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Gene & Protein in Disease FXR1 modulates gene expression in cancer

transcription reaction utilized oligo (dT) primers to genes, as well as their independent expression across
selectively synthesize cDNA from polyadenylated mRNA various cancer types and their impact on overall survival
transcripts. The reaction mixture was incubated at 37°C for (OS). The RBPsuite tool (http://www.csbio.sjtu.edu.cn/
45 min, followed by enzyme inactivation at 85°C for 5 s. The bioinf/RBPsuite/) was used to identify potential FXR1
resulting cDNA was diluted 1:5 in RNase-free water and binding sites on the 3’UTR of target genes. Correlation
stored at -20°C for subsequent PCR or qRT-PCR analysis. analysis between FXR1 and its target genes was conducted
To evaluate the expression levels of target mRNAs, using the Gene Multiple Association Network Integration
qRT-PCR was conducted using Applied Biosystems SYBR Algorithm (GeneMANIA) database. Gene expression
Green Master Mix (Vazyme, China). Each qRT-PCR data and survival analysis were obtained from The Cancer
Genome Atlas (TCGA) and Genotype-Tissue Expression
reaction was performed in a 10 µL volume containing 1 µL
of diluted cDNA, 5 µL of SYBR Green Master Mix, 0.5 µL (GTEx) using the Gene Expression Profiling Interactive
of each forward and reverse primer (10 µM), and 3 µL Analysis (GEPIA) database (http://gepia.cancer-pku.cn/).
of RNase-free water. The reactions were run in a 96-well Pairwise correlation analyses were performed to assess the
association between FXR1 and the selected target genes,
plate on an Applied Biosystems Real-Time PCR System while their expression levels in tumor versus normal tissues
under the following conditions: initial denaturation at
95°C for 5 min, followed by 40 cycles of denaturation at were visualized using boxplots. The Kaplan-Meier survival
95°C for 10 s, annealing at 60°C for 30 s, and extension curves were generated to evaluate the impact of high and
at 72°C for 30 s. Melting curve analysis was performed at low gene expression on OS, and statistical significance was
determined using log-rank tests. Correlation coefficients
the end of each run to confirm the specificity of the PCR were computed to quantify the strength of the association
products, ensuring no primer-dimer formation or non- between FXR1 and the expression levels of target genes.
specific amplification. Relative mRNA expression levels
were calculated using the 2 −ΔΔCt method, with GAPDH or 3. Results
β-actin as the internal control for normalization. Primer
sequences for the target mRNA transcripts are provided 3.1. Investigating FXR1 binding to mRNAs of selected
in Table 1. Each sample was run in triplicate, and all genes through RNA immunoprecipitation assay
experiments were repeated at least three times to ensure To explore whether FXR1 directly binds to the mRNAs
the reproducibility and reliability of the results. of key genes, we selected several of the significantly
For western blot analysis, cells were lysed in RIPA lysis upregulated and downregulated genes for further analysis.
buffer supplemented with a complete protease inhibitor These genes were chosen based on prior computational
cocktail (ab271306, Abcam, United Kingdom). Protein predictions and their relevance to the biological pathways
concentration was determined using a bicinchoninic acid under investigation. we performed RNA-IP using an
reagent (Cat no. 23225, Thermo Scientific, United States). FXR1-specific antibody on SH-SY5Y cells (Figure 1A).
Equal amounts of each protein sample were separated This cell line was selected due to its well-characterized
using electrophoresis on 10% sodium dodecyl nature and high expression levels of FXR1, making it
sulfate-polyacrylamide gels and then transferred to a ideal for studying RNA-protein interactions and post-
polyvinylidene fluoride (PVDF) membrane on ice. The transcriptional regulatory roles.
PVDF membranes were blocked with 5% fat-free milk The RNA-IP results revealed that the mRNAs of several
powder in tris-buffered saline containing 0.1% Tween- selected genes, including SHISAL1, SLC43A3, NBAT1,
20 (Sigma-Aldrich, Germany) for an hour. Subsequently, PDZK1IP1, ACKR3, KCCN3, NECAB2, ANO5, ATOH8,
the protein blots were incubated overnight at 4°C with IGFBP7, LEMD1, GPR35, WNT7A, and F2RL3, showed
the respective primary antibodies. Following incubation, significant binding to FXR1 compared to the IgG control
the blots were incubated with secondary antibodies. After beads. This suggests that these mRNAs are likely targets
incubation with secondary antibodies, the protein bands of FXR1 and may be involved in post-transcriptional
were visualized using the enhanced chemiluminescence regulation mediated by FXR1. However, a subset of
technique (Millipore, USA). β-actin served as the internal mRNAs analyzed in the same experiment, MIR3142HG,
control, and untreated cells were used as the blank. MUSK, SLPI, CA1, CALB2, and PAEP, did not show
binding to the FXR1 protein under identical conditions.
2.6. Bioinformatics analysis of FXR1 correlation and This discrepancy could be attributed to variations in FXR1
target gene expression
binding affinity, the presence of specific sequence motifs, or
We performed a comprehensive bioinformatics analysis structural properties of these mRNAs that hinder efficient
to evaluate the correlation between FXR1 and its target recognition by FXR1. The results of the RNA-IP experiment

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