miR-107 reverses the multidrug resistance of gastric cancer by targeting the CGA/EGFR/GATA2 positive feedback circuit

Abstract

Chemotherapy is still the main therapeutic strategy for gastric cancer (GC). However, most patients eventually acquire multidrug resistance (MDR). Hyperactivation of the EGFR signaling pathway contributes to MDR by promoting cancer cell proliferation and inhibiting apoptosis. We previously identified the secreted protein CGA as a novel ligand of EGFR and revealed a CGA/EGFR/GATA2 positive feedback circuit that confers MDR in GC. Herein, we outline a microRNA-based treatment approach for MDR reversal that targets both CGA and GATA2. We observed increased expression of CGA and GATA2 and increased activation of EGFR in GC samples. Bioinformatic analysis revealed that miR-107 could simultaneously target CGA and GATA2, and the low expression of miR-107 was correlated with poor prognosis in GC patients. The direct interactions between miR-107 and CGA or GATA2 were validated by luciferase reporter assays and Western blot analysis. Overexpression of miR-107 in MDR GC cells increased their susceptibility to chemotherapeutic agents, including fluorouracil, adriamycin, and vincristine, in vitro. Notably, intratumor injection of the miR-107 prodrug enhanced MDR xenograft sensitivity to chemotherapies in vivo. Molecularly, targeting CGA and GATA2 with miR-107 inhibited EGFR downstream signaling, as evidenced by the reduced phosphorylation of ERK and AKT. These results suggest that miR-107 may contribute to the development of a promising therapeutic approach for the treatment of MDR in GC.

Keywords: CGA; EGFR signaling; GATA2; gastric cancer; miRNA; multidrug resistance.

Figure 1

Validation of the CGA/EGFR/GATA2 positive feedback loop in GC patients and screening for miRNAs targeting CGA and GATA2.A, multiplex immunohistochemistry of CGA, GATA2, EGFR, p-EGFR, and CK in two paired GC patients. B, statistical analysis of CGA, GATA2, and p-EGFR expression in GC tissues and adjacent normal tissues (n = 20). C, diagram of screening for CGA- and GATA2-targeting miRNAs. D, expression levels of candidate miRNAs measured by RT‒PCR (n = 3), the data in the bar plots are expressed as the mean ± S.D. E, Kaplan-Meier (https://kmplot.com/analysis/) analyses of correlations between the expression of candidate miRNAs and overall survival of GC patients. Scale bars represent 20 μm. Significant differences were assessed among multiple groups using one-way ANOVA (D) and between two groups using a t test (B). ∗p< 0.05, ∗∗p< 0.01. CI, confidence interval; GC, gastric cancer; HR, hazard ratio.

Figure 2

miR-107 directly targets CGA and GATA2 in MDR GC cells.A, immunoblots and quantitative analysis of the CGA and GATA2 proteins in SGC7901^ADR and SGC7901^VCR cells. Blots are representative of three independent experiments. a.u., arbitrary unit. The data are presented as the means ± S.D.s from three independent experiments. B, predicted binding sites of miR-107 in the 3′UTR of CGA and GATA2 mRNA. C, relative luciferase reporter activity in HEK293T cells cotransfected with WT or mutated (Mut) reporter plasmids and miR-107, miR-107 inhibitor, and their controls. D, protein expression of CGA and GATA2 after treatment with different concentrations of miR-107 in SGC7901^ADR and SGC7901^VCR cells. E, protein expression of CGA and GATA2 after transfection of the CGA or GATA2 plasmid containing the WT 3′UTR or lacking the 3′UTR (no 3′UTR) along with miR-107 or miR-ctrl in HEK293T cells. The data in the bar plots are expressed as the mean ± S.D. Significant differences were assessed by using one-way ANOVA (A) and the t test (C). ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001. ns, not significant.

Figure 3

miR-107 sensitizes MDR GC cells to chemotherapeutic drugs in vitro.A, proliferation of SGC7901^ADR and SGC7901^VCR cells after transfection with miR-107 or miR-ctrl in the presence of chemotherapeutic drugs. B, IC50 values in SGC7901^ADR and SGC7901^VCR cells after transfection with miR-107 or miR-ctrl in the presence of chemotherapeutic drugs. C and D, apoptosis of SGC7901^ADR and SGC7901^VCR cells after transfection with miR-107 or miR-ctrl in the presence of chemotherapeutic drugs. E, Western blot analysis of Bax and Bcl2 in SGC7901^ADR and SGC7901^VCR cells (upper) or SGC7901 cells stably expressing CGA or GATA2 (lower) after transfection with miR-107 or miR-ctrl. F, proliferation of SGC7901 cells after transfection with miR-107, miR-ctrl, CGA, or the GATA2 vector in the presence of chemotherapeutic drugs. G, apoptosis of SGC7901 cells after transfection with miR-107, miR-ctrl, and CGA and GATA2 vector in the presence of chemotherapeutic drugs. The data in the bar plots are expressed as the mean ± S.D. (n = 3). Significant differences were assessed in multiple groups using one-way ANOVA (G) or repeated-measures ANOVA (A and F) and in two groups using the t test (B and C). ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001. Scale bar represents 50 μm.

Figure 4

miR-107 sensitizes MDR xenografts to chemotherapeutic drugs in vivo.A-C, nude mice (n = 5) were subcutaneously implanted with SGC7901^ADR cells. When the tumor size reached 100 mm³, the mice received the indicated treatment every 3 days: (A) adriamycin, 8 mg/kg, i.p. injection; fluorouracil, 20 mg/kg, i.p. injection; miR-107 prodrug, 1 nmol/mouse intratumoral injection. Tumor volume (B) and tumor weight (C) were measured. D, representative images of tumors from each group. E, representative IHC staining of Ki67, PCNA, Bax, and cleaved Caspase-3 in xenografts harvested from the indicated groups. Scale bar represents 50 μm. The percentages of staining-positive cells were measured. F, mouse body weights for each group were recorded. G, representative H&E staining images of xenografts, livers, and kidneys from each group. Scale bars represent 50 μm. The data in the bar plots are expressed as the mean ± S.D, (E) or mean ± S.E.M. (B, C, and F) (n = 5). Significant differences were assessed in multiple groups using repeated-measures ANOVA test (B) or one-way ANOVA test (C) and two groups using the t test (E). ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001. MSA, negative control of the miR-107 prodrug. IHC, immunohistochemistry.

Figure 5

miR-107 inhibits EGFR downstream signaling in MDR GC cells and xenografts.A, IHC staining of CGA, GATA2, p-EGFR, p-ERK, and p-AKT in xenografts from mice given the indicated treatments. The percentages of staining-positive cells were measured. B and C, Western blotting analysis of total and phosphorylated EGFR, AKT, and ERK after transfection of GC cells with miR-107 or miR-ctrl. D, proposed mechanism model in this study, drawn from the website “https://www.biorender.com/”. Scale bars represent the following: upper, 20 μm; lower, 50 μm. The data in the bar plots are expressed as the mean ± S.D. (n = 5). Significant differences were assessed by the t test (A). ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001. IHC, immunohistochemistry; ns, not significant.