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. 2019 Aug;23(8):5165-5175.
doi: 10.1111/jcmm.14385. Epub 2019 Jun 26.

MiR-629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

Mei Zhao  1 , Ni Chen  2 , Xuelian Li  3 , Ling Lin  4
Affiliations

MiR-629 regulates hypoxic pulmonary vascular remodelling by targeting FOXO3 and PERP

Mei Zhao et al. J Cell Mol Med. 2019 Aug.

Abstract

Pulmonary arterial hypertension (PAH) is featured by the increase in pulmonary vascular resistance and pulmonary arterial pressure. Despite that abnormal proliferation and phenotypic changes in human pulmonary artery smooth muscle cells (HPASMCs) contributing to the pathophysiology of PAH, the underlying molecular mechanisms remain unclear. In the present study, we detected the expression of miR-629 in hypoxia-treated HPASMCs and explored the mechanistic role of miR-629 in regulating HPASMC proliferation, migration and apoptosis. Hypoxia time-dependently induced up-regulation of miR-629 and promoted cell viability and proliferation in HPASMCs. Treatment with miR-629 mimics promoted HPASMCs proliferation and migration, but inhibited cell apoptosis; while knockdown of miR-629 suppressed the cell proliferation and migration but promoted cell apoptosis in HPASMCs. The bioinformatics prediction revealed FOXO3 and PERP as downstream targets of miR-629, and miR-629 negatively regulated the expression of FOXO3 and PERP via targeting the 3' untranslated regions. Enforced expression of FOXO3 or PERP attenuated the miR-629 overexpression or hypoxia-induced enhanced effects on HPASMC proliferation and proliferation, and the suppressive effects on HPASMC apoptosis. Furthermore, the expression of miR-629 was up-regulated, and the expression of FOXO3 and PERP mRNA was down-regulated in the plasma from PAH patients when compared to healthy controls. In conclusion, the present study provided evidence regarding the novel role of miR-629 in regulating cell proliferation, migration and apoptosis of HPASMCs during hypoxia.

Keywords: FOXO3; PAH; PERP; PHASMCs; hypoxia; miR-629.

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Conflict of interest statement

The authors declare that there are no conflict of interest.

