Journal of Pharmacy and Pharmacology 10 (2022) 173-189

doi: 10.17265/2328-2150/2022.06.001
D DAVID PUBLISHING

Clinical Significance of METTL Family Molecules in

Hepatocellular Carcinoma

Weijie Kong, Fuping Li and Wenqi Zhang

Shaanxi University of Chinese Medicine, Xianyang 712046, Shaanxi Province, China

Abstract: Objective: to search for hepatocellular carcinoma (HCC) in tumor subdivision from different aspects, such as transcripts,
proteins, gene mutations, protein interactions, signal pathways and functions. To explore the correlation between METTL1-6 protein
family and hepatocellular carcinoma (HCC), and to determine whether METTL family proteins are valuable as potential biomarkers
for tumor progression and prognosis of HCC. Methods: We try to use data collection technique to extract the data needed in medical
analysis. The correlation between the MRNA expression level of METTL family proteins and the prognosis of HCC patients was
obtained from TCGA data. METTL data and clinical data of protein METTL family in tissue samples of HCC patients were obtained
from Kaplan-Meier Plotter database for correlation analysis. The immunohistochemical data of METTL family protein 1, 2A, 2B, 3,
4, 5, 6 in normal liver tissue and HCC tissue were obtained from TIMER database. The protein network of METTL proteome was
obtained by using STRING database, and the heat map enrichment analysis of proteome interaction between KEGG and GO was
done by software. Results: 6 of the 7 METTL protein members were highly expressed in HCC tissues, and were positively correlated
with the grade and clinical analysis of HCC tumors.

Key words: METTL, hepatocellular carcinoma, tumor progression, prognosis, data collection, big data.

1. Introduction In the development and progression of HCC in

As data collection technique spread widely [1],
medical and such technique also began to be
associated [2]. So we try to use intelligent data
collection technique to extract the data needed in
medical analysis.
Primary liver cancer has been one of the most
common malignant tumors with high morbidity and
mortality. Liver cancer accounts for 45% of all cancer
deaths. Among all primary liver cancer, hepatocellular
carcinoma (HCC) is the most, and the male-to-female
ratio in China is 5: 1. According to histological
classification, primary liver cancer can be divided into
hepatocyte type, bile duct cell type and mixed type.
Hepatitis B and C virus, long-term heavy drinking,
ingestion containing nitrosamines or accidental
ingestion with aflatoxin and family inheritance are the
high risk factors of liver cancer.
Corresponding author: Wenqi Zhang, Chief physician,
Supervisor of Master studnets, research field: clinical medicine.
Email: zhangwenqi2519@163.com.
hepatocellular carcinoma, genetic and expression
changes trigger repeated fibrosis of dysplastic
hepatocytes and tissues, and repeated fibrotic
hepatocytes undergo a series of genetic and protein
translation changes, resulting in the formation of HCC
[3, 4].
The methyltransferase-like protein (METTL) family
regulates the concentration of s-Adenosylmethionine
for methylation modification [5, 6]. Members of the
METTL family are ubiquitous in mammalian cells and
participate in the methylation of RNA, DNA or
proteins, in which the methylation of RNA is the most
important work. In recent years, as an academic
hotspot, the METTL family has gradually been
studied out of the principle pathway and function.
In this paper, through data analysis, it is suggested
that the selected METTL family may be involved in
the progression of hepatocellular carcinoma and can
be used as one of the indexes for early diagnosis of
hepatocellular carcinoma.
174 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma
2. Data Collection and Methods
Search the TCGA database (http://ualcan.path.uab.
edu/analysis.html), search the seven METTL families
selected this time (1, 2A, 2B, 3, 4, 5, 6) select the
Liver hepatocellular carcinoma in TCGA dataset, and
obtain the expression sample data of these seven
METTL family members in HCC.
In the TCGA database, select Individual cancer stages
to get the relationship between the expression degree of
the molecules selected by METTL and the cancer stage.
In the TCGA database, select Tumor grade to get the
relationship between the expression degree of the
molecules selected by METTL and the tumor grade. In
the TCGA database, select TP53mutationstatus to get
the relationship between the mutation state of the
molecule selected by METTL and the tumor grade.
In the Kaplan-Meier Plotter data (http://kmplot.com/
analysis/index.php?p=service), the selected 7 molecules
were searched for survival analysis. Select Liver
Cancer in the classification column, enter 7 kinds of
METTL molecules in the gene column, and search OS,
RFS, PFS and DSS in the survival column to get the
survival curve of the corresponding molecules. The
lines were drawn and analyzed with probability as
ordinate, survival time as Abscissa, high expression
and low expression.
The relationship between the expression of METTL
family molecules and immune cell infiltration in HCC
patients was analyzed by TIMER database
Fig. 1 Expression pattern of input genes in Liver hepatocellular carcinoma (LIHC).
(https://cistrome.shinyapps.io/timer/). Select the Gene
item in the TIMER database to fill in the selected
family molecules (1-6) and select LIHC (Live
Hepatocellular Carcinoma) Immune Infiltrates in the
Types option to select B cells, CD8+ T cells, CD4+ T
cells, macrophages, neutrophils and dendritic cells.
Then output the results and analyze them.
The STRING database (https://www.string-db.org/)
was used to find the potential protein molecules of
the selected METTL family molecules, and the first
50 protein molecules were selected to construct the
PPI network map, and the GO and KEGG analysis
were carried out to draw the enrichment analysis heat
map.
3. Results
3.1 Differences in Expression of 7 Selected METTL
Molecules in HCC Tissues and Normal Liver Tissues
In the tissues of HCC patients, the hint can be used
as an indicator of HCC detection. We also compared
the relative expression level of the selected molecules
in hepatocellular carcinoma tissues and found that
among the 7 selected METTL molecules we evaluated,
the relative expression level of METTL5 was the
highest, as shown in Figure 1.
The expression of 7 kinds of METTL molecules in
HCC tissues and normal liver tissues was significantly
higher than that in normal liver tissues (p < 0.0001),
as shown in Figure 2..
 Clinical Significance
e of METTL Family
F Molecu
ules in Hepattocellular Carrcinoma 1755

