miR-155通过调控PKG1影响滋养细胞生物学功能并参与子痫前期的机制探讨

doi:10.11958/20221869

天津医药 ›› 2023, Vol. 51 ›› Issue (9): 928-934.doi: 10.11958/20221869

miR-155通过调控PKG1影响滋养细胞生物学功能并参与子痫前期的机制探讨

岳巾晶¹(), 曾莹¹, 郭晓珮¹, 董越¹, 姬若楠¹, 彭瑞², 罗晓华²^,^△()

1.郑州大学第三附属医院妇产科（邮编450000）
2.郑州大学第三附属医院科研办公室（邮编450000）

收稿日期:2022-11-18 修回日期:2023-03-02 出版日期:2023-09-15 发布日期:2023-09-13
通讯作者: △E-mail：Luoxiaohua620@163.com
作者简介:岳巾晶（1997），女，硕士在读，主要从事子痫前期的诊断和治疗方面研究。E-mail：1508362194@qq.com
基金资助:
河南省科技攻关项目(202102310473)

MiR-155 affects the biological functions of trophoblastic cells through regulating cGMP-dependent kinase 1 and is involved in the mechanism of preeclampsia

YUE Jinjing¹(), ZENG Ying¹, GUO Xiaopei¹, DONG Yue¹, JI Ruonan¹, PENG Rui², LUO Xiaohua²^,^△()

1. Department of Gynaecology and Obstetrics, the Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China
2. Department of Scientific Research Office, the Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China

Received:2022-11-18 Revised:2023-03-02 Published:2023-09-15 Online:2023-09-13
Contact: △E-mail：Luoxiaohua620@163.com

岳巾晶, 曾莹, 郭晓珮, 董越, 姬若楠, 彭瑞, 罗晓华. miR-155通过调控PKG1影响滋养细胞生物学功能并参与子痫前期的机制探讨[J]. 天津医药, 2023, 51(9): 928-934.

YUE Jinjing, ZENG Ying, GUO Xiaopei, DONG Yue, JI Ruonan, PENG Rui, LUO Xiaohua. MiR-155 affects the biological functions of trophoblastic cells through regulating cGMP-dependent kinase 1 and is involved in the mechanism of preeclampsia[J]. Tianjin Medical Journal, 2023, 51(9): 928-934.

摘要/Abstract

摘要：

目的探究miR-155和环磷酸鸟苷依赖性蛋白激酶1（PKG1）在子痫前期患者胎盘组织中的表达及miR-155在核因子（NF）-κB的介导下通过抑制PKG1对滋养细胞HTR-8/SVneo功能的影响。方法收集剖宫产分娩的正常产妇（NPE组）以及子痫前期产妇（PE组）的胎盘各20个,体外培养滋养细胞HTR-8/SVneo，分为NC组、mimics组、inhibitor组、siRNA NC组、PKG1 siRNA组、siRNA+inhibitor组；用NF-κB抑制剂PDTC处理细胞，分为Control组、PDTC组、PDTC+NC组、PDTC+mimics组。采用qPCR检测胎盘和滋养细胞HTR-8/SVneo中miR-155和PKG1 mRNA的表达；Western blot检测PKG1蛋白的表达；分别采用CCK-8法、Transwell法、流式细胞术检测细胞的增殖、迁移、凋亡能力。结果 PE组胎盘组织中miR-155的表达升高，而PKG1的表达降低（P<0.05）。体外实验表明，与NC组相比，miR-155 mimics组中PKG1的表达降低，滋养细胞迁移、增殖能力减弱，凋亡能力增强，miR-155 inhibitor组中PKG1表达则升高，滋养细胞迁移、增殖能力增强，凋亡能力减弱（P<0.05）；与Control组相比，PDTC组miR-155的表达降低，滋养细胞迁移、增殖能力增强，凋亡能力减弱（P<0.05）。结论 miR-155在子痫前期中表达上调，并且可在NF-κB的介导下通过下调PKG1影响滋养细胞的增殖、迁移和凋亡。

关键词: 先兆子痫, 细胞运动, 细胞增殖, 细胞凋亡, NF-κB, miR-155, PKG1, 滋养细胞

Abstract:

