Genome‑wide analysis and prediction of functional long noncoding RNAs in osteoblast differentiation under simulated microgravity

Abstract

Long noncoding RNAs (lncRNAs) have been regarded as important regulators in numerous biological processes during cell development. However, the holistic lncRNA expression pattern and potential functions during osteoblast differentiation under simulated microgravity remain unknown. In the present study, a high throughput microarray assay was performed to detect lncRNA and mRNA expression profiles during MC3TC‑E1 pre‑osteoblast cell osteo‑differentiation under simulated microgravity. The expression of 857 lncRNAs and 2,264 mRNAs was signi-ficantly altered when MC3T3‑E1 cells were exposed to simulated microgravity. A relatively consistent distribution pattern on the chromosome and a co‑expression network were observed between the differentially‑expressed lncRNAs and mRNAs. Genomic context analysis further identified 132 differentially‑expressed lncRNAs and nearby coding gene pairs. Subsequently, 3 lncRNAs were screened out for their possible function in osteoblast differentiation, based on their co‑expression association and potential cis‑acting regulatory pattern with the deregulated mRNAs. The present study aimed to provide a comprehensive understanding of and a foundation for future studies into lncRNA function in mechanical signal‑mediated osteoblast differentiation.

Figure 1.

Expression profile of lncRNAs and mRNAs in MC3T3-E1 cells exposed to simulated microgravity. (A) Osteogenic differentiation was assessed by reverse transcription-quantitative polymerase chain reaction analysis of osteoblast marker genes and activity analysis of ALP. n=3. *P<0.05, **P<0.01. (B) Expression profiles of ncRNAs in MC3T3-E1 cells when exposed to simulated microgravity. Green represents downregulated ncRNAs, and red represents upregulated ncRNAs. n=3. (C) The deregulated lncRNAs with a length exceeding 200 nucleotides were screened out and exhibited on a scatter plot. (D) Expression profile of mRNAs in MC3T3-E1 cells when exposed to simulated microgravity. n=3. lncRNA, long noncoding RNA; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; OSX, transcription factor Sp7.

Figure 2.

Chromosome location, GO and pathway analyses of deregulated lncRNAs and mRNAs. (A) Location distributions of deregulated lncRNAs and mRNAs on chromosomes. The co-expressed genes of deregulated lncRNAs were submitted for GO and pathway enrichment analyses. (B) The top 10 enriched GO terms (biological process) and (C) signal pathways are presented in bar diagrams. The value of -log₁₀ (P-value) was calculated to reflect the significance of enrichment. GO, Gene Ontology; lncRNA, long noncoding RNA.

Figure 3.

GO and pathway analyses of genes located near the deregulated long noncoding RNAs. (A) The top 10 enriched GO terms (biological process). (B) The top 10 enriched signal pathways. GO, Gene Ontology.

Figure 4.

Association between target lncRNAs and co-expressed or nearby genes. (A) The co-expression network was rebuilt between the target lncRNAs and their co-expressed deregulated mRNAs. Red nodes represent upregulated genes and green nodes represent downregulated genes. A solid line between two nodes represents a positive association while a dotted line represents a negative association. (B) The positional association on the chromosome of the target lncRNAs and their adjacent protein-coding genes. (C) Reverse transcription-quantitative polymerase chain reaction analysis of lncRNAs and their nearby genes in MC3T3-E1 cells exposed to simulated microgravity for 48 h. n=3. *P<0.05, **P<0.01. lncRNA, long noncoding RNA; Ptbp2, polypyrimidine tract-binding protein 2; Ext1, exostosin-1; Tnpo1, transportin-1; CON, control; MG, microgravity.