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中华妇幼临床医学杂志(电子版) ›› 2022, Vol. 18 ›› Issue (02) : 165 -174. doi: 10.3877/cma.j.issn.1673-5250.2022.02.007

论著

微小RNA-195靶向趋化因子5抑制滋养细胞增殖、迁移和侵袭及其机制研究
程慧, 李妍雨, 张蓓, 成杰, 张艳玲†()   
  1. 徐州市中心医院妇产科　221009
  • 收稿日期:2021-04-08 修回日期:2022-02-27 出版日期:2022-04-01
  • 通信作者: 张艳玲

MicroRNA-195 targeting chemokine 5 inhibits proliferation, metastasis and invasion of trophoblast cells and its mechanism

Hui Cheng, Yanyu Li, Bei Zhang, Jie Cheng, Yanling Zhang†()   

  1. Department of Obstetrics and Gynecology, Xuzhou Central Hospital, Xuzhou 221009, Jiangsu Province, China
  • Received:2021-04-08 Revised:2022-02-27 Published:2022-04-01
  • Corresponding author: Yanling Zhang
  • Supported by:
    Scientific Research Plan of Jiangsu Provincial Health and Family Planning Commission(F201662)
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引用本文：

程慧, 李妍雨, 张蓓, 成杰, 张艳玲. 微小RNA-195靶向趋化因子5抑制滋养细胞增殖、迁移和侵袭及其机制研究[J]. 中华妇幼临床医学杂志(电子版), 2022, 18(02): 165-174.

Hui Cheng, Yanyu Li, Bei Zhang, Jie Cheng, Yanling Zhang. MicroRNA-195 targeting chemokine 5 inhibits proliferation, metastasis and invasion of trophoblast cells and its mechanism[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2022, 18(02): 165-174.

目的

探讨微小RNA(miR)-195通过靶向调控趋化因子(CCL)5对胎盘滋养细胞增殖、侵袭的影响及其分子机制。

方法

采用随机数字表法，选取2018年8月至2019年8月在徐州市中心医院定期产前检查的70例孕妇，按照是否合并子痫前期(PE)，将其分为PE组(n＝40)和对照组(n＝30)。选取2组受试者分娩时自胎盘母-胎界面组织中分离的滋养细胞株HTR-8/SVneo细胞为研究对象，针对PE组胎盘滋养层细胞分别转染miR-195及RNA阴性对照序列。根据转染结果，将PE组胎盘滋养层细胞分为miR-195 mimics亚组、miR-NC亚组、miR-195+pcDNA亚组、miR-195+pcDNA-CCL5亚组。采用实时荧光定量聚合酶链式反应(RT-PCR)检测研究组与对照组胎盘组织中miR-195、CCL5 mRNA相对表达水平；MTT实验检测各亚组滋养细胞增殖能力，Transwell侵袭实验观察细胞侵袭能力，细胞划痕实验观察各亚组滋养细胞迁移能力，Western blotting法检测CCL5、PI3K及AKT蛋白相对表达水平；Pearson相关性分析对miR-195及CCL5表达相关性进行分析；荧光素酶实验验证miR-195与CCL5的靶向关系。本研究遵循的程序符合徐州市中心医院伦理委员会规定，通过该伦理委员会审查，并获得批准(审批文号：XZXY-LI-20181215-031)。所有受试者均签署临床研究知情同意书。

结果

①PE组和对照组孕妇年龄、孕龄等一般临床资料比较，差异无统计学意义(P>0.05)。2组孕妇血压及尿蛋白水平比较，差异有统计学意义(P<0.05)。②PE组患者胎盘组织中miR-195 mRNA相对表达水平高于对照组(t＝10.932，P<0.001)；PE组患者胎盘组织中CCL5 mRNA、蛋白相对表达水平低于对照组(t＝11.125、13.253，P<0.001)。③miR-195 mimics亚组滋养细胞中miR-195相对表达水平高于miR-NC亚组(P<0.001)，miR-195 mimics亚组第1、2、3、4天细胞活力低于miR-NC亚组，并且差异均有统计学意义(P<0.05)。④细胞培养72 h后，miR-195 mimics亚组侵袭细胞数、细胞迁移率均低于miR-NC亚组，并且差异均有统计学意义(t＝11.327，18.357，P<0.001)。⑤miR-195 mimics亚组细胞CCL5蛋白相对表达水平低于miR-NC亚组(P<0.001)；miR-195 mRNA水平与CCL5蛋白相对表达水平呈负相关关系(r＝-0.326，P<0.001)。⑥miR-195 mimics亚组细胞中PI3K和AKT蛋白相对表达水平低于miR-NC亚组；miR-195+pcDNA-CCL5亚组细胞中PI3K和AKT蛋白相对表达水平均高于miR-195+pcDNA亚组，并且差异均有统计学意义(P<0.05)。