Objective To investigate the expression levels of miR-155 and cGMP-dependent protein kinase 1 (PKG1) in placental tissue of patients with preeclampsia, and the effect of miR-155 on the function of trophoblasts HTR-8/SVneo by inhibiting PKG1 under the mediation of nucleus factor kappa B (NF-κB). Methods Twenty placentas were collected from normal pregnant women and pre-eclampsia pregnant women who delivered by cesarean section. In vitro trophoblasts HTR-8/SVneo were cultured and divided into the NC group, the mimics group, the inhibitor group, the siRNA NC group, the PKG1 siRNA group and the siRNA+inhibitor group. Cells were treated with NF-κB inhibitor PDTC and divided into the control group, the PDTC group, the PDTC+NC group and the PDTC+mimics group. Real-time quantitative polymerase chain reaction (qPCR) was performed to detect the expression of miR-155 and PKG1 mRNA in placentas and HTR-8/SVneo cells. Western blot assay was performed to measure the level of PKG1 protein. The cell proliferation, migration and apoptosis were assessed by CCK-8 assay, Transwell assay and flow cytometry. Results The expression of miR-155 was significantly upregulated in placental tissue of the PE group, while the expression of PKG1 decreased significantly (P<0.05). The vitro experiments showed that compared with the NC group, the expression of PKG1 was significantly reduced in the miR-155 mimics group (P<0.05). The migration and proliferation ability of trophoblast was significantly weakened, the apoptotic ability was significantly enhanced (P<0.05). The expression of PKG1 was significantly increased in the miR-155 inhibitor group, the migration and proliferation ability of trophoblast was significantly enhanced, and the apoptotic ability was significantly weakened. Compared with the control group, the expression of miR-155 was significantly reduced in the PDTC group, the migration and proliferation ability of trophoblast was significantly enhanced, and the apoptotic ability was significantly weakened (P<0.05). Conclusion Results indicate that the expression of miR-155 is upregulated in preeclampsia, and can affect proliferation, migration and apoptosis of trophoblast cells by down-regulating PKG1 mediated by NF-κB.

Key words: pre-eclampsia, cell movement, cell proliferation, apoptosis, NF-kappa B, miR-155, PKG1, trophoblast

中图分类号:

R714.244

图/表 16

表1 qPCR引物序列

Tab.1 Oligonucleotide primer sequences for qPCR

基因名称	引物序列（5′→3′）	产物大小/bp
U6	上游：CTCGCTTCGGCAGCACA	96
	下游：AACGCTTCACGAATTTGCGT	96
miR-155	上游：ACACTCCAGCTGGGTTAATGCT AATCGTGATA	60
	下游：TTAATGCTAATCGTGATAGGGG	60
GAPDH	上游：TGTTCGTCATGGGTGTGAAC	154
	下游：ATGGCATGGACTGTGGTCAT	154
PKG1	上游：AACTCCACAAATGCCAGTCG	272
	下游：GCAACTGTCCTTGCCATACT	272

表2 2组产妇一般资料比较（n=20，$\bar{x}\pm s$）

Tab.2 Comparison of clinical characteristics between the two groups of patients

组别	年龄/岁	BMI/（kg/m2）	SBP/mmHg
PE组	29.7±3.5	29.1±2.3	155.3±11.2
NPE组	31.0±3.1	28.3±1.9	107.3±6.2
t	1.187	1.193	16.756^＊＊
组别	DPB/mmHg	分娩孕周/周	胎儿出生体质量/kg
PE组	98.0±9.4	36.6±2.1	2.6±0.7
NPE组	67.8±6.3	37.0±1.8	3.0±0.5
t	11.957^＊＊	0.730	3.235^＊

表3 2组胎盘组织中miR-155及PKG1 mRNA表达水平比较（n=20，$\bar{x}\pm s$）

Tab.3 Comparison of expression levels of miR-155 and PKG1 mRNA in placental tissue between the two groups

组别	miR-155	PKG1
PE组	4.41±2.06	1.52±0.90
NPE组	2.07±0.61	4.04±2.03
t	4.782^＊＊	5.076^＊＊

图1 Western blot检测2组胎盘组织中PKG1蛋白的表达量

Fig.1 Expression of PKG1 protein in placenta of two groups detected by Western blot assay

表4 miR-155 mimics和miR-155 inhibitor处理后各组miR-155、PKG1 mRNA和蛋白水平比较（n=3，$\bar{x}\pm s$）

Tab.4 Comparison of miR-155 and PKG1 mRNA expression levels after treatment with miR-155 mimics and miR-155 inhibitor between the three groups

组别	miR-155	PKG1 mRNA	PKG1蛋白
NC组	1.00±0.07	1.00±0.23	0.49±0.10
miR-155 mimics组	12.41±1.02^a	0.41±0.03^a	0.13±0.01^a
miR-155 inhibitor组	0.29±0.02^a	2.76±0.31^a	1.19±0.07^a
F	400.478^＊＊	135.734^＊＊	185.053^＊＊

图2 Western blot检测各组滋养细胞HTR-8/SVneo中PKG1蛋白的表达 A：NC组；B：miR-155 mimics组；C：miR-155 inhibitor组。

Fig.2 Expression of PKG1 protein in each group of trophoblast HTR-8/SVneo detected by Western blot assay

表5 miR-155 mimics和miR-155 inhibitor处理后各组细胞增殖、迁移及凋亡水平的比较（n=3，$\bar{x}\pm s$）

Tab.5 Comparison of cell proliferation, migration and apoptosis after treatment with miR-155 mimics and miR-155 inhibitor between the three groups