Figures

Figure 1
Figure 1
The effects of hypoxia on miR‐629 expression, cell viability and cell proliferation in HPASMCs. A, The expression of miR‐629 in HPASMCs after being treated with hypoxia for 12, 24 and 48 h was determined by qRT‐PCR. B, Cell viability of HPASMCs after being treated with hypoxia for 12, 24 and 48 h was determined by CCK‐8 assay. C, Cell proliferation of HPASMCs after being treated with hypoxia for 12, 24 and 48 h was determined by BrdU assay. Data are expressed as mean ± standard deviation; n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control groups
Figure 2
Figure 2
Overexpression of miR‐629 promoted cell proliferation, cell migration and inhibited cell apoptosis of HPASMCs. A, The expression of miR‐629 in HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by qRT‐PCR. B, Cell viability and (C) cell proliferation of HPASMCs after being transfected with mimics NC or miR‐629 mimics was detected by CCK‐8 assay and BrdU assay respectively. (D and E) The expression of PCNA mRNA and protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics were determined by qRT‐PCR and western blot respectively. (F) Cell migration of HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by transwell migration assay. (G and H) The caspase‐3 activity and the expression of caspase‐3 protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics were determined by Caspase‐3 activity assay and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups
Figure 3
Figure 3
Knockdown of miR‐629 suppressed cell proliferation, cell migration and induced cell apoptosis of HPASMCs during hypoxia. A, The expression of miR‐629 in HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors was determined by qRT‐PCR. B, Cell viability and (C) cell proliferation of hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors was detected by CCK‐8 assay and BrdU assay respectively. (D and E) The expression of PCNA mRNA and protein in hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors were determined by qRT‐PCR and western blot respectively. F, Cell migration of hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors was determined by transwell migration assay. (G and H) The caspase‐3 activity and the expression of caspase‐3 protein in hypoxia‐treated HPASMCs after being transfected with inhibitors NC or miR‐629 inhibitors were determined by Caspase‐3 activity assay and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups
Figure 4
Figure 4
miR‐629 directly targets FOXO3. A, The predicted binding sites between 3′UTR of FOXO3 and miR‐629 as determined by the targetscan software. B, The relative luciferase activity of the reporter vector containing wide‐type (WT) 3′UTR of FOXO3 was determined at 48 h after transfection with mimics NC or miR‐629 mimics. C, The relative luciferase activity of the reporter vector containing mutant (MUT) 3′UTR of FOXO3 was determined at 48 h after transfection with mimics NC or miR‐629 mimics. (D and E) The relative luciferase activity of the reporter vector containing mutant 3′UTR of FOXO3 was determined at 48 h after transfection with inhibitors NC or miR‐629 inhibitors in hypoxia‐treated HPASMCs. (F and G) The expression of FOXO3 mRNA and protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by qRT‐PCR and western blot respectively. (H and I) The expression of FOXO3 mRNA and protein in HPASMCs after exposing to hypoxia or normoxia was determined by qRT‐PCR and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control groups
Figure 5
Figure 5
MiR‐629 directly targets PERP. A, The predicted binding sites between 3′UTR of PERP and miR‐629 as determined by the targetscan software. B, The relative luciferase activity of the reporter vector containing wide‐type (WT) 3′UTR of PERP was determined at 48 h after transfection with mimics NC or miR‐629 mimics. C, The relative luciferase activity of the reporter vector containing mutant (MUT) 3′UTR of PERP was determined at 48 h after transfection with mimics NC or miR‐629 mimics. (D and E) The relative luciferase activity of the reporter vector containing mutant 3′UTR of PERP was determined at 48 h after transfection with inhibitors NC or miR‐629 inhibitors in hypoxia‐treated HPASMCs. (F and G) The expression of PERP mRNA and protein in HPASMCs after being transfected with mimics NC or miR‐629 mimics was determined by qRT‐PCR and western blot respectively. (H and I) The expression of PERP mRNA and protein in HPASMCs after exposing to hypoxia or normoxia was determined by qRT‐PCR and western blot respectively. Data are expressed as mean ± standard deviation; n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control groups
Figure 6
Figure 6
Overexpression of FOXO3 and PERP reversed the effects of miR‐629 overexpression on cell proliferation, migration and apoptosis of HPASMCs. (A and B) The expression of FOXO3 mRNA and protein in HPASMCs after being transfected with pcDNA3.1 or pcDNA3.1‐FOXO3 was determined by qRT‐PCR and western blot respectively. (C and D) The expression of PERP mRNA and protein in HPASMCs after being transfected with pcDNA3.1 or pcDNA3.1‐PERP was determined by qRT‐PCR and western blot respectively. (E) Cell viability and (F) cell proliferation of HPASMCs after being co‐transfected with miRNAs and plasmids were determined by CCK‐8 assay and BrdU assay respectively. G, The expression of PCNA mRNA in HPASMCs after being co‐transfected with miRNAs and plasmids was determined by qRT‐PCR. H, Cell migration of HPASMCs after being co‐transfected with miRNAs and plasmids was measured by transwell migration assay. I, Cell migration of HPASMCs after being co‐transfected with miRNAs and plasmids was measured by transwell migration assay. Data are expressed as mean ± standard deviation; n = 3. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control groups
Figure 7
Figure 7
Overexpression of FOXO3 and PERP reversed the hypoxia‐induced effects on cell proliferation, migration and apoptosis of HPASMCs. HPASMCs under the normoxia condition served as negative controls. In other groups, HPASMCs were treated with hypoxia for 48 h, (A) cell viability, (B) cell proliferation, (C) expression of PCNA mRNA, (D) cell migration and (E) caspase‐3 activity were determined by respective in vitro functional assays at 24 h post‐transfection with pcDNA3.1, pcDNA3.1‐FOXO3 or pcDNA3.1‐PERP. Data are expressed as mean ± standard deviation; n = 3. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control groups
Figure 8
Figure 8
The expression of miR‐629, FOXO3 and PERP mRNA in clinical samples. A, The expression of miR‐629 in the plasma from healthy controls (n = 14) and PAH patients (n = 14) was determined by qRT‐PCR. B, The expression of FOXO3 mRNA in the plasma from healthy controls (n = 14) and PAH patients (n = 14) was determined by qRT‐PCR. C, The expression of PERP mRNA in the plasma from healthy controls (n = 14) and PAH patients (n = 14) was determined by qRT‐PCR. *P < 0.05 and **P < 0.01 versus control groups
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