人环状RNA_0114428对脂多糖诱导的人肾小管上皮HK-2细胞损伤的作用及其机制

doi:10.13481/j.1671-587X.20230113

摘要/Abstract

摘要：

目的探讨人环状RNA_0114428（circ_0114428）对脂多糖（LPS）诱导的人肾小管上皮HK-2细胞损伤的作用，阐明circ_0114428通过调节微小RNA-139-5p（miR-139-5p）/转化生长因子β受体2（TGFBR2）轴改善HK-2细胞损伤的可能分子机制。方法体外培养HK-2细胞，双荧光素酶报告基因实验验证miR-139-5p与circ_0114428和TGFBR2的靶向调控关系，CCK-8法筛选合适的LPS干预浓度和时间。给予10 mg·L^－1 LPS干预12 h构建HK-2细胞损伤模型，HK-2细胞分为对照组、LPS组、LPS+circ_0114428沉默对照（LPS+si-NC）组、LPS+circ_0114428沉默（LPS+si-circ_0114428）组、LPS+circ_0114428沉默+miR-139-5p抑制剂对照（LPS+si-circ_0114428+in-NC）组和LPS+circ_0114428沉默+miR-139-5p抑制剂（LPS+si-circ_0114428 +in-miR-139-5p）组，实时荧光定量PCR（RT-qPCR）法检测各组细胞中circ_0114428、miR-139-5p和TGFBR2 mRNA表达水平，流式细胞术检测各组细胞凋亡率，Western blotting法检测各组细胞中TGFBR2、B细胞淋巴瘤2 （Bcl-2）和Bcl-2相关X蛋白（Bax）蛋白表达水平，酶联免疫吸附试验（ELISA）法检测各组细胞上清液中白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）水平。结果双荧光素酶报告基因实验，miR-139-5p与circ_0114428和TGFBR2存在靶向调控关系（t=10.171，P<0.05；t=7.682，P<0.05）。CCK-8法检测LPS干预的最佳浓度为10 mg·L^－1，最佳干预时间为12 h。与对照组比较，LPS组HK-2细胞中circ_0114428表达水平、TGFBR2 mRNA和蛋白表达水平、Bax蛋白表达水平、细胞凋亡率及细胞上清液中IL-6、TNF-α和IL-1β水平均明显升高（P<0.05），miR-139-5p表达水平和Bcl-2蛋白表达水平明显降低（P<0.05）。与LPS+si-NC组比较，LPS+si-circ_0114428组HK-2细胞中circ_0114428表达水平、TGFBR2 mRNA和蛋白表达水平、Bax蛋白表达水平、细胞凋亡率及细胞上清液中IL-6、TNF-α和IL-1β水平均明显降低（P<0.05），miR-139-5p表达水平和Bcl-2蛋白表达水平明显升高（P<0.05）。与LPS+si-circ_0114428+in-NC组比较，LPS+si-circ_0114428+in-miR-139-5p组HK-2细胞中circ_0114428表达水平、TGFBR2 mRNA和蛋白表达水平、Bax蛋白表达水平、细胞凋亡率及细胞上清液中IL-6、TNF-α和IL-1β水平均明显升高（P<0.05），miR-139-5p表达水平和Bcl-2蛋白表达水平明显降低（P<0.05）。结论 circ_0114428能减轻LPS诱导的HK-2细胞损伤，其作用机制可能与调节miR-139-5p/TGFBR2轴有关。

关键词: 环状RNA_0114428, miR-139-5p, 转化生长因子β受体2, 脂多糖, 人肾小管上皮细胞

Abstract:

Objective To investigate the effect of human circular RNA_0114428 （circ_0114428） on the lipopolysaccharide （LPS）-induced injury of human renal tubular epithelial HK-2 cells， and to clarify the possible molecular mechanism of circ_0114428 in improving the injury of HK-2 cells by regulating microRNA-139-5p （miR-139-5p）/transforming growth factor β receptor 2 （TGFBR2） axis. Methods The HK-2 cells were cultured in vitro， the dual luciferase reporter gene experiment was used to verify the targeted regulation relationship between miR-139-5p and circ_0114428 or TGFBR2， and the CCK-8 method was used to screen the appropriate LPS intervention time and concentration. The HK-2 cell injury model was established by administering 10 mg·L^－1 LPS for 12 h， and the HK-2 cells were divided into control group， LPS group， LPS+circ_0114428 silence control （LPS+si-NC） group， LPS+circ_0114428 silence （LPS+si-circ_0114428） group， LPS+circ_0114428 silence+miR-139-5p inhibitor control （LPS+si-circ_0114428+in-NC） group， and LPS+circ_0114428 silence+miR-139-5p inhibitor（LPS+si-circ_0114428+in-miR-139-5p） group. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of circ_0114428， miR-139-5p and TGFBR2 mRNA in the cells in various groups， flow cytometry was used to detect the apoptotic rates of the cells in various groups， and Western blotting method was used to detect the expression levels of TGFBR2， B-cell lymphoma-2 （Bcl-2） and Bcl-2 associated X protein （Bax） proteins in the cells in various groups；enzyme linked immunosorbent assay（ELISA） was used to detect the levels of interleukin-6（IL-6）， tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in the cell supernatant in various groups. Results The results of dual luciferase reporter gene experiment showed that miR-139-5p had a targeted regulatory relationship with circ_0114428 and TGFBR2 （t=10.171， P<0.05；t=7.682， P<0.05）.The CCK-8 method results showed that the concentration of LPS induction was 10 mg·L^－1 and the optimal time was 12 h.Compared with control group， the expression levels of circ_0114428， expression levels of TGFBR2 mRNA and protein， and expression level of Bax protein， the apoptotic rate， and levels of IL-6， TNF-α and IL-1β in cell supernatant of the HK-2 cells in LPS group were significantly increased （P<0.05），and the expression levels of miR-139-5p and Bcl-2 protein were significantly decreased （P<0.05）. Compared with LPS+si-NC group， the expression levels of circ_0114428， expression levels of TGFBR2 mRNA and protein， and expression level of Bax protein， the apoptotic rate， and the levels of IL-6， TNF-α and IL-1β in cell supernatant of the HK-2 cells in LPS+si-circ_0114428 group were significantly decreased （P<0.05），and the expression levels of miR-139-5p and Bcl-2 protein were significantly increased （P<0.05）. Compared with LPS+si-circ_0114428+in-NC group， the expression levels of circ_0114428， expression levels of TGFBR2 mRNA and protein， and expression level of Bax protein， the apoptotic rate， and the levels of IL-6， TNF-α and IL-1β in cell supernatant of the HK-2 cells in LPS+si-circ_0114428+in-miR-139-5p group were significantly increased （P<0.05）， and the expression levels of miR-139-5p and Bcl-2 protein were significantly decreased （ P<0.05）. Conclusion Circ_0114428 can alleviate the LPS-induced HK-2 cell injury， and its mechanism may be related to the regulation of the miR-139-5p/TGFBR2 axis.

