LncRNA CRNDE通过miR-181a-5p/SOX6轴调节脂多糖诱导人肺泡上皮细胞的炎症反应和细胞凋亡

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

目的

探索长链非编码RNA （LncRNA）CRNDE通过微小RNA-181a-5p （miR-181a-5p）/转录因子性别决定区Y-box 6 （SOX6）轴对脂多糖（LPS）诱导人肺泡上皮细胞炎症反应和细胞凋亡的影响。

方法

将所有质粒和寡核苷酸分别转染A549细胞，并进行LPS处理，并以未经处理的A549细胞为对照。qRT-PCR检测细胞中CRNDE、miR-181a-5p以及SOX6 mRNA的表达水平；流式细胞仪、ELISA法检测细胞凋亡及肿瘤坏死因子-α （TNF-α）、炎症因子白细胞介素-1β （IL-β）和IL-6水平；双荧光素酶实验分别验证CRNDE和miR-181a-5p以及miR-181a-5p与SOX6的靶向关系；Western blot检测磷酸化（p）-核因子κB （NF-κB） p65/NF-κB p65、SOX6、活化的半胱氨酸天冬氨酸酶3 （cleaved caspase-3）的表达水平。两组间比较采用独立样本t检验；各组数据均满足正态分布，当方差齐时采用单因素方差分析，进一步两组间比较采用SNK-q检验，方差不齐时采用Games-Howell法检验。

结果

与对照比较，LPS的细胞凋亡率[（41.11 ± 4.11）%比（6.11 ± 0.61）%]、TNF-α [（353.21 ± 35.31）比（200.15 ± 20.02）pg/mg]、IL-1β [（286.91 ± 28.71）比（35.76 ± 3.58）pg/mg]、IL-6[（642.31 ± 64.23）比（32.64 ± 3.26）pg/mg]、CRNDE （0.66 ± 0.06比0.33 ± 0.03）、SOX6 （0.68 ± 0.06比0.28 ± 0.03）、cleaved caspase-3 （0.76 ± 0.07比0.24 ± 0.02）、p-NF-κB p65/NF-κB p65 （0.85 ± 0.08比0.26 ± 0.03）升高，miR-181a-5p表达（0.32 ± 0.03比0.64 ± 0.06）降低（P < 0.05）；与si-NC组相比，干扰CRNDE降低LPS诱导细胞的凋亡率[（15.58 ± 1.55）%比（41.09 ± 4.09）%]、TNF-α[（200.15 ± 20.02）比（356.51 ± 35.65）pg/mg]、IL-1β[（99.32 ± 9.94）比（288.88 ± 28.89 ） pg/mg]、IL-6[（211.34 ± 21.14）比（639.89 ± 63.99）pg/mg]、CRNDE（0.35 ± 0.03比0.65 ± 0.06）、SOX6 （0.39 ± 0.04比0.64 ± 0.07）的表达（P < 0.05），干扰SOX6降低LPS诱导细胞的凋亡率[（15.34 ± 1.53）%比（41.09 ± 4.09）%]、TNF-α[（192.37 ± 19.24）比（356.51 ± 35.65）pg/mg]、IL-1β [（100.02 ± 10.01）比（288.88 ± 28.89）pg/mg]、IL-6[（204.86 ± 20.49）比（639.89 ± 63.99）pg/mg]、SOX6 （0.36 ± 0.03比0.64 ± 0.07）的表达（P < 0.05）；与miR-NC相比，过表达miR-181a-5p降低LPS诱导细胞的凋亡率[（15.34 ± 1.53）%比（40.89 ± 4.09）%]、TNF-α [（189.45 ± 19.01）比（356.68 ± 35.67）pg/mg]、IL-1β[（95.37 ± 9.54）比（288.67 ± 28.87） pg/mg]、IL-6[（209.84 ± 20.98）比（637.93 ± 63.81）pg/mg]水平，miR-181a-5p （0.55 ± 0.05比0.35 ± 0.03）表达增加（P < 0.05）。抑制miR-181a-5p表达可逆转干扰CRNDE对LPS诱导A549细胞的保护作用；上调SOX6可逆转干扰CRNDE、过表达miR-181a-5p对LPS诱导A549细胞的保护作用。

