- Academic Editor
†These authors contributed equally.
Background: N6-methyladenosine (m
Gastric cancer (GC) ranks as the fifth most common and fourth most deadly cancer globally, posing a serious threat to human health [1]. GC is particularly common in China, where the proportion of new cases and deaths accounts for almost half of the world’s total case load. Patients with GC are usually diagnosed at a late stage, and have poor treatment options and prognosis [2]. The 5-year overall survival rate of patients with advanced distant metastases remains less than 5% [3]. Therefore, it is urgent to better understand the molecular mechanisms that control GC development and progression to reduce GC morbidity and mortality.
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This study aims to analyze the expression and prognostic relationship of YTHDF1 in GC and adjacent tissues in clinical samples. We also report the effects of knockdown and overexpression of YTHDF1 on the phenotype of GC cells. The downstream target genes and pathways of YTHDF1 were also studied by analyzing RNA-seq, RNA immunoprecipitation sequencing (RIP-seq), and m6A-seq data with the hope that these findings may provide a basis for a novel molecular targeted therapy for GC.
A total of 24 paired GC tissues and corresponding adjacent normal tissues were collected from the First Affiliated Hospital of Chongqing Medical University (Chongqing, China). Pathological examination was performed to confirm the resected samples were GC tissues. Human ethnics approval was obtained from the Ethics committee of the First Affiliated Hospital of Chongqing Medical University. The informed consents were obtained from all patients.
RNA-seq data was acquired from the Cancer Genome Atlas (TCGA) database. This data included 27 patient-matched tumor and normal samples, 32 unpaired normal samples, and 375 unpaired tumor samples.
The GC cell lines MGC803 and AGS were purchased from the Type Culture Collection
of the Chinese Academy of Sciences (Beijing, China). All cell lines were
validated by STR profiling and tested negative for mycoplasma. Cells were
cultured in a humidified incubator at 37 °C with 5% CO
Total RNA from tissues and cells was isolated using Trizol reagent (#9108, Takara, Osaka, Japan) according to manufacturer’s protocol. The concentration and quality of RNA was tested by using a nanodrop (#K5600C, KAIAO, Beijing, China). The RNA was used in cDNA synthesis reactions using PrimeScript RT reagent kit (#RR037A, Takara, Osaka, Japan). Relative RNA levels of YTHDF1 was determined using Ultra SYBR Mixture (#RR42WR, Takara, Osaka, Japan) and GAPDH mRNA was used as internal reference. Primers used in qRT-PCR are listed in Supplementary Table 1.
Small interfering RNA (siRNA) were used to knockdown gene expression, and siRNA targeting YTHDF1 and TCF7 was purchased from Sangon Biotech (Sangon Biotech, Shanghai, China).
Small hairpin RNA (shRNA) targeting YTHDF1 were ligated into the psi-LVRU6GP plasmid and a full-length YTHDF1 cDNA was cloned into the phosphoglycerate dehydrogenase-Cytomegalovirus (pCDH-CMV) plasmid. All plasmids were transfected into cells using Lipofectamine 2000 (#11668019, Invitrogen, Carlsbad, CA, USA). YTHDF1 overexpression and knockdown plasmids, together with psPAX2, pMD2.G plasmids, were transfected into 293T cells to generate lentiviral particles, and these lentivirus were subsequently used to transduce GC cell lines. Puromycin (4 µg/µL, Beyotime, Shanghai, China) was used to select stably transduced GC cells. Information relevant to siRNA, shRNA, and primers used is shown in Supplementary Table 2.
Flow cytometry was conducted at Chongqing Medical University, College of Life
Sciences and was used to evaluate cell cycle dynamics and apoptosis of GC cells.
For CCK-8 assays, cells were seeded in 96-well plates at a density of 2
RIP was conducted according to the manufacturer’s instructions included in the Magna RIP Kit (Merck Millipore, Billerica, MA, USA). RIP experiments were conducted with anti-YTHDF1 antibody (Proteintech, Wuhan, China). Co-precipitated TCF7 was analyzed using qRT-PCR.
