CircACTR2调节miR-23a-3p/TBL1X轴对高糖诱导的滋养层细胞损伤的影响

doi:10.11958/20230156

摘要/Abstract

摘要：

目的探讨环状RNA肌动蛋白相关蛋白2（circACTR2）调节miR-23a-3p/转导素β1X连锁蛋白（TBL1X）轴对高糖诱导的滋养层细胞损伤的影响。方法将人绒毛膜滋养层细胞HTR-8/Svneo分为NG组（5.5 mmol/L葡萄糖）、HG组（25 mmol/L葡萄糖）、si-NC组（25 mmol/L葡萄糖+转染si-NC）、si-circACTR2组（25 mmol/L葡萄糖+转染si-circACTR2）、si-circACTR2+inhibitor-NC组（25 mmol/L葡萄糖+si-circACTR2和inhibitor-NC共转染）、si-circACTR2+miR-23a-3p inhibitor组（25 mmol/L葡萄糖+si-circACTR2和miR-23a-3p inhibitor共转染）。实时荧光定量PCR（qPCR）检测细胞中circACTR2、miR-23a-3p的表达；CCK-8法检测细胞增殖；流式细胞仪检测细胞凋亡；划痕实验检测细胞迁移；酶联免疫吸附试验检测丙二醛（MDA）水平和乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）活性；Western blot检测细胞中TBL1X、增殖细胞核抗原（PCNA）、基质金属蛋白酶（MMP）-2、MMP-9、胱天蛋白酶3（caspase-3）的表达。双萤光素酶报告基因实验分别验证circACTR2、TBL1X和miR-23a-3p的靶向关系。结果与NG组相比，HG组HTR-8/Svneo细胞miR-23a-3p表达、增殖能力、划痕愈合率、PCNA、MMP-2、MMP-9表达、SOD活性降低，circACTR2、TBL1X和caxpase-3表达、MDA含量、LDH活性、凋亡率升高（P<0.05）；与HG组和si-NC组相比，si-circACTR2组HTR-8/Svneo细胞中miR-23a-3p表达、增殖能力、划痕愈合率、PCNA、MMP-2、MMP-9表达、SOD活性升高，circACTR2、TBL1X和caxpase-3表达、MDA含量、LDH活性、凋亡率降低（P<0.05）；在敲低circACTR2的基础上，下调miR-23a-3p可明显减弱circACTR2敲低对高糖诱导的HTR-8/Svneo细胞的增殖和迁移的促进作用，增强细胞凋亡和氧化应激能力。双萤光素酶报告基因实验结果显示，circACTR2靶向负调控miR-23a-3p表达，miR-23a-3p靶向负调控TBL1X表达。结论敲低circACTR2可调控miR-23a-3p/TBL1X轴，进而通过抑制高糖诱导的滋养层细胞损伤来发挥保护作用。

Abstract:

Objective To investigate the influence of circular RNA actin related protein 2 (circACTR2) on high glucose-induced trophoblast cell injury by regulating miR-23a-3p/transducin β1X-linked protein (TBL1X) axis. Methods Human chorionic trophoblast cells HTR-8/Svneo were grouped into the NG group (5.5 mmol/L glucose), the HG group (25 mmol/L glucose), the si-NC group (25 mmol/L glucose+transfected with si-NC), the si-circACTR2 group (25 mmol/L glucose+transfected with si-circACTR2), the si-circACTR2+inhibitor-NC group (25 mmol/L glucose+co-transfected with si-circACTR2 and inhibitor-NC) and si-circACTR2+miR-23a-3p inhibitor group (25 mmol/L glucose+co-transfected with si-circACTR2 and miR-23a-3p inhibitor). Real-time quantitative PCR (qPCR) was performed to measure expression levels of circACTR2 and miR-23a-3p in cells. Cell proliferation was detected by CCK-8 assay. Cell apoptosis was detected by flow cytometry. Scratch assay was performed to detect cell migration. Enzyme-linked immunosorbent assay was applied to detect malondialdehyde (MDA) level, lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities. Western blot assay was used to detect expressions of TBL1X, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-2 (MMP-2), MMP-9 and cysteine protease-3 (caspase-3) in cells. Dual-luciferase reporter gene experiment was performed to verify the targeting relationship between circACTR2, TBL1X and miR-23a-3p respectively. Results Compared with the NG group, the HTR-8/Svneo cell expression of miR-23a-3p, proliferative ability, scratch healing rate, expression of PCNA, MMP-2, MMP-9 and SOD activity were reduced in the HG group, and the expression of circACTR2, TBL1X, caxpase-3, MDA content, LDH activity and apoptosis rate were increased (P<0.05). Compared with the HG group and the si-NC group, the miR-23a-3p expression, proliferative ability, scratch healing rate, expression of PCNA, MMP-2 and MMP-9, and SOD activity in HTR-8/Svneo cells were increased in the si-circACTR2 group, and the expression of circACTR2, TBL1X and caxpase-3, MDA content, LDH activity and apoptosis rate were decreased (P<0.05). On the basis of knocking down circACTR2, the down-regulation of miR-23a-3p significantly attenuated the promotion of circACTR2 knockdown on the proliferation and migration of high glucose-induced HTR-8/Svneo cells, and enhanced the ability of apoptosis and oxidative stress. The results of dual-luciferase reporter gene assay showed that circACTR2 targeted to negatively regulate the expression of miR-23a-3p, and miR-23a-3p targeted to negatively regulate the expression of TBL1X. Conclusion Knocking down circACTR2 can inhibit high glucose-induced trophoblast cell injury and exert a protective effect by regulating miR-23a-3p/TBL1X axis.

