N6-methyladenosine (m6A) reader IGF2BP2 promotes gastric cancer progression via targeting SIRT1.

N6-methyladenosine (m6A) modification acts as the most prevalent internal modification in eukaryotic mRNA. Emerging evidence shows the critical biological roles of m6A key enzymes in human cancers. However, the roles of m6A binding protein IGF2BP2 in gastric cancer (GC) progression are still unclear. In this study, we confirmed that IGF2BP2 was highly expressed in GC cell lines and tumor tissues. Knocking down of IGF2BP2 suppressed cell proliferation and migration, and repressed xenograft tumor growth in vivo, while IGF2BP2 overexpression promoted the proliferation and migration. Mechanistically, we identified that IGF2BP2 regulated GC the proliferation/migration through recognizing the m6A modification sites of SIRT1 mRNA. In general, our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner, suggesting that m6A may be a therapeutic target for GC.

Highlights

IGF2BP2was highly expressed in GC cell lines and tumor tissues.

Knocking down of IGF2BP2 suppressed cell proliferation and migration, and repressed xenograft tumor growth in vivo, while IGF2BP2 overexpression promoted the proliferation and migration.

Mechanistically, we identified that IGF2BP2 regulated GC the proliferation/migration through recognizing the m6A modification site of SIRT1 mRNA.

Introduction

Gastric cancer (GC) is one of the most common cancers, meanwhile the second-leading fatal-related cancer worldwide [1,2]. Over the years, comprehensive treatments, including radiotherapy, chemotherapy, and surgery, have made great progress; however, the poor prognosis remains for GC patients and the 5-year survival rate is pessimistic [3]. Thus, the key to develop effective targeted therapies is to uncover the molecular mechanisms underneath GC metastasis [4,5]. Recently, emerging evidence identifies that genetic and epigenetic changes may make a correlation with GC progress.

N [6]-Methyladenosine (m6A) is a critical chemical modification occurred on RNA. M6A is a reversible modification catalyzed by methyltransferase complexes (‘writers’), demethylase (‘erasers’) and binding proteins (‘readers’) [6-8]. METTL3, KIAA1429, WTAP, and METTL14 constitute the methyltransferase complex. Existing literature suggests that m6A plays crucial regulatory roles on human cancer biological functions and regulates various pathological/physiological processes [9,10]. For example, METTL3 and the expression of m6A are both upregulated in human GC tissues and cells, and METTL3 silencing inhibits the proliferation and migration via regulating m6A methylation on YAP1 mRNA level [11]. FTO knockdown significantly down-regulates the mRNA and protein levels ITGB1 expression through positively augmenting ITGB1 mRNA m6A modification level [12]. Overall, these evidences illustrate the potential role of m6A on GC.

Here, we found that IGF2BP2 level upregulated in the GC tissue and cells. Moreover, the upregulated IGF2BP2 closely correlated to the proliferation. migration of GC cells and the poor prognosis of GC patients. The present research tried to evaluate the function of IGF2BP2 in GC and probe into the mechanism of IGF2BP2s involvement in GC. We identified that IGF2BP2 regulated GC the proliferation/migration through recognizing the m6A modification site of SIRT1 mRNA. In general, our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner, suggesting that m6A may be a therapeutic target for GC.

Materials and methods

Tissue specimens

The samples (primary GC tissue specimens and adjacent matched normal tissues) were obtained from 50 GC patients without lymph node metastasis who underwent surgery at the Tianjin Institute of Hepatobiliary Disease between April 2018 and July 2019. In this investigation, no patients received chemotherapy before surgery. Tumor histology and grading were identified according to WHO guidelines. The clinicopathologic data were showed in Table 1.

Table 1.

GC patients’ clinicopathological characteristic with IGF2BP2 expression

		50	IGF2BP2		p-value
			Low	High
Age	<55	27	12	15	0.395
	≥55	23	13	10
Gender	Male	28	15	13	0.776
	Female	22	10	12
TNM stage	I/II	19	13	6	0.041*
	III/IV	31	12	19
Lymph metastasis	Yes	32	15	17	0.556
	No	18	10	8
Distant metastasis	Yes	14	6	8	0.528
	No	36	19	17
Tumor differentiation	Well	10	3	7	0.617
	Moderate	11	5	6
	Poor	29	17	12

TNM, tumor-node-metastasis. Well: Well-differentiated adenocarcinoma; Moderate, moderately differentiated adenocarcinoma; Poor, poorly differentiated adenocarcinoma.