Tissues from GC patients and xenografts were fixed using 10% paraformaldehyde and subsequently embedded in paraffin. Immunohistochemistry staining was conducted as previously described [19]. Primary antibodies used were anti-YTHDF1 (1:200, Proteintech, Wuhan, China) and anti-Ki-67 (1:500, Cell Signaling Technology, Boston, USA). IHC images were captured with an inverted Leica microscope (#Leica DMI3000B, Leica, Wetzlar, Germany).
Proteins from cell and tissue samples were extracted using RIPA lysis buffer (1:100) containing PMSF (Beyotime, Shanghai, China). Proteins were subsequently added to SDS-PAGE gels prior to conducting electrophoresis. Proteins in SDS-PAGE gels were electrotransferred to PVDF membranes (#03010040001, Millipore, USA). Then, the PVDF membrane was blocked using 5% skim milk (Beyotime, Shanghai, China) for 1 hours. The primary antibodies used in this study are anti-GAPDH (1:6000, Proteintech, Wuhan, China), anti-YTHDF1 (1:3000, Proteintech, Wuhan, China), anti-MYC (1:8000, Proteintech, Wuhan, China), anti-Cyclin D1 (1:5000, Proteintech, Wuhan, China), anti-MMP7 (1:2500, Proteintech, Wuhan, China), and anti-TCF7 (1:5000, Proteintech, Wuhan, China). These antibodies were incubated with PVDF membranes at 4 °C for 12 hours. The next day, the membrane was incubated with corresponding secondary antibody (1:6000, Proteintech, Wuhan, China) for 2 hours. Proteins were visualized using an enhanced chemiluminescence solution ECL (#K-12049-D50, Advansta, CA, USA).
Six female nude mice were bought from the Animal Experimental Laboratory of
Chongqing Medical University. MGC803 cells stably overexpressing YTHDF1 and
control cells were injected (7
All statistical analyses were conducted using GraphPad Prism 5 (GraphPad
Software, San Diego, CA, USA), SPSS 19.0 (SPSS, Chicago, IL, USA) and R (version
4.1.0, Auckland, New Zealand) software. Data are expressed as mean
To explore a potential role for YTHDF1 in GC, we first accessed gene expression and survival data from the TCGA database. We compared the expression level of YTHDF1 in 375 tumor tissues and 32 normal tissues and result indicated that the mRNA level of YTHDF1 is significantly higher in tumor tissues (Fig. 1A). Next, we compared expression of YTHDF1 between 27 paired GC tissues and adjacent normal tissues and similar results showing higher expression in GC tumors was observed in these tissue pairs (Fig. 1B). We then compared overall survival differences between the low YTHDF1 expression group and the high YTHDF1 expression groups. Here, we observed that GC patients with higher YTHDF1 expression have poorer overall survival (Fig. 1C).
YTHDF1 is differentially expressed in GC. (A,B) RNA
level of YTHDF1 in unpaired and paired GC tissues and adjacent normal tissues in
the TCGA database. (C) Survival differences of GC patients with high or
low expression of YTHDF1 were analyzed using Kaplan-Meier analyses. (D) RNA
expression level of YTHDF1 in 24 patient-matched GC and normal tissues. (E,F) The
protein level of YTHDF1 in 24 paired GC tissues and corresponding normal tissues
were detected using immunohistochemical and western blot assays (scale
bar (200
We also collected 24 paired GC cancer/normal tissues from our hospital as an independent validation of the differential expression of YTHDF1 observed in the TCGA dataset. Results demonstrated that YTHDF1 mRNA was elevated in most GC tissues compared with adjacent normal tissues (Fig. 1D). In addition, immunohistochemistry and western blotting were used to directly evaluate the level of YTHDF1 protein in these tissues. Results obtained showed that YTHDF1 was upregulated in 20 out of 24 GC tissues in our panel when compared with adjacent normal tissues (Fig. 1E,F).
To further explore the role of YTHDF1 in GC, we constructed GC cell lines stably overexpressing YTHDF1. qRT-PCR and western blot assays were utilized to confirm overexpression (Fig. 2A).