Fig. 2 Expreession of MET

TTL family molecules in HCC
C and normal liver
l tissues (***p < 0.0001).
176 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma
3.2 Analysis of the Relationship between Molecular
Expression and HCC Staging
Figure 3 showed that the expression of selected
molecules in stage 1-3 was higher than that in stage 0,
and was proportional to the grade (P < 0.01). It was
statistically significant, and the expression of selected
METTL molecules increased with the progress of the
disease. It is suggested that it can be used as a
potential marker of disease stage.
3.3 Data Analysis of the Relationship between
Molecular Expression and HCC Grading
Figure 4 showed that the molecular expression of
1-4 grades was higher than 0 grades, and was
proportional to the grade (P < 0.01). It's statistically
significant. It is suggested that the selected METTL
molecules are involved in the progression of HCC and
can be used as a potential diagnostic marker for
grading the severity of the disease.
Besides the relationship between molecular
expression and TP53 gene mutation in HCC tissues,
TP53 gene mutation is common in all kinds of tumors
and is an important tumorigenesis factor. Therefore,
the relationship between the expression of 7 molecules
selected in TCGA database and TP53 gene mutation
in HCC tissues was further analyzed. The results of
data analysis (as shown in Figure 5) showed that the
expression of METTL1/METTL2B/METTL3/METTL4/
METTL5/METTL6 in mutated HCC tissues was
significantly higher than that in TP53 non-mutated
HCC tissues (P < 0.0001). The expression of
METTL2A in mutated tissues was higher than that in
non-mutated tissues (P < 0.01). The results were
statistically significant.
3.4 Data Analysis of the Relationship between
Molecular Expression and Survival and Prognosis
Among the 7 METTL molecules selected, the
patients with high expression of METTL1/METTL2A/
METTL3/METTL4/METTL5/METTL6 in OS/RFS/
PFS/DSS had a lower survival rate, especially in the
first three stages. As shown in Figure 6.
3.5 Data Analysis of the Relationship between
Molecular Expression and Immune Cell Infiltration
The immune cell infiltration of selected METTL
molecules in HCC patients participated in
inflammatory response and immune cell infiltration,
thus affecting the clinical outcome of HCC patients.
Therefore, we used TIMER database to study the
relationship between differentially expressed METTL
chemokine and immune cell infiltration. The
expression of 7 kinds of METTL molecules selected
were all correlated with the clearness of immune cells
in stem cell carcinoma, and the correlation between
METTL3 and METTL4 was the strongest. As shown
in Figure 7.
3.6 Data Analysis Molecular Protein Interaction
Network and Enrichment Analysis
Construction and analysis of protein interaction
network of selected METTL molecules using STRING
database (https://www.string-db.org/) to find the
potential protein molecules that interact with
the selected METTL family molecules, and select the
first 50 protein molecules to construct the PPI
network map, and carry out GO and KEGG analysis,
draw an enrichment analysis heat map. GO analysis
showed that. The functional enrichment of the seven
potential interacting proteins of METTL molecules is
mainly related to ribonucleic acid metabolism, nucleic
acid activity catalysis, MRNA metabolism, RNA
localization, ribonucleoprotein complex assembly,
positive regulation of telomere protein localization,
translation, ATP hydrolytic activity and chromatin
binding, which is also consistent with the results that
selected METTL molecules participate in gene
regulation by regulating protein expression. KEGG
analysis showed that the selected METTL molecules
were related to splice bodies, RNA transporters and
 Clinical Significance
e of METTL Family
F Molecu
ules in Hepattocellular Carrcinoma 1777

