LncRNA CRNDE regulates lipopolysaccharide-induced inflammatory response and apoptosis in human alveolar epithelial cells via miR-181a-5p/SOX6 axis

doi:10.3877/cma.j.issn.2095-1221.2023.03.001

Abstract

Abstract:

Objective

To explore the effects of long non-coding RNA (lncRNAs) CRNDE on lipopolysaccharide (LPS) -induced inflammatory response and apoptosis of human alveolar epithelial cells via the microRNA-181a-5p (miR-181a-5p) /transcription factor sex-determining region Y-box 6 (SOX6) axis.

Methods

All plasmids and oligonucleotides were transfected into A549 cells and treated with LPS. Untreated A549 cells were used as the control. QRT-PCR was performed to detect the expression levels of CRNDE, miR-181a-5p and SOX6 mRNA in cells; flow cytometry and ELISA were performed to detect cell apoptosis and the levels of tumor necrosis factor-α (TNF-α) , inflammatory factors interleukin-1β (IL-1β) and IL-6; dual luciferase experiment was performed to verify the targeting relationship between CRNDE and miR-181a-5p, and the relationship between miR-181a-5p and SOX6; Western blot was performed to detect the expression levels of phosphorylated (p) -nuclear factor κB (NF-κB) p65/NF-κB p65, SOX6 and cleaved caspase-3.

Results

Compared with the control, the apoptosis rate (41.11%± 4.11%) vs (6.11% ± 0.61%) , TNF-α (353.21 ± 35.31) vs (200.15 ± 20.02) pg/mg, IL-1β (286.91 ± 28.71) vs (35.76 ± 3.58) pg/mg, IL-6 (642.31 ± 64.23 vs 32.64 ± 3.26) pg/mg, the expression of CRNDE (0.66 ± 0.06 vs 0.33 ± 0.03) , SOX6 (0.68 ± 0.06 vs 0.28 ± 0.03) , cleaved caspase-3 (0.76 ± 0.07 vs 0.24 ± 0.02) , p-NF-κB p65/NF-κB p65 (0.85 ± 0.08 vs 0.26 ± 0.03) were visibly raised in the LPS group, and the expression of miR-181a-5p (0.32 ± 0.03 vs 0.64 ± 0.06) was visibly decreased (P < 0.05) ; Compared with the si-NC group, knockdown CRNDE decreased the apoptosis rate (15.58%± 1.55% vs 41.09%± 4.09%) , TNF-α[ (200.15 ± 20.02) vs (356.51 ± 35.65) pg/mg], IL-1β[ (99.32 ± 9.94) vs (288.88 ± 28.89) pg/mg], IL-6[ (211.34 ± 21.14) vs (639.89 ± 63.99) pg/mg], CRNDE (0.35 ± 0.03 vs 0.65 ± 0.06) , SOX6 (0.39 ± 0.04 vs 0.64 ± 0.07) induced by LPS (P < 0.05) , Knockdown SOX6 inhibited the apoptosis rate (15.34%± 1.53% vs 41.09%± 4.09%) , TNF-α[ (192.37 ± 19.24) vs (356.51 ± 35.65) pg/mg], IL-1β[ (100.02 ± 10.01) vs (288.88 ± 28.89) pg/mg]. The expression of IL-6[(204.86 ± 20.49) vs (639.89 ± 63.99) pg/mg], SOX6 (0.36 ± 0.03 vs 0.64 ± 0.07) induced by LPS (P < 0.05) ; Compared with miR-NC, overexpression of miR-181a-5p reduced apoptosis (15.34%± 1.53% vs 40.89%± 4.09%) , TNF-α[ (189.45 ± 19.01) vs (356.68 ± 35.67) pg/mg], IL-1β[ (95.37 ± 9.54) vs (288.67 ± 28.8 7) pg/mg], IL-6[ (209.84 ± 20.98) vs (637.93 ± 63.81) pg/mg]induced by LPS, and the expression of miR-181a-5p (0.55 ± 0.05 vs 0.35 ± 0.03) was significantly increased (P < 0.05) ; inhibiting the expression of miR-181a-5p could reverse the protective effect of CRNDE on LPS-induced A549 cells; up-regulation of SOX6 could reverse the protective effects of CRNDE interference and miR-181a-5p overexpression on LPS-induced A549 cells.

