Highly Expressed lncRNA GAS5 in the Serum of Children with Mycoplasma pneumoniae Pneumonia and Its Effect on LAMPs-Induced Apoptosis and Inflammation

Abstract

The aim of the study was to explore the serum expression of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in Mycoplasma pneumoniae pneumonia (MPP) and its effect on lipid-associated membrane proteins (LAMPs)-induced apoptosis and inflammation. Totally, 56 children with MPP (MPP group) and 56 healthy children (NC group) were enrolled. lncRNA GAS5 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Serum levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were detected using ELISA, and the high mobility family protein B1 (HMGBl) was detected by qRT-PCR. The methylated binding protein 2 (MECP2) was inhibited by gene silencing, and the expression of MECP2, TNF-α, IL-6, HMGBl, p-p65, and p-IκBα was measured. lncRNA GAS5 and TNF-α, IL-6, and HMGBl in the peripheral blood of the MPP group were positively correlated (P < 0.05). The expression of TNF-α, IL-6, HMGBl, and lncRNA GAS5 showed a positive correlation with that of LAMPs. The GAS5-siRNA group showed an increased cell survival rate compared with the scrambled-RNAi group (P < 0.05) while showing decreased apoptosis and cell death rates (P < 0.05). In addition, the expression of IL-6, TNF-α, HMGBl, p-p65, and p-IκBα was significantly reduced (P < 0.05). lncRNA GAS5 is highly expressed in the serum of children with MPP and inhibits LAMPs-induced apoptosis and alveolar macrophage inflammation.

Figure 1

Comparison of lncRNA GAS5 and cytokine expression in children with MPP (mean ± SD, n = 56). (a) TNF-α; (b) IL-6; (c) HMGB1; and (d) lncRNA GAS5. Compared with the NC group, ^∗∗∗P < 0.001. lncRNA GAS5: long noncoding RNA growth arrest-specific transcript 5; MPP: Mycoplasma pneumoniae pneumonia; TNF-α: tumor necrosis factor α; IL-6: interleukin-6; HMGB1: mobility family protein B1.

Figure 2

Changes in the expression of inflammatory factors after LAMPs stimulation in MH-S cells (mean ± SD, the experiment was repeated three times). (a) TNF-α; (b) IL-6; (c) HMGBl; and (d) lncRNA GAS5. Compared with the NC group at the same time point, ^#P < 0.05, ^###P < 0.001. LAMPs: lipid-associated membrane proteins; TNF-α: tumor necrosis factor α; IL-6: interleukin-6; HMGB1: mobility family protein B1; lncRNA GAS5: long noncoding RNA growth arrest-specific transcript 5.

Figure 3

Validation of the lncRNA GAS5 interference expression vector and its effect on the survival rate of LAMPs-induced MH-S cells (mean ± SD, the experiment was repeated three times). (a) Validation of the lncRNA GAS5 interference expression vector; (b) effect of lncRNA GAS5-siRNA inhibition on the survival rate of LAMPs-induced MH-S cells. Compared with the NC group, ^∗∗∗P < 0.001; compared with the LAMPs group, ^###P < 0.001; compared with the scrambled-RNAi group, ^&&&P < 0.001. lncRNA GAS5: long noncoding RNA growth arrest-specific transcript 5; LAMPs: lipid-associated membrane proteins.

Figure 4

Effect of lncRNA GAS5-siRNA on LAMPs-induced apoptosis in MH-S cells (mean ± SD, the experiment was repeated three times). (a) Flow cytograms of MH-S cell apoptosis and (b) bar graphs showing the comparison of apoptosis rates. Compared with the control group, ^∗∗∗P < 0.001; compared with the LAMPs group, ^###P < 0.001; compared with the scrambled-RNAi group, ^&&&P < 0.001. lncRNA GAS5: long noncoding RNA growth arrest-specific transcript 5; LAMPs: lipid-associated membrane proteins.

Figure 5

lncRNA GAS5-siRNA inhibits LAMPs-induced inflammatory responses in MH-S cells (mean ± SD, the experiment was repeated three times). (a) TNF-α; (b) IL-6; and (c) HMGBl. Compared with the control group, ^∗∗∗P < 0.001; compared with the LAMPs group, ^###P < 0.001; compared with the scrambled-RNAi group, ^&&&P < 0.001. lncRNA GAS5: long noncoding RNA growth arrest-specific transcript 5; LAMPs: lipid-associated membrane proteins; TNF-α: tumor necrosis factor α; IL-6: interleukin-6; HMGB1: mobility family protein B1.

Figure 6

lncRNA GAS5-siRNA inhibits LAMPs-induced protein expression of p-p65 and p-IκBα in MH-S cells (mean ± SD, the experiment was repeated three times). (a) Western blots of p-p65 and p-IκBα protein expression in MH-S cells; (b) bar graphs of p-p65 and p-IκBα protein expression. (1) Control group; (2) LAMPs group; (3) scrambled-RNAi group. Compared with the control group, ^∗∗∗P < 0.001; compared with the LAMPs group, ^###P < 0.001. lncRNA GAS5: long noncoding RNA growth arrest-specific transcript 5; LAMPs: lipid-associated membrane proteins.