Cell culture and culture

GC cell lines (SNU-216, MKN45, AGS) and the gastric epithelial cells (GES-1) were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and ATCC. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, HyClone) and antibiotics at 37°C under 5% CO2.

Cell transfection

For the construction of vector expression of IGF2BP2, the full-length coding region of IGF2BP2 was cloned into pLVX-IRES-Puro plasmids. To knock down the expression of IGF2BP2, the lentivirus constructs were generated by GenePharma (Shanghai, China). GC cells were infected stably with IGF2BP2-knockdown lentivirus (sh-IGF2BP2#1, sh-IGF2BP2#2) and pLKDCMV-G&PR-U6 negative control vectors (sh-NC), following the manufacturer’s instructions.

Reverse transcription and quantitative real-time PCR (qRT-PCR)

Total RNAs of GC cells were extracted using TRIzol (Thermo, USA). Reverse transcription was performed using PrimeScript RT Master Mix (Takara). Real-time qPCR was performed by SYBR Green PCR kit (TaKaRa, Dalian, China) on Applied Biosystems 7500. GAPDH was used as an internal control for mRNA. The relative RNA expression level was calculated with 2−ΔΔct. The sequences were listed in Table S1.

Western blots and antibodies

GC cells were harvested and lysed in RIPA lysis buffer (Roche, Diagnostics, Mannheim, Germany) containing protease inhibitor. The total protein concentration was determined by BCA protein assay kit (Vazyme, Nanjing, China). Equal amounts of protein samples were separated by SDS-PAGE (12%, sodium dodecyl sulphate polyacrylamide gel electrophoresis) and transferred to PVDF membranes (Merck Millipore, Germany). After being blocked with nonfat skim milk, the target protein expressions were performed primary antibody (anti-IGF2BP2/IMP2, ab124930, 1:1000) and secondary antibodies. GAPDH antibody acted as the internal control.

Cell viability assays

For the cell viability, GC cells (2 × 103/well) were plated into 96-well plates to detect the absorbance value at 450 nm. Cell growth curves was plotted according to the absorbance value.

Cell migration assays

Cell migration was detected using the Matrigel migration Chamber (BD Biosciences). GC cells (2 × 105) were seeded into the upper chamber in serum-free DMEM medium. DMEM with 10% FBS was added into the lower chamber. 48 h later, cells that successfully migrated through the chamber membrane were stained with 0.5% crystal violet and then the cell quantity was analyzed.

Apoptosis analysis

Cell apoptosis was analyzed by flow cytometry and detected as described previously. In brief, transfected GC cells were harvested at 80% confluence. Double staining by fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) was performed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s protocols. The dead cells were calculated and detected the relative ratio of early/lately apoptotic cells.

RNA stability analysis

For the RNA stability analysis, GC cells were treated with actinomycin D (Act D, 2 μg/ml). At indicated time point, the RNA was extracted using Trizol reagent (Invitrogen, Grand Island, NY, USA) and then reverse transcription was performed to measure the relative levels of SIRT1 mRNA using qRT-PCR.

RIP-qPCR

GC cells were cultured in plates and then washed with ice-cold PBS twice. Cells were centrifuged and re-suspended in an equal volume of completed RIP lysis buffer (Merck Millipore). In immunoprecipitation buffer (pH 7.5, 20 mM Tris-HCl, 140 mM NaCl, 0.05% TritonX-100), antibody (5 μg, anti-IGF2BP2) was pre-bound to Protein A/G magnetic beads over night at 4°C with rotation. After immunoprecipitation, the RNA was eluted and dissolved with RNase-free water. The enrichment of SIRT1 mRNA fragment was determined by real-time qPCR.

