RNA that hardly encodes protein.Recent studies have shown that lncRNA is widely involved in the regulation of cell proliferation and differentiation.Revealing the role of lncRNA in hBMSCs osteogenic differentiation has a positive significance for repairing maxillofacial bone defects using bone tissue engineeringObjectiveIn previous high-throughput sequencing experiments,we found that lncRNA-1708 showed differential expression after hBMSCs osteogenic differentiation.This study verified the differential expression of lncRNA-1708 in osteogenic differentiation through in vitro induced osteogenic differentiation and PCR detection.We transfected lentivirus in vitro to construct hBMSCs with stable high and low lncrna-1708 expression,so as to investigate the effect of lncrna-1708 on osteogenic differentiation of hBMSCs in vitro.Methods1.hBMSCs were expanded to P3 in vitro and cultured in osteogenic differentiation medium for 2 weeks.Then,the quantitative quantitative reverse transcription-polymerase chain reaction(qRT-PCR)method was used to detect the change of the relative expression of lncRNA-1708 before and after osteogenic differentiation of hBMSCs,and to analyze its relationship with osteogenic differentiation of hBMSCs.2.The lncRNA-1708 overexpression retrovirus and shlncRNA-1708 interference lentiviral groups were constructed and transfected separately to obtain hBMSCs cell lines capable of stably differentially expressing lncRNA-1708.3.Osteogenic differentiation was induced for 2 weeks,and the expression levels of Runt-related transcription factor 2(RUNX2)and alkaline phosphatase(ALP)mRNA were detected by real-time light quantitative PCR(RT-PCR).And alkaline phosphatase staining and alizarin red staining were used to identify the differences in bone differentiation of different hBMSCsResults1.After 2 weeks of hBMSCs osteogenic differentiation,the relative expression of lncRNA-1708 decreased significantly(P<0.001).2.The expression level of lncrna-1708 in the hBMSCs cell lines with stable high expression lncrna-1708 and low expression lncrna-1708 was significantly different from that in the corresponding negative control(lncrna-1708 overexpressed cell line,P<0.05).Lncrna-1708 low expression cell line,P<0.01).3.Compared with the negative control group,the expression levels of osteogenesis-related genes RUNX2 and ALP in hBMSCs overexpressed with lncRNA-1708 were reduced(P<0.01),alkaline phosphatase staining became lighter,and calcium nodules were relatively reduced.The expression levels of RUNX2 and ALP in hBMSCs transfected with shlncRNA-1708 were significantly higher than those of the corresponding negative control group(P<0.01),alkaline phosphatase staining was deeper,and calcium nodules were relatively increased.Conclusions1.LncRNA-1708 expression was reduced in hBMSCs osteogenic differentiation in vitro.2.LncRNA-1708 can inhibit osteogenic differentiation of hBMSCs in vitro.