Role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine beta-synthase (CBS) axis in regulating trophoblast invasion: an in vitro study

Abstract

IntroductionPlacenta expresses many ubiquitous as well as specific miRNAs which regulate trophoblast cell differentiation, proliferation, apoptosis, invasion, migration, and angiogenesis. Aberrant miRNAs expression (miR-22) has been linked to pregnancy complications, such as preeclampsia. Specificity protein 1 (Sp1) was identified a direct target of miR-22 and an inverse linear correlation between expression of miR-22 and Sp1 mRNA in gastric tumour has been reported. Also, Sp1 has a critical and indispensable role in the regulation of cystathionine {beta}-synthase (H2S synthesizing enzyme). Exogenous H2S could promote cell proliferation and invasion by upregulating the expression of MMP-2 and MMP-9 in human bladder cancer EJ cells. Worldwide studies reported significant role of MMP-2 and MMP-9 in the regulation of trophoblast invasion. Aims and ObjectivesTo determine the role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine {beta}-synthase (CBS) axis in regulating trophoblast invasion in the first-trimester trophoblast cells. Materials and methodsThe human first trimester trophoblast cell line (HTR-8/SVneo Cell line) was procured from American Type Culture Collection. Gain/Loss of function studies were conducted on HTR-8/SVneo cells and the expression of miR-22 (pre miR-22 and miR-22-3p), Sp1, CBS, MMP-2 and MMP-9 was observed. Invasion capacity of the cells was assessed by transwell invasion assay. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated regulated mRNA and protein expression of Sp1, CBS and and MMPs 2, 9 was observed when the cells were transfected with miR-22-3p mimic in comparison to miR-22-3p inhibitor transfected cells. Expression of CBS and MMPs 2, 9 was found to be significantly up regulated when the cells were transfected with Sp1 plasmid as compared to Sp1 inhibitor (2,4,5 trifluoroaniline) treated cells at both transcription and translation levels. MMP-2 and MMP-9 levels were found to be significantly elevated when the cells were subjected to NaHS [sodium hydrogen sulphide (H2S donor)] treatment as compared to AOAA (aminooxyacetic acid) treated cells. ConclusionThis is the first study which implies that miR-22 via targeting Sp1 must have influenced the mRNA and protein expression of CBS, MMP-2 and MMP-9 in order to regulate the trophoblast invasion.