The effect of LncRNA SNHG14 on high glucose induced podocyte injury by targeting miR-30a-5p

doi:10.11958/20251830

Abstract

Abstract:

Objective To investigate the effect of long non-coding RNA small nucleolar RNA host gene 14 (LncRNA SNHG14) on high glucose-induced podocyte injury by targeting microRNA-30a-5p (miR-30a-5p). Methods Podocytes were cultured in vitro and were divided into the following groups: the standard glucose (NG) group, the high glucose (HG) group, the si-NC+HG group, the si-SNHG14+HG group, the miR-NC+HG group, the miR-30a-5p mimics+HG group, the si-SNHG14+inhibitor NC+HG group and the si-SNHG14+miR-30a-5p inhibitor+HG group. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed to detect expression levels of LncRNA SNHG14 and miR-30a-5p. Flow cytometry was used to determine cell apoptosis. Enzyme-linked immunosorbent assay (ELISA) was applied to measure levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1β. Xanthine oxidase method, ammonium molybdate colorimetry and thiobarbituric acid method were respectively used to detect superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA). Dual-luciferase reporter gene was conducted to verify the targeting relationship between LncRNA SNHG14 and miR-30a-5p. Western blot assay was performed to detect expression levels of apoptosis-related proteins. Results Compared with the NG group, the HG group exhibited increased expression levels of LncRNA SNHG14, cell apoptosis rate, as well as levels of TNF-α, IL-6, IL-1β and MDA, whereas the expression level of miR-30a-5p and levels of SOD and CAT were decreased (P<0.05). Compared with the HG group and the si-NC+HG group, the si-SNHG14+HG group exhibited decreased expression levels of LncRNA SNHG14, apoptosis rate, levels of TNF-α, IL-6, IL-1β and MDA, as well as expression levels of Bax and cleaved caspase-3 proteins, while the expression level of miR-30a-5p, levels of SOD and CAT and the expression level of Bcl-2 protein were increased (P<0.05). Compared with the HG group and the miR-NC+HG group, the miR-30a-5p mimics+HG group showed no significant difference in the expression level of LncRNA SNHG14 (P>0.05). Meanwhile, the expression level of miR-30a-5p, levels of SOD and CAT, and expression level of Bcl-2 protein were increased, whereas the cell apoptosis rate, levels of TNF-α, IL-6, IL-1β and MDA, as well as expression levels of Bax and cleaved caspase-3 proteins were decreased (P<0.05). Compared with the si-SNHG14+HG group and the si-SNHG14+inhibitor NC+HG group, the si-SNHG14+miR-30a-5p inhibitor+HG group showed no significant difference in the expression level of LncRNA SNHG14 (P>0.05), meanwhile, the expression level of miR-30a-5p, levels of SOD and CAT, and expression level of Bcl-2 protein were reduced, whereas the cell apoptosis rate, levels of TNF-α, IL-6, IL-1β and MDA, as well as expression levels of Bax and cleaved caspase-3 proteins were increased (P<0.05). The dual-luciferase reporter gene assay confirmed that LncRNA SNHG14 targeted and negatively regulated miR-30a-5p. Conclusion The inhibition of LncRNA SNHG14 can target miR-30a-5p to alleviate high glucose induced podocyte injury.

Key words: diabetic nephropathies, podocytes, apoptosis, inflammation, LncRNA SNHG14, miR-30a-5p, high glucose

CLC Number:

R341.6

Figures/Tables 10

References 23

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基因名称	引物序列（5′→3′）	产物大小/bp
miR-30a-5p	上游：AAAGTGGAATTTGTAGAGA 下游：CAGGTACAGACGGATATCTTGC	83
U6	上游：GGTCGGGCAGGAAAGAGGGC 下游：GCTAATCTTCTCTGTATCGTTCC	95
LncRNA SNHG14	上游：AGCTAGAGCTATAGCTAGC 下游：TCGAGATACCGATAGCTAA	245
GAPDH	上游：CGCGATATAGATAGATCGTA 下游：CGCGATAGGCTATAGCTAGA	218

基因名称	引物序列（5′→3′）	产物大小/bp
miR-30a-5p	上游：AAAGTGGAATTTGTAGAGA 下游：CAGGTACAGACGGATATCTTGC	83
U6	上游：GGTCGGGCAGGAAAGAGGGC 下游：GCTAATCTTCTCTGTATCGTTCC	95
LncRNA SNHG14	上游：AGCTAGAGCTATAGCTAGC 下游：TCGAGATACCGATAGCTAA	245
GAPDH	上游：CGCGATATAGATAGATCGTA 下游：CGCGATAGGCTATAGCTAGA	218

组别	LncRNA SNHG14	miR-30a-5p	细胞凋亡率/%
NG组	1.02±0.12	1.01±0.11	2.05±0.23
HG组	2.56±0.29	0.34±0.04	35.17±3.91
t	12.019^＊＊	14.021^＊＊	20.713^＊＊
组别	TNF-α/（ng/L）	IL-6/（ng/L）	IL-1β/（ng/L）
NG组	39.25±4.12	21.37±2.26	13.59±1.58
HG组	106.15±11.23	90.20±9.35	59.83±6.22
t	13.699^＊＊	17.527^＊＊	17.649^＊＊
组别	SOD/（U/mL）	CAT/（U/mL）	MDA/（mol/L）
NG组	36.52±3.98	27.36±2.98	5.22±0.58
HG组	7.13±0.81	8.01±0.85	28.76±3.07^a
t	17.725^＊＊	15.295^＊＊	18.456^＊＊

组别	LncRNA SNHG14	miR-30a-5p	细胞凋亡率/%
NG组	1.02±0.12	1.01±0.11	2.05±0.23
HG组	2.56±0.29	0.34±0.04	35.17±3.91
t	12.019^＊＊	14.021^＊＊	20.713^＊＊
组别	TNF-α/（ng/L）	IL-6/（ng/L）	IL-1β/（ng/L）
NG组	39.25±4.12	21.37±2.26	13.59±1.58
HG组	106.15±11.23	90.20±9.35	59.83±6.22
t	13.699^＊＊	17.527^＊＊	17.649^＊＊
组别	SOD/（U/mL）	CAT/（U/mL）	MDA/（mol/L）
NG组	36.52±3.98	27.36±2.98	5.22±0.58
HG组	7.13±0.81	8.01±0.85	28.76±3.07^a
t	17.725^＊＊	15.295^＊＊	18.456^＊＊

组别	LncRNA SNHG14	miR-30a-5p	细胞凋亡率/%
HG组	2.56±0.29	0.34±0.04	35.17±3.91
si-NC+HG组	2.49±0.26	0.35±0.04	35.33±4.02
si-SNHG14+HG组	1.23±0.14^ab	0.89±0.11^ab	12.08±1.37^ab
miR-NC+HG组	2.53±0.27	0.33±0.04	35.78±4.11
miR-30a-5p mimics+ HG组	2.52±0.26	0.78±0.08^ac	14.23±1.56^ac
si-SNHG14+ inhibitor NC+HG组	1.26±0.15	0.87±0.10	12.35±1.42
si-SNHG14+miR-30a-5p inhibitor+HG组	1.21±0.14	0.56±0.10^de	15.31±1.63^de
F	56.647^＊＊	63.547^＊＊	101.610^＊＊