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Gene & Protein in Disease FXR1 modulates gene expression in cancer

and the negative control shRNA (shNC, 5’-GTTCT 1 mM EDTA, 1 mM EGTA, 0.5% triton X-100, and a
CCGAACGTGTCACGTT-3’) obtained from the Public protease inhibitor cocktail). The lysates were pre-cleared
Protein/Plasmid Library. The FXR1 overexpression using centrifugation at 12,000× g for 10 min at 4°C. For
construct was generated by cloning the human FXR1 RNA-IP, a polyclonal FXR1 antibody (rabbit, Proteintech,
coding sequence (NM_001013439.3) into a vector, which China) was utilized, with an isotype-matched rabbit IgG
was subsequently packaged into a lentivirus. antibody (Proteintech, China) serving as the negative
control to account for non-specific binding.
2.3. Stable transfection protocol
The pre-cleared cell lysates were incubated with the
For stable gene knockdown, proliferating SH-SY5Y cells antibody for 4 h at 4°C to allow binding of FXR1 and
were seeded in a 6-well plate and transduced with lentiviral associated mRNAs. All steps were performed under
particles containing shRNAs targeting the gene of interest. RNase-free conditions to preserve RNA integrity. The
The lentiviral supernatant was added directly to the cells samples were gently rotated during the incubation to
at a final concentration of approximately 30 µL of virus- facilitate interaction between the FXR1-mRNA complexes.
containing medium in serum-free MEM. To enhance The protein A/G+ agarose beads (Thermo Fisher, United
transduction efficiency, polybrene was added to the culture States) were added to the cell lysate and incubated at 4°C
medium at a final concentration of 5 – 8 µg/mL. The cells for 40 min on an orbital shaker. After incubation, the beads
were incubated with the lentivirus for 6 h to facilitate efficient were washed extensively with RNA-IP buffer to remove
viral uptake and gene silencing. Following this incubation unbound material. To prevent reabsorption of mRNA
period, the medium was replaced with MEM/F12 medium and ensure maximum efficiency of the RNA-IP, the RNA-
supplemented with 10% FBS, antibiotics (100 µg/mL bound complexes were resuspended in 1 mL of proteinase
streptomycin, 100 U/mL penicillin), and additional nutrients K buffer (containing 10 mM Tris-HCl, pH 8, 50 mM EDTA,
to support cell recovery. The cells were cultured for 48 h to 0.5% SDS, 50 mM NaCl), supplemented with proteinase
allow stable integration of the shRNA-expressing constructs. K enzyme at a ratio of 4:100 (v/v) and RNase inhibitor
To select successfully transduced cells, puromycin (1:100 v/v). The mixture was then incubated for 30 min at
(2 µg/mL) was added to the culture medium, with daily 50°C to degrade proteins bound to the beads and release
media changes until non-transduced cells were eliminated the RNA for subsequent analysis. The resulting RNA was
and only puromycin-resistant clones remained. Selection extracted using the TRIzol extraction method and purified
typically lasted 5 – 7 days and was considered complete for downstream analysis, such as polymerase chain reaction
when no further cell death was observed. Stable cell lines (PCR), quantitative reverse transcription (qRT)-PCR, or
were maintained in the puromycin-containing medium for RNA sequencing, to identify mRNA targets bound by FXR1.
downstream experiments.
2.5. RNA extraction, quantitative reverse
For overexpression experiments, SH-SY5Y cells were transcription polymerase chain reaction, and
transduced with lentiviral vectors carrying the gene of western blotting
interest. The lentiviral supernatant was added directly to
the cells at an appropriate concentration in serum-free Total RNA was isolated using TRI reagent (Takara, China),
MEM. Polybrene was also included at a final concentration following the manufacturer’s guidelines. Briefly, cells were
of 5 – 8 µg/mL to improve transduction efficiency. The lysed in TRI reagent, and the homogenized lysate was
cells were incubated with the lentivirus for 6 h to ensure phase-separated using chloroform. After centrifugation
efficient transduction and gene expression. After this at 12,000 × g for 15 min at 4°C, the aqueous phase
incubation, the medium was replaced with MEM/F12 containing RNA was carefully collected and precipitated
medium supplemented with 10% FBS and antibiotics to with isopropanol. The RNA pellet was washed with 75%
promote cell recovery. Cells were cultured for an additional ethanol, air-dried, and resuspended in RNase-free water.
48 h to ensure optimal gene expression before proceeding The concentration and purity of the isolated RNA were
with further experimental assays. Both protocols were determined using a NanoDrop spectrophotometer by
carried out using cells in the logarithmic growth phase to measuring absorbance at 260/280 nm and 260/230 nm
maximize transduction efficiency and minimize variability. ratios. RNA integrity was assessed through agarose
gel electrophoresis to ensure high-quality RNA for
2.4. FXR1 RNA immunoprecipitation assay downstream applications.
FXR1 RNA immunoprecipitation (RNA-IP) was performed For complementary DNA (cDNA) synthesis, 1 µg
with slight modifications as previously described. Briefly, of total RNA was reverse transcribed into cDNA using
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SH-SY5Y cells were lysed in RNase inhibitor-supplemented the 1 Strand cDNA Synthesis Kit (Vazyme, China)
st
RIP lysis buffer (20 mM Tris-HCl, pH 8.2, 200 mM NaCl, according to the manufacturer’s instructions. The reverse

Volume 4 Issue 1 (2025) 3 doi: 10.36922/gpd.5068