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Exosomal MiR‐500a‐3p promotes cisplatin resistance and stemness via negatively regulating FBXW7 in gastric cancer

Lin, Hao, Zhang, Liang, Zhang, Caihua, Liu, Pengpeng·2020·Journal of Cellular and Molecular Medicine
DOI 10.1111/jcmm.15524PMC PMC7417713
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Abstract

Chemoresistance has been a major challenge in advanced gastric cancer (GC) therapy. Exosomal transfer of oncogenic miRNAs implicates important effects in mediating recipient cell chemoresistance by transmitting active molecules. In this study, we found that microRNA‐500a‐3p was highly expressed in cisplatin (DDP) resistant GC cells (MGC803/DDP and MKN45/DDP) and their secreted exosomes than that in the corresponding parental cells. MGC803/DDP‐derived exosomes enhance DDP resistance and stemness properties of MGC803 recipient cells via exosomal delivery of miR‐500a‐3p in vitro and in vivo through targeting FBXW7. However, reintroduction of FBXW7 in MGC803 cells reverses miR‐500a‐3p‐mediated DDP resistance as well as stemness properties. Furthermore, elevated miR‐500a‐3p in the plasma exosomes of GC patients is correlated with DDP resistance and thereby results in poor progression‐free prognosis. Our finding highlights the potential of exosomal miR‐500a‐3p as an potential modality for the prediction and treatment of GC with chemoresistance.

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FIGURE 1 DDP resistant MGC803 and MKN45 cells display stem cell‐like features. A, B, Proliferation assay analysis and IC50 value of DDP resistant GC cells (MGC803/DDP and MKN45/DDP) and corresponding parental cells (MGC803 and MKN45). C, D, Representative images showed the bioluminescent signals of abdominal tumorigenesis by indicated GC cells under DDP treatment (left panel). Comparison of bioluminescent signals on the day 21 was analysed (right panel), five nude mice in each group. E, F, Comparison of abdominal metastatic nodules by indicated GC cells under DDP treatment. G, Expression level of stemness markers CD133, CD44 and SOX2 in indicated GC cells by confocal microscopy. H, Sphere formation assay in indicated GC cells. *P < 0.05, **P < 0.01
FIGURE 1 DDP resistant MGC803 and MKN45 cells display stem cell‐like features. A, B, Proliferation assay analysis and IC50 value of DDP resistant GC cells (MGC803/DDP and MKN45/DDP) and corresponding parental cells (MGC803 and MKN45). C, D, Representative images showed the bioluminescent signals of abdominal tumorigenesis by indicated GC cells under DDP treatment (left panel). Comparison of bioluminescent signals on the day 21 was analysed (right panel), five nude mice in each group. E, F, Comparison of abdominal metastatic nodules by indicated GC cells under DDP treatment. G, Expression level of stemness markers CD133, CD44 and SOX2 in indicated GC cells by confocal microscopy. H, Sphere formation assay in indicated GC cells. *P < 0.05, **P < 0.01CC BY · PubMed Central
FIGURE 2 MGC803/DDP‐derived exosomes disseminate DDP resistance and promote CSCs properties in recipient MGC803 cells. A, Electron microscopy and NTA images of exosomes isolated from MGC803 and MGC803/DDP cells medium. B, C, Identical quantity of MGC803 and MGC803/DDP cells were cultured 30 h and then their exosomes concentrations and positive markers (CD63, HSP81) were analysed by NTA (B) and Western blot (C). D, MGC803 cells were incubated with PHK67‐labelled exosomes from MGC803 and MGC803/DDP cells for 30 h, and the internalization of exosomes was detected by confocal microscopy. E, Uptake efficiency of PHK67‐labelled exosomes was examined by confocal microscopy. F, Proliferation assay analysis and IC50 value of MGC803 cells co‐cultured with PBS, MGC830 exosomes or MGC803/DDP exosomes under DDP treatment. G, Representative bioluminescent images and microscopy observations showed effects of PBS, MGC803 exosomes or MGC803/DDP exosomes on abdominal tumorigenesis under DDP treatment. H, I, Comparison of bioluminescent signals and abdominal metastatic nodules after indicated treatment, five nude mice in each group. J, K, Effect of MGC803 exosomes and MGC803/DDP exosomes on sphere formation (J) and expression levels of stemness markers (K) in MCG803 cells by Western blot. *P < 0.05, **P < 0.01