Fig. 3 Analyysis of the corrrelation betweeen the expresssion level of METTL

M familly molecules and HCC stagiing. Note: Thee
relationship between
b the deegree of molecu
ular expression
n and differentt cancer stagess (***p < 0.00001; **p < 0.01; *p < 0.05).
178 Clinical Significance
e of METTL Family
F Molecu
ules in Hepattocellular Carrcinoma

Fig. 4 Analyysis of the corrralation betweeen the expresssion level of METTL

M familyy molecules an
nd HCC gradiing. Note: Thee
relationship between
b the deggree of molecullar expression and different pathological
p graades of HCC (****p < 0.0001; **p < 0.01; *p
p
< 0.05) accordding to patholoogical classificaation, HCC waas divided into
o 1-4 grades (Edmondson-Steeiner I-IV).
 Clinical Significance
e of METTL Family
F Molecu
ules in Hepattocellular Carrcinoma 1799

Fig. 5 Analyysis of the relationship betweeen the expresssion level of METTL

M family molecules and
d the mutated HCC
H tissues off
TP53 gene.
180 Clinical Significance
e of METTL Family Molecu
ules in Hepattocellular Carrcinoma

OS
Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma 181

RFS
182 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma

PFS
 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma 183

DSS
Fig. 6 Analysis of the relationship between the expression of METTL family molecules in tumor tissues and survival
prognosis of patients with HCC. Note: P < 0.05, the results were statistically significant.
184 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma
Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma 185
186 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma

Fig. 7 Analysis of the relationship between the expression of METTL family molecules and imm infiltration in tumor tissues of patients with HCC.
 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma 187

Fig. 8 Protein interaction network and thermographic enrichment analysis of METTL molecules.

ribosomal biogenesis in eukaryotes. This reflects that with the occurrence of HCC. It shows that the selected
the enrichment of the selected molecules in the members may potentially play an important regulatory
channels related to HCC has a positive relationship role in the occurrence of HCC. As shown in Figure 8.
188 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma

