Gu Xiuge, Li Mengying, Jin Ye, Liu Dongxu, Wei Fulan
Department of Orthodontics, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Wenhua Xi Road No. 44-1, Jinan, Shandong, 250012, People's Republic of China.
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, People's Republic of China.
BMC Genet. 2017 Dec 2;18(1):100. doi: 10.1186/s12863-017-0569-4.
Researchers have been exploring the molecular mechanisms underlying the control of periodontal ligament stem cell (PDLSC) osteogenic differentiation. Recently, long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) were shown to function as competitive endogenous RNAs (ceRNAs) to regulate the effect of microRNAs (miRNAs) on their target genes during cell differentiation. However, comprehensive identification and integrated analysis of lncRNAs and circRNAs acting as ceRNAs during PDLSC osteogenic differentiation have not been performed.
PDLSCs were derived from healthy human periodontal ligament and cultured separately with osteogenic induction and normal media for 7 days. Cultured PDLSCs were positive for STRO-1 and CD146 and negative for CD31 and CD45. Osteo-induced PDLSCs showed increased ALP (alkaline phosphatase) activity and up-regulated expression levels of the osteogenesis-related markers ALP, Runt-related transcription factor 2 and osteocalcin. Then, a total of 960 lncRNAs and 1456 circRNAs were found to be differentially expressed by RNA sequencing. The expression profiles of eight lncRNAs and eight circRNAs were measured with quantitative real-time polymerase chain reaction and were shown to agree with the RNA-seq results. Furthermore, the potential functions of lncRNAs and circRNAs as ceRNAs were predicted based on miRanda and were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. In total, 147 lncRNAs and 1382 circRNAs were predicted to combine with 148 common miRNAs and compete for miRNA binding sites with 744 messenger RNAs. These mRNAs were predicted to significantly participate in osteoblast differentiation, the MAPK pathway, the Wnt pathway and the signaling pathways regulating pluripotency of stem cells. Among them, lncRNAs coded as TCONS_00212979 and TCONS_00212984, as well as circRNA BANP and circRNA ITCH, might interact with miRNA34a and miRNA146a to regulate PDLSC osteogenic differentiation via the MAPK pathway.
This study comprehensively identified lncRNAs/circRNAs and first integrated their potential ceRNA function during PDLSC osteogenic differentiation. These findings suggest that specific lncRNAs and circRNAs might function as ceRNAs to promote PDLSC osteogenic differentiation and periodontal regeneration.
研究人员一直在探索牙周膜干细胞（PDLSC）成骨分化调控的分子机制。最近，长链非编码RNA（lncRNA）和环状RNA（circRNA）被证明作为竞争性内源RNA（ceRNA）在细胞分化过程中调节微小RNA（miRNA）对其靶基因的作用。然而，尚未对PDLSC成骨分化过程中作为ceRNA的lncRNA和circRNA进行全面鉴定和综合分析。
从健康人牙周膜中分离出PDLSC，并分别在成骨诱导培养基和正常培养基中培养7天。培养的PDLSC对STRO-1和CD146呈阳性，对CD31和CD45呈阴性。成骨诱导的PDLSC显示碱性磷酸酶（ALP）活性增加，以及成骨相关标志物ALP、Runt相关转录因子2和骨钙素的表达水平上调。然后，通过RNA测序发现共有960个lncRNA和1456个circRNA差异表达。通过定量实时聚合酶链反应检测了8个lncRNA和8个circRNA的表达谱，结果与RNA测序结果一致。此外，基于miRanda预测了lncRNA和circRNA作为ceRNA的潜在功能，并使用基因本体论和京都基因与基因组百科全书分析进行了研究。总共预测147个lncRNA和1382个circRNA与148个常见miRNA结合，并与744个信使RNA竞争miRNA结合位点。这些mRNA预计会显著参与成骨细胞分化、丝裂原活化蛋白激酶（MAPK）途径、Wnt途径以及调节干细胞多能性的信号通路。其中，编码为TCONS_00212979和TCONS_00212984的lncRNA，以及circRNA BANP和circRNA ITCH，可能与miRNA34a和miRNA146a相互作用，通过MAPK途径调节PDLSC的成骨分化。
本研究全面鉴定了lncRNA/circRNA，并首次整合了它们在PDLSC成骨分化过程中的潜在ceRNA功能。这些发现表明，特定的lncRNA和circRNA可能作为ceRNA促进PDLSC的成骨分化和牙周组织再生。
