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. 2022 Jan 11;74(1):51–64. doi: 10.1007/s10616-021-00506-y

Exosomal circ_PTPRA inhibits tumorigenesis and promotes radiosensitivity in colorectal cancer by enriching the level of SMAD4 via competitively binding to miR-671-5p

Ying Yang 1, Nengwen Yang 2, Jun Jiang 1,✉
PMCID: PMC8817021  PMID: 35185285

Abstract

Accumulating evidence supports that exosomal RNAs are crucial in tumor microenvironment and may be used as diagnostic biomarkers for cancers. This study aimed to determine the role of exosomal circular RNA_protein tyrosine phosphatase receptor type A (circ_PTPRA) in colorectal cancer (CRC). The morphology of exosomes was identified by transmission electron microscopy (TEM), and several exosome-specific proteins were quantified by western blot. The expression of circ_PTPRA, miR-671-5p and SMAD family member 4 (SMAD4) was detected using quantitative polymerase chain reaction (qPCR). Cell cycle was assessed using flow cytometry assay. Cell proliferation was assessed by MTT assay. Radiosensitivity was observed according to colony growth and cell apoptosis rate by colony formation assay and flow cytometry assay. The protein levels of proliferation- and apoptosis-related markers and SMAD4 were measured by western blot. The predicted relationship between miR-671-5p and circ_PTPRA or SMAD4 was verified by dual-luciferase reporter assay. Animal study was performed to investigate the role of exosomal circ_PTPRA in vivo. Circ_PTPRA expression was declined in serumal exosomes from CRC patients and CRC cell lines. Exosomal circ_PTPRA induced CRC cell cycle arrest and inhibited cell proliferation. Besides, exosomal circ_PTPRA promoted radiosensitivity of CRC cells, leading to inhibitory colony formation and increased apoptotic rate. In mechanism, circ_PTPRA functioned as a competing endogenous RNA (ceRNA) to increasing SMAD4 level by binding to miR-671-5p. Rescue experiments concluded that circ_PTPRA inhibited CRC growth and radioresistance by decreasing miR-671-5p expression, and miR-671-5p inhibition also inhibited CRC growth and radioresistance by enriching SMAD4 expression. Moreover, exosomal circ_PTPRA blocked tumor growth in vivo. Exosomal circ_PTPRA enhanced CRC cell radiosensitivity and inhibited CRC malignant development partially by regulating the miR-671-5p/SMAD4 pathway, hinting that exosomal circ_PTPRA might be used as a potential predicted and therapeutic target for CRC.

Keywords: Exosomes, circ_PTPRA, miR-671-5p, SMAD4, Colorectal cancer

Introduction

Colorectal cancer (CRC) has developed to be the third most common malignancy (Kuipers et al. 2015). According to cancer statistics, there are 101,420 estimated new cases and 51,020 estimated deaths of CRC in 2019 in the USA (Siegel et al. 2019). Currently, the expected increase in the incidence and mortality of CRC is worrying. It is estimated that by 2035, the death toll from colon cancer and rectal cancer is expected to increase by 60.0% and 71.5%, respectively (Araghi et al. 2019). CRC is generally diagnosed at an advanced stage, and the development of chemoresistance and radioresistance fails to improve the outcomes of CRC (Beshara et al. 2020; Wu et al. 2018). It is necessary to elucidate CRC pathogenesis and explore new biomarkers for detection and treatment to improve outcomes.