Overexpression of YTHDF1 promotes proliferation and inhibits
apoptosis of GC Cells. (A) qRT-PCR and western blot assays was conducted to
evaluate the over expression efficiency of YTHDF1 in GC cells. (B,C) CCK-8
results proved that YTHDF1 promote the proliferation ability of GC cells. (D,E)
Colony formation assays were used to evaluate the effect of YTHDF1 on
proliferation ability of GC cells. (F,G) Flow cytometry was utilized to assess
the cell cycle of GC cells after over expression of YTHDF1. (H,I) EdU staining
was performed to evaluate the DNA synthesis abilities of GC cells after over
expression of YTHDF1 (scale bar = 120 µm). (J,K) Flow cytometry was conducted
to determine whether YTHDF1 effects the apoptotic population in cultured GC
cells. *, p
CCK-8 and colony forming assays indicated that proliferative ability of GC cells was increased following YTHDF1 overexpression (Fig. 2B–D). Flow cytometry indicated that YTHDF1 overexpression induced a higher proportion of S phase cells than that seen in normal control (NC) cells (Fig. 2E–G). In addition, EdU staining was utilized to measure cell proliferation in GC cells overexpressing YTHDF1. Consistent with other results, YTHDF1 overexpression significantly enhanced the rates of DNA synthesis in GC cells (Fig. 2H,I).
We also examined whether YTHDF1 could affect apoptosis in GC cells. The results suggested that YTHDF1 overexpression partially inhibited GC cell apoptosis (Fig. 2J,K). Taken together, these results suggest that YTHDF1 may act in promoting GC tumor growth.
To further examine the potential tumor promoting role of YTHDF1, we designed siRNAs targeting YTHDF1. Knockdown efficiency was measured using qRT-PCR and western blotting (Fig. 3A). CCK-8 and colony formation assays were used to evaluate the proliferative ability of GC cells. We observed that YTHDF1 knockdown significantly inhibited GC cell growth (Fig. 3B–D). Flow cytometry results demonstrated that MGC803 and AGS cells transfected with siRNA specific for YTHDF1 were arrested in the G0/G1 phase of the cell cycle (Fig. 3E–G). Result of EdU assay also showed that proliferation ability of the YTHDF1 knockdown group was significantly inhibited when compared to EdU incorporation than the NC cell group (Fig. 3H,I). Analysis of apoptosis by flow cytometry showed that the number of apoptotic GC cells was increased after knockdown of YTHDF1 (Fig. 3J,K). Taken together, these results suggest that YTHDF1 exert a crucial role in establishing/maintaining GC cell malignancy.
Knock down of YTHDF1 inhibits proliferation and promotes
apoptosis of GC Cells. (A) Knockdown efficiency of YTHDF1 in GC cells was
measured using qRT-PCR and western blot analysis. (B,C) CCK-8 assays indicate
that knockdown of YTHDF1 inhibits the proliferation of GC cells. (D,E) Colony
formation assays were used to evaluate the effect of YTHDF1 knockdown on GC cell
colony formation ability. (F,G) Flow cytometry was utilized to assess the effect
of YTHDF1 knockdown on the cell cycle of GC cells. (H,I) EdU staining was
performed to evaluate the proliferation status of GC cells after knockdown of
YTHDF1 (scale bar = 120 µm). (J,K) Flow cytometry was conducted to determine
the effects of YTHDF1 knockdown on GC cell apoptosis. *, p
To further explore the underlying mechanism mediated by YTHDF1, we conducted
transcriptome sequencing on GC cells with YTHDF1 knockdown (Supplementary
Table 3). Using the filter condition of (
Identification of potential target pathways regulated by YTHDF1. (A,B) Transcriptome sequencing was conducted after YTHDF1 knockdown. (C) mRNA expression differences between NC and YTHDF knockdown groups were visualized using a heatmap and a volcano map. GO and KEGG enrichment analyses were conducted on the differently expressed genes between the NC group and the YTHDF knock down group. (D) meRIP-seq, RNA-seq and RIP-seq data collected on MGC803 cells in a previously published study were acquired and interogated to obtain the potential target genes of YTHDF1. (E) GO and KEGG enrichment results of 991 YTHDF1 potential target genes from the above analyses. (F) Cytoscape software was utilized to visualize core genes involved in top four signal pathways and two signal pathways correlated with GC. mRNA, messenger RNA; NC, normal control; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; meRIP-seq, methylated RNA immunoprecipitation sequencing.