结论

miR-195可能通过抑制CCL5的表达，进而抑制其下游PI3K/AKT信号通路的活化，最终影响滋养细胞的侵袭及迁移能力，从而导致PE的发生。

关键词: 微RNAs, 微小RNA-195, 趋化因子CCL5, 滋养细胞, 细胞增殖, 侵袭, 先兆子痫
Objective

To investigate effects of microRNA (miR)-195 on the proliferation and invasion of trophoblast cells by targeting chemokine (CCL)5 and its molecular mechanism.

Methods

Forty pre-eclampsia (PE) pregnant women (PE group) and 30 healthy pregnant women (control group) were selected by randon number table method. The trophoblast cell line HTR-8/SVneo was transfected with miR-195 and RNA-negative control sequences, respectively. According to the transfer results, placental trophoblast cells in PE group were divided into miR-195 mimics subgroup, miR-NC subgroup, miR-195+ pcDNA subgroup, miR-195+ pcDNA-CCL5 subgroup. Real-time polymerase chain reaction (RT-PCR) was used to detect the expression level of miR-195, CCL5 mRNA. The cell proliferation ability of each group was detected by MTT. The cell invasion ability was detected by Transwell invasion experiment. The ability of cell migration in each group was observed by cell scratch test. The expression of CCL5, PI3K and AKT protein in each group were detected by Western blotting. The correlation between the expressions of miR-195 and CCL5 was analyzed by Pearson correlation analysis. Luciferase experiment was used to verify the targeting relationship between miR-195 and CCL5. The procedure followed in this study met the standards formulated by the Ethics Review Committee of Xuzhou Central Hospital and has been approved by it (Approval No. KS2204). Informed consent was obtained from each participant.

Results

①There was no significant difference between PE group and control group in general clinical data such as age, gestational age (P>0.05). The blood pressure and urine protein levels of pregnant women in two groups were significantly different (P<0.05). ②The relative expression level of miR-195 mRNA in placental tissue of PE group was higher than that of control group (t=10.932, P<0.001), and the relative expression of CCL5 mRNA and protein in placental tissue of PE group were lower than those of control group (t=11.125, 13.253; P<0.001). ③The level of miR-195 in miR-195 mimics subgroup was higher than that in miR-NC subgroup (P<0.001). The activity of cells on 1, 2, 3, 4 d in miR-195 mimics subgroup were lower than those in miR-NC subgroup (P<0.05). ④After the cells were cultured for 72 h, the number of invasive cells and cell mobility in miR-195 mimics subgroup were lower than those in miR-NC subgroup, and the differences were statistically significant (t=11.327, 18.357, P<0.001). ⑤The relative expression level of CCL5 protein in miR-195 mimics subgroup was lower than that in miR-NC subgroup (P<0.001). The relative expression level of miR-195 mRNA was negatively correlated with the relative expression level of CCL5 protein (r=-0.326, P<0.001). ⑥The relative expression level of PI3K and AKT in miR-195 mimics subgroup was lower than that in miR-NC subgroup (P<0.05). The relative expression level of PI3K and AKT in miR-195+ pcDNA-CCL5 subgroup was higher than that in miR-195 pcDNA subgroup (P<0.05).

Conclusions

miR-195 may inhibit the activation of PI3K/AKT signaling pathway by inhibiting the expression of CCL5, affecting the invasion and migration ability of trophoblasts contributing to the development of PE.

