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

doi:10.11958/20240046

Abstract

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

CLC Number:

R587.2

Figures/Tables 11

References 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^＊＊