The prognostic value of METTL3 in cancer patients: a meta-analysis

Abstract

Background: M6A methylation modification of RNA can regulate the development of tumor cells. METTL3 is one of the modified methyltransferases. A growing number of studies have shown that high expression of METTL3 may be associated with the unfavorable prognosis in cancer patients and may become a new biomarker. Therefore, this meta-analysis was used to evaluate the prognostic value of METTL3 in cancer patients through the available literature information.
Methods: Relevant studies were retrieved from electronic literature databases including six English databases and three Chinese databases. Correlation analysis was conducted by Stata SE 15.1 and RevMan 5.3 software. We analyzed 11 eligible studies with a total of 1638 cancer patients in this meta-analysis. We took advantage of HRs and ORs with 95% confidence intervals to evaluate the prognostic value of METTL3 in tumors. Besides,the article was qualified by MOOSE check lists and PRISMA Checklist.
Results: The results of the meta-analysis indicated that high expression of METTL3 was associated with low overall survival (OS) (HR=2.67, 95%CI:2.19-3.25, P<0.00001) and disease-free survival (DFS) (HR=2.23, 95%CI:1.60-3.11, P<0.00001) in various cancers.Stratified analysis of cancer types showed that over expressed METTL3 was related to poor prognosis in digestive system cancer patients, including CRC(HR=2.29, 95%CI:1.50-3.49，P=0.0001) ,GC(HR=2.96，95%CI:2.13-4.12，P＜0.00001)，and liver cancer（HR=2.70，95%CI:1.88-3.88，P＜0.00001). Meanwhile, the elevated METTL3 expression also affected the lymph node metastasis (OR=3.40，95%CI=1.58-7.33，p=0.002) ，vascular invasion (OR=2.04，95%CI=1.06-3.95，p=0.03) and the tumors progression (III/IV vs. I/II: OR = 3.72, 95% CI: 1.94 - 7.13, P < 0.0001).
Conclusion: The existing analysis indicates that METTL3 is associated with low OS and DFS in cancer patients and may serve as a biomarker to assess prognostic value.

Full text

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The prognostic value of METTL3 in cancer patients:
a meta-analysis
Xingzhu Pan
The First School of Clincial Medicine, Southern Medical University
Haolin Li
The First School of Clinical Medicine, Southern Medical University
Yuyuan Xu
State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis
Research, Department of Hepatology Unit and Infectious Diseases, Nanfang Hospital, Southern Medical
University, Guangzhou, China
Chengcheng He
Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang
Hospital, Southern Medical University, Guangzhou, Guangdong, China
Qiuhua Lai
Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang
Hospital, Southern Medical University, Guangzhou, Guangdong, China
Xinke Wang
Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gsatroenterology, Nanfang
Hospital, Southern Medical University, Guangzhou, Guangdong, China
Qingyuan Li (  liqingyuan09@smu.edu.cn )
Southern Medical University https://orcid.org/0000-0002-5601-8467
Research article
Keywords: Cancer, Prognosis, METTL3, meta-analysis
DOI: https://doi.org/10.21203/rs.3.rs-31588/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Background: M6A methylation modication of RNA can regulate the development of tumor cells.
METTL3 is one of the modied methyltransferases. A growing number of studies have shown that high
expression of METTL3 may be associated with the unfavorable prognosis in cancer patients and may
become a new biomarker. Therefore, this meta-analysis was used to evaluate the prognostic value of
METTL3 in cancer patients through the available literature information.
Methods: Relevant studies were retrieved from electronic literature databases including six English
databases and three Chinese databases. Correlation analysis was conducted by Stata SE 15.1 and
RevMan 5.3 software. We analyzed 11 eligible studies with a total of 1638 cancer patients in this meta-
analysis.We took advantage of HRs and ORs with 95% condence intervals to evaluate the prognostic
value of METTL3 in tumors. Besides,the article was qualied by MOOSE check lists and PRISMA
Checklist.
Results: The results of the meta-analysis indicated that high expression of METTL3 was associated with
low overall survival (OS) (HR=2.67, 95%CI:2.19-3.25, P<0.00001) and disease-free survival (DFS)
(HR=2.23, 95%CI:1.60-3.11, P<0.00001) in various cancers.Stratied analysis of cancer types showed that
over expressed METTL3 was related topoor prognosis indigestive systemcancer patients, including
CRC(HR=2.29, 95%CI:1.50-3.49P=0.0001) ,GC(HR=2.9695%CI:2.13-4.12P0.00001)and liver
cancerHR=2.7095%CI:1.88-3.88P0.00001). Meanwhile, the elevated METTL3 expression also affected
the lymph node metastasis (OR=3.4095%CI=1.58-7.33p=0.002) vascular invasion (OR=2.04
95%CI=1.06-3.95p=0.03) and the tumors progression (III/IV vs. I/II: OR = 3.72, 95% CI: 1.94 - 7.13, P <
0.0001).
Conclusion: The existing analysis indicates that METTL3 is associated with low OS and DFSin cancer
patients and may serve as a biomarker to assess prognostic value.
Background
Cancer, a major public health problem, is a great threat to human health in the 21st century. In the past
decade, the death rate of many cancers has increased, including liver cancer, male pancreatic cancer,
uterine cancer, small intestine cancer, reproductive system cancer ,as well as the cancers related to
human papillomavirus (HPV). [1]. Numbers of cancer patients and deaths are increasing worldwide,
especially in low and middle-income countries [2]. Early diagnosis of many tumors and long-term survival
rate after treatment are not encouraging. Despite according to recent studies ,advances has been made in
the molecular aspects of tumorigenesis and development, the specic mechanism is not fully clear [3-5].
Therefore, what we need to explore is a new tumor marker that is conducive to early diagnosis, treatment
and prognosis evaluation .
The key to controlling gene expression at different levels is chemical modication of the nucleobases,
which can affect the translation of related mRNA, proteins and regulate signal pathways[6]. Among more

