Microarray analysis reveals a potential role of LncRNAs expression in cardiac cell proliferation

Abstract

Background: Long non-coding RNAs (LncRNAs) have been identified to play important roles in epigenetic processes that underpin organogenesis. However, the role of LncRNAs in the regulation of transition from fetal to adult life of human heart has not been evaluated.

Methods: Immunofiuorescent staining was used to determine the extent of cardiac cell proliferation. Human LncRNA microarrays were applied to define gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Pathway analysis was performed to predict the function of differentially expressed mRNAs (DEM). DEM related to cell proliferation were selected to construct a lncRNA-mRNA co-expression network. Eight lncRNAs were confirmed by quantificational real-time polymerase chain reaction (n = 6).

Results: Cardiac cell proliferation was significant in the fetal heart. Two thousand six hundred six lncRNAs and 3079 mRNAs were found to be differentially expressed. Cell cycle was the most enriched pathway in down-regulated genes in the adult heart. Eight lncRNAs (RP11-119 F7.5, AX747860, HBBP1, LINC00304, TPTE2P6, AC034193.5, XLOC_006934 and AL833346) were predicted to play a central role in cardiac cell proliferation.

Conclusions: We discovered a profile of lncRNAs differentially expressed between the human fetal and adult heart. Several meaningful lncRNAs involved in cardiac cell proliferation were disclosed.

Keywords: Cardiac cell proliferation; Human heart; Long non-coding RNA; Microarray.

Fig. 1

Extent of cardiomyocyte proliferation in fetal and adult hearts. Immunofluorescent staining shows the proliferating Ki-67+ cardiac cell in fetal (a) and adult heart (b) respectively. Scale bar: 50 μm. The yellow lines indicate the position within the image of projections given in the lower and right. Percentage of Ki-67 positive cardiomyocyte in all positive cells was shown respectively (c). Data are expressed as the mean ± standard deviation (SD) of three independent experiments (*P < 0.01)

Fig. 2

Differential expression of lncRNAs between fetal and adult heart. Hierarchical clustering analysis shows differential expression of LncRNAs (FC ≥ 5) using a heat map (a). ‘Red’ indicates higher expression, whereas ‘blue’ indicates lower expression. The bar code represents the color scale of log2 transformed values. The scatterplot (b) is a visualization used for assessing the variation between the chips. The X and Y axes in the scatterplot represent the normalized signal values of each group (log2 scaled). The green lines represent FC (FC = 2.0) lines

Fig. 3

Pathway analysis. The pathways that exhibited significant differences between fetal and adult hearts are listed. A shows the down-regulated genes pathways and B the up-regulated genes pathways in adult group. Higher -lg P-value indicates higher significance level

Fig. 4

Establishment of lncRNA-mRNA co-expression network. Nodes represent genes and an edge is used to connect two correlated genes. Solid lines represent positive correlation and dotted lines negative correlation. Blue nodes represent up-regulated genes and red nodes represent down-regulated genes in adult heart. Square nodes represent lncRNAs, while circular nodes represent mRNAs. The larger the node is, the higher the degree would be

Fig. 5

qRT-PCR validation of differential expressions of LncRNAs. a Eight LncRNAs confirmed by qRT-PCR show to have significant changes between fetal and adult groups. Data are expressed as the mean ± standard deviation (SD) of three independent experiments (*P < 0.01). b qRT-PCR patterns of seven LncRNAs except for LINC00304 are completely consistent with those of microarray data. Data of gene chip group are expressed as the mean ± standard deviation (SD) (n = 4, normalized data). The Y-axis represents FCs (A/F)