Conclusion

Interfering with CRNDE could inhibit the inflammatory response and apoptosis of A549 cells induced by LPS, which may be related to the regulation of the miR-181a-5p/SOX6 axis.

Key words: LncRNA CRNDE, miR-181a-5p/SOX6 axis, LPS, Inflammatory response, Apoptosis

Chunwen Deng, Song Chen, Pei Zhong, Shiqiang Min, Jian Wan. LncRNA CRNDE regulates lipopolysaccharide-induced inflammatory response and apoptosis in human alveolar epithelial cells via miR-181a-5p/SOX6 axis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(03): 129-136.

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Figures/Tables 16

References 21

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基因	序列（5'→3'）	预期扩增引物长度（bp）
CRNDE	上游TGAAGGAAGGAAGTGGTGCA	186
	下游TCCAGTGGCATCCTACAAGA
miR-181a-5p	上游AACAUUCAACGCUGUCGGUGAGU	246
	下游UUUUGUAAGUUGCGACAGCCACU
SOX6	上游CCCCTCTGAACATGGTGGTGGC	113
	下游TGAGACTGCCCCTGCCGAGT
β-actin	上游AGAAAATCTGGCACCACACC	153
	下游CCATCTCTTGCTCGAAGTCC
U6	上游CTCGCTTCGGCAGCACA	105
	下游AACGCTTCACGAATTTGCGT

基因	序列（5'→3'）	预期扩增引物长度（bp）
CRNDE	上游TGAAGGAAGGAAGTGGTGCA	186
	下游TCCAGTGGCATCCTACAAGA
miR-181a-5p	上游AACAUUCAACGCUGUCGGUGAGU	246
	下游UUUUGUAAGUUGCGACAGCCACU
SOX6	上游CCCCTCTGAACATGGTGGTGGC	113
	下游TGAGACTGCCCCTGCCGAGT
β-actin	上游AGAAAATCTGGCACCACACC	153
	下游CCATCTCTTGCTCGAAGTCC
U6	上游CTCGCTTCGGCAGCACA	105
	下游AACGCTTCACGAATTTGCGT

分组	实验次数	CRNDE	miR-181a-5p	SOX6 mRNA
对照	6	0.33 ± 0.03	0.64 ± 0.06	0.28 ± 0.03
LPS	6	0.66 ± 0.06	0.32 ± 0.03	0.68 ± 0.06
t值		12.050	11.685	14.606
P值		< 0.001	< 0.001	< 0.001

分组	实验次数	CRNDE	miR-181a-5p	SOX6 mRNA
对照	6	0.33 ± 0.03	0.64 ± 0.06	0.28 ± 0.03
LPS	6	0.66 ± 0.06	0.32 ± 0.03	0.68 ± 0.06
t值		12.050	11.685	14.606
P值		< 0.001	< 0.001	< 0.001

分组	实验次数	CRNDE	SOX6 mRNA	凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
对照	6	0.33 ± 0.03	0.28 ± 0.03	6.11 ± 0.61	88.45 ± 8.85	35.76 ± 3.58	32.64 ± 3.26
LPS	6	0.66 ± 0.06^a	0.68 ± 0.06^a	41.11 ± 4.11^a	353.21 ± 35.31^a	286.91 ± 28.71^a	642.31 ± 64.23^a
LPS+si-NC	6	0.65 ± 0.06^a	0.64 ± 0.07^a	41.09 ± 4.09^a	356.51 ± 35.65^a	288.88 ± 28.89^a	639.89 ± 63.99^a
LPS+si-CRNDE	6	0.35 ± 0.03^bc	0.39 ± 0.04^abc	15.58 ± 1.55^abc	200.15 ± 20.02^abc	99.32 ± 9.94^abc	211.34 ± 21.14^abc
LPS+si-SOX6	6	0.33 ± 0.03^bc	0.36 ± 0.03^abc	15.34 ± 1.53^abc	192.37 ± 19.24^abc	100.02 ± 10.01^abc	204.86 ± 20.49^abc
F值		92.363	80.420	203.425	118.497	222.138	255.907
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

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