4. Discussion and Conclusion a marker of HCC and as a basis for the prognosis of
the disease.
METTL3 as the first discovered M6A
In addition, with the progression of the disease,
methyltransferase (regulating the transcription and
patients with high expression of METTL family
translation of mRNA, which is closely related to
molecules had lower survival rates. These data suggest
carcinogenesis). Therefore, in recent years, METTL3,
that the seven METTL family molecules selected may
as the most concerned molecule in the METTL family,
play an important role in HCC. In view of the
has been found to be related to digestive tract cancer,
differences in the expression of various molecules in
prostate cancer, osteosarcoma and hematopoietic stem
HCC, we analyzed the data of GO and KEGG
cells [5]. With the discovery of more METTL family
pathways by enrichment analysis, and focused on the
molecules with technological advances, it has also
function of potential interacting proteins of selected
been confirmed that METTL family members are
METTL molecules. We found that METTL molecules
involved in other cancers [6-8]. However, the
are related to ribonucleic acid metabolism, nucleic
probability of gene mutation in HCC is higher, and
acid activity catalysis, MRNA metabolism, RNA
there is a potential interaction between METTL family
localization, ribonucleoprotein complex assembly,
molecules and TP53 [9]. UALCAN analysis showed
positive regulation of telomere protein localization,
that there was a high correlation between METTL1
translation, ATP hydrolysis activity and chromatin
expression of METTL family molecules in TCGA
binding. This happens to be related to the partial
database and cancer. The strategy of clinical diagnosis
occurrence channel of HCC. Previous studies have
and treatment of HCC is early diagnosis and early
shown that the METTL family is involved in m6A
treatment, and most of the methods rely on the
methyltransferase and mRNA transcription and
transformation of de-differentiate hepatoblast
translation, which is related to the progression of
progenitor cells into hepatocellular carcinoma cells to
various cancers. At the same time, the database results
express progenitor cells, or through the production of
suggest that the METTL molecular family can be used
iCCA [10]. The improvement of early diagnosis plays
as a potential target for drug therapy.
a decisive role in the prognosis of patients with HCC
Through the analysis of Oncomine and TCGA
[11].
database, it was found that 7 kinds of METTL family
The most important role of the METTL molecular
molecules (1/2A/2B/3/4/5/6) were highly expressed in
family is to form M6A methyltransferase, which
HCC tissues, and the survival rate of patients with
indirectly regulates protein production, and the
high expression was decreased. The selected METTL
METTL protein family forms an interaction group
family may be involved in the progression of
[12]. The expression of METTL family members is
hepatocellular carcinoma and can be used as one of
closely related to the occurrence of cancer [13-15]. In
the indicators for early diagnosis of hepatocellular
recent years, METTL18 has been proved to be a
carcinoma.
potential prognostic marker.
In this paper, we first analyzed the relationship Conflicts of Interest
between the expression, pathological grade and
The authors declare no conflict of interest.
staging of 7 kinds of METTL molecules and
hepatocellular carcinoma (HCC). We found that the References
expression of METTL in HCC patients was higher [1] Wang, W., & Wei, C. 2020. “Advances in the Early
than that in normal tissues, and was proportional to Diagnosis of Hepatocellular Carcinoma.” Genes Dis 7
each other. It is suggested that METTL can be used as (3): 308-319.
 Clinical Significance of METTL Family Molecules in Hepatocellular Carcinoma
[2] Levrero, M., & Zucman-Rossi, J. 2016. “Mechanisms of
HBV-induced Hepatocellular Carcinoma.” J Hepatol 64
(1 Suppl): S84-S101.
[3] Wong, J. M., & Eirin-Lopez, J. M. 2021. “Evolution of
Methyltransferase-like (METTL) Proteins in Metazoa: A
Complex Gene Family Involved in Epitranscriptomic
Regulation and Other Epigenetic Processes.” Mol Biol
Evol 38 (12): 5309-5327.
[4] Kostyusheva, A., Brezgin, S., Glebe, D., et al. 2021.
“Host-cell Interactions in HBV Infection and
Pathogenesis: the Emerging role of m6A Modification.”
Emerg Microbes Infect 10 (1): 2264-2275.
[5] Tian, Q-H., Zhang, M-F., Zeng, J-S., et al. 2019.
“METTL1 Overexpression is correlated with Poor
Prognosis and Promotes Hepatocellular Carcinoma via
PTEN.” J Mol Med (Berl) 97 (11): 1535-1545.
[6] Liu, D., Li, W., Zhong, F., et al. 2020. “METTL7B Is
Required for Cancer Cell Proliferation and
Tumorigenesis in Non-Small Cell Lung Cancer.” Front
Pharmacol doi: 10.3389/fphar.2020.00178.
[7] Li, T-H., Qin, C., Zhao, B-B., et al. 2021. “Identification
METTL18 as a Potential Prognosis Biomarker and
Associated with Immune Infiltrates in Hepatocellular
Carcinoma.” Front Oncol doi:
10.3389/fonc.2021.665192.
[8] Liu, S., Hausmann, S., Carlson, S. M., et al. 2019.
“METTL13 Methylation of eEF1A Increases Translational
Output to Promote Tumorigenesis.” Cell 176 (3):
189
491-504.e21.
[9] Si, W., Li, Y., Ye, S., et al. 2020. “Methyltransferase 3
Mediated miRNA m6A Methylation Promotes Stress
Granule Formation in the Early Stage of Acute Ischemic
Stroke.” Front Mol Neurosci 13: 103.
[10] Sia, D., Villanueva, A., Friedman, S. L., & Llovet, J. M.
2017. “Liver Cancer Cell of Origin, Molecular Class, and
Effects on Patient Prognosis.” Gastroenterology 152 (4):
745-761.
[11] Shiani, A., Narayanan, S., Pena, L., & Friedman, M. 2017.
“The Role of Diagnosis and Treatment of Underlying
Liver Disease for the Prognosis of Primary Liver Cancer.”
Cancer Control doi: 10.1177/1073274817729240.
[12] Ignatova, V. V., Jansen, P. W. T. C., Baltissen, M. P., et
al. 2019. “The Interactome of a Family of Potential
Methyltransferases in Hela Cells.” Sci Rep 9 (1): 6584.
[13] Wu, S., Zhang, S., Wu, X, & Zhou, X. 2020. “m6A RNA
Methylation in Cardiovascular Diseases.” Mol Ther 28
(10): 2111-2119.
[14] Song, Y., Pan, Y., Wu, M., et al. 2021. “Correction to:
METTL3-Mediated lncRNA m6A Modification in the
Osteogenic Differentiation of Human Adipose-derived
Stem Cells Induced by NEL-like 1 Protein.” Stem Cell
Rev Rep 17 (6): 2366-2367.
[15] Arcidiacono, O. A., Krejí, J., & Bártová, E. 2020. “The
Distinct Function and Localization of
METTL3/METTL14 and METTL16 Enzymes in
Cardiomyocytes.” Int J Mol Sci 21 (21): 8139.