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than 100 RNA chemical modications identied, N6- methyl- adenosine (m6A) was the most common
internal modication of mRNA and long non-coding RNA in eukaryotes. [7, 8]. Including RNA processing,
splicing, migration, translation and decay, m6A modication affects many aspects of RNA metabolism [9-
11]. The occurrence of m6A modication is encoded by methyltransferase complex. It can add, remove
and read m6A sites respectively through the writer, eraser and reader[12-15].New evidences showed that
by controlling the expression of oncogenes or tumor suppressor genes, m6A modication could regulate
the occurrence, differentiation and metastasis of tumors[14, 16, 17].
METTL3 (methyltransferase like 3) is one of N6 methyladenosylmethy transferases, which plays an
important role in mRNA pre-splicing, 3 '- terminal processing and translation regulation[18-21].According
to recent studies, METTL3 can also inuence the tumorigenesis and growth of tumor by regulating the
m6A modication [21-23].This mechanism has been found in a variety of tumor tissues[24-26]. Striking
discoveries indicated that METTL3 played a carcinogenic role in tumors. METTL3 promoted the growth
and invasion of human lung cancer cells by enhancing the translation of mRNA, and played a role of
oncogene in the tumorigenesis of gastric cancer[27-29]. In addition, recent studies have indicated that
METTL3 is up-regulated in many cancer tissues and associated with poor prognosis of patients [30, 31].
It suggests that METTL3 may be a potential biomarker for cancer patients. Therefore, it is important to
research the prognostic value of METTL3 expression in cancer patients. We conducted a meta-analysis
of relevant studies to obtain the relationship between the expression of METTL3 and long-term survival
of cancer patients.
Methods
Methods and materials
This meta-analysis is based on the meta-analysis report standard of observational research(MOOSE
check list)(Additional le 1.)
Literature search
We searched six English databases including Pubmed, Wed of Science, springer, Embase, Cochrane
Library and ScienceDirect, and three Chinese databases including Weipu and Wanfang and CNKI
database. No lower limit was set for the retrieval date, and the deadline was April 21,2020.We used
retrieval formula: (“methyltransferase-like 3”OR “METTL3” OR “Writers”) AND (“carcinoma”OR “cancer”
OR “tumor”OR “neoplasm”) AND (“prognosis”OR “prognostic”) and their Chinese forms to search in the
above online databases.After retrieving all eligible studies, we manually searched the references cited in
the study to nd more publications that were relevant.
Inclusion and exclusion criteria
Eligible studies should meet the following criteria: 1) The patients in the studies were denitely diagnosed
with cancer; 2) To study the effect of METTL3 on survival outcomes of cancer patients; 3) Information