Exosomes, nanosized extracellular vesicles, are widely found in body fluids, such as blood, urine and saliva (Lim and Kim 2019). Exosomes reach the site of action through systemic circulation and exert their physiological effects through direct contact or release of inclusions (Jiang et al. 2019; Guo et al. 2017), such as proteins, DNA, mRNA and non-coding RNAs. A growing number of studies have shown that exosomes play important roles in tumor diagnosis and treatment, and targeted drug research based on the properties of exosomal carriers is also developing rapidly (Haney et al. 2015). Recently, the presence of circular RNAs (circRNAs) in exosomes has aroused much attention, which contributes to distinguishing cancer patients from healthy groups according to the level of exosomal circRNAs (Fanale et al. 2018). CircRNAs are produced from precursor mRNAs through a special mechanism, called “back-splicing” (Fanale et al. 2018). CircRNAs are widely and stably expressed in all eukaryotic cells, and sufficient evidence demonstrates that circRNAs exert vital effects in number biological processes, such as cell proliferation, migration, invasion, chemosensitivity and radiosensitivity in cancer development (Guan et al. 2020; Hong et al. 2020). CircRNA protein tyrosine phosphatase receptor type A (circ_PTPRA) had been identified to be downregulated in bladder cancer, and it functioned as a tumor suppressor in bladder cancer (Li et al. 2017; He et al. 2019). However, little is known about the role of circ_PTPRA in CRC.

Importantly, circRNAs are known to function as sponges of target microRNAs (miRNAs), and circRNAs may compete with downstream mRNAs for the same miRNAs in a competing endogenous RNA (ceRNA)-dependent manner (Cui et al. 2018). In particular, the bioinformatics tool shows that miR-671-5p is a potential target of circ_PTPRA, and miR-671-5p has been reported to promote proliferation, migration and invasion of colon cancer cells (Jin et al. 2019). Thus, it is reasonable to speculate that circ_PTPRA regulates CRC progression partly by mediating the expression of miR-671-5p. However, it is not confirmed yet. Moreover, the circRNA-miRNA-mRNA regulatory networks have been widely proposed to explain the mechanism of cancer development (Liu et al. 2020; Bai et al. 2020; Liang et al. 2020). It is also necessary to establish the similar networks of circ_PTPRA to introduce the potential mechanism of circ_PTPRA action in CRC.

In the present study, we investigated the expression of circ_PTPRA in serumal exosomes from CRC patients and CRC cells. The function of exosomal circ_PTPRA was explored on CRC cell growth and radiosensitivity in vitro, as well as CRC tumor growth in vivo. In mechanism, we constructed the circ_PTPRA-miR-671-5p-SMAD4 regulatory network to partly clarify the role of circ_PTPRA in CRC. Our aim was to explore more exosomes-related biomarkers for CRC diagnosis and treatment.

Materials and methods

Serum samples

A total of 10 mL blood samples were collected from 25 CRC patients and 10 healthy volunteers (HV) who were recruited from Hunan Cancer Hospital. All these subjects approved the use of these samples with the written informed consent. Serum samples were obtained by centrifugalizing from blood samples. Then, serum samples were stored at − 80 °C conditions until use. Our study was implemented with the approval of the Ethics Committee of Hunan Cancer Hospital.

Exosomes isolation and exosome identification

Blood samples (1 mL for each sample) were centrifuged (2400 g) for 10 min at 4 °C, and supernatants were collected and again centrifuged (800 g) for 10 min at 4 °C. Subsequently, serum samples were used to extract exosomes using the exoEasy Maxi Kit (Qiagen, Duesseldorf, Germany) according to the manufacturer’s instruction. The morphology of exosomes was monitored by transmission electron microscopy (TEM). In brief, exosome pellets were fixed with 4% glutaraldehyde, followed by fixation using osmium tetroxide at 4 °C. After eluting with ethanol, pellets were embedded and sectioned. The ultrathin sections were stained with uranyl acetate and lead citrate. The morphology of exosomes was observed, and the images were captured using a TEM (Hitachi H7500; Tokyo, Japan).

