The long non-coding RNA PVT1 represses ANGPTL4 transcription through binding with EZH2 in trophoblast cell

Abstract

Despite progress in diagnostics and treatment for preeclampsia, it remains the foremost cause of maternal and foetal perinatal morbidity and mortality worldwide. Over recent years, various lines of evidence have emphasized long non-coding RNAs (lncRNAs) which function as an innovative regulator of biological behaviour, as exemplified by proliferation, apoptosis and metastasis. However, the role of lncRNAs has not been well described in preeclampsia. Here, we identified a lncRNA, PVT1, whose expression was down-regulated in qRT-PCR analyses in severe preeclampsia. The effects of PVT1 on development were studied after suppression and overexpression of PVT1 in HTR-8/SVneo and JEG3 cells. PVT1 knockdown notably inhibited cell proliferation and stimulated cell cycle accumulation and apoptosis. Exogenous PVT1 significantly increased cell proliferation. Based on analysis of RNAseq data, we found that PVT1 could affect the expression of numerous genes, and then investigated the function and regulatory mechanism of PVT1 in trophoblast cells. Further mechanistic analyses implied that the action of PVT1 is moderately attributable to its repression of ANGPTL4 via association with the epigenetic repressor Ezh2. Altogether, our study suggests that PVT1 could play an essential role in preeclampsia progression and probably acts as a latent therapeutic marker; thus, it might be a useful prognostic marker when evaluating new therapies for patients with preeclampsia.

Keywords: PVT1; PRC2; preeclampsia; proliferation.

Figure 1

Relative PVT1 expression in preeclampsia. (A and B) LncRNA PVT1 levels are lessened in PE placentas. The expression of PVT1 was significantly lower in PE samples (n = 52) than that in normal placentas (log2). (C) The PVT1 expression in BeWo, WISH, JEG‐3 and HTR‐8/SVneo was normalized to that in HUVEC‐C. At least three times of biological replicates have been performed and presented. *P < 0.05, **P < 0.01.

Figure 2

PVT1 promotes Cells proliferation in vitro. (A and B) MTT assays were implemented to detect the viability of si‐PVT1‐1#‐treated or pcDNA3.1‐PVT1‐treated Trophoblast Cells. The cell viability after transfected with si‐PVT1 was significantly lower than that treated with the control. (C and D). Proliferating Trophoblast Cells were labelled using Edu. The Click‐it reaction shown Edu staining (red). Cell nuclei were stained with DAPI (blue). *P < 0.05, **P < 0.01.

Figure 3

Effect of PVT1 on apoptosis and cycle in Trophoblast cells. Trophoblast Cells were treated with siRNAs or pcDNA3.1‐PVT1. (A) The apoptotic rates of cells were measured by Flow cytometry. LL, dead cells; UL, viable cells; LR, early apoptotic cells; UR, terminal apoptotic cells. (B) Cell cycle analyses by Flow cytometry in vitro. *P < 0.05, **P < 0.01.

Figure 4

Effect of PVT1 on Trophoblast cell migration and invasion in vitro. (A and B) Transwell assays were performed to detect the role of PVT1 on trophoblast cell migration and invasion in vitro. The migration and invasion viability of the trophoblast cell transfected with si‐PVT1 were meaningfully lower than that of the control. The cells on the lower chamber were stained by crystal violet and displayed, and cell number in random five perspectives was analysed. *P < 0.05; **P < 0.01.

Figure 5

PVT1 knockdown increases genes expression which contributed to the cell proliferation and migration. (A) RNA transcriptome sequencing analysis was performed to analyse gene expression profiling in HTR‐8/SVneo cells following PVT1 knockdown. The picture showed the all of different expressed gene. (B) GO analysis for these genes with abnormal levels between the treating with si‐NC or si‐PVT1 in trophoblast cells. (C and D) qRT‐PCR analysis reveal abnormal mRNA expression of genes contributed to cell proliferation and migration in trophoblast cells after knockdown or overexpression of PVT1. (E) WB assay of ANGPTL4 level after transfecting with si‐PVT1 or Plasmid vectors (pcDNA3.1‐PVT1) into trophoblast cells. *P < 0.05, **P < 0.01.

Figure 6

PVT1 binds to Ezh2 to suppress ANGPTL4 expression. (A) Using qPCR, relative PVT1 levels are mostly located in nucleus, in which GAPDH and U1 acted as the marker of cytoplasm and nucleus, respectively. (B) RIP assays established that PVT1 could interact with Ezh2. (C) Knockdown EZH2 triggered ANGPTL4 expression at the mRNA levels by qPCR and protein levels by WB. (D) ChIP assays uncovered that Ezh2 and H3K27me3 were enriched in the promoter region of ANGPTL4, and this enrichment was reduced after PVT1 knockdown. *P < 0.05, **P < 0.01.

Figure 7

ANGPTL4 expression in preeclampsia women. (A) Results are presented as the fold change in preeclampsia placental tissues relative to normal tissues, and ANGPTL4 expression was classified into two groups (log2). (B) immunohistochemistry was performed to determine ANGPTL4 protein abundance. *P < 0.05, **P < 0.01.