FIGURE 2 MGC803/DDP‐derived exosomes disseminate DDP resistance and promote CSCs properties in recipient MGC803 cells. A, Electron microscopy and NTA images of exosomes isolated from MGC803 and MGC803/DDP cells medium. B, C, Identical quantity of MGC803 and MGC803/DDP cells were cultured 30 h and then their exosomes concentrations and positive markers (CD63, HSP81) were analysed by NTA (B) and Western blot (C). D, MGC803 cells were incubated with PHK67‐labelled exosomes from MGC803 and MGC803/DDP cells for 30 h, and the internalization of exosomes was detected by confocal microscopy. E, Uptake efficiency of PHK67‐labelled exosomes was examined by confocal microscopy. F, Proliferation assay analysis and IC50 value of MGC803 cells co‐cultured with PBS, MGC830 exosomes or MGC803/DDP exosomes under DDP treatment. G, Representative bioluminescent images and microscopy observations showed effects of PBS, MGC803 exosomes or MGC803/DDP exosomes on abdominal tumorigenesis under DDP treatment. H, I, Comparison of bioluminescent signals and abdominal metastatic nodules after indicated treatment, five nude mice in each group. J, K, Effect of MGC803 exosomes and MGC803/DDP exosomes on sphere formation (J) and expression levels of stemness markers (K) in MCG803 cells by Western blot. *P < 0.05, **P < 0.01CC BY · PubMed Central
FIGURE 3 MGC803/DDP‐derived exosomes enhance DDP resistance of MGC803 recipient cells via exosome‐mediated delivery of miR‐500a‐3p in vitro and in vivo. A, B, Real‐time qRT‐PCR analysis of miR‐500a‐3p expression in MGC803, MGC803/DDP and their corresponding exosomes. C, D, Real‐time qRT‐PCR analysis of miR‐500a‐3p expression in MGC803 cells treated by indicated conditions and their corresponding proliferation assay analysis as well as IC50 value under DDP treatment. E, F, Real‐time qRT‐PCR analysis of miR‐500a‐3p or pre‐miR‐500a‐3p expression in MGC803 cells treated by MGC803 exosomes or MGC803/DDP exosomes at indicated time. G, Real‐time qRT‐PCR analysis of miR‐500a‐3p expression in the culture medium (CM) of MGC803/DDP after treatment with RNase (2 μg/mL) alone or combined with Triton X‐100 (0.1%) for 20 min. H, MGC803 or MGC803/DDP cells transfected with the PHK67‐miR‐500a‐3p mimic (green fluorescence) were placed in the upper chamber and coincubated with MGC803 cells seeded in the lower chamber in a transwell system with a 0.4 μm pore membrane. After coincubation for 30 h, MGC803 cells in the lower chamber were examined by the fluorescence microscope. I, Representative bioluminescent images and microscopy observations showed effects of CM without exosomes or indicated MGC803 cell‐derived exosomes on abdominal tumorigenesis under DDP treatment. J, K, Comparison of bioluminescent signals and abdominal metastatic nodules after indicated treatment, five nude mice in each group. *P < 0.05, **P < 0.01