结论

干扰CRNDE可以抑制LPS诱导A549细胞的炎症反应和细胞凋亡，可能与调控miR-181a-5p/SOX6轴有关。

关键词: LncRNA CRNDE, miR-181a-5p/SOX6轴, LPS, 炎症反应, 细胞凋亡

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

表1 qRT-PCR引物序列信息

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

表1 qRT-PCR引物序列信息

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

表2 比较CRNDE、miR-181a-5p以及SOX6 mRNA表达（ ± s）

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

表2 比较CRNDE、miR-181a-5p以及SOX6 mRNA表达（ ± s）

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

图1 流式细胞术检测细胞凋亡注：a ~ g图分别为对照、LPS、LPS+si-NC、LPS+si-CRNDE、LPS+si-SOX6、miR-NC、LPS+miR-181a-5p

表3 分别干扰CRNDE、SOX6对LPS诱导的细胞凋亡、炎症因子以及CRNDE表达的影响（ ± s）

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

表3 分别干扰CRNDE、SOX6对LPS诱导的细胞凋亡、炎症因子以及CRNDE表达的影响（ ± s）

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

表4 上调miR-181a-5p对细胞凋亡、炎症因子以及miR-181a-5p表达的影响（ ± s）

分组	实验次数	miR-181a-5p	凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
LPS	6	0.32 ± 0.03	41.02 ± 4.09	358.43 ± 35.84	288.14 ± 28.81	642.67 ± 64.31
miR-NC	6	0.35 ± 0.03	40.89 ± 4.09	356.68 ± 35.67	288.67 ± 28.87	637.93 ± 63.81
miR-181a-5p	6	0.55 ± 0.05^ab	15.34 ± 1.53^ab	189.45 ± 19.01^ab	95.37 ± 9.54^ab	209.84 ± 20.98^ab
F值		65.442	109.977	58.107	127.430	128.575
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表4 上调miR-181a-5p对细胞凋亡、炎症因子以及miR-181a-5p表达的影响（ ± s）

分组	实验次数	miR-181a-5p	凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
LPS	6	0.32 ± 0.03	41.02 ± 4.09	358.43 ± 35.84	288.14 ± 28.81	642.67 ± 64.31
miR-NC	6	0.35 ± 0.03	40.89 ± 4.09	356.68 ± 35.67	288.67 ± 28.87	637.93 ± 63.81
miR-181a-5p	6	0.55 ± 0.05^ab	15.34 ± 1.53^ab	189.45 ± 19.01^ab	95.37 ± 9.54^ab	209.84 ± 20.98^ab
F值		65.442	109.977	58.107	127.430	128.575
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

图2 Western blot检测各组细胞p-NF-κB p65、NF-κB p65、SOX6、cleaved caspase-3蛋白表达注：1为对照；2为LPS；3为LPS+si-NC；4为LPS+si-CRNDE；5为LPS+si-SOX6；6为LPS+miR-NC；7为LPS+miR-181a-5p

表5 分别干扰CRNDE、SOX6或上调miR-181a-5p对相关蛋白表达的影响（ ± s）

分组	实验次数	SOX6/β-actin	p-NF-κB p65/NF-κB p65	cleaved caspase-3/β-actin
对照	6	0.31 ± 0.03	0.26 ± 0.03	0.24 ± 0.02
LPS	6	0.67 ± 0.06^a	0.85 ± 0.08^a	0.76 ± 0.07^a
LPS+si-NC	6	0.66 ± 0.06^a	0.82 ± 0.08^a	0.78 ± 0.08^a
LPS+miR-NC	6	0.65 ± 0.06^a	0.84 ± 0.08^a	0.80 ± 0.08^a
LPS+si-CRNDE	6	0.35 ± 0.03^bcd	0.34 ± 0.03^bcd	0.29 ± 0.03^bcd
LPS+si-SOX6	6	0.32 ± 0.03^bcd	0.32 ± 0.03^bcd	0.34 ± 0.03^bcd
LPS+miR-181a-5p	6	0.29 ± 0.03^bcd	0.35 ± 0.03^bcd	0.32 ± 0.03^bcd
F值		98.760	143.500	127.300
P值		< 0.001	< 0.001	< 0.001

