Differential circRNA expression profiles during the BMP2-induced osteogenic differentiation of MC3T3-E1 cells.
Recent studies have indicated that circular RNAs (circRNAs) might play important roles in various diseases. However, little is known about the functions of circRNAs in the skeletal system, and the role of circRNAs in the mechanism by which bone morphogenetic protein 2 (BMP2) promotes bone differentiation remains unknown. Here, we performed RNA-seq to analyze differential expression of circRNA during different osteoblast differentiation stages and investigated the relevant mechanisms.
Alkaline phosphatase (ALP) staining and activity were performed to assess osteogenic differentiation in MC3T3-E1 cells. The expression of osteogenic markers in MC3T3-E1 cells and the differential expression levels of circRNAs were measured and validated by qRT-PCR. Osteogenic marker proteins were measured by western blot. RNA-seq was performed to detect differential expression of circRNAs during the osteogenic differentiation of MC3T3-E1 cells induced by BMP2. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER pathway analyses were performed to predict the functions of differentially expressed circRNAs and potentially co-expressed target genes. The microRNA (miRNA) targets of the circRNAs and circRNA-miRNA interactions were predicted by miRanda. The circRNA-miRNA co-expression network was constructed based on the correlation analysis between the differentially expressed circRNAs and miRNAs. A graph of the circRNA-miRNA network was created using Cytoscape 3.01.
The Cell Counting Kit 8 (CCK-8) assay showed that BMP2 promoted the proliferation of osteoblasts in vitro. Both the intracellular ALP content and activity were increased in BMP2-treated MC3T3-E1 cells. In addition, the mRNA and protein levels of the osteoblastic markers ALP, Sp7 transcription factor (SP7) and runt-related transcription factor 2 (RUNX2) were substantially up-regulated. In the present study, 158 circRNAs were differentially expressed by a fold-change ≥2.0, P<0.05 and false discovery rate <0.05. Among these, 74 circRNAs were up-regulated, while 84 circRNAs were down-regulated. In addition, the expression levels of circRNA.5846, circRNA.19142 and circRNA.10042 were significantly increased in the BMP2 group. Furthermore, by analyzing the target mRNAs of miR-7067-5p using GO and PANTHER pathway analyses, circ19142 and circ5846 were found to be not only strongly associated with the biological process of the positive regulation of developmental processes but also related to the fibroblast growth factor, epidermal growth factor, platelet-derived growth factor and Wnt signaling pathways, which are involved in cell growth and differentiation.
The present study identified circ19142 and circ5846 as being associated with osteoblast differentiation and BMP2 may induce osteogenic differentiation through a circ19142/circ5846-targeted miRNA-mRNA axis.
Qian DY
，Yan GB
，Bai B
，Chen Y
，Zhang SJ
，Yao YC
，Xia H
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Comprehensive circRNA expression profile and function network in osteoblast-like cells under simulated microgravity.
Circular RNAs (circRNAs) are a new class of non-coding RNA with a stable structure formed by special loop splicing. Research increasingly suggests that circRNAs play a vital role in the pathogenesis and progression of various diseases. However, the roles of circRNAs in osteoblast differentiation under microgravity remain largely unknown. Here, we investigated the roles and mechanobiological response of circRNAs in osteoblasts under simulated microgravity.
Differential circRNA and mRNA expression profiles of MC3T3-E1 cells during exposure to microgravity were screened by RNA transcriptome sequencing technology (RNA-seq). The selected RNAs were validated using quantitative real-time polymerase chain reaction (qRT-PCR). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were applied for gene function analyses.
A total of 427 circRNAs and 1912 mRNAs were differentially expressed along with osteogenic differentiation in the simulated microgravity group (SMG) compared to the control group (CON). Of these, 232 circRNAs and 991 mRNAs were upregulated, whereas 95 circRNAs and 921 mRNAs were downregulated (fold change ≥ 2, p < 0.05). The results showed that the parental genes of circRNAs and mRNAs were mainly enriched in anatomical structure morphogenesis, anchoring junction and protein binding. KEGG analysis results showed that the differentially expressed mRNAs were enriched in the regulation of the actin cytoskeleton, focal adhesion, and Ras signalling pathway. Subsequently, 9 core regulatory genes, including 6 mRNAs and 3 circRNAs, were identified based on their possible function in osteoblast differentiation. Based on this analysis, circ_014154 was selected as the target circRNA, which likely plays important roles in osteogenic differentiation processes under microgravity. The circRNA-miRNA-mRNA network showed that circRNAs might act as miRNA sponges to regulate osteoblast differentiation.
By presenting a better understanding of the molecular mechanisms of genes and circRNAs in simulated microgravity, the present study will provide a novel view of circRNAs in the regulation of osteogenic differentiation and bone formation.
Cao Z
，Zhang Y
，Wei S
，Zhang X
，Guo Y
，Han B
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A comprehensive evaluation of skin aging-related circular RNA expression profiles.
Circular RNAs (circRNAs) have been shown to play important regulatory roles in a range of both pathological and physiological contexts, but their functions in the context of skin aging remain to be clarified. In the present study, we therefore, profiled circRNA expression profiles in four pairs of aged and non-aged skin samples to identify identifying differentially expressed circRNAs that may offer clinical value as biomarkers of the skin aging process.
We utilized an RNA-seq to profile the levels of circRNAs in eyelid tissue samples, with qRT-PCR being used to confirm these RNA-seq results, and with bioinformatics approaches being used to predict downstream target miRNAs for differentially expressed circRNAs.
In total, we identified 571 circRNAs with 348 and 223 circRNAs being up and downregulated that were differentially expressed in aged skin samples compared to young skin samples. The top 10 upregulated circRNAs in aged skin sample were hsa_circ_0123543, hsa_circ_0057742, hsa_circ_0088179, hsa_circ_0132428, hsa_circ_0094423, hsa_circ_0008166, hsa_circ_0138184, hsa_circ_0135743, hsa_circ_0114119, and hsa_circ_0131421. The top 10 reduced circRNAs were hsa_circ_0101479, hsa_circ_0003650, hsa_circ_0004249, hsa_circ_0030345, hsa_circ_0047367, hsa_circ_0055629, hsa_circ_0062955, hsa_circ_0005305, hsa_circ_0001627, and hsa_circ_0008531. Functional enrichment analyses revealed the potential functionality of these differentially expressed circRNAs. The top 3 enriched gene ontology (GO) terms of the host genes of differentially expressed circRNAs are regulation of GTPase activity, positive regulation of GTPase activity and autophagy. The top 3 enriched KEGG pathway ID are Lysine degradation, Fatty acid degradation and Inositol phosphate metabolism. The top 3 enriched reactome pathway ID are RAB GEFs exchange GTP for GDP on RABs, Regulation of TP53 Degradation and Regulation of TP53 Expression and Degradation. Six circRNAs were selected for qRT-PCR verification, of which 5 verification results were consistent with the sequencing results. Moreover, targeted miRNAs, such as hsa-miR-588, hsa-miR-612, hsa-miR-4487, hsa-miR-149-5p, hsa-miR-494-5p were predicted for circRna-miRna interaction networks.
Overall, these results offer new insights into circRNA expression profiles, potentially highlighting future avenues for research regarding the roles of these circRNAs in the context of skin aging.
Wang L
，Si X
，Chen S
，Wang X
，Yang D
，Yang H
，He C
... -
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ResearchGate
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钛学术
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