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containing survival curve or providing HR and 95% condence intervals (CI). Exclusion is based on the
following criteria: 1) Studies lacking survival outcomes; 2)Reviews, non-human studies, case reports,
correspondence articles, and other studies without original data; 3) Repeated studies.
Data extraction
The two authors carefully read the included texts, extracted the data, checked and discussed . If there was
any difference, an investigator (LQY) would arbitrate it to reach a consensus. The necessary information
for all eligible articles includes: the name of the rst author; year of publication; type of cancer; type of
studies;survival analysis type;cancer stage;form of specimen;cut-off; total number of cases; follow-up;
METTL3 detection method; the source of the HR.The survival information directly get from the original
dates or obtain from the Kaplan–Meier survival curves data with the Enguage Digitizer (Version 4.1)
software by the previously described method [32-34].
Quality assessment
Two authors (PXZ and LHL) carefully reviewed the details of the each included study independently. The
enrolled literature were then qualied based on the Newcastle-Ottawa Scale, which included three
aspects: queue selection, comparability and results [35]. In the selection and result categories, a
maximum of one score can be obtained for each item. In the category of comparability, a maximum of
two scores can be given. According to the scores, the research level was rated as low quality, medium
quality and high quality [36]. All the included studies were of medium and above quality.
Statistical analysis
All analyses were performed using Stata SE 15.1 and RevMan 5.3 software. Potential publication bias
was evaluated by funnel plot and Egger’s test [37]. The heterogeneity of the included studies was
assessed by I2 statistical test, with I2>50% as evidence of heterogeneity. According to the test results, if
I2>50%, the random effect model should be selected; if I2<50%, the xed effect model should be
selected[33, 34, 38]. A sensitivity analysis was performed to assess the impact of a single study on
overall HR. P value less than 0.05 was statistically signicant.
Results
Eligible researches
The studies were screened according to the following procedure [19,39-48](Fig. 1).At rstWe retrieved a
total of 349 articles from the online database.After preliminary screening and duplicate checking, 253
articles were retained.Through reading the title and abstract of the articles, the articles were further
screened.Studies of 207 related to METTL3 expression and cancer prognosis were excluded according to
exclusion criteria. After reading the rest 46 articles carefully, 35 articles were eliminated.The remained
articles were then evaluated for quality,nally, 11 articles were included in this study.

