
天津医药 ›› 2023, Vol. 51 ›› Issue (9): 928-934.doi: 10.11958/20221869
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,△(
)
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: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:基金资助:
YUE Jinjing1(
), ZENG Ying1, GUO Xiaopei1, DONG Yue1, JI Ruonan1, PENG Rui2, LUO Xiaohua2,△(
)
Received:2022-11-18
Revised:2023-03-02
Published:2023-09-15
Online:2023-09-13
Contact:
△E-mail: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:
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。
中图分类号:
| 基因名称 | 引物序列(5′→3′) | 产物大小/bp |
|---|---|---|
| U6 | 上游:CTCGCTTCGGCAGCACA | 96 |
| 下游:AACGCTTCACGAATTTGCGT | ||
| miR-155 | 上游:ACACTCCAGCTGGGTTAATGCT AATCGTGATA | 60 |
| 下游:TTAATGCTAATCGTGATAGGGG | ||
| GAPDH | 上游:TGTTCGTCATGGGTGTGAAC | 154 |
| 下游:ATGGCATGGACTGTGGTCAT | ||
| PKG1 | 上游:AACTCCACAAATGCCAGTCG | 272 |
| 下游:GCAACTGTCCTTGCCATACT |
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 | ||
| miR-155 | 上游:ACACTCCAGCTGGGTTAATGCT AATCGTGATA | 60 |
| 下游:TTAATGCTAATCGTGATAGGGG | ||
| GAPDH | 上游:TGTTCGTCATGGGTGTGAAC | 154 |
| 下游:ATGGCATGGACTGTGGTCAT | ||
| PKG1 | 上游:AACTCCACAAATGCCAGTCG | 272 |
| 下游:GCAACTGTCCTTGCCATACT |
| 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* |
| 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** |
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** |
| 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±1.02a | 0.41±0.03a | 0.13±0.01 a |
| miR-155 inhibitor组 | 0.29±0.02a | 2.76±0.31a | 1.19±0.07a |
| F | 400.478** | 135.734** | 185.053** |
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±1.02a | 0.41±0.03a | 0.13±0.01 a |
| miR-155 inhibitor组 | 0.29±0.02a | 2.76±0.31a | 1.19±0.07a |
| 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
| 组别 | 增殖能力(A450) | ||
|---|---|---|---|
| 24 h | 48 h | 72 h | |
| NC组 miR-155 mimics组 miR-155 inhibitor组 | 0.26±0.02 0.25±0.01a 0.29±0.01a | 0.43±0.21 0.32±0.03a 0.50±0.03a | 0.64±0.19 0.46±0.02a 0.87±0.04a |
| 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±10.02a 205.33±8.02a | 11.58±1.14 17.56±1.44a 5.80±0.54a | |
| F | 153.205** | 85.114** | |
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) | ||
|---|---|---|---|
| 24 h | 48 h | 72 h | |
| NC组 miR-155 mimics组 miR-155 inhibitor组 | 0.26±0.02 0.25±0.01a 0.29±0.01a | 0.43±0.21 0.32±0.03a 0.50±0.03a | 0.64±0.19 0.46±0.02a 0.87±0.04a |
| 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±10.02a 205.33±8.02a | 11.58±1.14 17.56±1.44a 5.80±0.54a | |
| 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)
| 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±0.04a | 3.06±0.19a | 0.83±0.03a |
| 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±0.07b | 0.64±0.08b | 0.34±0.00b |
| F | 10.933* | 12.000* | 67.230** |
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±0.04a | 3.06±0.19a | 0.83±0.03a |
| 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±0.07b | 0.64±0.08b | 0.34±0.00b |
| 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
| 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±0.05a | 3.24±0.23a | 1.00±0.04a |
| PDTC+NC组 | 0.57±0.01 | 3.13±0.25 | 1.01±0.05 |
| PDTC+ mimics组 | 10.11±0.28b | 0.50±0.01b | 0.11±0.03b |
| F | 3 233.927** | 182.535** | 311.788** |
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±0.05a | 3.24±0.23a | 1.00±0.04a |
| PDTC+NC组 | 0.57±0.01 | 3.13±0.25 | 1.01±0.05 |
| PDTC+ mimics组 | 10.11±0.28b | 0.50±0.01b | 0.11±0.03b |
| 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
| 组别 | 增殖能力(A450) | ||
|---|---|---|---|
| 24 h | 48 h | 72 h | |
| Control组 | 0.26±0.01 | 0.42±0.04 | 0.63±0.02 |
| PDTC组 | 0.29±0.02a | 0.59±0.04a | 0.93±0.04a |
| PDTC+NC组 | 0.30±0.03 | 0.53±0.03 | 0.90±0.06 |
| PDTC+mimics组 | 0.24±0.02b | 0.33±0.01b | 0.45±0.03b |
| 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±8.62a | 6.25±1.06a | |
| PDTC+NC组 | 156.67±4.17 | 6.59±1.09 | |
| PDTC+mimics组 | 64.00±6.25b | 15.56±0.06b | |
| F | 171.173** | 69.012** | |
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) | ||
|---|---|---|---|
| 24 h | 48 h | 72 h | |
| Control组 | 0.26±0.01 | 0.42±0.04 | 0.63±0.02 |
| PDTC组 | 0.29±0.02a | 0.59±0.04a | 0.93±0.04a |
| PDTC+NC组 | 0.30±0.03 | 0.53±0.03 | 0.90±0.06 |
| PDTC+mimics组 | 0.24±0.02b | 0.33±0.01b | 0.45±0.03b |
| 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±8.62a | 6.25±1.06a | |
| PDTC+NC组 | 156.67±4.17 | 6.59±1.09 | |
| PDTC+mimics组 | 64.00±6.25b | 15.56±0.06b | |
| F | 171.173** | 69.012** | |
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