Hypoxic tumour cell-derived exosomal miR-340-5p promotes radioresistance of oesophageal squamous cell carcinoma via KLF10 - PubMed (original) (raw)

Hypoxic tumour cell-derived exosomal miR-340-5p promotes radioresistance of oesophageal squamous cell carcinoma via KLF10

Fangyu Chen et al. J Exp Clin Cancer Res. 2021.

Abstract

Background: Radiotherapy resistance is a major obstacle in the treatment of oesophageal squamous cell carcinoma (OSCC). Hypoxia is a critical cause of radioresistance. However, the communication between hypoxic cells and aerobic cells via exosomes during the transfer of radiation resistance remains unclear.

Methods: Exo-miR-340-5p levels were analysed by RNA-seq and qRT-PCR. We co-cultured OSCC cells with isolated normoxic and hypoxic exosomes to study their impact on radiosensitivity. We used a specific exo-miR-340-5p mimic and knock-down retrovirus to explore the role of this miRNA in the transfer of radioresistance from hypoxic to normoxic cells. Dual-luciferase reporter and RIP assays were used to verify KLF10 as a putative target of miR-340-5p. Several in vitro assays were conducted and xenograft models were established to investigate the effect of exo-miR-340-5p on OSCC radiosensitivity. The plasma exo-miR-340-5p levels in OSCC patients were analysed to study the clinical value of this parameter.

Results: Hypoxic exosomes alleviated radiation-induced apoptosis and accelerated DNA damage repair. miR-340-5p was highly expressed in hypoxic exosomes and was transferred into normoxic cells, where it induced radioresistance. Overexpression of miR-340-5p in normoxic OSCC cells mimicked the radioresistance of cells co-cultured with hypoxic exosomes. Knockdown of miR-340-5p in hypoxic exosomes reversed the radioresistance effect, indicating that exo-miR-340-5p is critical for hypoxic EV-transferred radioresistance. KLF10 was identified as the direct target of miR-340-5p. Moreover, metformin was found to increase the expression of KLF10 and enhance the radiosensitivity of OSCC. Higher levels of miR-340-5p in the plasma exosomes from OSCC patients are related to a poorer radiotherapy response and prognosis.

Conclusions: Hypoxic tumour cell-derived exosomal miR-340-5p confers radioresistance in OSCC by targeting KLF10/UVRAG, suggesting that miR-340-5p could be a potential biomarker and therapeutic target for the enhancement of radiosensitivity in OSCC. Metformin can increase KLF10 expression, which ameliorates the radioresistance induced by exo-miR-340-5p transfer. Therefore, metformin could be further investigated as a therapeutic option for the treatment of OSCC.

Keywords: Extracellular vesicles; Hypoxia; Oesophageal cancer; Radiotherapy; miRNA.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

EVs derived from hypoxic OSCC cells promote radioresistance. a-b EVs derived from OSCC cells in a hypoxic environment contain higher levels of protein (a) and RNA (b). c-d Coculture with H-EVs drastically decreased OSCC cells apoptosis induced by irradiation. e-f H-EV-treated OSCC cells expressed lower levels of the DNA damage repair marker γ-H2AX after irradiation (scale bar = 20 μm). g Cell survival curve constructed from the colony formation assay data. Cells were supplemented with N-EVs or H-EVs and ionizing radiation as indicated. h The expression of γ-H2AX in irradiated OSCC cells was related to H-EV supplementation in a dose-dependent manner (scale bar = 20 μm). i Apoptosis of OSCC cells caused by irradiation was decreased by coculture with H-EVs in a dose-dependent manner. j Schematic diagram of the experimental design used to establish the animal model. k Images of tumours in each group (n = 6). l Alterations in the tumour volume in each group (n = 6). m Time to triple the tumour volume after the first day of EV injection in each group (n = 6). n-o H-EV-treated OSCC xenografts exhibited fewer TUNEL positive cells after irradiation

Fig. 2

miR-340-5p promotes radioresistance in OSCC. a qRT-PCR confirmed the effect of miR-340-5p mimic transfection. b miR-340-5p overexpression suppressed irradiation-induced apoptosis in OSCC cells. c The expression of γ-H2AX was decreased in miR-340-5p overexpressing OSCC cells after irradiation (scale bar = 20 μm). d Western blot assays demonstrated that miR-340-5p blocked the expression of the DNA damage repair proteins γ-H2AX and p-DNApkcs after irradiation in OSCC cells. e Images of tumours in each group (n = 6). f Alterations in the tumour volume in each group (n = 6). g-h miR-340-5p-overexpressing OSCC xenografts exhibited fewer TUNEL positive cells after irradiation. NC: cells transfected with negative control lentiviral vectors; miR-340-5p: cells transfected with miR-340-5p mimic lentiviral vectors