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Features of articles included
The meta-analysis included 11 studies from 9 publishers, with a total of 1638 patients (Table 1). The
expression of METTL3 in frozen or formalin xed paran embedded specimens was detected by qRT-
PCR. The cancer tissue samples in the study were from ovarian carcinoma, hepatocellular carcinoma,
hepatoblastoma, thyroid carcinoma, bladder cancer, gastric cancer and colorectal cancer.The cut-off
values of METTL 3 were different in these studies.Of the 11 studies, 10 studies evaluated the relationship
between METTL3 expression and OS, three of which evaluated the relationship between METTL3 and OS
and DFS at the same time, and one evaluated PFS. Table 1 summarizes the main details of these studies.
The relationship between METTL3 expression and survival outcome
Because there was no signicant heterogeneity in the nine studies related to OS( I2= 0%)the xed effect
model was used to calculate the combined effect. The results showed that the increased expression of
METTL3 was signicantly related to the low OS ( HR=2.67,95%CI:2.19-3.25P<0.00001,Fig.2A) and DFS
(HR=2.23,95%CI:1.60-3.11P<0.00001,Fig 2.B) of various cancers.
Stratied analysis
Then we analyzed the subgroups according to the sample size,specimen types, degree of differentiation,
patient ages, quality scores, cancer types, lymph node metastasis, vascular invasion and tumor stage.
Because the vascular inltration ( I2= 57%) and tumor stage ( I2= 51%) subgroups had heterogeneity, the
random effect model were used, and the other subgroups used xed models to combine the effect
values.In stratied analysis of the different cancer types ,we found that elevated expression of METTL3
was related to poor prognosis in digestive system cancer patients, including CRC (HR=2.29, 95%CI:1.50-
3.49P=0.0001) ,GC(HR=2.9695%CI:2.13-4.12P0.00001)and liver cancerHR=2.7095%CI:1.88-3.88P
0.00001) (Fig. 3A). Subgroup analysis showed that patients with high expression of METTL3 had a
higher risk of lymph node metastasis, indicating that METTL3 might be related to lymph node
metastasis(OR=3.4095%CI=1.58-7.33p=0.002, Fig. 4A). We found the same result between vascular
invasion and METTL3 (Fig. 4B). In addition, the improvement of METTL3 level was related to tumor
progression(III/IV vs. I/II: OR = 3.72, 95% CI: 1.94 - 7.13, P < 0.0001, Fig. 4C).
Heterogeneity & sensitivity analysis
There was no heterogeneity in this meta-analysis. In order to determine the impact of each study on the
combined HRs, we conducted a sensitivity analysis. The results showed that excluding any of the studies
had no signicant effect on the METTL3 combined HR (Fig. 5).
Publication bias
In order to evaluate the publication bias of this meta-analysis, we evaluated the funnel plot and Egger's
test, and found that the funnel plot was basically symmetrical (Fig. 6A). The examination results of Egger

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's test showed that there was no signicant publication bias (P = 0.127, Fig. 6B). Therefore, there was no
obvious publication bias in our results.
Discussion
RNA methylation plays an important role in many biological processes and is widely involved in human
development and disease occurrence[49]. N6-methyladenosine (m6A) is a conserved internal
modication of most eukaryotic nuclear RNA, which is closely related to the splicing , stability and
translation eciency of mRNA and noncoding RNA [28,50].With further studies, M6A can catalyse the
M6A modication in the mRNA of oncogenes or tumor suppressor genes,and recognize the changes
through molecular biological mechanism to regulate the expression of oncogene and tumor suppressor
gene[51].The evidence showed that the change of m6A level was involved in the occurrence and
development of tumor by affecting the expression of tumor-related genes BRD4, SOCS2 and EGFR[52].
METTL3 was considered to be a methyltransferase, part of the methyltransferase complex, and was
responsible for the m6A modication[27,53].METTL3 was involved in many signal pathways such as
PI3K / Akt [31,54-58], MAPK [22] ,Wnt /beta-Catenin [47,59,60] and p38 / ERK [61]pathways,which were all
associated with tumor deterioration. In addition, METTL3 could inuence the development of tumor by
regulating some transcription factors or important oncogenes.Studies had found that METTL3 could
positively regulate the expression of oncogene EZH2 [24,25,62].It promoted the expression of MYC as well
as increased stability of protein by regulating the m6A methylation of MYC mRNA to lead to
carcinogenesis in PCA and gastric cancer, promote the occurrence of OSCC tumor and affect the growth
and invasion of BCA cells[40,63-66].When METTL3 was silenced, it inhibited the activity of the Wnt
pathway by reducing the m6A methylation level of LEF1 mRNA and reducing protein expression [59,67].
Besides, some studies have shown that METTL3 can be used as a new marker to evaluate the prognosis
of multiple tumors.Xiao Li et al. found that the survival time of RCC patients with positive METTL3
expression was signicantly longer, and the expression of METTL3 in RCC is lower than that in
paracancerous tissues[68]. Deng et al. noted that the positive expression of METTL3 was an obstacle to
the proliferation, migration and invasion of colorectal cancer cells [61].On the contrary,it had been
reported that the high expression of METTL3 was a factor of poor prognosis, which might be a new
prognosis or treatment target for lung cancer, gastric cancer and ovarian cancer[39,41,42,54,69].It can be
seen that the expression and role of METTL3 in different tumors are still controversial, which may be due
to differences in tumor type, extracellular microenvironment, and samples’ source and size.Up to now, the
prognostic value of METTL3 in various tumors has not been systematically analyzed, so according to the
existing reports, we studied the prognostic value of METTL3 in cancer.
A total of 11 studies, including 1638 patients, are included in this meta-analysis, which is reasonable to
believe that the analysis results are reliable.This study included the relationship between the high
expression of METTL3 and the prognosis of ovarian cancer, HCC, hepatoblastoma, thyroid carcinoma,
bladder cancer, gastric cancer, colorectal cancer.The results showed that METTL3 expression could affect

