MiR-135a-5p suppresses trophoblast proliferative, migratory, invasive, and angiogenic activity in the context of unexplained spontaneous abortion

Abstract

Background: Spontaneous abortions (SA) is amongst the most common complications associated with pregnancy in humans, and the underlying causes cannot be identified in roughly half of SA cases. We found miR-135a-5p to be significantly upregulated in SA-associated villus tissues, yet the function it plays in this context has yet to be clarified. This study explored the function of miR-135a-5p and its potential as a biomarker for unexplained SA.

Method: RT-qPCR was employed for appraising miR-135a-5p expression within villus tissues with its clinical diagnostic values being assessed using ROC curves. The effects of miR-135a-5p in HTR-8/SVneo cells were analyzed via wound healing, Transwell, flow cytometry, EdU, CCK-8, and tube formation assays. Moreover, protein expression was examined via Western blotting, and interactions between miR-135a-5p and PTPN1 were explored through RIP-PCR, bioinformatics analyses and luciferase reporter assays.

Results: Relative to normal pregnancy (NP), villus tissue samples from pregnancies that ended in unexplained sporadic miscarriage (USM) or unexplained recurrent SA (URSA) exhibited miR-135a-5p upregulation. When this miRNA was overexpressed in HTR-8/SVneo cells, their migration, proliferation, and cell cycle progression were suppressed, as were their tube forming and invasive activities. miR-135a-5p over-expression also downregulated the protein level of cyclins, PTPN1, MMP2 and MMP9. In RIP-PCR assays, the Ago2 protein exhibited significant miR-135a-5p and PTPN1 mRNA enrichment, and dual-luciferase reporter assays indicated PTPN1 to be a bona fide miR-135a-5p target gene within HTR-8/SVneo cells.

Conclusion: miR-135a-5p may suppress trophoblast migratory, invasive, proliferative, and angiogenic activity via targeting PTPN1, and it may thus offer value as a biomarker for unexplained SA.

Keywords: PTPN1(PTP1B); Trophoblast; Unexplained spontaneous abortion (SA); miR-135a-5p.

Fig. 1

miR-135a-5p offers promise as a diagnostic biomarker for unexplained SA. (a) Heat maps demonstrating relative miR-135a-5p expression in first-trimester villous tissue samples from NP (n = 5) and URSA (n = 5) patients. (b) MiR-135a-5p expression in the villous tissues from NP (n = 32) and USM (n = 32) patients. (c) MiR-135a-5p offered good diagnostic potential for USM. (d) MiR-135a-5p expression in the villous tissues from NP (n = 18) and URSA (n = 18) patients. (e) MiR-135a-5p offered good diagnostic potential for URSA. NP: normal pregnancy; SA: spontaneous abortion; USM: unexplained sporadic miscarriage; URSA: unexplained recurrent spontaneous abortion; ROC: Receiver operating characteristic; AUC: Area under the curve. *P < 0.05, **P < 0.01, *** P < 0.001

Fig. 2

MiR-135a-5p mimics inhibit HTR-8/SVneo cell proliferation and cell cycle progression. (a) MiR-135a-5p expression in HTR-8/SVneo following miR-135a-5p mimics or NC mimics transfection. (b) MiR-135a-5p overexpression inhibited cell proliferation in CCK-8 assays, with similar results in EdU assays (c-d). (e-f) MiR-135a-5p overexpression inhibited G0/G1 to S phase progression of the cell cycle within HTR-8/SVneo cells. (g-i) Cyclin D1 and cyclin B1 protein levels were reduced following the overexpression of miR-135a-5p within HTR-8/SVneo cells, with GAPDH and β-Tubulin serving as reference controls. Outcomes are means ± SD from at least three experiments. NC: negative control; CCK-8: Cell counting Kit-8; EdU: 5-Ethynyl-2′-deoxyuridine. *P < 0.05, **P < 0.01, *** P < 0.001

Fig. 3

miR-135a-5p mimics suppress migratory and invasive activity in HTR-8/SVneo cells. (a-b) Overexpressing miR-135a-5p inhibited HTR-8/SVneo cell migration in a wound healing assay. (c-e) Overexpressing miR-135a-5p suppressed HTR-8/SVneo cell migration and invasion in Transwell assessments. (f-g) Western blotting revealed E-cad upregulation and the downregulation of MMP2, MMP9, and Vimentin, with GAPDH as a reference control. Data are means ± SD from at least three experiments. E-cad: E-cadherin. *P < 0.05, **P < 0.01, *** P < 0.001

Fig. 4

miR-135a-5p overexpression suppresses HTR-8/SVneo cell angiogenic activity. The overexpression of miR-135a-5p was found to inhibit HTR-8/SVneo cell angiogenic activity in a tube forming assay

Fig. 5

miR-135a-5p targets PTPN1 in HTR-8/SVneo cells. (a) Intersecting candidate miR-135a-5p target genes were identified using three independent miRNA target databases and downregulated differentially expressed genes in our sequencing dataset. (b) The expression of four putative target genes in HTR-8/SVneo cells were assessed following miR-135a-5p overexpression, revealing significant PTPN1 downregulation. (c) PTPN1 protein levels were reduced following miR-135a-5p overexpression in HTR-8/SVneo cells, with GAPDH as a normalization control. (d) Ago2 RIP-PCR revealed the preferential enrichment of miR-135a-5p and PTPN1 mRNA in samples precipitated with Ago2. (e) Alignment of miR-135a-5p with the predicted binding site in the PTPN1 3’-UTR. (f) HTR-8/Svneo cells were co-transfected with PTPN1 3’-UTR constructs and miR-135a-5p mimics or NC mimics, with the ratio of Firefly/Renilla luciferase activity then being quantified. Outcomes are means ± SD from three assessments. PTPN1: Protein Tyrosine Phosphatase Non-Receptor Type 1; Ago2-RIP: Ago2-RNA immunoprecipitation; UTR: untranslated region; ns: non-significant; NC: negative control. *P < 0.05, **P < 0.01, *** P < 0.001