tRF-1:30对高糖诱导的肾小管上皮细胞炎性因子表达的影响

doi:10.11958/20240046

天津医药 ›› 2024, Vol. 52 ›› Issue (6): 561-566.doi: 10.11958/20240046

• 细胞与分子生物学 • 下一篇

tRF-1:30对高糖诱导的肾小管上皮细胞炎性因子表达的影响

夏雨薇¹(), 乔云阳¹, 刘雪薇¹, 施会敏¹, 曲高婷¹, 张爱青², 甘卫华¹^,^△()

1 南京医科大学第二附属医院儿科（邮编210003）
2 南京医科大学第四附属医院儿科

收稿日期:2024-01-08 修回日期:2024-02-01 出版日期:2024-06-15 发布日期:2024-06-06
通讯作者: ^△E-mail：weihuagan@njmu.edu.cn
作者简介:夏雨薇（2000），女，硕士在读，主要从事小儿肾脏疾病诊治及机制方面研究。E-mail：13218006127@163.com
基金资助:
江苏省医学会儿科医学科研专项基金项目(SYH-32034-0073);江苏省医学会儿科医学科研专项基金项目(SYH-32034-0085);南京市卫生科技发展专项资金项目(YKK23209);南京市卫生科技发展专项资金项目(YKK23288);南京市卫生科技发展专项资金项目(YKK23289)

Effect of tRF-1:30 on the expression of inflammatory factors in renal tubular epithelial cells induced by high glucose

XIA Yuwei¹(), QIAO Yunyang¹, LIU Xuewei¹, SHI Huimin¹, QU Gaoting¹, ZHANG Aiqing², GAN Weihua¹^,^△()

1 Department of Pediatric Nephrology, the Second Affiliated Hospital of Nanjing Medical University, Nanjing 210003, China
2 Department of Pediatric Nephrology, the Fourth Affiliated Hospital of Nanjing Medical University

Received:2024-01-08 Revised:2024-02-01 Published:2024-06-15 Online:2024-06-06
Contact: ^△E-mail: weihuagan@njmu.edu.cn

夏雨薇, 乔云阳, 刘雪薇, 施会敏, 曲高婷, 张爱青, 甘卫华. tRF-1:30对高糖诱导的肾小管上皮细胞炎性因子表达的影响[J]. 天津医药, 2024, 52(6): 561-566.

XIA Yuwei, QIAO Yunyang, LIU Xuewei, SHI Huimin, QU Gaoting, ZHANG Aiqing, GAN Weihua. Effect of tRF-1:30 on the expression of inflammatory factors in renal tubular epithelial cells induced by high glucose[J]. Tianjin Medical Journal, 2024, 52(6): 561-566.

摘要/Abstract

摘要：

目的探讨tRF-1:30（tRF-1:30-Gln-CTG-4）对高糖（HG）诱导的肾小管上皮细胞（RTECs）中炎性因子表达的影响及分子机制。方法将小鼠RTECs分为Control组、HG组、HG+tRF-1:30 mimic组、HG+tRF-1:30 NC组、HG+si-IKZF2组（IKAROS家族锌指2，tRF-1:30抑制剂）、HG+si-NC组。实时荧光定量PCR检测tRF-1:30、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、单核细胞趋化蛋白-1（MCP-1）和IKZF2 mRNA的水平。酶联免疫吸附试验检测炎性因子水平，Western blot检测IKZF2蛋白表达水平，双萤光素酶报告实验验证tRF-1:30和IKZF2的关系。结果在HG诱导的RTECs中炎性因子的表达水平显著升高，而tRF-1:30表达水平显著降低。过表达tRF-1:30显著降低HG诱导的RTECs中炎性因子的表达水平。IKZF2在HG诱导的RTECs中显著高表达，进一步敲低IKZF2可抑制炎性因子的释放，而过表达tRF-1:30后IKZF2的表达水平下调。双萤光素酶报告实验进一步验证tRF-1:30与IKZF2可能存在靶向关系。结论过表达tRF-1:30可能通过负向调控IKZF2的表达进而抑制HG诱导的RTECs炎性因子的释放。

