microRNA-98 protects sepsis mice from cardiac dysfunction, liver and lung injury by negatively regulating HMGA2 through inhibiting NF-κB signaling pathway

Abstract

Recently, MicroRNA-98 (miR-98) works as a biomarker in some diseases, such as lung cancer, Schizophrenia, and breast cancer, but there still lack of studies on the function of miR-98 during sepsis. Thus, our study is conducted to figure out the function of miR-98 for the regulation of cardiac dysfunction, liver and lung injury in sepsis mice. Cecum ligation and puncture was used to establish the sepsis mice model. Next, miR-Con and agomiR-98 were injected into the tail vein of mice 48 h after modeling. Then, expression of miR-98, HMGA2, NF-κB, inflammatory factors, apoptosis-related proteins in myocardial, liver and lung tissues of septic mice were determined. Moreover, other indices that were associated with cardiac dysfunction, liver and lung injury in septic mice were detected. Finally, bioinformatics analysis and luciferase activity assay were utilized to validate the binding site between miR-98 and HMGA2. miR-98 was poorly expressed, while HMGA2, NF-κB pathway-related proteins were highly expressed in myocardial, liver, and lung tissues of mice with sepsis. Upregulated miR-98 inhibited HMGA2, NF-κB, TNF-α, IL-6, Bcl-2 and increased IL-10, Cleaved caspase-3 and Bax expression in myocardial, liver, and lung tissues of septic mice. Upregulation of miR-98 decreased LVEDP, CTn-I, BNP, ALT, AST, TBIL, LDH, and PaCO₂ while increased +dp/dt max, -dp/dt max, pH and PaO₂ in sepsis mice. miR-98 was a direct target gene of HMGA2. Our study provides evidence that miR-98 protects sepsis mice from cardiac dysfunction, liver and lung injury by negatively mediating HMGA2 via the inhibition of the NF-κB signaling pathway.

Keywords: HMGA2; MicroRNA-98; sepsis.

Figure 1.

Downregulated miR-98, upregulated HMGA2 and NF-κB pathway-related proteins are found in liver tissues of mice with sepsis. (a) Expression of miR-98 and HMGA2 mRNA in myocardial tissue of mice in each group; (b) Immunoblots of HMGA2 and NF-κB in myocardial tissues of mice in each group; (c) HMGA2 and NF-κB protein expression in myocardial tissues of mice in each group. * P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, data was expressed as mean ± standard deviation; one-way ANOVA was used for data analysis; LSD-t test was used after pairwise comparison.

Figure 2.

Upregulated miR-98 declined LVEDP, CTn-I and BNP, and elevated +dp/dt max and-dp/dt max in mice with sepsis. (a) Comparison of LVEDP in mice of each group; (b) Comparison of +dp/dt max in mice of each group; (c) Comparison of -dp/dt max in mice of every group; (d) Comparison of CTn-I level in mice of each group; (e) Comparison of BNP level in mice of each group; * P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 3.

Upregulated miR-98 alleviated cardiac pathological injury and inhibited TNF-α, IL-6 expression and promoted IL-10 expression in septic mice. (a) The myocardial tissues of mice was observed by HE staining (× 400, scale bar: 25 μm); (b) the ultrastructure of cardiomyocytes of mice was observed by transmission electron microscopy (× 10 000, scale bar: 1 μm). N = 15; (c) The mRNA expression levels of inflammatory factors in myocardial tissues of each group of mice; (d) protein expression level of TNF-α in myocardial tissues of each group; (e) protein expression level of IL-6 in myocardial tissue of each group of mice; (f) IL-10 protein expression level in myocardial tissues of each group of mice; * P< 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 4.

Upregulated miR-98 inhibited apoptosis of cardiomyocytes in mice with sepsis. (a) TUNEL staining was used to observe the apoptosis of cardiomyocytes in mice of each group (× 400, scale bar = 25 μm); (b) Apoptotic index of cardiomyocytes in mice of each group; (c) Immunoblots of Cleaved caspase-3, Bax and Bcl-2 proteins in myocardial tissues of mice in each group; (d-f) Cleaved caspase-3, Bax and Bcl-2 protein expression in myocardial tissues of mice in each group. * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 5.