Fig. 3

miR-340-5p is essential for the hypoxic EV-induced radioresistance shift in OSCC. a The miR-340-5p level was greatly elevated in H-EV-treated OSCC cells but not in N-EV-treated OSCC cells. b qRT-PCR revealed miR-340-5p expression in OSCC cells after coculture with RNase A-treated EVs. c miR-340-5p levels in OSCC cells treated with RNase A or with RNase A and Triton X-100 together, as detected by qRT-PCR assays. d-e The expression of miR-340-5p in OSCC cells was associated with H-EV supplementation in a time-dependent (d) and dose-dependent (e) manner. f qRT-PCR confirmed the effect of miR-340-5p knockdown in OSCC cells. g No significant changes were observed by qRT-PCR between OSCC cells cocultured with PBS or H-sh-miR-340-5p-EVs. h-i Knockdown of miR-340-5p in hypoxic EVs rescued the suppression of IR-induced apoptosis caused by H-EVs. j Knockdown of miR-340-5p in hypoxic EVs reversed the H-EV-induced decrease in γ-H2AX expression after irradiation in OSCC cells (scale bar = 20 μm). k-l Knockdown of miR-340-5p in hypoxic EVs reversed the decreased in TUNEL-positive cell numbers in OSCC xenografts after irradiation. m Alterations in the tumour volume in each group (n = 6). n Images of tumours in each group (n = 6). NC: cells transfected with negative control lentiviral vectors

Fig. 4

KLF10 is a direct target for miR-340-5p. a Illustration of the putative predicted miR-340-5p binding site in the KLF10 mRNA 3′-UTR. b-c Expression of KLF10 in OSCC cells transfected with relevant lentiviral vectors was detected by qRT-PCR (b) and Western blotting (c). d Expression of KLF10 in OSCC cells was decreased after cocultured with hypoxic EVs. e Expression of KLF10 was detected by IHC in xenografts transfected with the relevant lentiviral vectors (scale bar = 200 μm). f Luciferase reporter assays were conducted to verify that miR-340-5p binds to the 3′-UTR region of KLF10 directly. g RIP assays confirmed the binding status between miR-340-5p and KLF10 in treated and untreated OSCC cells. NC: cells transfected with negative control lentiviral vectors; miR-340-5p: cells transfected with miR-340-5p mimic lentiviral vectors

Fig. 5

miR-340-5p induces radioresistance by affecting KLF10. a KLF10 knockdown attenuated apoptosis caused by irradiation in OSCC cells. b KLF10 knockdown decreased the expression of the DNA damage repair protein γ-H2AX (scale bar = 20 μm). c Western blot analysis was used to detect the expression of several key factors involved in apoptosis and DNA damage repair in OSCC cells transfected with KLF10 knockdown and control lentiviral vectors. d-f Overexpression of KLF10 in OSCC cells transfected with the miR-340-5p mimic lentiviral vectors rescued the apoptosis (d), DNA damage repair (e, scale bar = 20 μm), and the expression of several key proteins after irradiation (f). NC: cells transfected with negative control lentiviral vectors; sh-KLF10: cells transfected with KLF10 knockdown lentiviral vectors; miR-340-5p: cells transfected with miR-340-5p mimic lentiviral vectors; miR-340-5p + KLF10: cells cotransfected with miR-340-5p mimic and KLF10 mimic lentiviral vectors

Fig. 6

Metformin increased KLF10 expression and enhanced the radiosensitivity of OSCC cells. a-b Metformin reversed the decreased apoptosis (a) and accelerated DNA damage repair (b, scale bar = 20 μm) after irradiation caused by coculture with H-EVs. c Metformin increased KLF10 expression and regulated several key factors in irradiated OSCC cells treated with H-EVs. d Images of tumours in each group (n = 6). e Alterations in the tumour volume in each group (n = 6). f TUNEL-positive cell numbers were decreased after metformin supplementation

Fig. 7

Upregulation of miR-340-5p indicates radioresistance and correlates with a poor response to radiotherapy in OSCC patients. a qRT-PCR analysis of miR-340-5p levels in tissues and plasma exosomes of OSCC patients with and without in-field recurrence within 3 years post radiotherapy. b-c Kaplan-Meier survival analysis of in-field recurrence free survival (b) and overall survival (c) of OSCC patients with different histological and plasma exosomal miR-340-5p levels 3 years post radiotherapy. d Expression of plasma exosomal miR-340-5p in 10 OSCC patients receiving neoadjuvant chemoradiotherapy. e Expression of KLF10 was negatively correlated with the plasma exosomal miR-340-5p levels in 40 OSCC patients. f-g Expression of KLF10 in OSCC tissues (f) and plasma EVs (g) was detected by qRT-PCR in clinical samples of patients with different miR-340-5p levels. h IHC showing the expression of KLF10 in clinical specimens expressing different miR-340-5p levels. i Schematic diagram showing that metformin attenuates hypoxic tumour cell-orchestrated radioresistance cascades

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Hypoxic tumour cell-derived exosomal miR-340-5p promotes radioresistance of oesophageal squamous cell carcinoma via KLF10 - PubMed (original) (raw)