Key words: MicroRNAs, microRNA-195, Chemokine CCL5, Trophoblast cells, Cell proliferation, Invasion, Pre-eclampsia
表1 CCL5、miR-195、U6目标基因引物序列
目标基因 上游引物序列 下游引物序列
CCL5 5′-CAGCAAACGCAAGGATGTC-3′ 5′-AAGGCGTAGAGAACGGGATT-3′
miR-195 5′-ATAACCTACCAGCGCCGAATTCGGGACCA-3′ 5′-ACCACCGACTTGTGACA-3′
U6 5′-TATTGACATTAGTTAAT-3′ 5′-TTTAGAAGACTCCCTAGAGTA-3′
表1 2组受试者一般临床资料比较(±s)
组别 例数 年龄(岁) 孕龄(周)
PE组 40 30.3±2.6 37.5±3.5
对照组 30 29.4±3.1 37.6±2.7
t值   1.235 1.002
P值   0.159 0.202
表2 2组孕妇血压及尿蛋白水平比较(±s)
组别 例数 收缩压(mmHg) 舒张压(mmHg) 24 h尿蛋白(g/24 h)
PE组 40 169.6±6.8 114.5±4.3 5.7±1.1
对照组 30 111.7±8.1 69.1±5.2 0±0
t值   9.032 12.549 14.861
P值   <0.001 <0.001 <0.001
表3 2组孕妇胎盘组织中miR-195 mRNA、CCL5 mRNA及CCL5蛋白相对表达水平比较(%，±s)
组别 例数 miR-195 mRNA CCL5 mRNA CCL5蛋白
PE组 40 89.2±11.3 28.8±1.4 1.3±0.5
对照组 30 40.2±6.4 61.1±5.3 4.3±0.8
t值   10.932 11.125 13.253
P值   <0.001 <0.001 <0.001
表4 胎盘滋养细胞中miR-195过表达对滋养细胞增殖的影响(±s)
组别 miR-195 细胞活力
0 d 1 d 2 d 3 d 4 d
miR-195 mimics亚组 3.803±0.243 0.401±0.038 0.513±0.182 0.593±0.078 0.792±0.304 0.903±0.331
miR-NC亚组 0.267±0.023 0.398±0.044 0.611±0.284 0.803±0.053 1.258±0.195 1.583±0.477
t值 49.834 0.162 2.834 3.023 4.103 4.834
P值 <0.001 0.624 0.026 0.013 0.008 0.003
表5 胎盘滋养细胞中miR-195对滋养细胞侵袭、迁移的影响(±s)
组别 侵袭细胞数(个) 细胞迁移率(%)
miR-195 mimics亚组 78.3±6.3 22.1±2.5
miR-NC亚组 114.1±5.6 51.8±5.4
t值 11.327 18.357
P值 <0.001 <0.001
图1 miR-195与CCL5的靶向关系(图1A：双荧光素酶结果；图1B：miR-195与CCL5的靶向结合位点预测图)注：CCL5 WT为野生型CCL5基因序列质粒，CCL5 MUT1为突变型CCL5基因序列质粒1，CCL5 MUT2为突变型CCL5基因序列质粒2，miR-195 mimics为miR-195过表达质粒，CCL5 mut为突变型CCL5序列，miR-195为微小RNA-195，CCL5为趋化因子5
图2 胎盘滋养细胞中miR-195过表达对CCL5蛋白的影响(图2A：Western blotting法检测miR-195 mimics亚组与miR-NC亚组滋养细胞CCL5蛋白表达电泳条带图；图2B：miR-195 mimics亚组与miR-NC亚组滋养细胞CCL5蛋白相对表达水平；图2C：miR-195 mRNA相对表达水平与CCL5蛋白相对表达水平相关性分析散点图)注：①为miR-195 mimics亚组，②为miR-NC亚组；miR-195为微小RNA-195，CCL5为趋化因子5
图3 miR-195调控CCL5影响滋养细胞PI3K/AKT通路(图3A：Western blotting法检测4个亚组滋养细胞PI3K和AKT蛋白表达电泳条带图；图3B：4个滋养细胞亚组PI3K和AKT蛋白相对表达水平)注：①为miR-195 mimics亚组，②为miR-195+pcDNA亚组，③为miR-NC亚组，④为miR-195+pcDNA-CCL5亚组。miR-195为微小RNA-195，CCL5为趋化因子5
图4 miR-195调控CCL5影响滋养细胞p-PI3K、p-AKT蛋白作用(图4A：Western blotting法检测4个亚组滋养细胞p-PI3K和p-AKT蛋白表达电泳条带图；图4B：4个亚组滋养细胞p-PI3K和p-AKT蛋白相对表达水平)注：①为miR-195 mimics亚组，②为miR-195+pcDNA亚组，③为miR-NC亚组，④为miR-195+pcDNA-CCL5亚组。miR-195为微小RNA-195，CCL5为趋化因子5
表6 4个亚组滋养细胞PI3K、p-PI3K和AKT、p-AKT蛋白相对表达水平(%，±s)
组别 PI3K p-PI3K AKT p-AKT
miR-195 mimics亚组 30.4±7.2 26.4±6.7 11.3±3.3 24.4±4.2
miR-195+pcDNA亚组 43.2±10.1 35.3±8.3 21.1±6.3 30.9±5.5
miR-NC亚组 68.2±1.3 51.8±9.3 37.4±2.2 49.2±7.1
miR-195+pcDNA-CCL5亚组 70.1±11.6 69.7±10.2 41.7±9.3 58.7±8.4
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