表5 分别干扰CRNDE、SOX6或上调miR-181a-5p对相关蛋白表达的影响（ ± s）

分组	实验次数	SOX6/β-actin	p-NF-κB p65/NF-κB p65	cleaved caspase-3/β-actin
对照	6	0.31 ± 0.03	0.26 ± 0.03	0.24 ± 0.02
LPS	6	0.67 ± 0.06^a	0.85 ± 0.08^a	0.76 ± 0.07^a
LPS+si-NC	6	0.66 ± 0.06^a	0.82 ± 0.08^a	0.78 ± 0.08^a
LPS+miR-NC	6	0.65 ± 0.06^a	0.84 ± 0.08^a	0.80 ± 0.08^a
LPS+si-CRNDE	6	0.35 ± 0.03^bcd	0.34 ± 0.03^bcd	0.29 ± 0.03^bcd
LPS+si-SOX6	6	0.32 ± 0.03^bcd	0.32 ± 0.03^bcd	0.34 ± 0.03^bcd
LPS+miR-181a-5p	6	0.29 ± 0.03^bcd	0.35 ± 0.03^bcd	0.32 ± 0.03^bcd
F值		98.760	143.500	127.300
P值		< 0.001	< 0.001	< 0.001

图3 Western blot检测各组细胞p-NF-κB p65、NF-κB p65、SOX6和cleaved caspase-3蛋白表达注：1为LPS+si-CRNDE+anti-miR-NC；2为LPS+si-CRNDE+anti-miR-181a-5p；3为LPS+si-CRNDE+oe-NC；4为LPS+si-CRNDE+oe-SOX6；5为LPS+miR-181a-5p+oe-NC；6为LPS+miR-181a-5p+oe-SOX6

图4 流式细胞术检测细胞凋亡注：a ~ f图分别为LPS+si-CRNDE+anti-miR-NC、LPS+si-CRNDE+anti-miR-181a-5p、LPS+si-CRNDE+oe-NC、LPS+si-CRNDE+oe-SOX6、LPS+miR-181a-5p+oe-NC、LPS+miR-181a-5p+oe-SOX6

表6 抑制miR-181a-5p可逆转干扰CRNDE对LPS诱导A549细胞凋亡、炎症因子的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
LPS+si-CRNDE+anti-miR-NC	6	40.01 ± 4.01	333.21 ± 33.31	256.45 ± 25.64	634.32 ± 63.43
LPS+si-CRNDE+anti-miR-181a-5p	6	55.24 ± 5.52	456.32 ± 45.63	376.42 ± 37.65	868.31 ± 86.82
t值		5.468	5.338	6.451	5.331
P值		< 0.001	< 0.001	< 0.001	< 0.001
分组	实验次数	SOX6/β-actin	p-NF-κB p65/NF-κB p65	cleaved caspase-3/β-actin
LPS+si-CRNDE+anti-miR-NC	6	0.65 ± 0.06	0.83 ± 0.08	0.75 ± 0.07
LPS+si-CRNDE+anti-miR-181a-5p	6	0.94 ± 0.09	1.08 ± 0.10	0.96 ± 0.09
t值		6.567	4.782	4.512
P值		< 0.001	0.001	0.001

