RNA-sequencing analysis reveals the potential contribution of lncRNAs in palmitic acid-induced insulin resistance of skeletal muscle cells

Abstract

Insulin resistance (IR) has been considered as the common pathological basis and developmental driving force for most metabolic diseases. Long noncoding RNAs (lncRNAs) have emerged as pivotal regulators in modulation of glucose and lipid metabolism. However, the comprehensive profile of lncRNAs in skeletal muscle cells under the insulin resistant status and the possible biological effects of them were not fully studied. In this research, using C2C12 myotubes as cell models in vitro, deep RNA-sequencing was performed to profile lncRNAs and mRNAs between palmitic acid-induced IR C2C12 myotubes and control ones. The results revealed that a total of 144 lncRNAs including 70 up-regulated and 74 down-regulated (|fold change| > 2, q < 0.05) were significantly differentially expressed in palmitic acid-induced insulin resistant cells. In addition, functional annotation analysis based on the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases revealed that the target genes of the differentially expressed lncRNAs were significantly enriched in fatty acid oxidation, lipid oxidation, PPAR signaling pathway, and insulin signaling pathway. Moreover, Via qPCR, most of selected lncRNAs in myotubes and db/db mice skeletal muscle showed the consistent expression trends with RNA-sequencing. Co-expression analysis also explicated the key lncRNA-mRNA interactions and pointed out a potential regulatory network of candidate lncRNA ENSMUST00000160839. In conclusion, the present study extended the skeletal muscle lncRNA database and provided novel potential regulators for future genetic and molecular studies on insulin resistance, which is helpful for prevention and treatment of the related metabolic diseases.

Keywords: Insulin resistance; LncRNA; Palmitic acid; Skeletal muscle.

Figure 1. Profile of differentially expressed lncRNAs and mRNAs between PA-treated (PA group) and control C2C12 myotubes (C group)

(A and C) Hierarchical clustering of RNA sequencing indicated lncRNA and mRNA expression profiles in C2C12 myotubes between PA-treated and control samples (three samples per group). Data were expressed as FPKM. Red: relatively high expression; Green: relatively low expression. (B and D) The scatter plot, a visualization method, was applied for assessing lncRNA and mRNAs expression variation between the two compared groups. The red dots indicated up-regulated RNA and blue dots indicated down-regulated RNA in PA-treated C2C12 myotubes (|fold change| >2, q < 0.05).

Figure 2. GO analysis and pathway analysis of differentially expressed mRNAs in control and PA-treated C2C12 myotubes

(A) Top 15 GO terms of differentially expressed mRNAs were listed. (B) Top 15 pathway terms of dysregulated mRNAs were listed.

Figure 3. qPCR verification of differentially expressed lncRNAs in PA-treated C2C12 myotubes

Differentiated C2C12 myotubes were incubated in either the presence or absence of 0.75 mM concentrations of PA in 2% BSA for 24 h (n = 6 per group). The expression levels of 12 differentially expressed lncRNAs were evaluated by qPCR in C2C12 myotubes exposed to PA. (A–F and J–L): 9 lncRNAs were up-regulated in RNA sequencing and (G–I) 3 lncRNAs were down-regulated. 2^−ΔC_T was used to show the expression levels of lncRNAs and PPIA expression was used as the internal control for lncRNA expression analysis. Values are the means ± SEM (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4. qPCR verification of differentially expressed lncRNAs in skeletal muscle tissues

Skeletal muscle tissues were isolated from male C57BLKS/J db/db mice at the age of 12 weeks (n = 8) and age-matched WT controls (n = 8). (A–E) The differentially expressed lncRNAs selected from the PA-treated C2C12 cells results were validated in skeletal muscle tissues by qPCR analysis. 2^−ΔC_T was used to show the expression levels of lncRNAs and PPIA expression was used as the internal control for lncRNA expression analysis. Values are the means ± SEM (n = 8 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5. Construction of a co-expression network between the differentially expressed lncRNAs and mRNAs

Circular and square nodes represent mRNAs and lncRNAs, respectively. Red represents up-regulation and green represents down-regulation. Furthermore, edges represent interactions between lncRNAs and mRNAs (P value< 0.05 and correlation coefficient > 0.90)