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The functional role of LOXL3 in astrocytomas

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Author(s):
Talita de Sousa Laurentino
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
Defense date:
Examining board members:
Sueli Mieko Oba Shinjo; Erico Tosoni Costa; Maria Lucia Cardillo Correa Giannella
Advisor: Sueli Mieko Oba Shinjo
Abstract

Astrocytomas are neoplasms originated from astrocytic cells of the central nervous system and are the most common tumors among gliomas (glial cell tumors). Initially, the World Health Organization classified astrocytomas according to malignancy, taking into account histological characteristics (astrocytomas of grade I to IV). However, recently, molecular characteristics such as mutations and other chromosomal alterations have been incorporated into the classification. Glioblastoma (GBM), grade IV, is the most common glioma and with the worst prognosis. For a better understanding of gliomagenesis process, our laboratory compared genes with greater expression in GBM in relation to grade I astrocytoma, in order to identify new therapeutic targets. The gene that codes for the enzyme lysyl oxidase (LOX) was one of the genes with higher expression in GBM. The LOX enzyme belongs to a family with five members, LOX, LOXL1, LOXL2, LOXL3 and LOXL4, and acts on the catalysis of crosslinks of collagen and elastin, playing an important role in the stiffness of the extracellular matrix. Among the LOX family members, LOXL3 expression influenced on the prognosis of GBM cases: patients with higher LOXL3 expression had a lower mean survival rate than those with the lowest LOXL3 expression. In the present study, transient LOXL3 silencing with two siRNA sequences was performed in the GBM U87MG cell line. The knock down efficiency of LOXL3 for siRNA1 and siRNA2 was 84.70% and 50.9% respectively at the transcript level, and 41.5% and 39.2%, respectively, at the protein level in relation to the control. In functional assays, LOXL3 silencing promoted a decrease in cell proliferation (36.9% and 26.2% for siRNA1 siRNA2, respectively), as well as an increase in apoptosis without (13.43% and 7.04% for the siRNA1 siRNA2, respectively) and with (32.8% and 24.64% for the siRNA1 siRNA2, respectively) treatment with temozolomide. Moreover, it was also demonstrated by immunofluorescence that LOXL3 colocalized with mitochondria in U87MG cells. In addition to the colocalization, cells with LOXL3 silencing showed an increase in mitochondrial marker fluorescence 5-fold higher than the control cells. Moreover, cells with LOXL3 knock down had an increase in the number of mitochondrial DNA copy number of 18.24% for siRNA1. Furthermore, RNA-seq analysis showed that U87MG cells with LOXL3 knock down had a decrease in the expression of genes related to the process related to autophagic flux, suggesting that LOXL3 probably plays a role in the autophagy/mitophagy process. In addition, genes related to mtDNA replication are also an increase in expression. Additionally, genes related to the apoptosis pathway are also overexpressed, corroborating the data obtained. Therefore, transient silencing of LOXL3 in the U87MG cell line promoted a decrease in cell viability, and consequently an increase in apoptosis level. In addition, LOXL3 colocalized with mitochondria in the U87MG cell. Low LOXL3 expression promotes increased mitochondrial marker fluorescence and, additionally, mitochondrial DNA copy number exhibits an increase compared to control. This work suggests that LOXL3 may be involved with the mitochondrial dynamics process, favoring the autophagy / mitophagy pathway, and inhibiting cell death in the U87MG strain. There are no papers that describe the role of LOXL3 in astrocytomas and neither the involvement with mitochondria and autophagy and / or mitophagy. However, there is no paper that has described the role of LOXL3 in astrocytomas and neither its involvement with mitochondria and autophagy and/or mitophagy. Our data suggest a new role of LOXL3 in astrocytomas (AU)

FAPESP's process: 16/05777-1 - Functional role of enzyme LOXL3 in astrocytoma
Grantee:Talita de Sousa Laurentino
Support type: Scholarships in Brazil - Master