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the prognosis of OS (HR = 2.67, 95% CI: 2.19-3.25, P < 0.00001) and DFS (HR = 2.23, 95% CI: 1.60-3.11, P
< 0.00001), and METTL3 could predict the prognosis of digestive system cancers, including CRC
(HR=2.29, 95%CI:1.50-3.49P=0.0001) ,GC(HR=2.9695%CI:2.13-4.12P0.00001)and liver
cancerHR=2.7095%CI:1.88-3.88P0.00001) .In other subgroups of METTL3 expression and overall
survival,we performed analysis based on sample size, specimen type, and quality evaluation(The detailed
results are shown in table 2).According to the result in group of sample size, we discovered that METTL3
in small sample group (≤100) had a more signicant prognosis than in large sample group (> 100)(Fig.
3B), which indicated that the sample size might lead to the instability of results.In addition, METTL3 was
associated with tumor progression (III / IV vs. I / II: OR = 3.72, 95% CI: 1.94 - 7.13, P < 0.0001).Subgroup
analysis showed that METTL3 had a potential role in the prediction of lymph node metastasis(OR=3.40
95%CI=1.58-7.33p=0.002) and vascular invasion(OR=2.0495%CI=1.06-3.95p=0.03),but more articles
need to be included for further study. However, the expression of METTL3 was not related to the age of
the patients or the degree of tumor differentiation(Table 3.)
However, this meta-analysis had some limitations.First of all, the small number of studies included and
the insucient sample size might affect the reliability of the results and hinder the stability of the results
of further stratied analysis. Secondly, although the statistical method did not detect publication bias,
there might be potential bias.Besides, the accuracy of the method of extracting HR from the survival
curve was not high,which would affect the combined HRs.Finally,the cancer patients in our study were
Asian, and the results were a good indication of the association between METTL3 and prognosis in Asian
patients, but the association with patients of other races was not clear.In a word, in order to make the
conclusion more convincing, further research is needed.
Conclusions
In summary, our meta-analysis showed that high expression of METTL3 was associated with poor OS
and DFS in cancer patients, vascular invasion ,as well as with lymph node metastasis of the
tumor.Cancer patients would be still at risk of recurrence or metastasis after treatment.Therefore, it is of
great signicance to nd effective prognostic markers to solve the problems faced by patients in the
process of treatment.In the future, we need to further explore the prognostic value of METTL3 in large-
scale, standard and multi-ethnic clinical researches, in order to apply METTL3 as a novel marker of
prognosis in the clinical guidance of cancer patients as soon as possible.
Abbreviations

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METTL3 methyltransferase like 3
m6A N6- methyladenosine
OS overall survival
DFS disease free survival
PFS progression free survival
HR hazard ratio
OR odds ratio
95%CI 95% condence interval
NOS Newcastle-Ottawa Scale
NA not available
qRT-PCR quantitative reverse transcription PCR
IHC Immunohistochemistry
TNM Tumor Node Metastasis
FIGO Federation International of Gynecology and
Obstetrics
KM Kaplan-Meier
Uni Univariate
N number
OC ovarian carcinoma
GC gastric cancer
CRC colorectal cancer
BCA bladder cancer
HCC hepatocellular carcinoma
HB hepatoblastoma
THCA thyroid carcinoma
OSCC oralsquamous cell cancer
RCC renal cell carcinoma
PCA prostatic cancer
BRD4 bromodomain-containing protein 4
SOCS2 suppressor of cytokine signaling 2

