An exploratory study into the role of miR-204-5p in pregnancy-induced hypertension

Abstract

The molecular mechanism that leads to pregnancy-induced hypertension (PIH), a pregnancy-specific syndrome, remains poorly understood. It has been suggested that microRNAs (miRNAs) may be potentially useful biomarkers for severe preeclampsia (PE), which is an important condition associated with PIH. The aim of the present study was to identify miR-204 by verifying differentially expressed serum miRNAs in patients with PIH during pregnancy compared with normal controls. Subsequently, the effects of miR-204 on proliferation and apoptosis of human choriocarcinoma (JAR) cells in hypoxic microenvironment were investigated. Previous studies indicated a number of miRNA candidates and the present study validated the expression of eight miRNAs in serum samples using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A higher expression of miR-204 was identified in patients with PIH. To assess the impact of miR-204 inhibition on hypoxic JAR cells function in vitro, cell proliferation was detected using a Cell Counting Kit-8 assay. The rate of apoptosis and cell cycle progression was then examined by flow cytometry. RT-qPCR confirmed that serum miR-204-5p is more highly expressed in patients with PIH. Further statistical analysis indicated that the survival ratio of JAR cells in hypoxic microenvironments was increased in the miR-204-5p inhibitor group. However, the miR-204-5p inhibitor protected hypoxic JAR cells from apoptosis. The analysis of cell-cycle status demonstrated that the percentage of cells in the G2/G1 phase was larger compared with the control group. The results of the present study suggest that low levels of miR-204-5p may increase cell proliferation and reduce cell apoptosis with cell cycle changes in vitro. Therefore, serum miR-204-5p may be used as a notable biomarker for the diagnosis, prevention and treatment of PIH.

Keywords: cell apoptosis; cell proliferation; miR-204-5p; preeclampsia; pregnancy-induced hypertension.

Figure 1.

Expression of miRNAs in serum samples of healthy controls and patients with PIH, detected using reverse transcription-quantitative polymerase chain reaction. Expression of (A) miR-197-3p, (B) miR-92b-3p, (C) miR-342-3p, (D) miR-26a-5p, (E) miR-198, (F) miR-204-5p, (G) miR-296-5p and (H) miR-95-5p in serum samples of healthy normal controls and patients with PIH. Data are presented as the mean ± standard deviation. All experiments were repeated independently three times. ***P<0.001 vs. control group. miR, microRNA; PIH, pregnancy-induced hypertension.

Figure 2.

Expression of miR-197-3p, miR-92b-3p, miR-26a-5p, miR-198 and miR-204-5p in human choriocarcinoma JAR cells following hypoxia pre-treatment for 0, 48 and 72 h respectively, detected using reverse transcription-quantitative polymerase chain reaction. Data are presented as the mean ± standard deviation. All experiments were repeated independently three times. *P<0.05; **P<0.01; ***P<0.001 vs. 0 h. miR, microRNA.

Figure 3.

miR-204-5p inhibitor protects growth of JAR cells in the hypoxic environment in vitro. Cell viability was measured using a Cell Counting Kit-8 assay. Cells were counted at 48, 72 and 96 h. Data are presented as the mean ± standard deviation. All experiments were repeated independently three times. ***P<0.001 vs. NC group. NC, normal control group, OD, optical density; miR, microRNA.

Figure 4.

Induction of JAR apoptosis. (A) Cell apoptosis following hypoxia treatment for 24, 48 and 72 h. (B) Percentage of JAR cells undergoing apoptosis at different time points during hypoxia. Data are presented as the mean ± standard deviation. All experiments were repeated independently three times. **P<0.01 vs. NC inhibitor group. NC, normal control; PI, propidium iodide; FITC, fluorescein isothiocyanate; miR, microRNA.

Figure 5.

Induction of JAR cell cycle change. (A) Cell cycle distribution following hypoxia treatment for 24, 48 and 72 h. (B) Percentage of JAR cells in cycle in S, G1 and G2 phases following 48 h of hypoxia. Data are presented as the mean ± standard deviation. All experiments were repeated independently three times. *P<0.05 vs. G1 phase arrest in NC group at the same time point. NC, normal control; miR, microRNA.