The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts†

Abstract

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

A microRNA component of the hypoxic response

“…An additional difference was in the experimental conditions employed by the different groups: hypoxia mimetics, 22 versus 1% oxygen for 1 h, 23 versus 5% oxygen for 8 h, 23 versus 0.2% for various periods of time from 8-48 h. 21 In addition to the microRNAs that respond to hypoxia by upregulation, a set of microRNAs were identified as downregulated in hypoxic cells, including miR-15b, 16, 19a, 20a, 20b, 29b, 30b, 30e-5p, 101, 141, 122a, 186, 320 and 197. [22][23][24] In our study, we have also detected microRNAs that exhibited downregulation at the level of microarrays (miR-126, 128, 138, 323, 326); however, the changes seemed restricted to one cell line and were not pursued at this stage (R Kulshreshtha et al, unpublished).…”

“…Three additional articles reported microRNAs that respond to low oxygen with some notable similarities, including miR-210, miR-30b, 93 and 181b. [22][23][24] It is also true, however, that a significant number of microRNAs differed between the studies, which is not necessarily surprising, given the differences in the cellular backgrounds and technology employed, both being a recognized source of variability. An additional difference was in the experimental conditions employed by the different groups: hypoxia mimetics, 22 versus 1% oxygen for 1 h, 23 versus 5% oxygen for 8 h, 23 versus 0.2% for various periods of time from 8-48 h. 21 In addition to the microRNAs that respond to hypoxia by upregulation, a set of microRNAs were identified as downregulated in hypoxic cells, including miR-15b, 16, 19a, 20a, 20b, 29b, 30b, 30e-5p, 101, 141, 122a, 186, 320 and 197.…”

Regulation of erythroid differentiation by miR-376a and its targets

“…Ago2 demonstrates nucleolytic activity in siRNA and miRNA biogenesis [25,26]. To date, two labs have reported a dicer-independent miRNA biogenesis pathway that requires Ago2 catalysis [27,28].…”

hsa‐miR‐210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer

“…10 One previous study found no up-regulation of microRNA biosynthesis machinery at the protein level 36 in placental tissues from women with pre-eclampsia, but this regulation may be different in cancer cells, as they are genetically different from normal tissue. This would be in keeping with multiple other studies showing dysregulation of parts of the pathway in cancer cell lines and tissues.…”