Key words: actin-related protein 2, pregnancy-specific beta 1-glycoproteins, trophoblasts, apoptosis, miR-23a-3p, high glucose, human billous trophoblasts HTR-8/Svneo

中图分类号:

R349.64

图/表 9

表1 各组HTR-8/Svneo细胞中circACTR2、miR-23a-3p表达比较 (n=6, $\bar{x}±s$)

Tab.1 Comparison of expression of circACTR2 and miR-23a-3p in HTR-8/Svneo cells between the six groups

组别	circACTR2	miR-23a-3p
NG组	1.00±0.00	1.00±0.00
HG组	1.62±0.14^a	0.47±0.08^a
si-NC组	1.61±0.15	0.48±0.07
si-circACTR2组	1.15±0.12^bc	0.83±0.06^bc
si-circACTR2+inhibitor-NC组	1.16±0.13	0.82±0.04
si-circACTR2+ miR-23a-3p inhibitor组	1.15±0.14	0.64±0.06^de
F	27.181^＊＊	80.931^＊＊

表2 各组HTR-8/Svneo细胞增殖能力比较（n=6，OD450值，$\bar{x}±s$）

Tab.2 Comparison of proliferative ability of HTR-8/Svneo cells between the six groups

组别	24 h	48 h	72 h
NG组	1.05±0.12	1.26±0.13	1.54±0.14
HG组	0.51±0.06^a	0.63±0.08^a	0.82±0.11^a
si-NC组	0.52±0.07	0.64±0.07	0.83±0.13
si-circACTR2组	0.88±0.05^bc	1.02±0.05^bc	1.24±0.06^bc
si-circACTR2+inhibitor- NC组	0.89±0.07	1.01±0.06	1.23±0.08
si-circACTR2+ miR-23a-3p inhibitor组	0.67±0.06^de	0.85±0.07^de	1.04±0.07^de
F	51.681^＊＊	54.934^＊＊	43.465^＊＊

图1 各组HTR-8/Svneo细胞凋亡情况及凋亡率比较 A：各组HTR-8/Svneo细胞凋亡情况；B：各组HTR-8/Svneo细胞凋亡率比较（n=6），1—6分别为NG组、HG组、si-NC组、si-circACTR2组、si-circACTR2+inhibitor-NC组、si-circACTR2+miR-23a-3p inhibitor组，a与NG组比较，b与HG组比较，c与si-NC组比较，d与si-circACTR2组比较，e与si-circACTR2+inhibitor-NC组比较，P<0.05；图2B同。

Fig.1 Comparison of HTR-8/Svneo cell apoptosis and apoptosis rate between the six groups

图2 各组HTR-8/Svneo细胞迁移情况及其划痕愈合率比较

Fig.2 Comparison of HTR-8/Svneo cell migration and scratch healing rate between the six groups

表3 各组HTR-8/Svneo细胞MDA水平以及LDH、SOD活性比较 (n=6,$\bar{x}±s$)

Tab.3 Comparison of MDA level, LDH, SOD activities of HTR-8/Svneo cells between the six groups

