lncRNA-SNHG14 Promotes Atherosclerosis by Regulating ROR α Expression through Sponge miR-19a-3p

Abstract

Coronary heart disease (CHD) is the most common cardiovascular disease with high prevalence, disability, and mortality. The balance between proliferation and apoptosis of vascular smooth muscle cells (VSMCs) plays a key role in the initiation of atherosclerosis. In this study, we found a significant decrease in the expression of lncRNA-SNHG14 in atherosclerotic plaque tissues of ApoE-/- mice. Overexpression of lncRNA-SNHG14 can inhibit VSMC proliferation while promoting apoptosis. There is a potential reciprocal regulatory relationship between lncRNASNHG14 and miR-19a-3p, which inhibit each other's expression in vascular smooth muscle cells. In addition, the luciferase reporter gene analysis results showed that there was a direct interaction between miR-19a-3p and the 3'UTR of RORα. The results of qRT-PCR showed that the level of RORα mRNA was significantly increased in the aortas treated with miR-19a-3p and SNHG14 compared with that treated with miR-19a-3p alone. In conclusion, we demonstrated that lncRNA-SNHG14 regulates the apoptosis/proliferation balance of VSMCs in atherosclerosis.

Figure 1

Expression of lncRNA-SNHG14 in atherosclerotic plaques. (a) Expression of lncRNA-SNHG14 in mouse atherosclerotic plaques. WT: 5 normal C57BL/6 mice; ApoE-/-: 5 ApoE-/- knockout mice, ^∗P < 0.05. (b) Detection of lncRNA-SNHG14 expression in RAW264.7/HA-VSMC cells. Cntl-siRNA was the irrelevant sequence siRNA control transfection group, si-mlncRNA-SNHG14 was the siRNA transfection group for mouse lncRNA-SNHG14, si-hlncRNA-SNHG14 was the siRNA transfection group for human lncRNA-SNHG14, ^∗P < 0.05.

Figure 2

Detection of the role of lncRNA-SNHG14 in cell proliferation and apoptosis. (a) Cell counts of RAW264.7/HA-VSMC cells at different time points after lncRNA-SNHG14 silencing. Left: RAW264.7 cells; Right: HA-VSMC cells. The ordinate represents the relative cell number; the harvesting time of 0, 24, 48, 72 cells after transfection, in hours. (Cntl-siRNA was the irrelevant sequence siRNA control transfection group, si-mlncRNA-SNHG14 was the siRNA transfection group for mouse lncRNA-SNHG14, si-hlncRNA-SNHG14 was the siRNA transfection group for human lncRNA-SNHG14, ^∗P < 0.05). (b) Cell proliferation detection of RAW264.7/HA-VSMC cells at different time points after lncRNA-SNHG14 silencing. Left: RAW264.7 cells; Right: HA-VSMC cells. The ordinate represents the relative absorbance value; the harvesting time of 0, 24, 48, 72 cells after transfection, in hours. (Ctl-siRNA was the irrelevant sequence siRNA control transfection group, si-mlncRNA-SNHG14 was the siRNA transfection group for mouse lncRNA-SNHG14, si-hlncRNA-SNHG14 was the siRNA transfection group for human lncRNA-SNHG14, ^∗P < 0.05. (c) Detection of apoptosis in RAW264.7/HA-VSMC cells after lncRNA-SNHG14 silencing. Flow cytometry two-dimensional dot plot and Flow cytometry two-dimensional dot plot data statistical graph. The ordinate of the two-dimensional dot plot of flow cytometry data is the percentage of apoptotic cells. Cntl-siRNA was an irrelevant sequence siRNA control transfection group, and si-lncRNA-SNHG14 was a siRNA transfection group for mouse or human lncRNA-SNHG14, ^∗P < 0.05.

Figure 3

Interaction between lncRNA-SNHG14 and miR-19a-3p. (a) Targeted binding between LncRNA SNHG14 and miR-19a-3p. (b). Left: the expression result of miR-19a-3p when overexpressing LncRNASNHG14; Right: the expression result of lncRNASNHG14 when overexpressing miR-19a-3p; ^∗P < 0.05, ^∗∗P < 001. (c) Analysis results of fluorescence data after cotransfection of mimic with dual luciferase plasmid; (d) RNA pull-down validates the binding of miR-19a-3p to lncRNA SNHG14; ^∗P < 0.05, ^∗∗P < 0.01.

Figure 4

SNHG14/miR-19a-3p promotes VSMC proliferation and inhibits its apoptosis by targeting RORα. (a) Patterns of construction of wild-type and mutant dual-luciferase plasmids; the red part is the mutated base site; (b) Analysis results of fluorescence value data after cotransfection of mimic and dual-luciferase plasmids; ^∗∗P < 0.1. (c) Relative expression of RORalpha protein in cells; ^∗P < 0.05, ^∗∗P < 0.01.

Figure 5

Overexpression of SNHG14 reverses the inhibition of RORalpha mRNA (a) and protein (b) levels in ApoE-/- mouse aorta by miR-19a-3p. Note: ^∗P < 0.05 vs. miR-NC group; ^#P < 0.05 vs. miR-19a-3p group.