Page 148 - GPD-4-1

Gene & Protein in Disease FXR1 modulates gene expression in cancer

1. Introduction mediating host defense mechanisms against pathogenic
challenges. 24,25 In this study, we demonstrate that FXR1
Post-transcriptionally, RNA-binding proteins (RBPs) overexpression enhances the oncogenic activity of several
regulate the transcript levels of various RNAs by interacting cancer-related genes while decreasing the levels of tumor-
with specific regions, particularly within the 3’-untranslated suppressing genes. Conversely, FXR1 knockdown results
region (UTR). Accumulating evidence suggests that in reduced oncogene activity and increased expression of
dysregulation of RBPs significantly contributes to tumor suppressors. Our results reveal that FXR1 directly
carcinogenesis. In this context, fragile X-related protein 1 regulates the expression of oncogenes, such as SLC43A3,
(FXR1) is increasingly recognized as an important RBP in ACKR3, KCNN3, LEMD1, GPR35, WNT7A, F2RL3, and
various cancers. Due to its diverse RNA-binding domains ANO5, while also targeting tumor suppressor genes, such
1-5
and functional flexibility, FXR1 is implicated in controlling as NBAT1, PDZK1IP1, NECAB2, ATOH8, and IGFBP7.
multiple post-transcriptional processes, including mRNA These findings provide new insights into the role of FXR1
transport, translation, and degradation, by binding to in cancer progression and highlight its potential as a
adenylate/uridylate-rich elements and G-quartet regions therapeutic target.
of RNA. In addition to its RNA-binding capabilities,
6-8
FXR1 is known to interact with unbound ribosomes and 2. Materials and methods
polysomes. Like many other RBPs, FXR1 is primarily
9
localized in the cytoplasm, but it can undergo nuclear 2.1. Cell culture
10
reorientation under certain conditions. 11 The SH-SY5Y cell line (Fenghbio Biological Ltd., China)
was cultured in a 1:1 mixture of Minimum Essential
Several types of cancers, including head and neck
squamous cell carcinoma (HNSCC) and lung squamous cell Medium (MEM) and F12 media. The growth medium
was supplemented with 10% fetal bovine serum (FBS),
carcinoma (LSCC), exhibit elevated FXR1 expression. 12,13 100 µg/mL streptomycin, and 100 U/mL penicillin to
FXR1 deficiency has been shown to induce apoptosis in ensure adequate nutrition for cell growth and protection
LSCC, while in HNSCC, it promotes cellular senescence. against contamination. Cells were maintained at 37°C in a
11
13
Previous research has demonstrated that FXR1 targets the humidified incubator with an atmosphere of 5% CO₂ and
3’-UTR of the tumor suppressor gene p21 for degradation, 95% air. Cell passages were performed when the cultures
thereby facilitating the development and proliferation of reached approximately 80 – 90% confluence, and the media
HNSCC by evading senescence. In addition, FXR1 has was refreshed every 2 – 3 days to sustain optimal growth
12
been shown to prevent the degradation of miR301a-3p by conditions. Regular morphological assessments were
exoribonucleases. In FXR1-deficient cells, the exonuclease performed to monitor cellular health and morphology
polyribonucleotidyltransferase 1 degrades miR301a-3p, throughout the culture period. All experiments utilized
resulting in increased p21 transcript levels in squamous cell cells between passages 5 and 15 to ensure reproducibility
carcinoma. These findings suggest that inhibiting FXR1, and minimize genetic drift.
14
combined with anti-microRNA (miRNA) oligonucleotide
therapies and chemotherapy, could represent a more 2.2. Construction of plasmid and lentiviral
effective strategy for treating HNSCC. 15-17 packaging
FXR1 interacts with a diverse array of mRNA Lentiviral packaging was conducted using Lipofectamine
transcripts, regulating their stability and translation, 3000 transfection reagent (Thermo Fisher, United States).
which is essential for various physiological functions. HEK293T cells were seeded in 10 cm dishes to achieve 40 –
In muscle development, FXR1 regulates the expression 50% confluency. A total of 12 µg plasmid DNA, comprising
of key myogenic factors, such as MyoD and myogenin, the target plasmid (pLVX-shRNA2-puro or pCDH-
thereby facilitating myogenesis. In addition, FXR1 plays GFP+puro), helper plasmid psPAX2, and helper plasmid
18
a pivotal role in cellular stress responses, particularly pMD2.G in ratios of 5:4:1 or 4:3:2, was transfected in a
through the regulation of heat shock protein mRNAs, serum- and antibiotic-free medium. After 6 h, the medium
which are crucial for cell survival under oxidative was replaced with a fresh culture medium. The supernatant
stress conditions. 19,20 Furthermore, FXR1 contributes was collected 72 h post-transfection, centrifuged at
significantly to neuronal function by modulating mRNAs 1,000 rpm for 4 min at room temperature, and filtered
encoding glutamate receptors, influencing synaptic through a 0.45 µm membrane to obtain the lentiviral
plasticity and cognitive processes, including learning and solution. The lentiviral solution was aliquoted and stored
memory. 21-23 Moreover, FXR1 has been implicated in the at −80°C. The sequences used for FXR1 knockdown were
immune response through its interaction with mRNAs shFXR1#1 (5’-GCTAGAGGTTTCTTGGAATTT-3’),
encoding inflammatory cytokines, suggesting a role in shFXR1#2 (5’-CGCCAGGTTCCATTTAATGAA-3’),

Volume 4 Issue 1 (2025) 2 doi: 10.36922/gpd.5068