组别	MDA/（μmol/L）	LDH/（U/L）	SOD/（U/mL）
NG组	5.23±0.46	11.37±1.15	28.76±2.35
HG组	24.36±2.15^a	36.54±2.32^a	8.54±1.24^a
si-NC组	23.74±2.13	35.83±2.16	9.36±1.32
si-circACTR2组	13.86±1.54^bc	17.46±2.21^bc	21.68±2.21^bc
si-circACTR2+ inhibitor-NC组	12.78±1.46	18.52±2.14	22.75±2.15
si-circACTR2+ miR- 23a-3p inhibitor组	19.43±1.37^de	24.36±2.13^de	17.58±2.14^de
F	123.683^＊＊	150.307^＊＊	99.442^＊＊

图3 Western blot检测HTR-8/Svneo细胞中TBL1X、PCNA、MMP-2、MMP-9、caspase-3蛋白表达 A：NG组；B：HG组；C：si-NC组；D：si-circACTR2组；E：si-circACTR2+inhibitor-NC组；F：si-circACTR2+miR-23a-3p inhibitor组。

Fig.3 Western blot detection of TBL1X, PCNA, MMP-2, MMP-9 and caspase-3 protein expression in HTR-8/Svneo cells

表4 各组HTR-8/Svneo细胞中TBL1X、PCNA、MMP-2、MMP-9、caspase-3蛋白表达比较 (n=6,$\bar{x}±s$)

Tab.4 Comparison of protein expression of TBL1X, PCNA, MMP-2, MMP-9 and caspase-3 in HTR-8/Svneo cells between the six groups

组别	TBL1X	PCNA	MMP-2	MMP-9	caspase-3
NG组	0.47±0.08	1.24±0.15	1.51±0.16	1.36±0.14	0.72±0.06
HG组	1.16±0.12^a	0.53±0.07^a	0.67±0.08^a	0.58±0.06^a	1.52±0.14^a
si-NC组	1.15±0.13	0.54±0.08	0.68±0.07	0.59±0.08	1.51±0.15
si-circACTR2组	0.63±0.07^bc	0.96±0.12^bc	1.23±0.14^bc	1.02±0.11^bc	1.04±0.08^bc
si-circACTR2+inhibitor-NC组	0.64±0.05	0.95±0.13	1.22±0.13	1.01±0.12	1.03±0.07
si-circACTR2+miR-23a-3p inhibitor组	0.82±0.06^de	0.76±0.06^de	0.93±0.08^de	0.79±0.07^de	1.25±0.09^de
F	61.368^＊＊	39.658^＊＊	51.248^＊＊	52.912^＊＊	53.429^＊＊

图4 circACTR2与miR-23a-3p的结合位点及双萤光素酶检测结果 A：circACTR2与miR-23a-3p的结合位点；B：双萤光素酶实验结果（n=6）。

Fig.4 Binding sites of circACTR2 to miR-23a-3p and results of dualluciferase assay