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EGFR epidermal growth factor receptor
MAPK mitogen-activated protein kinase
Wnt /β-Catenin Canonical Wnt/β-catenin pathway
EZH2 Enhancer Of Zeste Homolog 2
LEF1 Lymphoid enhancer-binding factor 1
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
All data used in the study are included in this article.
Competing interests
The authors declare that they have no competing interests in this section.
Funding
This work was supported by the Guangdong Medical Science and Technology Research Fund
(A2020143), Guangdong Gastrointestinal Disease Research Center (No.2017B020209003), and the
Foundation for the President of Nanfang Hospital of Southern Medical University (2018C027).Funding
agencies are not involved in research, collection, analysis and interpretation of data, nor in writing
manuscripts. The author is solely responsible for the content.
Authors’ contributions
QYL and XZP conceived and designed the experiments. XZP, HLL, YYX and CCH collected materials and
prepared the tools for analysis. QHL, XKW and QYL screened and analyzed the collected data. CCH, HLL,
XZP performed the experiments. XZP, HLL and YYX wrote this article. All authors read the nal
manuscript and approved it.
Acknowledgements
We are grateful to all researchers of enrolled studies.

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Tables
Due to technical limitations Table 1-3 are available as downloads in the Supplementary Files.
Table 1: Summary of the included studies.
Table 2: Meta-analysis of METTL3 expression and overall survival.
Table 3: The relationship between the expression of mettl3 and clinical characteristics.
Figures

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Figure 1
Flow chart of the process of searching and selecting articles.

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Figure 1
Flow chart of the process of searching and selecting articles.

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Figure 2
Forest plot of METTL3 and prognosis: A Forest plot for the relationship between the expression of
METTL3 and overall survival rate (OS). B Forest plot for the relationship between the expression of
METTL3 and disease-free survival(DFS).

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Figure 2
Forest plot of METTL3 and prognosis: A Forest plot for the relationship between the expression of
METTL3 and overall survival rate (OS). B Forest plot for the relationship between the expression of
METTL3 and disease-free survival(DFS).

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Figure 3

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Stratied analyses for the relationship between METTL3 expression and overall survival (OS): A
Subgroup analysis of HRs of OS by cancer type. B Subgroup analysis of OS by samples size.

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Figure 3

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Stratied analyses for the relationship between METTL3 expression and overall survival (OS): A
Subgroup analysis of HRs of OS by cancer type. B Subgroup analysis of OS by samples size.
Figure 4
Subgroup analysisA Forest plot for the relationship between METTL3 expression and lymph node
metastasis . B Forest plot for the relationship between METTL3 expression and vascular invasion . C
Forest plot for the relationship between METTL3 expression and TNM stage (III/IV vs. I/II).

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Figure 4
Subgroup analysisA Forest plot for the relationship between METTL3 expression and lymph node
metastasis . B Forest plot for the relationship between METTL3 expression and vascular invasion . C
Forest plot for the relationship between METTL3 expression and TNM stage (III/IV vs. I/II).

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Figure 5
Sensitivity analysis of the impact of a single study on combined HRs.

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Figure 5
Sensitivity analysis of the impact of a single study on combined HRs.

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Figure 6
Publication bias: A Funnel plot of the publication bias for overall survival. B Egger's test of the publication
bias for overall survival.

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Figure 6
Publication bias: A Funnel plot of the publication bias for overall survival. B Egger's test of the publication
bias for overall survival.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
Table2.xlsx
Table2.xlsx
Table1.xlsx
Table1.xlsx
Additionalle1.Moosechecklist.docx
Additionalle1.Moosechecklist.docx
Table3.xlsx
Table3.xlsx
Additionalle2.PRISMAChecklist.docx
Additionalle2.PRISMAChecklist.docx