表6 抑制miR-181a-5p可逆转干扰CRNDE对LPS诱导A549细胞凋亡、炎症因子的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
LPS+si-CRNDE+anti-miR-NC	6	40.01 ± 4.01	333.21 ± 33.31	256.45 ± 25.64	634.32 ± 63.43
LPS+si-CRNDE+anti-miR-181a-5p	6	55.24 ± 5.52	456.32 ± 45.63	376.42 ± 37.65	868.31 ± 86.82
t值		5.468	5.338	6.451	5.331
P值		< 0.001	< 0.001	< 0.001	< 0.001
分组	实验次数	SOX6/β-actin	p-NF-κB p65/NF-κB p65	cleaved caspase-3/β-actin
LPS+si-CRNDE+anti-miR-NC	6	0.65 ± 0.06	0.83 ± 0.08	0.75 ± 0.07
LPS+si-CRNDE+anti-miR-181a-5p	6	0.94 ± 0.09	1.08 ± 0.10	0.96 ± 0.09
t值		6.567	4.782	4.512
P值		< 0.001	0.001	0.001

表7 过表达SOX6可逆转干扰CRNDE对LPS诱导A549细胞凋亡、炎症因子的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）	SOX6/β-actin
LPS+si-CRNDE+oe-NC	6	39.84 ± 3.99	346.38 ± 34.64	235.71 ± 23.57	621.28 ± 62.13	0.66 ± 0.06
LPS+si-CRNDE+oe-SOX6	6	50.19 ± 5.02	462.31 ± 46.23	378.52 ± 37.85	871.64 ± 87.17	0.89 ± 0.09
t值		3.954	4.916	7.845	5.729	5.208
P值		0.003	0.001	< 0.001	< 0.001	< 0.001
分组	实验次数	p-NF-κB p65/NF-κB p65	cleaved-caspase-3/β-actin	SOX6 mRNA	CRNDE
LPS+si-CRNDE+oe-NC	6	0.82 ± 0.08	0.72 ± 0.07	0.39 ± 0.03	0.35 ± 0.04
LPS+si-CRNDE+oe-SOX6	6	1.11 ± 0.11	0.98 ± 0.09	0.56 ± 0.06	0.36 ± 0.03
t值		5.223	5.586	6.208	0.490
P值		< 0.001	< 0.001	< 0.001	0.6351

表7 过表达SOX6可逆转干扰CRNDE对LPS诱导A549细胞凋亡、炎症因子的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）	SOX6/β-actin
LPS+si-CRNDE+oe-NC	6	39.84 ± 3.99	346.38 ± 34.64	235.71 ± 23.57	621.28 ± 62.13	0.66 ± 0.06
LPS+si-CRNDE+oe-SOX6	6	50.19 ± 5.02	462.31 ± 46.23	378.52 ± 37.85	871.64 ± 87.17	0.89 ± 0.09
t值		3.954	4.916	7.845	5.729	5.208
P值		0.003	0.001	< 0.001	< 0.001	< 0.001
分组	实验次数	p-NF-κB p65/NF-κB p65	cleaved-caspase-3/β-actin	SOX6 mRNA	CRNDE
LPS+si-CRNDE+oe-NC	6	0.82 ± 0.08	0.72 ± 0.07	0.39 ± 0.03	0.35 ± 0.04
LPS+si-CRNDE+oe-SOX6	6	1.11 ± 0.11	0.98 ± 0.09	0.56 ± 0.06	0.36 ± 0.03
t值		5.223	5.586	6.208	0.490
P值		< 0.001	< 0.001	< 0.001	0.6351

表8 上调SOX6可逆转过表达miR-181a-5p对细胞凋亡、炎症因子的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
LPS+miR-181a-5p+oe-NC	6	40.18 ± 4.02	349.51 ± 34.96	248.72 ± 24.87	618.91 ± 61.91
LPS+miR-181a-5p+oe-SOX6	6	52.43 ± 5.24	467.43 ± 46.74	369.89 ± 36.99	875.34 ± 87.53
t值		7.462	4.949	6.659	8.710
P值		< 0.001	0.001	< 0.001	< 0.001
分组	实验次数	SOX6/β-actin	p-NF-κB p65/NF-κB p65	cleaved caspase-3/β-actin
LPS+miR-181a-5p+oe-NC	6	0.63 ± 0.06	0.84 ± 0.08	0.73 ± 0.07
LPS+miR-181a-5p+oe-SOX6	6	0.92 ± 0.09	1.09 ± 0.10	0.97 ± 0.09
t值		6.567	4.782	5.156
P值		< 0.001	0.001	< 0.001