Tumor xenograft implantation in nude mice

The study was approved by the Ethics Committee of Tianjin Institute of Hepatobiliary Disease. Five week-old nude mice (10 mice) were randomly divided into two groups (five mice per group) and cultured in pathogen-free sterile conditions with continuous sterile food and water. About 5 × 106 MKN45 cells stably transfected with IGF2BP2 shRNA or sh-NC vectors were subcutaneously injected into flank of nude mice. Tumor volume was monitored every three days and calculated according to formula (volume = length×width [2]/2). Animal experiments were performed according to NIH guidelines on animal welfare.

Statistical analysis

Result data was presented as mean ± S.D., and calculated in triplicates. The statistics was performed using SPSS 13.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 8.0 software. Student’s t-test was performed to compare the means between two groups. Survival was analyzed by the Kaplan–Meier survival curve and log-rank test. P < 0.05 was considered as statistically significant.

Results

In this study, we confirmed that IGF2BP2was highly expressed in GC cell lines and tumor tissues. Knocking down of IGF2BP2 suppressed cell proliferation and migration, and repressed xenograft tumor growth in vivo, while IGF2BP2 overexpression promoted the proliferation and migration. Mechanistically, we identified that IGF2BP2 regulated GC the proliferation/migration through recognizing the m6A modification site of SIRT1 mRNA. In general, our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner.

IGF2BP2 indicated the poor prognosis of GC patients

In the clinical samples, the level of IGF2BP2 was found to be upregulated in GC tissue samples as compared to the normal tissue (Figure 1a). In the GEPIA database (http://gepia.cancer-pku.cn/index.html), the IGF2BP2 level was upregulated in gastric adenocarcinoma cohort as compared to the normal cohort (Figure 1b). In the GC cell lines (SNU-216, MKN45, AGS), RT-PCR analysis showed that IGF2BP2 mRNA levels were upregulated compared to normal cell line (gastric epithelial cells, GES-1) (Figure 1c). Kaplan–Meier method following log-rank test indicated that the overall survival rate of the two clusters was significantly different. The group with high IGF2BP2 level had a lower survival for GC patients (Figure 1d). Overall, our data suggested that IGF2BP2 indicated the poor prognosis of GC patients

Figure 1.

IGF2BP2 indicated the poor prognosis of GC patients. (a) The level of IGF2BP2 was detected in the clinical samples as compared to the normal tissue. (b) The level of IGF2BP2 was found to be upregulated in gastric adenocarcinoma cohort as compared to the normal cohort based on GEPIA database (http://gepia.cancer-pku.cn/index.html). (c) RT-PCR analysis revealed the IGF2BP2 mRNA level in GC cell lines (SNU-216, MKN45, AGS) and normal cells (GES-1). (d) Kaplan–Meier method following log-rank test indicated the overall survival rate of GC patients with higher/lower IGF2BP2 level. *p < 0.05; **p < 0.01.

IGF2BP2 promoted the malignant phenotypes of GC cells

To explore the function of IGF2BP2 on the GC malignant progression, we utilized the MKN45 cells for IGF2BP2 overexpression and AGS cells for IGF2BP2 knockdown. The transfection efficiency was determined by RT-PCR and western blot, and results indicated that IGF2BP2 levels were significantly upregulated (Figure 2a) or silenced (Figure 2b). CCK-8 proliferative ability assay found that IGF2BP2 overexpression promoted the proliferation of MKN45 cells and IGF2BP2 silencing reduced the proliferation of AGS cells (Figure 2c). Migration assay using transwell illustrated that IGF2BP2 overexpression accelerated the migrative quantity of MKN45 cells and IGF2BP2 silencing decreased the migrative quantity of AGS cells (Figure 2d, 2e). Apoptosis using flow cytometry demonstrated that IGF2BP2 overexpression mitigated the apoptotic rate and IGF2BP2 silencing augmented the apoptosis (figure 2f, 2g). Taken together, these results clearly demonstrated that IGF2BP2 promoted the malignant phenotypes of GC cells.

Figure 2.