关键词: 糖尿病肾病, 端粒重复序列结合蛋白质1, tRF-1:30-Gln-CTG-4, 肾小管上皮细胞, 炎性因子, IKAROS家族锌指2

Abstract:

Objective To investigate the effect and molecular mechanism of tRF-1:30-Gln-CTG-4 (tRF-1:30) on the expression of inflammatory factors in high glucose (HG)-induced renal tubular epithelial cells (RTECs). Methods RTECs were divided into the control group, the HG group, the HG+tRF-1:30 mimic group, the HG+tRF-1:30 negative control (NC) group, the HG+si-IKZF2 group and the HG+si-NC group. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of tRF-1:30, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and IKAROS family zinc finger protein 2 (IKZF2). Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of TNF-α, IL-6 and MCP-1. Protein expression of IKZF2 was detected by Western blot assay. Dual-luciferase reporter assay was used to detect the targeting relationship between tRF-1:30 and IKZF2. Results The expression levels of inflammatory factors were elevated in HG-induced RTECs, and the expression level of tRF-1:30 was decreased (P<0.05). Overexpression of tRF-1:30 significantly decreased expression levels of inflammatory factors in HG-induced RTECs (P<0.05), and the expression level of IKZF2 was significantly increased (P<0.05). Further knockdown of IKZF2 can inhibit the release of inflammatory factors, and the expression level of IKZF2 was down-regulated after overexpression of tRF-1:30. Double luciferase reporting experiment further verified the possible targeting relationship between tRF-1:30 and IKZF2. Conclusion Overexpression of tRF-1:30 inhibits the expression of inflammatory factors in HG-induced RTECs by target binding and negatively regulating the expression of IKZF2.

Key words: diabetic nephropathies, telomeric repeat binding protein 1, tRF-1:30-Gln-CTG-4, renal tubular epithelial cells, inflammatory factors, IKAROS family zinc finger protein 2

中图分类号:

R587.2

图/表 11

参考文献 21

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基因名称	引物序列（5′→3′）	产物大小/bp
TNF-α	上游：CCTTATCTACTCCCAGGTTCTC	109
	下游：GAGGCTGACTTTCTCCTGGTATG	109
IL-6	上游：CTGCAAGAGACTTCCATCCAG	131
	下游：AGTGGTATAGACAGGTCTGTTGG	131
MCP-1	上游：TAAAAACCTGGATCGGAACCAAA	115
	下游：GCATTAGCTTCAGATTTACGGGT	115
IKZF2	上游：AAGGGGAACACGCCAATATGG	135
	下游：GCTGCCTGTCACACTCTTCA	135
β-actin	上游：CATCCGTAAAGACCTCTATGCCAAC	171
	下游：ATGGAGCCACCGATCCACA	171
tRF-1:30	上游：GGTTCCATGGTGTAATGGTGAGCACTCTGG	220
	下游：ACGCTTCACGAATTTGCGTGTC	220
U6	上游：CTCGCTTCGGCAGCACATATACT	93
	下游：ACGCTTCACGAATTTGCGTGTC	93

基因名称	引物序列（5′→3′）	产物大小/bp
TNF-α	上游：CCTTATCTACTCCCAGGTTCTC	109
	下游：GAGGCTGACTTTCTCCTGGTATG	109
IL-6	上游：CTGCAAGAGACTTCCATCCAG	131
	下游：AGTGGTATAGACAGGTCTGTTGG	131
MCP-1	上游：TAAAAACCTGGATCGGAACCAAA	115
	下游：GCATTAGCTTCAGATTTACGGGT	115
IKZF2	上游：AAGGGGAACACGCCAATATGG	135
	下游：GCTGCCTGTCACACTCTTCA	135
β-actin	上游：CATCCGTAAAGACCTCTATGCCAAC	171
	下游：ATGGAGCCACCGATCCACA	171
tRF-1:30	上游：GGTTCCATGGTGTAATGGTGAGCACTCTGG	220
	下游：ACGCTTCACGAATTTGCGTGTC	220
U6	上游：CTCGCTTCGGCAGCACATATACT	93
	下游：ACGCTTCACGAATTTGCGTGTC	93