FIGURE 3 MGC803/DDP‐derived exosomes enhance DDP resistance of MGC803 recipient cells via exosome‐mediated delivery of miR‐500a‐3p in vitro and in vivo. A, B, Real‐time qRT‐PCR analysis of miR‐500a‐3p expression in MGC803, MGC803/DDP and their corresponding exosomes. C, D, Real‐time qRT‐PCR analysis of miR‐500a‐3p expression in MGC803 cells treated by indicated conditions and their corresponding proliferation assay analysis as well as IC50 value under DDP treatment. E, F, Real‐time qRT‐PCR analysis of miR‐500a‐3p or pre‐miR‐500a‐3p expression in MGC803 cells treated by MGC803 exosomes or MGC803/DDP exosomes at indicated time. G, Real‐time qRT‐PCR analysis of miR‐500a‐3p expression in the culture medium (CM) of MGC803/DDP after treatment with RNase (2 μg/mL) alone or combined with Triton X‐100 (0.1%) for 20 min. H, MGC803 or MGC803/DDP cells transfected with the PHK67‐miR‐500a‐3p mimic (green fluorescence) were placed in the upper chamber and coincubated with MGC803 cells seeded in the lower chamber in a transwell system with a 0.4 μm pore membrane. After coincubation for 30 h, MGC803 cells in the lower chamber were examined by the fluorescence microscope. I, Representative bioluminescent images and microscopy observations showed effects of CM without exosomes or indicated MGC803 cell‐derived exosomes on abdominal tumorigenesis under DDP treatment. J, K, Comparison of bioluminescent signals and abdominal metastatic nodules after indicated treatment, five nude mice in each group. *P < 0.05, **P < 0.01CC BY · PubMed Central
FIGURE 4 MGC803/DDP‐derived exosomal miR‐500a‐3p confers DDP resistance in recipient MGC803 cells via inhibiting FBXW7. A, Sequences of miR‐500a‐3p and the potential miR‐500a‐3p‐binding sites at the 3′UTR of FBXW7, including nucleotides mutated in FBXW7‐3′‐UTR. Seed sequences are marked. Also shown effects of Blank, mimic or anti–miR‐500a‐3p and corresponding control on the luciferase activity of FBXW7 3′UTR‐Wild‐Type and FBXW7 3′UTR‐Mutant by dual‐luciferase reported assay in 293T cells. B‐D, Expression of FBXW7 in MGC803 or FBXW7 overexpressed MGC803 cells transfected with mimic‐control or mimic‐miR‐500a‐3p by real‐time qRT‐PCR (B) and Western blot analysis (D). Expression of FBXW7 in MGC803 or FBXW7 overexpressed MGC803 cells co‐cultured with CM without exosomes or MGC803/DDP exosomes by real‐time qRT‐PCR (C) and Western blot analysis (D). E, Proliferation assay analysis and IC50 value in MGC803 or FBXW7 overexpressed MGC803 cells transfected with mimic‐control or mimic‐miR‐500a‐3p. F, Proliferation assay analysis and IC50 value in MGC803 or FBXW7 overexpressed MGC803 cells co‐cultured with CM without exosomes or MGC803/DDP exosomes. G, Representative bioluminescent images and microscopy observations showed effects of CM without exosomes, MGC803/DDP exosomes on abdominal tumorigenesis by MGC803 or FBXW7 overexpressed MGC803 cells abdominal tumorigenesis under DDP treatment. H, J, Comparison of bioluminescent signals and abdominal metastatic nodules after indicated treatment, five nude mice in each group. *P < 0.05 and **P < 0.01
FIGURE 4 MGC803/DDP‐derived exosomal miR‐500a‐3p confers DDP resistance in recipient MGC803 cells via inhibiting FBXW7. A, Sequences of miR‐500a‐3p and the potential miR‐500a‐3p‐binding sites at the 3′UTR of FBXW7, including nucleotides mutated in FBXW7‐3′‐UTR. Seed sequences are marked. Also shown effects of Blank, mimic or anti–miR‐500a‐3p and corresponding control on the luciferase activity of FBXW7 3′UTR‐Wild‐Type and FBXW7 3′UTR‐Mutant by dual‐luciferase reported assay in 293T cells. B‐D, Expression of FBXW7 in MGC803 or FBXW7 overexpressed MGC803 cells transfected with mimic‐control or mimic‐miR‐500a‐3p by real‐time qRT‐PCR (B) and Western blot analysis (D). Expression of FBXW7 in MGC803 or FBXW7 overexpressed MGC803 cells co‐cultured with CM without exosomes or MGC803/DDP exosomes by real‐time qRT‐PCR (C) and Western blot analysis (D). E, Proliferation assay analysis and IC50 value in MGC803 or FBXW7 overexpressed MGC803 cells transfected with mimic‐control or mimic‐miR‐500a‐3p. F, Proliferation assay analysis and IC50 value in MGC803 or FBXW7 overexpressed MGC803 cells co‐cultured with CM without exosomes or MGC803/DDP exosomes. G, Representative bioluminescent images and microscopy observations showed effects of CM without exosomes, MGC803/DDP exosomes on abdominal tumorigenesis by MGC803 or FBXW7 overexpressed MGC803 cells abdominal tumorigenesis under DDP treatment. H, J, Comparison of bioluminescent signals and abdominal metastatic nodules after indicated treatment, five nude mice in each group. *P < 0.05 and **P < 0.01CC BY · PubMed Central
