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Gene & Protein in Disease m1A-mediated ESCCAL-1 promotes ESCA stemness

containing 4 mL of sphere-forming medium and 2.8. Statistical analysis
10,000 cells. Dulbecco’s modified eagle medium (DMEM)/ Statistical analysis and illustration of experimental
F12 medium contained 20 ng/mL epidermal growth factor data were completed by SPSS 19.0 and GraphPad 9.0.
(EGF), 10 ng/mL basic fibroblast growth factor (bFGF), 5 Comparison of experimental data between the two groups
μg/mL insulin, 0.4% bovine serum albumin (BSA), and was conducted by t-test, and P < 0.05 was considered
1× B27. The plates were placed in an incubator containing statistically significant.
5% carbon dioxide at 37°C for 7 – 12 days. Finally, the
spheres were photographed and the number of spheres 3. Results
were analyzed.
3.1. ESCCAL-1 is upregulated in ESCA and correlates
2.5. Colony formation assay with patient outcome
The transfected cells were uniformly inoculated in 12-well We previously identified the dysregulation of lncRNA
plates with 1000 cells per well, and placed in an incubator ESCCAL-1 in ESCA using RNA-seq from ten pairs of
containing 5% carbon dioxide at 37°C for 7 – 10 days. The tumor samples and adjacent normal samples and verified
[8]
cell clones were immobilized with paraformaldehyde and its elevation in multiple ESCA cohorts . In this study, we
[10]
stained with crystal violet solution. Finally, cell colonies analyzed the expression of ESCCAL-1 in 11 normal samples,
were photographed and the area of colonies in each well 80 EAC samples, and 81 ESCC samples by interrogating the
was analyzed. cancer database UALCAN. ESCCAL-1 was significantly
overexpressed in ESCA (Figure 1A). Moreover, the
2.6. Western blot analysis expression of ESCCAL-1 in ESCC was higher than in EAC
Total proteins were extracted by RIPA lysis buffer (EpiZyme, (Figure 1A). Then, using this study cohort, we analyzed the
China) containing protease inhibitors. After concentration relationship between ESCCAL-1 expression and race, sex,
and purity determination, the total proteins were denatured and tumor stage in ESCA patients. ESCCAL-1 was found
with loading buffer at 100°C for 10 min. Then, 10% to be more highly expressed in African Americans and
polyacrylamide gel was prepared, and an equal amount Asians than that in Caucasians (Figure 1B). In addition, the
of protein was added to each lane and electrophoresis was expression of ESCCAL-1 in male ESCA was higher than in
performed. Subsequently, the proteins on the gel were female ESCA (Figure 1C). To a certain extent, ESCCAL-1
transferred to the polyvinylidene fluoride membrane. expression increased with the progression of the ESCA
After blocking with skimmed milk and incubation with tumor stage (Figure 1D). ESCCAL-1 was reported to be
the primary antibody and secondary antibody, the ECL kit closely related to the outcome of ESCA patients in our
and the luminescence imaging analysis system were used previous study . Here, we further analyzed the relationship
[8]
to detect the target protein attached to the membrane. between ESCCAL-1 expression and ESCA prognosis
Primary antibodies include anti-GAPDH (1:5000, using the survival database KMP. It was found that the
Bioworld, China), anti-CD44 (1:2000, Bioss, China), and median survival time of patients with high ESCCAL-1
anti-KLF4 (1:2000, Bioss, China). expression was significantly shorter than that of patients
with low ESCCAL-1 expression in both EAC (16.5 months
2.7. Methylated RNA immunoprecipitation (MeRIP) vs. 46.7 months, Figure 1E) and ESCC (25.4 months vs.
N -methyladenosine (m A) modification on the ESCCAL-1 42.1 months, Figure 1F). These findings, in combination
1
1
transcript was detected by the m A MeRIP Kit (GenSeq, with our previous results, suggest that ESCCAL-1 is a bona
1
China). The experimental procedures were performed fide risk factor for ESCA progression and survival.
according to the user manual. In brief, the Trizol reagent
was used to extract total RNAs from cells and the RNA 3.2. ESCCAL-1 is essential for maintaining the
concentration was adjusted to 1 μg/μL with enzyme- stemness of ESCA cells
free water. The fragment buffer was used to process We have previously shown that knockdown of ESCCAL-1
RNA transcripts. Subsequently, m A antibody or control inhibits ESCA cell growth and metastasis, while
1
antibody IgG, fragmented RNAs and immunoprecipitation overexpression of ESCCAL-1 promotes these malignant
(IP) buffer were added to the prepared immunomagnetic phenotypes of ESCA [9,10] . As stem cell-like property is an
beads and incubated at 4°C for 1 h. Then, m A-labeled RNA intrinsic characteristic of tumors and closely related to
1
samples obtained by immunoprecipitation were dissolved malignant proliferation and metastasis [16,17] , we speculate
in enzyme-free water and detected by polymerase chain that ESCCAL-1 might be involved in the stemness
reaction (PCR). Specific primers of ESCCAL-1 transcript maintenance of ESCA. First, we detected the expression of
used for MeRIP-PCR are shown in Table S2. ESCCAL-1 at the ESCA cell level by qRT-PCR and found

Volume 2 Issue 1 (2023) 3 https://doi.org/10.36922/gpd.305