表8 上调SOX6可逆转过表达miR-181a-5p对细胞凋亡、炎症因子的影响（ ± s）

分组	实验次数	细胞凋亡率（%）	TNF-α （pg/mg）	IL-1β （pg/mg）	IL-6 （pg/mg）
LPS+miR-181a-5p+oe-NC	6	40.18 ± 4.02	349.51 ± 34.96	248.72 ± 24.87	618.91 ± 61.91
LPS+miR-181a-5p+oe-SOX6	6	52.43 ± 5.24	467.43 ± 46.74	369.89 ± 36.99	875.34 ± 87.53
t值		7.462	4.949	6.659	8.710
P值		< 0.001	0.001	< 0.001	< 0.001
分组	实验次数	SOX6/β-actin	p-NF-κB p65/NF-κB p65	cleaved caspase-3/β-actin
LPS+miR-181a-5p+oe-NC	6	0.63 ± 0.06	0.84 ± 0.08	0.73 ± 0.07
LPS+miR-181a-5p+oe-SOX6	6	0.92 ± 0.09	1.09 ± 0.10	0.97 ± 0.09
t值		6.567	4.782	5.156
P值		< 0.001	0.001	< 0.001

图5 LncBase Predicted v.2预测CRNDE与miR-181a-5p的关系

表9 LncBase Predicted v.2预测及靶向验证CRNDE与miR-181a-5p的关系

因素	实验次数	WT-CRNDE	MUT-CRNDE
miR-NC	6	1.11 ± 0.11	1.06 ± 0.11
miR-181a-5p	6	0.42 ± 0.03	1.09 ± 0.11
t值		14.609	0.472
P值		< 0.001	0.647

表9 LncBase Predicted v.2预测及靶向验证CRNDE与miR-181a-5p的关系

因素	实验次数	WT-CRNDE	MUT-CRNDE
miR-NC	6	1.11 ± 0.11	1.06 ± 0.11
miR-181a-5p	6	0.42 ± 0.03	1.09 ± 0.11
t值		14.609	0.472
P值		< 0.001	0.647

图6 Targetscan预测SOX6与miR-181a-5p的结合位点

表10 验证SOX6与miR-181a-5p的靶向关系（ ± s）

因素	实验次数	WT-SOX6	MUT-SOX6
miR-NC	6	1.12 ± 0.11	1.08 ± 0.11
miR-181a-5p	6	0.44 ± 0.03	1.13 ± 0.11
t值		14.609	0.787
P值		< 0.001	0.449

表10 验证SOX6与miR-181a-5p的靶向关系（ ± s）

因素	实验次数	WT-SOX6	MUT-SOX6
miR-NC	6	1.12 ± 0.11	1.08 ± 0.11
miR-181a-5p	6	0.44 ± 0.03	1.13 ± 0.11
t值		14.609	0.787
P值		< 0.001	0.449

