Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Hana Totary-Jain, Ph.D.

Committee Member

Jerome Breslin, Ph.D.

Committee Member

Caralina Marin De Evsikova, Ph.D.

Committee Member

Joshua Scallan, Ph.D.

Keywords

placenta, stem cells, differentiation, epigenetics

Abstract

Trophoblast differentiation and invasion is essential for normal implantation and establishment of the maternal-fetal interface, which allows for proper nutrient exchange and support of the fetus. For this to occur, cytotrophoblasts must undergo an epithelial to mesenchymal transition and differentiate into migratory and invasive extravillous trophoblasts (EVTs) that invade the maternal decidua and myometrium. Trophoblast differentiation, migration and invasion is highly regulated by a complex network of signaling pathways, adhesion molecules and transcription factors and is important for the remodeling of maternal spiral arteries from low flow, high resistance to high flow, low resistance vessels to allow optimal perfusion of the fetus and the placenta. Cytotrophoblasts also differentiate into syncytiotrophoblasts which form the outer layer of the placenta and are important for fetal-maternal gas exchange.

The chromosome 19 miRNA cluster (C19MC) and the RNA binding protein Lin28 are highly expressed in embryonic stem cells, the placenta and certain cancers. C19MC is the largest human miRNA cluster that spans over 100 kb. It is primate specific and contains 46 miRNA genes flanked by Alu elements. C19MC is regulated by genomic imprinting. The expression of C19MC often correlates closely with the RNA binding protein LIN28. LIN28 was originally discovered as a regulator of developmental timing due to its inhibitory effects on the expression of the let-7 miRNA family. Two paralogs, LIN28A and LIN28B exist in vertebrates, which were shown to regulate thousands of mRNAs transcripts involved in cellular processes including stem-cell self-renewal, cell differentiation, proliferation, migration, and cellular metabolism.

The role of C19MC miRNAs in the regulation of EMT, trophoblast differentiation and somatic cell reprogramming has not been fully determined. In this study, we hypothesized that hypoxic conditions during implantation and placentation downregulate C19MC expression, which in turn induces EMT genes, resulting in trophoblast differentiation toward EVTs.

In this study we found that C19MC is highly expressed in villous cytotrophoblasts but not in differentiated EVTs or decidual cells in human first trimester placenta tissues. CRISPR/Cas9 mediated overexpression of C19MC inhibited the expression of EMT markers SNAI1, CDH2, TWIST1 and SERPINE1, induced the expression of reprogramming factors OCT4 and FGF4, and enhanced the reprogramming of human fibroblasts to iPSCs. Importantly, hypoxic conditions decreased the expression of C19MC and increased the expression of EMT markers in iPSCs. This study demonstrates that overexpression of C19MC induces epithelial cell phenotype and enhances cell reprogramming. Furthermore, exposure to hypoxic conditions induces EMT and trophoblast differentiation at least in part by inhibiting C19MC and reprogramming factors.

Recently, C19MC was reported to be highly expressed in infantile hemangiomas (IH). IH are benign tumors of infancy characterized by rapid growth phase followed by spontaneous involution. During the proliferative phase, immature glucose transporter-1 (GLUT1) positive endothelial cells form highly disorganized blood vessels. IH lesions also highly express C19MC and the stem cell reprogramming factors Oct4, Sox2, Nanog and Myc. When GLUT1 and C19MC expressing endothelial cells isolated from proliferating IH are grown in culture, they lose expression of both GLUT1 and C19MC and undergo an endothelial to mesenchymal transition. Propranolol, a beta-adrenergic receptor blocker, has been shown to trigger early IH tumor involution and has subsequently become first line treatment for IH patients. The levels of circulating C19MC correlated with tumor phase and with propranolol treatment. Some studies have shown considerable similarities between IH and placental transcriptomes suggesting a placental origin for proliferating endothelial cells in IH. On the other hand, other studies have proposed that IH originate from Hemangioma stem cells. This theory is supported by studies which show that human CD133+ hemangioma stem cells recapitulate the entire IH life cycle when implanted subcutaneously into immunocompromised mice.

The expression of C19MC miRNAs have been strongly correlated with the expression of the RNA binding protein LIN28. Although LIN28/let-7 axis plays a central role in the regulation of stem cell self-renewal and tumorigenesis, the role of LIN28B/let-7 signaling in IH pathogenesis has not yet been elucidated. We hypothesized that the LIN28/let-7 signaling pathway is dysregulated in IH and is modulated by propranolol treatment during IH involution. We found that LIN28B is highly expressed in proliferative IH compared to involuted IH and propranolol treated IH. The increase in LIN28B correlated with C19MC expressed and inversely correlated with let-7. Overexpression of LIN28B in HEK293 cells increased the expression of miR-516b, a C19MC miRNA. Moreover, iPSCs treated with propranolol decreased the expression of C19MC miRNAs and LIN28B and increased the expression of let-7. Importantly, propranolol treatment decreased iPSC proliferation and increased the expression of EMT markers. These results describe for the first time, the role of the LIN28/let-7 switch in IH pathogenesis and identifies a novel mechanism by which propranolol induces IH involution.

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