FIGURE 5 FBXW7 reverse the DDP resistance of exosomal miR‐500a‐3p by inhibiting CSCs properties in MGC803 cells. A, B, Effect of CM without exosomes, MGC803/DDP exosomes on sphere formation in MGC803 or FBXW7 overexpressed MGC803 cells. C, Relative mRNA expression of stemness markers CD133, CD44 and SOX2 in MGC803 or FBXW7 overexpressed MGC803 cells co‐cultured with CM without exosomes or MGC803/DDP exosomes. D‐F, Expression level of stemness markers in indicated GC cells by confocal microscopy (D) and Western blot (E, F). *P < 0.05, **P < 0.01 and ***P < 0.001
FIGURE 5 FBXW7 reverse the DDP resistance of exosomal miR‐500a‐3p by inhibiting CSCs properties in MGC803 cells. A, B, Effect of CM without exosomes, MGC803/DDP exosomes on sphere formation in MGC803 or FBXW7 overexpressed MGC803 cells. C, Relative mRNA expression of stemness markers CD133, CD44 and SOX2 in MGC803 or FBXW7 overexpressed MGC803 cells co‐cultured with CM without exosomes or MGC803/DDP exosomes. D‐F, Expression level of stemness markers in indicated GC cells by confocal microscopy (D) and Western blot (E, F). *P < 0.05, **P < 0.01 and ***P < 0.001CC BY · PubMed Central
FIGURE 6 Plasma exosomal miR‐500a‐3p is related to DDP resistance in III stage GC patients. A, Plasma exosomal miR‐500a‐3p level was detected in III stage GC patients responding or not responding to DDP treatment by real‐time qRT‐PCR. B, Kaplan‐Meier analysis of 3 y progression‐free survival rate in III stage GC patients from high (n = 30) and low (n = 25) miR‐500a‐3p groups, according to the median exosomal miR‐500a‐3p level in pre‐gastrectomy plasma. C, ROC curve analysis of plasma exosomal miR‐500a‐3p expression for discriminating the DDP resistant group (n = 30) from the sensitive group (n = 25). AUC, area under the curve. D, Schematic diagram of the potential roles of exosomal miR‐500a‐3p in GC DDP resistance. Briefly, the DDP‐resistant GC cell‐secreted exosomes containing miR‐500a‐3p can be taken up by surrounding DDP sensitive GC cells and subsequently down‐regulate FBXW7 to enhance DDP resistance as well as CSCs properties of the recipient GC cells. ***P < 0.001
FIGURE 6 Plasma exosomal miR‐500a‐3p is related to DDP resistance in III stage GC patients. A, Plasma exosomal miR‐500a‐3p level was detected in III stage GC patients responding or not responding to DDP treatment by real‐time qRT‐PCR. B, Kaplan‐Meier analysis of 3 y progression‐free survival rate in III stage GC patients from high (n = 30) and low (n = 25) miR‐500a‐3p groups, according to the median exosomal miR‐500a‐3p level in pre‐gastrectomy plasma. C, ROC curve analysis of plasma exosomal miR‐500a‐3p expression for discriminating the DDP resistant group (n = 30) from the sensitive group (n = 25). AUC, area under the curve. D, Schematic diagram of the potential roles of exosomal miR‐500a‐3p in GC DDP resistance. Briefly, the DDP‐resistant GC cell‐secreted exosomes containing miR‐500a‐3p can be taken up by surrounding DDP sensitive GC cells and subsequently down‐regulate FBXW7 to enhance DDP resistance as well as CSCs properties of the recipient GC cells. ***P < 0.001CC BY · PubMed Central

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Metadata

Authors
Lin, H., Zhang, L., Zhang, C., & Liu, P.
Venue
Journal of Cellular and Molecular Medicine
Publisher
Wiley
Published
2020-06-25
Volume
24
Issue
16
Pages
8930-8941
ISSN
  • 1582-1838
  • 1582-4934
DOI
10.1111/jcmm.15524
PMC
PMC7417713
Cited by
56
References
3
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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