Influence of circACTR2 on high glucose-induced trophoblast cell injury by regulating miR-23a-3p/TBL1X axis

doi:10.11958/20230156

Abstract

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

CLC Number:

R349.64

Figures/Tables 9

References 18

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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^＊＊

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^＊＊

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^＊＊

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^＊＊

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^＊＊