IGF2BP2 promoted the malignant phenotypes of GC cells. (a, b) RT-PCR and western blotting analysis detected the IGF2BP2 mRNA levels or protein levels in MKN45 cells with IGF2BP2 overexpression or AGS cells with IGF2BP2 knockdown. (c) CCK-8 proliferative ability assay showed the proliferation of MKN45 cells for IGF2BP2 overexpression and AGS cells for IGF2BP2 silencing. (d, e) Migration assay using transwell illustrated the migrative quantity of MKN45 cells for IGF2BP2 overexpression and AGS cells for IGF2BP2 silencing. (f, g) Apoptosis using flow cytometry demonstrated the apoptotic rate of MKN45 cells and AGS cells. *p < 0.05; **p < 0.01.

SIRT1 acted as the direct target of IGF2BP2

Previous research illustrated that sirtuin 1 (SIRT1) participated in the GC progression, our team utilized the online predictive tools (SRAMP, http://www.cuilab.cn/sramp) to discover the m6A modification site on SIRT1. Fortunately, we found that there were several potential m6A sites on SIRT1 gene (Figure 3a). Clinical interaction analysis (GEPIA, http://gepia.cancer-pku.cn/index.html) found that IGF2BP2 was positively correlated to the level of SIRT1 levels in stomach adenocarcinoma (STAD) cohorts (Figure 3b). In the 3’-UTR of SIRT1 genome, we found that the m6A modification sites were ‘GGACU’ (chr10:69,676,212 ~ 69,676,257) (Figure 3c). The motif of IGF2BP2 was consistently identified as ‘GGACU’ sequence by RMBase v2.0 ([13]https://rna.sysu.edu.cn/rmbase/)[13[13]https://rna.sysu.edu.cn/rmbase/)[13] (Figure 3d). RNA binding protein immunoprecipitation (RIP) analysis indicated that, comparing with the control IgG, SIRT1 mRNA expression was remarkably enriched in the anti-IGF2BP2 antibody precipitation in GC cells (MKN45, AGS) (Figure 3e). Moreover, the IGF2BP2 overexpression promoted the binding within IGF2BP2/SIRT1, besides, IGF2BP2 silencing decreased the binding within IGF2BP2/SIRT1 (figure 3f). Thus, the above results indicated that SIRT1 acted as the direct target of IGF2BP2.

Figure 3.

SIRT1 acted as the target of IGF2BP2. (a) Online predictive tools (SRAMP, http://www.cuilab.cn/sramp) discovered that the m6A modification site on SIRT1 genome. (b) Clinical interaction analysis (GEPIA, http://gepia.cancer-pku.cn/index.html) found the positive correlation within IGF2BP2 with SIRT1 levels in stomach adenocarcinoma (STAD) cohorts. (c) The m6A modification sites were ‘GGACU’ (chr10:69,676,212 ~ 69,676,257) in the 3’-UTR of SIRT1 genome. (d) The motif of IGF2BP2 was consistently identified as ‘GGACU’ sequence by RMBase v2.0 (https://rna.sysu.edu.cn/rmbase/). (e) RNA binding protein immunoprecipitation (RIP) analysis indicated the enriched SIRT1 mRNA expression in the anti-IGF2BP2 antibody precipitation and control IgG in GC cells (MKN45, AGS). (f) RIP-PCR illustrated the role of IGF2BP2 on IGF2BP2 with SIRT1 in GC cells (MKN45, AGS). **p < 0.01.

IGF2BP2 enhanced the stability of SIRT1 mRNA

Previous work found that SIRT1 acted as the target of IGF2BP2, thus, subsequent studies research was performed to investigate the molecular mechanism. Firstly, RT-PCR analysis found that SIRT1 mRNA was increased upon IGF2BP2 overexpression (Figure 4a); however, SIRT1 mRNA was repressed upon IGF2BP2 knockdown (Figure 4b). RNA stability analysis revealed that the stability of SIRT1 mRNA was enhanced by IGF2BP2 overexpression (Figure 4c), while the stability of SIRT1 mRNA was inhibited in IGF2BP2 knockdown transfection (Figure 4d). Animal in vivo xenograft assay indicated that IGF2BP2 knockdown repressed the tumor volume (Figure 4e) and neoplasm weight (figure 4f). Taken together, these results clearly demonstrated that IGF2BP2 enhanced the stability of SIRT1 mRNA.