Next, we performed Kyoto Encyclopedia of Genes
and Genomes (KEGG) enrichment analysis on these differentially expressed
genes. We found that several tumor-associated signaling pathways such as MAPK
signaling, Hippo signaling, WNT signaling, and TGF-
Considering that YTHDF1 was reported to enhance the translational efficiency of its target genes, we speculated that YTHDF1 might promote target gene translation and thereby affect these tumor related pathways. To identify YTHDF1 direct target genes, we acquired the meRIP-seq, RNA-seq and RIP-seq datasets obtained from the analysis of MGC803 cells from a previously published study [20]. Our analysis of these data sets uncovered 991 genes that are potential direct targets of YTHDF1 (Fig. 4D). Furthermore, we found that these genes were also enriched in Hippo signaling, WNT signaling, and in cells responding to human papillomavirus (HPV) infection (Fig. 4E). To acquire the core genes mediated by YTHDF1, we queried the genes involved in the top six signaling pathways associated with GC. These efforts identified eight (8) genes as core components within these pathways (Fig. 4F).
Among these core genes, we noted that the transcription factor TCF7 was
associated with all six signaling pathways and, thus, TCF7 was selected for
subsequent analyses. We found that TCF7 has potential m
YTHDF1 regulate the translation efficiency of TCF7, thereby
affecting WNT pathway. (A) An online database (https://www.cuilab.cn/) was used
to visualize the predicted m
Further, we also observed that the mRNA level of TCF7 was not altered after YTHDF1 knockdown (Fig. 5E), and the TCF7 protein level was elevated after overexpression of recombinant YTHDF1 as judged by western blotting (Fig. 5F). Of note, we observed TCF7 protein level downregulation following YTHDF1 knockdown (Fig. 5G).
We also examined the effect of TCF7 on the WNT pathway. After knockdown of TCF7 expression, the mRNA expression levels of WNT downstream target genes, including c-myc, Cyclin D1, Survivin, MMP7 and CD44, were decreased (Fig. 5H). Consistent with these findings, YTHDF1 knockdown in MGC803 and AGS cells resulted in decreased expression of TCF7 protein as were the levels of Cyclin D1, MMP7, and MYC (Fig. 5G). Taken together, these results suggest that YTHDF1 activates the WNT pathway by upregulating TCF7, and that this molecular event likely promotes GC development and/or progression.
To directly verify that YTHDF1 serves in a tumor promoting role in vivo, MGC803 cells with stable knockdown of YTHDF1 were injected into 4-weeks female nude mice. These MGC803 knockdown cells were engineered by transducing cells with lentivirus encoding a YTHDF1-specific shRNA or normal control (NC) shRNA sequences. Tumor volume was measured every 7 days and after 28 days, we found that the tumor size in the YTHDF1 knockdown tumors was significantly smaller than those in the NC group (Fig. 6A) and that this was observed in four measurements (Fig. 6B). Tumor weight in the YTHDF1 knockdown group was also markedly lower compared with the NC group (Fig. 6C). In addition, compared with the NC group, expression of YTHDF1 and the proliferation marker Ki-67 were significantly lower in the YTHDF1 knockdown group as indicated by IHC staining (Fig. 6D). The protein level of YTHDF1, as well as its target gene TCF7, were also found to be lower in the YTHDF1 knockdown group than that of NC group as measured by western blotting (Fig. 6E). Taken together, these results strongly suggest that YTHDF1 contributes to GC malignancy both in vitro and in vivo.
YTHDF1 promote GC growth both in vivo. (A) Knockdown
of YTHDF1 effectively inhibits GC tumor growth in vivo. (B,C) Tumor
volume and tumor weight was decreased in the YTHDF1 knockdown group when compared
to the normal control group. (D) Expression of YTHDF1 and Ki-67 in NC and YTHDF1
knockdown tumors was determined by IHC staining (scale bar = 200 µm). (E)
Western blotting was performed to evaluate the protein level of YTHDF1 and TCF7
after YTHDF1 knockdown. *, p
Gastric cancer is one of the most common digestive tract malignancies and exhibits high morbidity and mortality on a worldwide basis [1, 21]. Despite great improvements in both the diagnostic and treatment strategies for GC developed in recent years, the mortality of this cancer type remains high. Mounting evidence indicates that epigenetic alteration, genetic mutation, and aberrant activation, or inactivation, of tumor-related signaling pathways are closely associated with gastric carcinogenesis and progression [21, 22]. Thus, exploring underlying mechanisms that contribute to gastric carcinogenesis and progression is of great significance as such knowledge may lead to new, effective prognostic biomarkers or potential therapeutic targets for GC patients.
