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. 2021 Sep 21:11:711207.
doi: 10.3389/fonc.2021.711207. eCollection 2021.

Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer

Libin Jiang  1 , Yingxia Li  1 , Yixin He  1 , Dapeng Wei  1 , Lvyin Yan  1 , Hongtao Wen  1
Affiliations

Knockdown of m6A Reader IGF2BP3 Inhibited Hypoxia-Induced Cell Migration and Angiogenesis by Regulating Hypoxia Inducible Factor-1α in Stomach Cancer

Libin Jiang et al. Front Oncol. .

Abstract

Hypoxia is a common feature of solid tumors including stomach cancer (SC) and is closely associated with cancer malignant progression. N6-methyladenosine (m6A), a common modification on RNA, is involved in the regulation of RNA fate and hypoxic responses in cancers. However, the interaction between m6A reader insulin-like growth factor-II mRNA-binding protein 3 (IGF2BP3) and SC hypoxic microenvironment is poorly defined. In the present study, expression levels of IGF2BP3 and hypoxia inducible factor-1α (HIF1A) were examined by bioinformatics analysis and RT-qPCR and western blot assays. Cell migratory ability was assessed through Transwell and wound healing assays. The angiogenic potential was evaluated by VEGF secretion, tube formation, and chick embryo chorioallantoic membrane (CAM) assays. The interaction between IGF2BP3 and HIF1A was explored using bioinformatics analysis and RIP and luciferase reporter assays. The results showed that IGF2BP3 and HIF1A were highly expressed in SC tissues and hypoxia-treated SC cells. IGF2BP3 knockdown inhibited hypoxia-induced cell migration and angiogenesis in SC. IGF2BP3 positively regulated HIF1A expression by directly binding to a specific m6A site in the coding region of HIF1A mRNA in SC cells. HIF1A overexpression abrogated the effects of IGF2BP3 knockdown on hypoxia-induced cell migration and angiogenesis in SC. In conclusion, IGF2BP3 knockdown inhibited hypoxia-induced cell migration and angiogenesis by down-regulating HIF1A in SC.

