Alteration of circRNA and lncRNA expression profile in exosomes derived from periodontal ligament stem cells undergoing osteogenic differentiation.

This study investigated circRNA and lncRNA expression profile in exosomes derived from periodontal ligament stem cell (PDLSC) before and after its osteogenic differentiation. Exosomes derived from PDLSCs before (EX0) and after osteogenic induction for 5 (EX5) and 7 (EX7) days were harvested and exosomal circRNAs and lncRNAs were analyzed by RNA sequencing. Certain RNAs showing significantly altered expression were selected for qRT-PCR verification. The circRNA-miRNA-mRNA network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. All groups of exosomes showed typical characteristics under nanoparticle tracking analysis, flow cytometry assay and transmission electron microscopy. 69-557 circRNAs and 2907-11581 lncRNAs were found in EX0, EX5 and EX7, which were broadly distributed across the 24 pairs of human chromosomes. Compared with EX0, 3 circRNAs and 2 lncRNAs were up-regulated and 39 circRNAs and 5 lncRNAs down-regulated consistently through out of EX5 and EX7, p < 0.05. qRT-PCR confirmed certain those consistently expressed RNAs, such as circ lysophosphatidic acid receptor 1 (LPAR1). KEGG analysis showed that those consistent expressed RNAs closely related to TGF-beta pathway, MAPK pathway, mTOR pathway and FoxO signaling pathways regulating pluripotency of stem cells. Exosomal circRNAs and lncRNAs had significant expression changes during the early phase of osteogenic differentiation of PDLSCs. Further study would be taken for understanding the roles of exosomal circRNAs and lncRNAs playing in osteogenic differentiation of PDLSCs.

Xie L ，Chen J ，Ren X ，Zhang M ，Thuaksuban N ，Nuntanaranont T ，Guan Z ... -  《-》

Identification and integrated analysis of differentially expressed lncRNAs and circRNAs reveal the potential ceRNA networks during PDLSC osteogenic differentiation.

Gu X ，Li M ，Jin Y ，Liu D ，Wei F ... -  《BMC GENETICS》

CircRNA, lncRNA, and mRNA profiles of umbilical cord blood exosomes from preterm newborns showing bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) represents a multifactorial chronic pulmonary pathology and a major factor causing premature illness and death. The therapeutic role of exosomes in BPD has been feverishly investigated. Meanwhile, the potential roles of exosomal circRNAs, lncRNAs, and mRNAs in umbilical cord blood (UCB) serum have not been studied. This study aimed to detect the expression profiles of circRNAs, lncRNAs, and mRNAs in UCB-derived exosomes of infants with BPD. Microarray analysis was performed to compare the RNA profiles of UCB-derived exosomes of a preterm newborn with (BPD group) and without (non-BPD, NBPD group) BPD. Then, circRNA/lncRNA-miRNA-mRNA co-expression networks were built to determine their association with BPD. In addition, cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of lipopolysaccharide (LPS)-induced human bronchial epithelial cells (BEAS-2B cells) and human umbilical vein endothelial cells (HUVECs). The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in LPS-induced BEAS-2B cells and HUVECs were assessed through Western blot analysis. Then, quantitative reverse transcription-polymerase chain reaction assay was used to evaluate the expression levels of four differentially expressed circRNAs (hsa_circ_0086913, hsa_circ_0049170, hsa_circ_0087059, and hsa_circ_0065188) and two lncRNAs (small nucleolar RNA host gene 20 (SNHG20) and LINC00582) detected in LPS-induced BEAS-2B cells or HUVECs. A total of 317 circRNAs, 104 lncRNAs, and 135 mRNAs showed significant differential expression in UCB-derived exosomes of preterm infants with BPD compared with those with NBPD. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to examine differentially expressed exosomal circRNAs, lncRNAs, and mRNAs. The results showed that the GO terms and KEGG pathways mostly involving differentially expressed exosomal RNAs were closely associated with endothelial or epithelial cell development. In vitro, CCK-8 and Western blot assays revealed that LPS remarkably inhibited the viability and promoted inflammatory responses (TNF-α and IL-1β) of BEAS-2B cells or HUVECs. The expression levels of circRNAs hsa_circ_0049170 and hsa_circ_0087059 were upregulated in LPS-induced BEAS-2B cells; the expression level of hsa_circ_0086913 was upregulated and that of hsa_circ_0065188 was downregulated in LPS-induced HUVECs. Moreover, the expression level of lncRNA SNHG20 was upregulated and that of LINC00582 was downregulated in LPS-induced BEAS-2B cells. Further, 455 circRNA/lncRNA-miRNA-mRNA interaction networks were predicted, including hsa_circ_0086913/hsa-miR-103a-3p/transmembrane 4 L six family member 1 (TM4SF1) and lncRNA-SNHG20/hsa-miR-6720-5p/spermine synthase (SMS) networks, which may take part in BPD. This study provided a systematic perspective on UCB-derived exosomal circRNAs and lncRNAs and laid an important foundation for further investigating the potential biological functions of exosomal circRNAs and lncRNAs in BPD. • BPD represents a multifactorial chronic pulmonary pathology and a major factor causing premature illness and death. • The therapeutic role of exosomes in BPD has been feverishly investigated, and exosomal RNAs were ignored. • The profiles of UCB-derived exosomal circRNAs, lncRNAs, and mRNAs were performed. • Several differentially expressed circRNAs and lncRNAs were identified in LPS-induced BEAS-2B cells and HUVECs.

Wang Y ，Wang X ，Xu Q ，Yin J ，Wang H ，Zhang L ... -  《-》

Integrative Analysis of ceRNA Networks in human periodontal ligament stem cells under hypoxia.

This study aims to investigate the regulatory effect of hypoxia on human periodontal ligament stem cells (PDLSCs) through RNA sequencing (RNA SEQ). Human PDLSCs were cultured in normoxia (20% O2 ) or hypoxia (2% O2 ). Total RNA was extracted and sequenced. The expression profiles of circRNAs, lncRNAs, and miRNAs were determined, and the lncRNA/circRNA-miRNA-mRNA networks were analyzed. In total, 15 miRNAs, 449 lncRNAs, and 53 circRNAs were differentially expressed. Among them, 21 circRNAs, 262 lncRNAs, 5 miRNAs, and 5 mRNAs were selected to construct competing endogenous RNA (ceRNA) networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to explore potential related pathways and regulatory functions. Several ceRNA axes (lncRNA-FTX/circRNA-FAT1-hsa-miR-4781-3p-SMAD5 and circRNA LPAR1-hsa-miR-342-3p-ADAR) may provide a theoretical basis on the study of osteogenic differentiation of PDLSCs under hypoxia. This study revealed that the expression profiles of circRNAs, lncRNAs, and miRNAs had changed significantly in PDLSCs cultured in 2% O2 ; specific circRNAs/lncRNAs may play a competitive role in the differentiation of PDLSCs.

Ye Y ，Fu L ，Liu L ，Xiao T ，Cuba Manduca AG ，Yu J ... -  《-》

Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells.

Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role. The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs. qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs. Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs. This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

Wang H ，Gao S ，Dissanayaka WL 《-》