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. 2020 Jan 30;40(1):79-86.
doi: 10.12122/j.issn.1673-4254.2020.01.13.

[miR-34a-5p regulates viability, invasion and apoptosis of placental trophoblastic cells via modulating CDK6 and PI3K/AKT pathway]

[Article in Chinese]
Qin Li  1 , Juanxiu Xu  2
Affiliations

[miR-34a-5p regulates viability, invasion and apoptosis of placental trophoblastic cells via modulating CDK6 and PI3K/AKT pathway]

[Article in Chinese]
Qin Li et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the roles of microRNA (miR)-34a-5p and cyclin-dependent kinase (CDK) 6 in the regulation of cell viability, apoptosis and invasion of human placental trophoblastic cells and the relationship between miR-34a-5p and CDK6.

Methods: We examined the expression of miR-34a-5p using RT-qPCR in cultured human trophoblast HTR-8/Svneo cells and human choriocarcinoma cell lines BeWo and JEG-3HTR-8/Svneo. HTR-8/Svneo cells transfected with a miR-34a-5p-mimic, the miR-34a-5p-inhibitor, or pcDNA-CDK6 along with the mimic group were analyzed for changes in cell proliferation using MTT assay; the apoptosis of the cells were assessed by detecting caspase 3 activity and cleaved caspase 3 protein expression, and the cell invasion was evaluated using Transwell assay. Western blotting was used to determine the protein levels of CDK6, cleaved caspase 3, and MMP-9 in the cells. The interaction between CDK6 and miR-34a-5p analyzed using a luciferase reporter assay.

Results: Transfection with the miR-34a-5p mimic significantly reduced the viability (P=0.000), suppressed the invasion (P=0.049), enhanced the cell apoptosis (P=0.018), down-regulated the expressions of MMP-9 (P=0.004) and CDK6 (P=0.014), and up-regulated caspase 3 activity (P=0.018) and cleaved caspase 3 expression (P=0.003) in cultured HTR-8/Svneo cells. CDK6 was confirmed as one of the target gene of miR-34a-5p. Transfection with pcDNA-CDK6 significantly reversed the effects of miR- 34a-5p overexpression on the cell viability (P=0.000), apoptosis (P=0.015), and invasion (P=0.046). Treatment of the cells with insulin-like growth factor 1 (IGF-1), an activator of the PI3K/AKT pathway, also significantly attenuated the effects of miR-34a- 5p overexpression on the cell viability (P=0.011), apoptosis (P=0.004), and invasion (P=0.002).

Conclusions: miR-34a-5p promotes apoptosis and inhibits the viability and invasion of human placental trophoblastic cells by down-regulating CDK6 and inactivating the PI3K/AKT pathway.

目的: 探讨miR-34a-5p及CDK6对滋养层细胞增殖，浸润和凋亡的作用和下游机制以及二者的调节关系。

方法: 培养滋养层细胞HTR-8/Svneo和人绒毛膜癌细胞系BeWo和JEG-3HTR-8/Svneo，使用RTqPCR法检测3种细胞中miR-34a-5p的表达差异。根据对HTR-8/Svneo细胞的不同处理进行如下分组：无处理的HTR-8/Svneo细胞（对照组）；miR-34a-5p拟似物mimic转染细胞（mimic组），pcDNA-CDK6和mimic共转染HTR-8/Svneo细胞（pcDNA-CDK6+mimic组），miR-34a-5p抑制剂inhibitor转染HTR-8/Svneo细胞（inhibitor组）。MTT法检测细胞增殖，ELISA法测定caspase 3活性检测细胞凋亡水平，Transwell实验检测细胞浸润能力；Western blot检测细胞周期依赖性蛋白激酶CDK6，凋亡标记物cleaved-caspase 3，浸润标记物MMP-9的表达；荧光素酶报告基因实验确认miR-34a-5p对CDK-6的直接靶向关系。

结果: 过表达miR-34a-5p抑制HTR-8/Svneo细胞的增殖活性（P=0.000）和浸润能力（P=0.049），下调细胞中MMP-9（P=0.004）和CDK6（P=0.014）的表达水平，上调caspase 3活性（P=0.018）和cleaved caspase 3的表达水平（P=0.003）；CDK6是miR-34a-5p的靶基因，过表达CDK6削弱miR-34a-5p对细胞增殖（P=0.000）、凋亡（P=0.015）和浸润（P=0.046）的作用；使用IFG-1激活PI3K/AKT通路部分逆转miR-34a-5p对细胞增殖（P=0.011）、凋亡（P=0.004）和浸润（P=0.002）的作用。

结论: miR-34a-5p通过调控CDK6表达和PI3K/AKT信号通路激活调节滋养层细胞的增殖，浸润和凋亡。

Keywords: apoptosis; cell proliferation; cyclin-dependent kinase 6; human placental trophoblastic cells; invasion; miR-34a-5p.

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Figures

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miR-34a-5p体外表达水平miR-34a-5p在培养的人滋养层细胞系HTR-8/Svneo及人绒毛膜癌细胞系BeWo和JEG-3中的表达水平 Expression of miR-34a-5p in cultured human trophoblast cell line HTR-8 /Svneo and human chorionic cancer cell lines BeWo and JEG-3. *P < 0.05 vs BeWo cells.
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miR-34a-5p对HTR-8/Svneo细胞增殖活性的作用 Effect of transfection with miR-34a-5p mimic on proliferation of HTR-8/Svneo cells. A: The miR-34a-5p mimic was used to induce miR-34a-5p overexpression in HTR-8/Svneo cells; B: Changes in survival rate of HTR-8 /Svneo cells; C: Changes in CDK6 protein expression level in HTR-8/Svneo cells. *P < 0.05 vs control group.
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miR-34a-5p对HTR-8/Svneo细胞凋亡的作用 Effect of transfection with miR-34a-5p mimic on apoptosis of HTR-8 /Svneo cells. A: Change in caspase 3 activity in HTR-8/Svneo cells; B: Change in cleaved caspase 3 protein expression levels in HTR-8 /Svneo cells. *P < 0.05 vs control group.
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miR-34a-5p对HTR-8/Svneo细胞浸润能力的作用 Effect of transfection with miR-34a-5p mimic on invasion capacity of HTR-8 /Svneo cells. A: Transwell assay to detect the changes in HTR-8/Svneo cell invasion (Original magnification: ×200); B: Changes in MMP-9 protein expression in HTR-8/Svneo cells. *P < 0.05 vs control group.
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CDK6是miR-34a-5p靶基因之一 CDK6 is one of the target genes of miR-34a-5p. A: Direct interaction between CDK6 and miR-34a-5p detected by luciferase reporter gene assay; B: Changes in survival rate of HTR-8/Svneo cells; C: Changes in caspase-3 activity in HTR-8/Svneo cells; D: Transwell assay to detect the changes in HTR-8/Svneo cell infiltration number (×200). *P < 0.05 vs negative control group or control group; #P < 0.05 vs mimic group.
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PI3K/AKT是miR-34a-5p发挥作用的关键信号通路 PI3K/AKT is the key signaling pathway for miR-34a-5p to function. A: An inhibitor was used to inhibit miR-34a-5p in HTR-8/Svneo cells (*P < 0.05 vs control group); B-E: PI3K/AKT signal protein expression bands detected by Western blotting (*P < 0.05 vs control group); F: The changes in the survival rate of HTR-8/Svneo cells; G: Changes in caspase 3 activity in HTR-8/Svneo cells; H: Transwell invasion assay for detecting the changes of HTR-8/Svneo cell invasion (×200). F-H: *P < 0.05 vs control group; #P < 0.05 vs mimic group.
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