In recent years, mounting evidence implies that m
Beyond these various YTH family members, YTHDF1 was found to be involved in the progression of multiple tumors including GC [15, 16, 17]. Most studies suggested that YTHDF1 promotes the translation of its target genes, thereby affecting tumor progression [15, 16, 17]. Although the function of YTHDF1 has been preliminary explored [17, 18], there were still much unknown in regard to the function of YTHDF1 in GC.
In this study, we initially explored the expression of YTHDF1 in GC using the TCGA-STAD data portal. We found that YTHDF1 was aberrantly overexpressed in GC compared to normal tissue. Subsequently, elevated expression of YTHDF1 was validated in 24 patient-matched GC/normal tissue samples. In addition, survival analysis indicated that patients with higher YTHDF1 levels exhibit poorer survival compared to those patients who do not have elevated YTHDF1 expression. Based on these results, we speculated that YTHDF1 might be involved in GC carcinogenesis or progression.
We overexpressed YTHDF1 in cultured GC cells and determined the effect of YTHDF1 on proliferation and apoptosis. We found that YTHDF1 could promote the proliferation and inhibit the apoptosis of GC cells. Conversely, knockdown of YTHDF1 was found to significantly inhibit the proliferation and promote apoptosis in these cells. These results suggested that YTHDF1 serves in an oncogene role in GC consistent with a previous study [30]. Another study found that YTHDF1 modulates transcription of the deubiquitinating enzyme USP14 and that this promoted GC tumorigenesis and metastasis [17].
While m
KEGG pathway analysis revealed that these genes were associated with several
tumor-related pathways. Among these pathways, Wnt signaling has been reported to
control multiple fundamental cell functions, including proliferation, migration,
differentiation, and stemness [31]. Underscoring its importance in GC, nearly
46% of GC tumors exhibit dysregulation of the WNT/
Another signaling axis that was revealed in our studies were signaling pathways that regulate pluripotency of stem cells. The stemness of GC cells confer upon tumors the capability for self-renewal and the generation of heterogeneous tumor cells [35]. Cancer stem cells play a pivotal role in cancer metastasis, recurrence, and resistance to chemotherapy [36]. It is evident that these pathways play crucial roles in the onset and progression of gastric cancer.
We further visualized the correlation of these GC-associated pathways and core
genes using cytoscape. The results identified eight genes, including FZD1, GSK3B,
FZD8, AXIN1, FZD6, FZD7, APC2 and TCF7, as the core genes of these pathways.
Interestingly, these genes are largely components of the WNT pathway [37].
However, the transcription factor TCF7 is associated with all six signaling
pathways leading us to further explore the relationship between YTHDF1 and TCF7.
Results indicated that YTHDF1 could modulate the translational efficiency of
TCF7. Moreover, m
In summary, we identified that YTHDF1 was highly expressed in GC tissues. YTHDF1 could promote the proliferation ability and inhibit the apoptosis of GC cells in vitro. In addition, YTHDF1 promotes GC tumor growth in vivo. Mechanistically, YTHDF1 activates WNT signaling via upregulation of TCF7 translation. Our results support a novel regulatory mechanism of YTHDF in GC, which might shed light on GC development and progression.
GC expression and survival data used in this study can be found in TCGA (https://portal.gdc.cancer.gov/). The project ID is “STAD”. All the datasets generated/or analyzed in our study are available from the corresponding author on reasonable request.
YQL, XLL and ZWW designed this study. YQL and XLL conducted the cell experiments. YQL and XG conducted the animal experiments. YQL wrote the draft. ZWW reviewed the article. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
The informed consents were obtained from all patients. The human study was carried out in accordance with the declaration of Helsinki. The animal study was conducted in accordance with the ARRIVE guidelines. The animal study was carried out in accordance with relevant guidelines and regulations of the Ethics committee of Chongqing Medical University. Human study and animal study was approved by the Ethics committee of the First Affiliated Hospital of Chongqing Medical University. The approval number is 2021-30.
Not applicable.
This study was funded by the National Natural Science Foundation of China (81974385).
The authors declare no conflict of interest.
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