Effect of miR-133 on apoptosis of trophoblasts in human placenta tissues via Rho/ROCK signaling pathway

W.-M. Zhang, P. Cao, L. Xin, Y. Zhang, Z. Liu, N. Yao, Y.-Y. Ma

Department of Obstetrics, Qilu Hospital of Shandong University, Jinan, China. zhangwangmeng009@126.com

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• Obstetrics

OBJECTIVE: The aim of this study was to explore the role of micro ribonucleic acid (miR)-133 in the apoptosis of human placental trophoblasts through the Ras homolog gene family (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway.
PATIENTS AND METHODS: The plasma samples were collected from 30 patients with pre-eclampsia (PE) undergoing treatment and 30 healthy subjects (control group) who received physical examination in our hospital. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was utilized to measure the expression of miR-133 in PE patients and healthy people. Meanwhile, blood pressure, urine protein content, liver function, and kidney function were detected in patients of both groups as well. Subsequently, the placental trophoblasts were extracted and transfected with inhibitors and miRNA mimics to suppress and overexpress miR-133, respectively. The transfection efficiency was determined by RT-PCR. The levels of interleukin-6 (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-α) were measured in both groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of trophoblasts. Next, the RT-PCR and Western blotting were carried out to detect the expressions of the Rho/ROCK pathway. Furthermore, the influence of miR-133 on the apoptosis of trophoblasts in human placenta tissues through Rho/ROCK was comprehensively observed.
RESULTS: In vivo experiments demonstrated that the urinary protein content, miR-133 level, systolic blood pressure, diastolic blood pressure, and liver function and renal function indexes were significantly elevated in pre-eclampsia (PE) patients in comparison with normal subjects (p<0.05). After transfection of mimics and inhibitors, the expression of miR-133 was remarkably up- and down-regulated, respectively. The content of the inflammatory factors in miR-133 mimics group was overtly higher than the other two groups. The TUNEL staining results showed that the number of apoptotic cells significantly increased and decreased in the miR-133 mimics group and miR-133 inhibitors group, respectively. Subsequent experiments indicated that the expressions of apoptosis gene Caspase3, pathway gene, and protein ROCKI were notably up-regulated in miR-133 mimics group. However, they were evidently down-regulated in miR-133 inhibitors group than in the control group. In addition, a consistent trend was observed in the protein expression level.
CONCLUSIONS: MiR-133 participates in the development and progression of PE through the Rho/ROCK signaling pathway, which may affect the apoptosis of trophoblasts in the placenta tissues.

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