This study investigated the relationship between MMP-9, TIMP-1, and sialic acid (NANA) in a human glial cell line and the effects of NANA on the expression of these genes. The study found that NANA upregulated the expression of both MMP-9 and TIMP-1 at lower concentrations in a way that maintained the MMP-9/TIMP-1 balance. However, at higher concentrations of 1000μM NANA, MMP-9 expression was upregulated to a significantly greater degree than TIMP-1 expression, causing an imbalance similar to reports in neurodegenerative diseases. This suggests NANA may be involved in signaling pathways regulating the expression of these genes linked to neuroinfl

1. Abstract
Surname: Shabani Sadr Name: Narjes Khatoun
Title: Study of MMP-9 and TIMP-1 gene expression in the presence of sialic acid
in human glial cell line
Supervisor/s: Mouhammad Shafiei, Hamid Galedari
Advisor/s: Alireza Kheirollah
Degree:Masters of Science
University: Shahid Chamran University of Ahvaz
Faculty: Science Department: Genetics
Keywords: MMPs, TIMPs, Sialic acids, N-acetylneuraminic acid,
neurodegenerative diseases, MS, AD, PD, ALS
Abstract:
Introduction: Matrix metalloproteases (MMPs) and tissue inhibitors of
metalloproteases (TIMPs) are involved in many cell signaling and Inflammatory
processes and produced by many cell types, including activated T cells,
macrophages, neurons, astrocytes and microglial(glial) cells. Nonregulated MMP
activity and an imbalance between metalloproteinases and their inhibitors might
contribute to various disorders, including neurodegenerative diseases such as
Multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD) and
Amyotrophic lateral sclerosis (ALS). For example, several lines of evidence suggest
that MMPs and TIMPs are involved in the pathogenic cascade of both experimental
autoimmune encephalomyelitis (EAE) and MS. Increased expression of MMP-9and
imbalance between MMP-9/TIMP-1 has been found in brain lesions and in
peripheral blood mononuclear cells from MS patients.
The surfaces of all vertebrate cells are decorated with a dense and complex array of
sugar chains, which are mostly attached to proteins and lipids. Sialic acids (N-
acetylneuraminic acid, NANA) are a diverse family of sugar units with a nine-
carbon backbone that are typically found attached to the outermost ends of these
chains. Given their location and ubiquitous distribution, sialic acids can mediate or
modulate a wide variety of physiological and pathological processes, such as
starting or inhibiting the immune response, the activating of the complement system
and cell signaling. For unknown reasons, the brain is the organ with the highest level
of sialic acids in the body, much of it in the form of sialylated glycolipids
(gangliosides). Sialic acids are the ligands for the Siglec receptor family (Sialic acid
binding immunoglobulin-like lectin) that play important role in immune defense and
may be target in several human diseases. For example, Mortem MS lesions showed
that PSA-NCAM, normally absent from analyzed regions, was re-expressed on
demyelinated axons and absent from re-myelinated axons and white matter.
Therefore, it has an important role in the regulation of intracellular physiological
and pathological processes. So, evidence suggests that both MMPs and NANA play
an important role in inflammatory neurodegenerative diseases. sialic acid
involvement in the signaling pathways leading to MMP-9 and TIMP-1 gene
expression in glial cells is unclear. Therefore, this study investigated the relationship

2. between MMP-9, TIMP-1 and NANA and the effects of these ligand on the
signaling processes of the neurodegeneration and inflammatory demyelination.
Materials and methods: Human Glial cell line was prepared from “Pasteur
Institute of Iran” and cultured in DMEM, supplemented with 10% FBS. The IC50
value of NANA was obtained by MTT assay. Glial cell line was treated with NANA
(300,500 and 1000 µg/ml) for 24h to investigate the effects of these ligand on the
expression of MMP-9 and TIMP-1. Then total cellular RNA was isolated from
approximately 10×106 Glial cells. 1000 ng of total RNA from each sample was used
into cDNA. Real-time PCR determined the expression level of the MMP-9 and
TIMP-1 transcripts and REST and SPSS software were used for analyze.
Results and conclusion: The results of MTT assay were analyzed by Excel
software and IC50 was obtained equivalent to 1273.3 µM. Therefore, concentrations
of sialic acid were selected for treatments that less than Ic50 (300,500 and 1000
µM). By analyzing Real-time data, it was found that MMP-9 and TIMP-1 mRNA
expression was up-regulated with treatment (300, 500 and 1000 µM). At
concentrations of 300 and 500µM, there wasn’t significant difference between the
up-regulated of two genes. It is concluded that along with the up-regulated of MMP-
9, TIMP-1 expression was also up-regulated. This event indicates cells attempt for
maintaining MMP-9/TIMP-1 balance. MMPs expression is strictly controlled at
different levels, to prevent their harmful effects and TIMPs are one of the MMPs
controller. But it was found a significant difference between up-regulated of MMP-
9 and up-regulated of TIMP-1, at 1000µM concentration of sialic acid. In this
concentration, was observed imbalance at MMP-9/TIMP-1, similar to clinical
reports of neurodegenerative diseases. These results indicate a possible involvement
NANA on signaling pathway those genes expression.