组别	mRNA
组别	TNF-α		IL-6		MCP-1		tRF-1:30
Control组	1.01±0.09		1.00±0.14		0.99±0.08		1.02±0.08
HG组	2.47±0.29		2.68±0.35		2.01±0.28		0.27±0.06
t	8.228^＊＊		7.690^＊＊		6.126^＊＊		13.300^＊＊
组别		蛋白/（ng/L）
组别		TNF-α		IL-6		MCP-1
Control组		70.33±8.67		42.33±5.20		30.67±5.16
HG组		202.50±26.47		165.50±20.42		65.17±4.73
t		8.224^＊＊		10.120^＊＊		8.538^＊＊

组别	mRNA
组别	TNF-α		IL-6		MCP-1		tRF-1:30
Control组	1.01±0.09		1.00±0.14		0.99±0.08		1.02±0.08
HG组	2.47±0.29		2.68±0.35		2.01±0.28		0.27±0.06
t	8.228^＊＊		7.690^＊＊		6.126^＊＊		13.300^＊＊
组别		蛋白/（ng/L）
组别		TNF-α		IL-6		MCP-1
Control组		70.33±8.67		42.33±5.20		30.67±5.16
HG组		202.50±26.47		165.50±20.42		65.17±4.73
t		8.224^＊＊		10.120^＊＊		8.538^＊＊

组别	mRNA				蛋白/（ng/L）
组别	TNF-α	IL-6	MCP-1	tRF-1:30	TNF-α	IL-6	MCP-1
Control组	1.00±0.04	0.99±0.11	0.97±0.06	1.02±0.27	70.33±4.04	42.53±6.15	32.30±4.30
HG组	1.97±0.17^a	1.65±0.19^a	1.54±0.09^a	0.27±0.06^a	203.70±23.16^a	153.90±15.12^a	58.86±6.40^a
HG+tRF-1:30 NC组	1.95±0.13^a	1.69±0.07^a	1.56±0.18^a	0.28±0.10^a	205.00±23.84^a	174.13±6.97^a	64.10±5.37^a
HG+tRF-1:30 mimic组	1.10±0.11^bc	1.09±0.13^bc	1.21±0.03^bc	1 294.00±233.50^bc	93.67±7.57^bc	63.57±13.46^bc	34.73±8.18^bc
F	54.470^＊＊	23.210^＊＊	20.860^＊＊	92.110^＊＊	43.450^＊＊	99.280^＊＊	12.400^＊＊

tRF-1:30对高糖诱导的肾小管上皮细胞炎性因子表达的影响

Effect of tRF-1:30 on the expression of inflammatory factors in renal tubular epithelial cells induced by high glucose

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组别	IKZF2蛋白	IKZF2 mRNA
Control组	1.00±0.05	1.00±0.09
HG组	1.45±0.01^a	1.53±0.06^a
HG+si-NC组	1.41±0.02^a	1.53±0.11^a
HG+si-IKZF2组	0.65±0.11^bc	0.19±0.03^bc
F	376.400^＊＊	191.400^＊＊

组别	IKZF2 mRNA	IKZF2蛋白
Control组	1.02±0.02	0.99±0.04
HG组	2.23±0.29^a	1.34±0.12^a
HG+tRF-1:30 NC组	2.06±0.31	1.53±0.07
HG+tRF-1:30 mimic组	1.54±0.11^bc	0.73±0.04^bc
F	18.910^＊＊	66.600^＊＊

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