图5 miR-23a-3p与TBL1X的结合位点及双萤光素酶检测结果 A：miR-23a-3p与TBL1X的结合位点；B：双萤光素酶实验结果（n=6）。

Fig.5 Binding sites of miR-23a-3p to TBL1X and results of dual-luciferase assay

参考文献 18

[1]	HUA Z, LI D, WU A, et al. miR-377 inhibition enhances the survival of trophoblast cells via upregulation of FNDC5 in gestational diabetes mellitus[J]. Open Med (Wars), 2021, 16(1):464-471. doi:10.1515/med-2021-0247.
[2]	PÉREZ-PÉREZ A, VILARIÑO-GARCÍA T, GUADIX P, et al. Leptin and Nutrition in gestational diabetes[J]. Nutrients, 2020, 12(7):1970. doi:10.3390/nu12071970.
[3]	TAN H X, YANG S L, LI M Q, et al. Autophagy suppression of trophoblast cells induces pregnancy loss by activating decidual NK cytotoxicity and inhibiting trophoblast invasion[J]. Cell Commun Signal, 2020, 18(1):73. doi:10.1186/s12964-020-00579-w.
[4]	SHANG J, LIN L, HUANG X, et al. Re-expression of circ_0043610 contributes to trophoblast dysfunction through the miR-558/RYBP pathway in preeclampsia[J]. Endocr J, 2022, 69(12):1373-1385. doi:10.1507/endocrj.EJ22-0153.
[5]	ZHU C, LIU Y, WU H. Overexpression of circACTR2 in gestational diabetes mellitus predicts intrauterine death,fetal malformation,and intrauterine infection[J]. Diabetes Metab Syndr Obes, 2021, 14:4655-4660. doi:10.2147/DMSO.S316043.
[6]	ZHENG T, CHEN H. Resveratrol ameliorates the glucose uptake and lipid metabolism in gestational diabetes mellitus mice and insulin-resistant adipocytes via miR-23a-3p/NOV axis[J]. Mol Immunol, 2021, 137:163-173. doi:10.1016/j.molimm.2021.06.011.
[7]	DING R, GUO F, ZHANG Y, et al. Integrated transcriptome sequencing analysis reveals role of miR-138-5p/TBL1X in placenta from gestational diabetes mellitus[J]. Cell Physiol Biochem, 2018, 51(2):630-646. doi:10.1159/000495319.
[8]	田雨青, 任宏丽, 梁阿娟. 黄芩苷通过调控miR-451对高糖诱导滋养层细胞损伤的保护作用[J]. 中成药, 2022, 44(4):1314-1317.
	TIAN Y Q, REN H L, LIANG A J. Protective effect of baicalin on trophoblast cell damage induced by high glucose by regulating miR-451[J]. China Journal of Chinese Materia Medica, 2022, 44(4):1314-1317. doi:10.3969/j.issn.1001-1528.2022.04.053.
[9]	FISHER J J, VANDERPEET CL, BARTHO LA, et al. Mitochondrial dysfunction in placental trophoblast cells experiencing gestational diabetes mellitus[J]. J Physiol, 2021, 599(4):1291-1305. doi:10.1113/JP280593.
[10]	李琴, 谢莹莺. 胰岛素生长因子2甲基化对高糖诱导滋养层细胞凋亡及PI3K/Akt通路的影响[J]. 安徽医科大学学报, 2020, 55(5):665-670.
	LI Q, XIE Y Y. Effects of insulin growth factor 2 methylation on high glucose induced trophoblast cell apoptosis and PI3K/Akt pathway[J]. Acta Universitatis Medicinalis Anhui, 2020, 55(5):665-670. doi:10.19405/j.cnki.issn1000-1492.2020.05.003.
[11]	BAO D, ZHUANG C, JIAO Y, et al. The possible involvement of circRNA DMNT1/p53/JAK/STAT in gestational diabetes mellitus and preeclampsia[J]. Cell Death Discov, 2022, 8(1):121. doi:10.1038/s41420-022-00913-w.
[12]	CHEN H, ZHANG S, WU Y, et al. The role of circular RNA circ_0008285 in gestational diabetes mellitus by regulating the biological functions of trophoblasts[J]. Biol Res, 2021, 54(1):14. doi:10.1186/s40659-021-00337-3.
[13]	WANG H, ZHOU W, SHE G, et al. Downregulation of hsa_circ_0005243 induces trophoblast cell dysfunction and inflammation via the β-catenin and NF-κB pathways[J]. Reprod Biol Endocrinol, 2020, 18(1):51. doi:10.1186/s12958-020-00612-0.
[14]	WEN S, LI S, LI L, et al. circACTR2:A novel mechanism regulating high glucose-induced fibrosis in renal tubular cells via pyroptosis[J]. Biol Pharm Bull, 2020, 43(3):558-564. doi:10.1248/bpb.b19-00901.
[15]	陈春来, 王毅, 舒静. microRNA-195-5p在妊娠糖尿病患者血浆中的表达及其对细胞增殖和凋亡的影响[J]. 中国临床药理学杂志, 2021, 37(18):2397-2400.
	CHEN C L, WANG Y, SHU J. Expression of microRNA-195-5p in plasma of pregnant women with diabetes and its effect on cell proliferation and apoptosis[J]. The Chinese Journal of Clinical Pharmacology, 2021, 37(18):2397-2400. doi:10.13699/j.cnki.1001-6821.2021.18.004.
[16]	WANG J, PAN Y, DAI F, et al. Serum miR-195-5p is upregulated in gestational diabetes mellitus[J]. J Clin Lab Anal, 2020, 34(8):e23325. doi:10.1002/jcla.23325.
[17]	ZHANG L, ZENG M, TANG F, et al. Circ-PNPT1 contributes to gestational diabetes mellitus (GDM) by regulating the function of trophoblast cells through miR-889-3p/PAK1 axis[J]. Diabetol Metab Syndr, 2021, 13(1):58. doi:10.1186/s13098-021-00678-9.
[18]	LI Y, ZHUANG J. miR-345-3p serves a protective role during gestational diabetes mellitus by targeting BAK1[J]. Exp Ther Med, 2021, 21(1):2. doi:10.3892/etm.2020.9434.