1	Li J, Lu K, Sun F, et al. Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway[J]. J Transl Med, 2021, 19(1):96.
2	亓水芹, 冯向功. miR-221靶向AdipoR1基因对LPS诱导的肺泡上皮细胞A549炎症分泌及凋亡影响[J]. 中国免疫学杂志, 2020, 36(5):538-542.
3	Qiu N, Xu X, He Y. LncRNA TUG1 alleviates sepsis-induced acute lung injury by targeting miR-34b-5p/GAB1[J]. BMC Pulm Med, 2020, 20(1):49. doi:10.1186/s12890-020-1084-3.
4	Zhu-Ge D, Yang YP, Jiang ZJ. Knockdown CRNDE alleviates LPS-induced inflammation injury via FOXM1 in WI-38 cells[J]. Biomed Pharmacother, 2018, 103:1678-1687.
5	Alipoor SD, Adcock IM, Garssen J, et al. The roles of miRNAs as potential biomarkers in lung diseases[J]. Eur J Pharmacol, 2016, 791:395-404.
6	Qu Y, Zhang Q, Cai X, et al. Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation[J]. J Cell Mol Med, 2017, 21(10):2491-2502.
7	Yang L, Ma Y, Xin Y, et al. Role of the microRNA 181 family in glioma development[J]. Mol Med Rep, 2018, 17(1):322-329.
8	Sapoznikov A, Gal Y, Falach R, et al. Early disruption of the alveolar-capillary barrier in a ricin-induced ARDS mouse model: neutrophil-dependent and -independent impairment of junction proteins[J]. Am J Physiol Lung Cell Mol Physiol, 2019, 316(1):L255-L268.
9	Qu L, Chen C, He W, et al. Glycyrrhizic acid ameliorates LPS-induced acute lung injury by regulating autophagy through the PI3K/AKT/mTOR pathway[J]. Am J Transl Res, 2019, 11(4):2042-2055.
10	Li C, Liu JH, Su J, et al. LncRNA XIST knockdown alleviates LPS-induced acute lung injury by inactivation of XIST/miR-132-3p/MAPK14 pathway : XIST promotes ALI via miR-132-3p/MAPK14 axis[J]. Mol Cell Biochem, 2021, 476(12):4217-4229.
11	Zhu X, Du J, Yu J, et al. LncRNA NKILA regulates endothelium inflammation by controlling a NF-κB/KLF4 positive feedback loop[J]. J Mol Cell Cardiol, 2019, 126:60-69.
12	Ma X, Zhang W, Zhang R, et al. Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers[J]. Front Med, 2019, 13(3):330-343.
13	邹丽绢, 李磊, 肖政, 等. 骨髓间充质干细胞通过调控miR-142-3p保护LPS诱导大鼠肺上皮细胞RLE-6TN的炎症损伤与凋亡[J]. 中国免疫学杂志, 2021, 37(18):2194-2198.
14	Tang X, Wang T, Qiu C, et al. Long Non-Coding RNA (lncRNA) CRNDE Regulated Lipopolysaccharides (LPS)-Induced MRC-5 Inflammation Injury Through Targeting MiR-141[J]. Med Sci Monit, 2020, 26:e920928.
15	Su Y, Yuan J, Zhang F, et al. MicroRNA-181a-5p and microRNA-181a-3p cooperatively restrict vascular inflammation and atherosclerosis[J]. Cell Death Dis, 2019, 10(5):365. doi: 10.1038/s41419-019-1599-9.
16	Zhao H, Guo Y, Sun Y, et al. miR-181a/b-5p ameliorates inflammatory response in monocrotaline-induced pulmonary arterial hypertension by targeting endocan[J]. J Cell Physiol, 2020, 235(5):4422-4433.
17	Liu Y, Wang X, Li P, et al. Targeting MALAT1 and miRNA-181a-5p for the intervention of acute lung injury/acute respiratory distress syndrome[J]. Respir Res, 2021, 22(1):1.
18	Barbarani G, Fugazza C, Barabino SML, et al. SOX6 blocks the proliferation of BCR-ABL1+and JAK2V617F+leukemic cells[J]. Sci Rep, 2019, 9(1):3388.
19	Jia Z, Wang J, Shi Q, et al. SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition[J]. Apoptosis, 2016, 21(2):174-183.
20	Zeng Z, Liu Y, Zheng W, et al. MicroRNA-129-5p alleviates nerve injury and inflammatory response of Alzheimer's disease via downregulating SOX6[J]. Cell Cycle, 2019, 18(22):3095-3110.
21	Zhang W, Li J, Yao H, et al. Restoring microRNA-499-5p protects sepsis-induced lung injury mice via targeting Sox6[J]. Nanoscale Res Lett, 2021, 16(1):89.

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

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