Downregulated miR-98, upregulated HMGA2 and NF-κB pathway-related proteins are found in liver tissues of mice with sepsis. (a) The expression of miR-98 and HMGA2 mRNA in liver tissues of mice in each group; (b) Immunoblots of HMGA2 and NF-κB protein in liver tissues of mice of every group; (c) HMGA2 and NF-κB protein expression in liver tissues of mice in each group; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 6.

Upregulated miR-98 decreased ALT, AST, TBIL and LDH in mice with sepsis. (a) ALT expression of mice in every group; (b) AST expression of mice in each group; (c) LDH expression of mice from all group; (d) TBIL expression of mice in each group; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 7.

Upregulated miR-98 inhibited TNF-α, IL-6 expression and promoted IL-10 expression and alleviated histopathological changes of liver tissues in mice of each group. (a) Liver histopathological changes in each group of mice (HE staining, × 200, scale bar = 50 μm), N = 15. (b) The mRNA expression levels of inflammatory factors in liver tissues of mice in each group; (c) Protein expression levels of TNF-α in liver tissues of mice in each group; (d) Protein expression levels of IL-6 in liver tissues of mice in each group; (e) Protein expression levels of IL-10 in liver tissue of each group of mice; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 8.

Upregulated miR-98 decreased Cleaved caspase-3 and Bax and increased Bcl-2 in liver tissues of mice with sepsis. (a) Immunoblots of Cleaved caspase-3, Bax and Bcl-2 proteins in liver tissues of mice in each group; (b) Cleaved caspase-3 protein expression in liver tissues of mice in each group; (c) Bax protein expression in liver tissues of mice in each group; (d) Bcl-2 protein expression in liver tissues of mice in each group; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 9.

Downregulated miR-98, upregulated HMGA2 and NF-κB pathway-related proteins are found in lung tissues of mice with sepsis, and upregulated miR-98 decreased PaCO₂ and increased pH and PaO₂ in mice with sepsis. (a) Expression of miR-98 and HMGA2 mRNA in lung tissues of mice in each group; (b) Immunoblots of HMGA2 and NF-κB protein in lung tissues of mice in each group; (c) HMGA2 and NF-κB protein expression in lung tissues of mice in each group; (d) Comparison of the pH levels of the mice in each group; (e) Comparison of the levels of PaO₂ in each group of mice; (f) Comparison of the levels of PaCO₂ in each group of mice; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 10.

Upregulated miR-98 inhibited TNF-α, IL-6 expression and promoted IL-10 expression and alleviated histopathological changes of lung tissues in mice of each group. (a) HE staining was used to observe the lung tissues of mice (× 400, scale bar = 25 μm); (b) W/D ratio of lung tissues of mice in each group; (c) Pathological scores of lung injury of mice in each group; (d) The mRNA expression levels of inflammatory factors in lung tissues of mice in each group; (e) Protein expression levels of TNF-α in lung tissues of mice in each group; (f) protein expression levels of IL-6 in lung tissues of mice in each group; (g) IL-10 protein expression levels in lung tissue of mice in each group; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 11.

Upregulated miR-98 decreased Cleaved caspase-3 and Bax and increased Bcl-2 in mice with sepsis. (a) Immunoblots of Cleaved caspase-3, Bax and Bcl-2 protein in lung tissues of mice in each group; (b) Cleaved caspase-3 protein expression in lung tissue of mice in each group; (c) Bax protein expression in lung tissues of mice in each group; (d) Bcl-2 protein expression in lung tissues of mice in each group; * P < 0.05 vs. the sham group; # P< 0.05 vs. the CLP group. N = 15, the data was expressed in the form of mean ± standard deviation; one-way ANOVA was used for data analysis. LSD-t test was used after pairwise comparison.

Figure 12.

HMGA2 is the direct target gene of miR-98. (a) Online software predicted on the binding site of miR-98 and HMGA2; (b) Luciferase activity assay verified the targeting relationship between miR-98 and HMGA2; * P < 0.05 vs. the agomiR NC group.