Keywords: HIF-1A; IGF2BP3; angiogenesis; hypoxia; migration; stomach cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
M6A regulator IGF2BP3 was highly expressed in SC tissues. (A) Global m6A methylation level in total RNA was measured in 20 pairs of SC tumor tissues and adjacent normal tissues. (B) Expression heatmap of 24 m6A-related genes in 303 cases of SC tumor tissues and 32 cases of normal tissues. (C) Violin plots of 24 m6A-related genes in 27 pairs of SC tumor tissues and adjacent normal tissues. Violin plots were drawn using the bioinformatics website (http://www.bioinformatics.com.cn/plot_basic_vertical_horizontal_violin_plot_068). (D) Expression analysis of IGF2BP3 in SC tumor tissues (n=408) and normal tissues (n=211). The figure was drawn by the GEPIA website (http://gepia.cancer-pku.cn/). (E) Expression level of IGF2BP3 in 6 pairs of SC tumor tissues and adjacent normal tissues was measured by western blot assay. * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
IGF2BP3 knockdown hampered hypoxia-induced cell migration and angiogenesis in SC. (A) MKN-45 and HGC-27 cells were cultured in normoxic or hypoxic conditions for 24 h Next, the IGF2BP3 protein level was measured by western blot assay. (B) MKN-45 and HGC-27 cells were transfected with si-NC, si-IGF2BP3#1, si-IGF2BP3#2, or si-IGF2BP3#3. Next, the IGF2BP3 mRNA level was measured by RT-qPCR assay at 48 h after transfection. (C–F) MKN-45 and HGC-27 cells were transfected with si-NC or si-IGF2BP3#1 for 48 h and then maintained in hypoxic conditions for another 24 h Cells in the normoxia group were maintained in normoxic conditions for 72 h Cells in the hypoxia group were cultured in normoxia for 48 h and then exposed to hypoxia for an additional 24 h (C, D) Cell migratory potential was assessed by Transwell migration (C) and wound healing (D) assays. (E) VEGF level in cell culture supernatants was detected using a commercial kit. (F) The conditioned medium of MKN-45 and HGC-27 cells were collected after normoxia/hypoxia treatment or/and transfection. Next, HUVECs were cultured in a mixed medium of ECM and conditioned medium (volume ratio=1:1), followed by the measurement of tube formation ability at 12 h after incubation. * indicate that the difference is significant at 0.05 level. ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001 compared with the normoxia group.
Figure 3
Figure 3
HIF1A as a potential IGF2BP3 target was highly expressed in SC. (A) Venn analysis (http://jvenn.toulouse.inra.fr/app/example.html) for genes in Supplementary Tables 1, 2, and 3. List 1: Starbase-predicted mRNAs that could bind with IGF2BP3. List 2: m6A2Target-analyzed genes that could be positively regulated by IGF2BP3. List 3: GEPIA-analyzed differentially expressed genes in SC tissues versus normal tissues. (B) Expression analysis of HIF1A in 408 cases of SC tumor samples and 211 cases of normal samples by the GEPIA website (http://gepia.cancer-pku.cn/). (C) Expression analysis of HIF1A in primary SC tumor tissues (n=415) and normal tissues (n=34) by the UALCAN database (http://ualcan.path.uab.edu/). (D) Expression level of HIF1A in 6 pairs of SC tumor tissues and adjacent normal tissues was determined by western blot assay. * p < 0.05.
Figure 4
Figure 4
IGF2BP3 positively regulated HIF1A expression in an m6A-dependent manner. (A, B) MKN-45 and HGC-27 cells were transfected with si-NC or si-IGF2BP3#1 for 48 h and then maintained in hypoxic conditions for another 24 h Cells in the normoxia group were maintained in normoxic conditions for 72 h Cells in the hypoxia group were cultured in normoxia for 48 h and then exposed to hypoxia for an additional 24 h Next, HIF1A mRNA and protein levels were measured through RT-qPCR and western blot assays, respectively. (C) RIP assay was performed using IgG or IGF2BP3 antibody in MKN-45 cells. HIF1A level in IgG or IGF2BP3 immunoprecipitation complex was measured through RT-qPCR assay. (D, E) Putative m6A modification and IGF2BP3 binding site on HIF1A mRNA. (F) Luciferase activities were measured at 48 h post-transfection. ** p < 0.01, *** p < 0.001, # p < 0.05.
Figure 5
Figure 5
IGF2BP3 exerted its functions by up-regulating HIF1A. (A) MKN-45 and HGC-27 cells were transfected with pcDNA3.1 or pcDNA-HIF1A. Next, HIF1A mRNA level was measured by RT-qPCR assay at 48 h upon transfection. (B–E) MKN-45 and HGC-27 cells were transfected with si-NC+pcDNA3.1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h, followed by the examination of cell migratory potential (B, C) and VEGF secretion level (D). (E) MKN-45 and HGC-27 cells were transfected with si-NC+pcDNA3.1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h, followed by the collection of conditioned medium. Next, HUVECs were cultured in a mixed medium of ECM and conditioned medium (volume ratio=1:1) and tube formation potential was examined at 12 h after incubation. ** p < 0.01, *** p < 0.001, ## p < 0.01, ### p < 0.001.
Figure 6
Figure 6
IGF2BP3 knockdown inhibited hypoxia-induced angiogenesis by down-regulating HIF1A in vivo. MKN-45 and HGC-27 cells were transfected with si-NC, si-IGF2BP3#1, si-IGF2BP3#1+pcDNA3.1, or si-IGF2BP3#1+pcDNA-HIF1A for 48 h and then maintained in hypoxic conditions for another 24 h. Cells in the normoxia group were maintained in normoxic conditions for 72 h. Cells in the hypoxia group were cultured in normoxia for 48 h and then exposed to hypoxia for an additional 24 h. Next, the conditioned medium was collected and added to the exposed CAM. The angiogenic ability was assessed at 3 days after incubation. *** p < 0.001.
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