Figure 4.

IGF2BP2 enhanced the stability of SIRT1 mRNA. (a, b) RT-PCR analysis was performed to detect the level of SIRT1 mRNA in MKN45 cells transfected with IGF2BP2 overexpression (IGF2BP2 OE) and in AGS cells transfected with IGF2BP2 knockdown (sh-IGF2BP2), as well as their controls vectors. (c, d) RNA stability analysis was performed using the Act D administration (Actinomycin D, 2 μg/ml). MKN45 cells was transfected with IGF2BP2 overexpression (IGF2BP2 OE) or SIRT1 knockdown (si-SIRT1). AGS cells was transfected with IGF2BP2 knockdown (sh-IGF2BP2) or SIRT1 overexpression (SIRT1 OE). (e, f) Animal in vivo xenograft assay was performed in BALB/c nude mice with subcutaneously injection. Tumor volume (e) and neoplasm weight (f) were recorded. *p < 0.05; **p < 0.01.

Discussion

Acting as a leading cause of cancer-related deaths, gastric cancer (GC) had heterogeneous subtypes. Novel insights are inclined to propose new solutions based on epigenetics molecular mechanism. N [6]-methyladenosine (m6A) is one of the most common modifications of messenger RNAs (mRNAs) in eukaryotes [14,15]. Mountains of evidence have shown that m6A is involved in numerous pathophysiological process, including GC.

In this study, we identified that m6A reader IGF2BP2 is upregulated in the GC tissue and cells. Besides, the clinical analysis found that the high expression of IGF2BP2 was closely correlated with the low survival rate of GC patients. Functionally, cellular analysis found that IGF2BP2 promoted the GC cells’ proliferation, migration and reduced the apoptosis. These results illustrated that IGF2BP2 functioned as an oncogene in the GC progression.

In other cancers, IGF2BP2 has been reported to regulate numerous tumorigenesis [16-18]. For example, in hepatocellular carcinoma, researchers found that IGF2BP2 targets thousands of mRNA transcripts, which may be involved in the progression. The identified genes might provide novel insight and facilitate the development of potential biomarkers for hepatocellular carcinoma diagnosis [19]. Moreover, higher IGF2BP2 expression was associated with a poor prognosis of hepatocellular carcinoma patients. IGF2BP2 overexpression promotes the proliferation and directly recognizes the m6A site on FEN1 mRNA to enhance FEN1 mRNA stability [20]. In colorectal cancer, IGF2BP2 promotes the proliferation, invasion, and migration of cancer cells through activating the expression of ErbB2 by recognizing the m6A of YAP [21].

For the GC tumorigenesis, m6A participates in the progression [22]. For instance, m6A methyltransferase KIAA1429 catalyzes the m6A modification on LINC00958 loci and then LINC00958 interacts with GLUT1 mRNA through m [6]A-dependent manner to enhance GLUT1 mRNA transcript stability [23]. YTHDC2 recognizes the m [6]A-modified 5`-UTR of YAP mRNA to enhance the translation efficiency of YAP, and CRISPR-Cas9-mediated YTHDC2 knockout significantly inhibits the cell viability and proliferation of GC cells [24]. METTL16 is highly expressed in GC cells and associated with prognosis, and METTL16 down-regulation inhibits the proliferation via G1/S blocking and reduces the level of cyclin D1 [25]. Collectively, these data demonstrate that m6A regulates GC, which provides the theoretical basis for the strategy of targeting m6A for GC.

Conclusion

Our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner. In summary, we identified IGF2BP2 as a novel tumor promoter that is frequently increased in GC. Mechanistically, IGF2BP2 led to mRNA stability of SIRT1. Overall, our study revealed that IGF2BP2, acting as an oncogene, enhanced SIRT1 mRNA expression through an m6A/IGF2BP2-dependent mechanism in GC (Figure 5).

Figure 5.

Our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner.

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