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

1. Introduction 2. Materials and methods

Esophageal cancer (ESCA), a common digestive system 2.1. Cancer databases
malignancy, is the sixth leading cause of cancer-related UALCAN is a comprehensive cancer database containing
[1]
death worldwide . Esophageal squamous cell carcinoma multiple omics data (http://ualcan.path.uab.edu/index.
(ESCC) is the primary pathological type of ESCA in html). We used this database to verify the expression of
Asia, while esophageal adenocarcinoma (EAC) is more ESCCAL-1 in ESCA and its relationship with various
common in Western countries [2,3] . Although researchers clinical indicators of patients. Kaplan–Meier Plotter (KMP,
have discovered in recent years that genetic mutations http://kmplot.com/analysis/index.php), an online server
alter susceptibility to ESCA and that epigenetic changes designed to provide users with clinical data on pan-cancer,
contribute to the development of ESCA [4-6] , the detailed was used to analyze the relationship of patient survival
mechanisms that drive the tumorigenesis of ESCA are between ESCCAL-1 and ESCA. GEPIA (http://gepia.
still not well understood. Therefore, uncovering the cancer-pku.cn/about.html) is an online interactive website
molecular mechanism of ESCA is expected to contribute based on RNA-seq data, which is used to analyze the
to the development of new diagnostic and therapeutic
strategies. expression of ESCCAL-1 and ALKBH3 as well as their
correlation in ESCA.
Long non-coding RNAs (lncRNAs), non-coding
transcripts longer than 200 nucleotides, widely mediate 2.2. Cell culture and transfection
tumor development and influence disease prognosis . We Three ESCA cell lines, including TE1, KYSE70, EC1, and
[7]
previously used transcriptome sequencing technology to one immortalized esophageal epithelial cell line Het-1A,
detect differentially expressed lncRNAs in ten pairs of ESCA were cultured in an incubator containing 5% carbon
and adjacent normal tissues and identified esophageal dioxide at 37°C. All cells were maintained in RPMI 1640
squamous cell carcinoma associated long non-coding medium containing 10% fetal bovine serum and 1%
RNA 1 (ESCCAL-1) as an upregulated molecule closely penicillin-streptomycin solution. The lentivirus-based
related to ESCA . It was further found that ESCCAL-1 can recombinant vectors were purchased from Shanghai
[8]
promote the proliferation, metastasis, cycle progression, GeneChem Company (China) for knockdown (sh-AL1#1,
and apoptosis resistance of ESCA cells [9,10] , suggesting that sh-AL1#2) or overexpression (OE-AL1) of ESCCAL-1.
ESCCAL-1 may be a critical oncogenic lncRNA in ESCA The vectors were transfected into ESCA cells with the
occurrence. However, the molecules responsible for the transfection reagent HitransGA (GeneChem, China).
uncontrolled expression of ESCCAL-1 in ESCA and their ALKBH3 silencing siRNA was purchased from Shanghai
biological roles still need to be fully understood. GenePharma Company (China) and transfected into
N -methyladenosine (m A) methylation is one of ESCA cells with the transfection agent INTERFERin
1
1
the eukaryotic cell’s most common RNA modifications. (Polyplus, France).
Deregulated methylase regulates RNA stability, splicing,
translation, and other processes by affecting the m A 2.3. Real-time quantitative reverse transcription
1
modification of transcripts [11,12] . RNA m A modification polymerase chain reaction
1
controls intracellular gene expression profile at the post- Total RNAs were extracted from the cells using Trizol
transcriptional level and participates in the regulation reagent (Invitrogen, USA). After concentration and
of tumor initiation and development [13-15] . However, the purity measurements, 1 μg of RNAs in a 20-μL reaction
function of m A modification in ESCA and its regulation system were reverse-transcribed into cDNA using a
1
of lncRNA expression remains unclear. reverse transcription kit (Novoprotein, China). Finally,
In this study, we found that high expression of real-time quantitative reverse transcription polymerase
ESCCAL-1 was closely related to the progression and chain reaction (qRT-PCR) was performed using the
prognosis of ESCA. The absence of ESCCAL-1 inhibits SYBRGREEN kit (Novoprotein, China) and amplification
the stem-like properties of ESCA cells, and the forced system (Applied Biosystems, USA). The housekeeping
expression of ESCCAL-1 promotes the self-renewal gene GAPDH was used as the internal reference, and the
ability of ESCA. Alkylation repair homolog 3 (ALKBH3), relative expression level of the target gene was calculated
an RNA demethylase, erases the m A modification of by method 2 -ΔΔCt . The primers used are shown in Table S1.
1
ESCCAL-1 and causes the upregulation of the latter 2.4. Tumor sphere formation assay
expression in ESCA. ALKBH3/ESCCAL-1 axis is involved
in the stemness maintenance of ESCA, providing a new The transfected cells were uniformly inoculated in
therapeutic target for this disease. low-adhesion six-well plates (CORNING, USA), each

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