Key words: Circular RNA_0114428, MiR-139-5p, Transforming growth factor β receptor 2, Lipopolysaccharide, Human renal tubular epithelial cells

中图分类号:

R631

王亚静,贾宝辉,韩方方,韩迎东,赵兰兰,石莹,张华. 人环状RNA_0114428对脂多糖诱导的人肾小管上皮HK-2细胞损伤的作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(1): 101-109.

Yajing WANG,Baohui JIA,Fangfang HAN,Yingdong HAN,Lanlan ZHAO,Ying SHI,Hua ZHANG. Effect of human circular RNA_0114428 on lipopolysaccharide-induced injury of human renal tubular epithelial HK-2 cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(1): 101-109.

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Group	Luciferase activity
Group	Circ_0114428 WT	Circ_0114428 MUT	TGFBR2 3'-UTR WT	TGFBR2 3'-UTR MUT
Mimic NC	1.00±0.10	1.00±0.09	1.00±0.11	1.00±0.09
MiR-139-5p mimic	0.21±0.09^*	0.86±0.08	0.31±0.11^*	0.82±0.12

Group	Proliferation activity				F	P
Group	(t/h) 0	12	24	48	F	P
Control	100.00±11.34	89.13±14.18	72.25±10.59	63.44±7.42^*	6.536	0.016
LPS（mg·L^－1）
5	100.00±9.44	74.18±14.15	62.30±10.71^*	51.29±8.92^*	10.850	0.003
10	100.00±6.27	60.47±14.11^*△	44.51±10.55^*△	31.22±7.18^*△	26.565	0.000
20	100.00±11.41	46.72±14.71^*△	26.11±8.47^*△	17.99±6.33^*△	35.648	0.000

Group	Circ_0114428	MiR-139-5p	TGFBR2
Group	Circ_0114428	MiR-139-5p	mRNA	Protein
Control	1.00±0.13	1.00±0.11	1.00±0.22	1.00±0.18
LPS	3.15±0.41^*	0.37±0.05^*	2.69±0.37^*	2.24±0.14^*
LPS+si-NC	3.20±0.45^*	0.34±0.06^*	2.62±0.42^*	2.31±0.16^*
LPS+si-circ_0114428	1.54±0.26^△	0.82±0.12^△	1.41±0.18^△	1.36±0.11^△
LPS+si-circ_0114428+in-NC	1.62±0.27^△	0.80±0.09^△	1.50±0.25^△	1.33±0.17^△
LPS+si-circ_0114428+in-miR-139-5p	2.69±0.32^*#	0.49±0.07^*#	2.39±0.33^*#	1.98±0.20^*#
F	24.868	29.442	16.459	33.697
P	<0.01	<0.01	<0.01	<0.01

Group	Apoptotic rate（η/%）	Bax	Bcl-2
Control	4.23±0.95	1.00±0.23	1.00±0.17
LPS	35.12±3.24^*	2.68±0.24^*	0.34±0.08^*
LPS+si-NC	34.11±4.08^*	2.71±0.32^*	0.40±0.09^*
LPS+si-circ_0114428	10.41±2.98^△	1.47±0.19^△	0.86±0.10^△
LPS+si-circ_0114428+in-NC	13.16±2.64^△	1.32±0.10^△	0.83±0.15^△
LPS+si-circ_0114428+in-miR-139-5p	28.94±3.71^*#	2.34±0.10^*#	0.49±0.14^*#
F	55.513	37.298	14.443
P	<0.01	<0.01	<0.01

Group	IL-6	TNF-α	IL-1β
Control	61.37±15.59	106.77±22.38	64.92±18.25
LPS	213.63±20.09^*	264.82±25.50^*	224.51±24.10^*
LPS+si-NC	220.89±19.74^*	251.37±24.55^*	235.81±23.57^*
LPS+si-circ_0114428	120.75±17.35^*△	146.69±21.34^△	104.85±20.75^△
LPS+si-circ_0114428+in-NC	130.15±22.77^*△	151.33±24.10^△	115.15±21.04^△
LPS+si-circ_0114428+in-miR-139-5p	198.36±25.31^*#	221.72±21.08^*#	212.72±21.47^*#
F	29.098	23.004	34.388
P	<0.01	<0.01	<0.01