组别	增殖能力（A₄₅₀）
组别	24 h	48 h	72 h
NC组 miR-155 mimics组 miR-155 inhibitor组	0.26±0.02 0.25±0.01^a 0.29±0.01^a	0.43±0.21 0.32±0.03^a 0.50±0.03^a	0.64±0.19 0.46±0.02^a 0.87±0.04^a
F	6.048^＊	37.327^＊＊	160.188^＊＊
组别	迁移细胞数/（个/视野）		凋亡率/%
NC组 miR-155 mimics组 miR-155 inhibitor组	124.67±11.37 64.33±10.02^a 205.33±8.02^a		11.58±1.14 17.56±1.44^a 5.80±0.54^a
F	153.205^＊＊		85.114^＊＊

图3 miR-155 mimics和miR-155 inhibitor处理后各组细胞迁移情况（×200）

Fig.3 Cell migration of each group after treatment with miR-155 mimics and miR-155 inhibitor (×200)

图4 流式细胞术检测各组细胞凋亡情况

Fig.4 Detection of apoptosis in each group detected by flow cytometry

表6 抑制PKG1的表达后各组miR-155、PKG1 mRNA和蛋白水平比较（n=3，$\bar{x}\pm s$）

Tab.6 Comparison of miR-155, PKG1 mRNA and protein levels after inhibiting the expression of PKG1 between the five groups

组别	miR-155	PKG1 mRNA	PKG1蛋白
NC组	0.99±0.01	1.02±0.04	0.21±0.04
inhibitor组	0.32±0.04^a	3.06±0.19^a	0.83±0.03^a
siRNA NC组	1.07±0.09	1.00±0.03	0.25±0.05
PKG1 siRNA组	1.04±0.08	0.25±0.04	0.10±0.00
siRNA+inhibitor组	0.65±0.07^b	0.64±0.08^b	0.34±0.00^b
F	10.933^＊	12.000^＊	67.230^＊＊

图5 Western blot检测各组滋养细胞HTR-8/SVneo中PKG1蛋白的表达 A：NC组；B：inhibitor组；C：siRNA NC组；D：PKG1 siRNA组；E：siRNA+inhibitor组。

Fig.5 Expression of PKG1 protein in each group of trophoblast HTR-8/SVneo detected by Western blot assay

表7 抑制NF-κB表达后各组细胞中miR-155、PKG1 mRNA和蛋白表达水平比较（n=3，$\bar{x}\pm s$）

Tab.7 Comparison of miR-155 mimics and miR-155 mRNA expression levels after inhibiting the expression of NF-κB between the four groups

组别	miR-155 mRNA	PKG1 mRNA	PKG1蛋白
Control组	1.00±0.03	1.01±0.13	0.35±0.02
PDTC组	0.56±0.05^a	3.24±0.23^a	1.00±0.04^a
PDTC+NC组	0.57±0.01	3.13±0.25	1.01±0.05
PDTC+ mimics组	10.11±0.28^b	0.50±0.01^b	0.11±0.03^b
F	3 233.927^＊＊	182.535^＊＊	311.788^＊＊

图6 Western blot检测各组滋养细胞HTR-8/SVneo中PKG1蛋白的表达 A：Control组；B：PDTC组；C：PDTC+NC组；D：PDTC+ mimics组。

Fig.6 Expression of PKG1 protein in each group of trophoblast HTR-8/SVneo detected by Western blot assay

表8 抑制NF-κB表达后各组细胞增殖、迁移及凋亡率比较（n=3，$\bar{x}\pm s$）

Tab.8 Comparison of cell proliferation, migration and apoptosis after inhibiting the expression of NF-κB between the four groups

组别	增殖能力（A₄₅₀）
组别	24 h	48 h	72 h
Control组	0.26±0.01	0.42±0.04	0.63±0.02
PDTC组	0.29±0.02^a	0.59±0.04^a	0.93±0.04^a
PDTC+NC组	0.30±0.03	0.53±0.03	0.90±0.06
PDTC+mimics组	0.24±0.02^b	0.33±0.01^b	0.45±0.03^b
F	6.267^＊	38.309^＊＊	104.970^＊＊
组别	迁移细胞数/（个/视野）		凋亡率/%
Control组	116.00±5.29		10.13±0.96
PDTC组	169.67±8.62^a		6.25±1.06^a
PDTC+NC组	156.67±4.17		6.59±1.09
PDTC+mimics组	64.00±6.25^b		15.56±0.06^b
F	171.173^＊＊		69.012^＊＊

图7 抑制NF-κB的表达后各组细胞迁移情况（×200）

Fig.7 Cell migration of each group after inhibiting the expression of NF-κB (×200)

图8 流式细胞术检测抑制NF-κB表达后各组细胞凋亡情况

Fig.8 Detection of apoptosis in each group after inhibition of NF-κB expression by flow cytometry

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miR-155通过调控PKG1影响滋养细胞生物学功能并参与子痫前期的机制探讨

MiR-155 affects the biological functions of trophoblastic cells through regulating cGMP-dependent kinase 1 and is involved in the mechanism of preeclampsia

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