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

doi:10.3877/cma.j.issn.1673-5250.2022.02.007

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 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
组别	miR-195	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
组别	miR-195	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

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

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

[1]	漆洪波，童超．从母胎界面着眼探究子痫前期的发病机制[J]．中国实用妇科与产科杂志，2020，36(1): 61-64. DOI：10.19538/j.fk2020010115.
[2]	董环，郝冉，宋园园，等. 慢性疼痛相关MicroRNA的研究进展[J]. 实用医学杂志，2018，34(8): 1390-1393. DOI：10.3969/j.issn.1006-5725.2018.08.042.
[3]	MA YY, Liang XX, Wu H, et al. Long non-coding RNA NR_002794 is up-regulated in pre-eclampsia and regulates the proliferation, apoptosis and invasion of trophoblast cells[J]. Mol Med Rep, 2019, 20(5): 4567-4575. DOI: 10.3892/mmr.2019.10701
[4]	Han L, Luo QQ, Peng MG, et al. miR-483 is downregulated in pre-eclampsia via targeting insulin-like growth factor 1 (IGF1) and regulates the PI3K/Akt/mTOR pathway of endothelial progenitor cells[J]. J Obstet Gynaecol Res, 2020, 47(1): 63-72. DOI: 10.1111/jog.14412.
[5]	Wu L, Song WY, Xie Y, et al. miR-181a-5p suppresses invasion and migration of HTR-8/SVneo cells by directly targeting IGF2BP2[J]. Cell Death Dis, 2018, 9(2): 16. DOI: 10.1038/s41419-017-0045-0.
[6]	Zuo W, Zhang W, Xu F, et al. Long non-coding RNA LINC00485 acts as a microRNA-195 sponge to regulate the chemotherapy sensitivity of lung adenocarcinoma cells to cisplatin by regulating CHEK1[J]. Cancer Cell Int, 2019, 12(19)：240.
[7]	Yu X, Zhang Y, Wu B, et al. The miR-195 axis regulates chemoresistance through TUBB and lung cancer progression through BIRC5[J]. Mol Ther Oncolytics, 2019, 14: 288-298. DOI: 10.1016/j.omto.2019.07.004.
[8]	Do HTT, Lee CH, Cho J. Chemokines and their receptors: multifaceted roles in cancer progression and potential value as cancer prognostic markers[J]. Cancers (Basel), 2020, 12(2): 287. DOI: 10.3390/cancers12020287.
[9]	Eltounali SA, Moodley J, Naicker T. Role of kidney biomarkers [kidney injury molecule-1, calbindin, interleukin-18 and monocyte chemoattractant protein-1]in HIV associated pre-eclampsia[J]. Hypertens Pregnancy, 2017, 36(4): 288-294. DOI: 10.1080/10641955.2017.1385793.
[10]	谢幸，苟文丽．妇产科学[M].8版．北京：人民卫生出版社，2013: 64-71.
[11]	Han X, Niu C, Zuo Z, et al. MiR-342-3p inhibition promotes cell proliferation and invasion by directly targeting ID4 in pre-eclampsia[J]. J Obstet Gynaecol Res, 2020, 46(1): 49-57. DOI: 10.1111/jog.14150.
[12]	Tang H, Pan L, Xiong Y, et al. Down-regulation of the Sp1 transcription factor by an increase of microRNA-4497 in human placenta is associated with early recurrent miscarriage[J]. Reprod Biol Endocrinol, 2021, 19(1): 21. DOI: 10.1186/s12958-021-00701-8.
[13]	Niu ZR, Han T, Sun XL, et al. MicroRNA-30a-3p is overexpressed in the placentas of patients with preeclampsia and affects trophoblast invasion and apoptosis by its effects on IGF-1[J]. Am J Obstet Gynecol, 2018, 218(2): 249. e1-249. e12. DOI: 10.1016/j.ajog.2017.11.568.
[14]	Wang X, He C, Yang Z, et al. Dysregulation of long non-coding RNA SNHG12 alters the viability, apoptosis, and autophagy of prostate cancer cells by regulating miR-195/CCNE1 axis[J]. Int J Clin Exp Pathol, 2019, 12(4): 1272-1283.
[15]	Yang K, Tang H, Ding M, et al. Expression of miR-195 and MEK1 in patients with bladder cancer and their relationship to prognosis[J]. Int J Clin Exp Pathol, 2019, 12(3): 843-850.
[16]	Zhao XM, Dai LL, Yue QF, et al. MiR-195 inhibits migration, invasion and epithelial-mesenchymal transition (EMT) of endometrial carcinoma cells by targeting SOX4[J]．J Biosci, 2019, 44(6): 146. DOI：10.1007/s12038-019-9966-3.
[17]	彭美茜，徐丽云，杨丹，等. Drosha通过调控微RNA-195-5P促进胃癌细胞的迁移和侵袭[J]．肿瘤，2018，38(10): 925-932.
[18]	Naito Y, Yamamoto Y, Sakamoto N, et al. Cancer extracellular vesicles contribute to stromal heterogeneity by inducing chemokines in cancer-associated fibroblasts[J]. Oncogene, 2019, 38(28): 5566-5579. DOI: 10.1038/s41388-019-0832-4.
[19]	Sebrell TA, Hashimi M, Sidar B, et al. A novel gastric spheroid co-culture model reveals chemokine-dependent recruitment of human dendritic cells to the gastric epithelium[J]. Cell Mol Gastroenterol Hepatol, 2019, 8(1): 157-171. e3. DOI: 10.1016/j.jcmgh.2019.02.010.
[20]	康玲，彭诗元，秦源，等. CCL28通过增强MMP-2活力促进滋养细胞浸润功能[J]．现代妇产科进展，2017，26(5): 337-341. DOI: 10.13283/j.cnki.xdfckjz.2017.05.005.
[21]	Li H, Meng YH, Shang WQ, et al. Chemokine CCL24 promotes the growth and invasiveness of trophoblasts through ERK1/2 and PI3K signaling pathways in human early pregnancy[J]. Reproduction, 2015, 150(5): 417-427. DOI: 10.1530/REP-15-0119.
[22]	达成利，帕拉提·艾斯卡，比力克孜·玉素甫. 趋化因子CCL5在涎腺腺样囊性癌患者肿瘤组织和血清中的表达及临床意义[J]．实用癌症杂志，2019，34(6)：891-894. DOI: 10.3969/j.issn.1001-5930.2019.06.006.
[23]	梁亮，邓骞，聂治军，等. 趋化因子CCL5表达与肾透明细胞癌病理特征及预后的相关性研究[J]．现代肿瘤医学，2018，26(3)：405-408. DOI: 10.3969/j.issn.1672-4992.2018.03.020.
[24]	Liu GT, Chen HT, Tsou HK, et al. CCL5 promotes VEGF-dependent angiogenesis by down-regulating miR-200b through PI3K/Akt signaling pathway in human chondrosarcoma cells[J]. Oncotarget, 2014, 5(21): 10718-10731. DOI: 10.18632/oncotarget.2532
[25]	Chao CC, Lee CW, Chang TM, et al. CXCL1/CXCR2 paracrine axis contributes to lung metastasis in osteosarcoma[J]. Cancers (Basel), 2020, 12(2): 459. DOI: 10.3390/cancers12020459.
[26]	Zhou B, Sun C, Li N, et al. Cisplatin-induced CCL5 secretion from CAFs promotes cisplatin-resistance in ovarian cancer via regulation of the STAT3 and PI3K/Akt signaling pathways[J]. Int J Oncol, 2016, 48(5): 2087-2097. DOI: 10.3892/ijo.2016.3442.

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