LncRNA SNHG14靶向miR-30a-5p对高糖诱导的足细胞损伤的影响

doi:10.11958/20251830

摘要/Abstract

摘要：

目的探究长链非编码RNA小核仁RNA宿主基因14（LncRNA SNHG14）靶向调控微小RNA-30a-5p（miR-30a-5p）对高糖诱导的足细胞损伤的影响。方法体外培养足细胞，并将其分为标准糖（NG）组、高糖（HG）组、si-NC+HG组、si-SNHG14+HG组、miR-NC+HG组、miR-30a-5p mimics+HG组、si-SNHG14+inhibitor NC+HG组、si-SNHG14+miR-30a-5p inhibitor+HG组。实时荧光定量聚合酶链式反应（RT-qPCR）检测LncRNA SNHG14、miR-30a-5p表达；流式细胞术检测细胞凋亡；酶联免疫吸附试验（ELISA）检测肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-1β水平；分别采用黄嘌呤氧化法、钼酸铵比色法、硫代巴比妥酸法检测超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、丙二醛（MDA）水平；双萤光素酶报告基因检测LncRNA SNHG14与miR-30a-5p的靶向关系；Western blot检测凋亡相关蛋白表达。结果与NG组相比，HG组LncRNA SNHG14表达、细胞凋亡率、TNF-α、IL-6、IL-1β、MDA水平升高，miR-30a-5p表达、SOD、CAT水平降低（P<0.05）。与HG组、si-NC+HG组相比，si-SNHG14+HG组LncRNA SNHG14表达、细胞凋亡率、TNF-α、IL-6、IL-1β、MDA水平、Bcl-2相关X蛋白（Bax）、活化型胱天蛋白酶-3（cleaved caspase-3）蛋白表达水平降低，miR-30a-5p表达、SOD、CAT水平、B细胞淋巴瘤-2蛋白（Bcl-2）表达水平升高（P<0.05）；与HG组、miR-NC+HG组相比，miR-30a-5p mimics+HG组LncRNA SNHG14表达无变化（P>0.05），miR-30a-5p表达、SOD、CAT水平、Bcl-2蛋白表达升高，细胞凋亡率、TNF-α、IL-6、IL-1β、MDA水平、Bax、cleaved caspase-3蛋白表达水平降低（P<0.05）；与si-SNHG14+HG组、si-SNHG14+inhibitor NC+HG组相比，si-SNHG14+miR-30a-5p inhibitor+HG组LncRNA SNHG14表达无变化（P>0.05），miR-30a-5p表达、SOD、CAT水平、Bcl-2蛋白表达水平降低，细胞凋亡率、TNF-α、IL-6、IL-1β、MDA水平、Bax、cleaved caspase-3蛋白表达水平升高（P<0.05）。双萤光素酶报告基因实验证实LncRNA SNHG14靶向负调控miR-30a-5p。结论抑制LncRNA SNHG14可靶向miR-30a-5p减轻高糖诱导的足细胞损伤。

关键词: 糖尿病肾病, 足细胞, 细胞凋亡, 炎症, LncRNA SNHG14, miR-30a-5p, 高糖

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

中图分类号:

R341.6

图/表 10

参考文献 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^＊＊