Western blot

The protein levels of exosomes-related markers, including heat Shock Protein 70 (HSP70), tumor susceptibility gene 101 (TSG101) and CD63, the level of proliferation-related marker, Ki67, the levels of apoptosis-associated markers, including B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), and the level of SMAD4 were all detected by western blot. The protein samples were extracted from exosomes or cells using Total exosome RNA and Protein Isolation kit (Invitrogen, Carlsbad, CA, USA) or RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA, USA). The protein samples were subjected to 10% SDS-PAGE, and the separated proteins were transferred to PVDF membranes (Beyotime, Shanghai, China). After incubating with 5% skim milk, the membranes were cultured with the primary antibodies, including anti-HSP70 (ab2787; Abcam, Cambridge, MA, USA; dilution 1/1000), anti-TSG101 (ab125011; Abcam; dilution 1/5000), anti-CD63 (ab134045; Abcam; dilution 1/5000), anti-Ki67 (ab92742; Abcam; dilution 1/5000), anti-Bcl-2 (ab32124; Abcam; dilution 1/1000), anti-Bax (ab263897; Abcam; dilution 1/2000), anti-SMAD4 (ab110175; Abcam; dilution 1/1000) and anti-GAPDH (ab9485; Abcam; dilution 1/2500). After overnight-incubation, the membranes were cultured with the secondary antibodies (ab205718 and aba205719; Abcam; dilution 1/5000). Subsequently, the protein bands were visualized with the Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA).

Quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from exosomes or cells using the total exosome RNA and Protein Isolation kit (Invitrogen) or Trizol reagent (Invitrogen). Then, total RNA was reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) or TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems), followed by the qPCR amplification using the SYBR™ Green Master Mix (Applied Biosystems). The reactions were performed under the 7500 Fast Real-Time PCR System (Applied Biosystems). The relative expression was calculated using the 2−ΔΔCt method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or small nuclear RNA U6 as the internal reference. The primer sequences were:

circ_PTPRA, F: 5′-CCCTCCTTCAGATAAGCATGG-3′ and R: 5′-TTTGGGCTGAATGTTGGTGC-3′; PTPRA, F: 5′-CACTGAGTGGTAATGGATGATGC-3′ and R: 5′-ACTGCCGAGCAGAACAAGAA-3′; SMAD4, F: 5′-GCTGCAGAGCCCAGTTTAGA-3′ and R: 5′-CCCCAAAGCAGAAGCTACGA-3′; GAPDH, F: 5′-CCATGAGAAGTATGACAACAGC-3′ and R: 5′-ATGGACTGTGGTCATGAGTC-3′; miR-671-5p, F: 5′-GCCCGCAGGAAGCCCUGGAGGGGC-3′ and R: 5′-GTGCAGGGTCCGAGGT-3′; U6, F: 5′-GCUUCGGCAGCACAUAUACUAAAAU-3′ and R: 5′-CGCUUCACGAAUUUGCGUGUCAU-3′.

Cell lines

Human CRC cell lines (HCT116 and DLD1) and normal colonic epithelial cell line (NCM460) were purchased from Bena Culture Collection (Beijing, China). Next, HCT116 and NCM460 cells were cultured in 90% McCoy’s 5A medium (Bena Culture Collection) containing 10% fetal bovine serum (FBS), and DLD1 cells were cultured in RPMI1640 medium (Bena Culture Collection) containing 10% FBS under a humidified atmosphere at 37 °C with 5% CO2. Noticeably, in exosomes-related experiments, cells were resuspended in culture medium with exosome-depleted FBS.

RNase R treatment

The RNA samples were treated with RNase R (3 U/μg; Epicentre, Madison, WI, USA) for 15 min. Then, the treated RNA was transcribed into cDNA and subjected to qPCR reaction to detect the expression of circ_PTPRA and PTPRA.

Cell transfection and exosomes incubation

Circ_PTPRA overexpression vector, pCD-ciR-circ_PTPRA (circ_PTPRA), was assembled by Geenseed (Guangzhou, China), using empty vector (Vector) as the control. MiR-671-5p mimic (miR-671-5p) and miR-671-5p inhibitor (anti-miR-671-5p) were obtained from Ribobio (Guangzhou, China) for miR-671-5p overexpression or inhibition, using miR-NC or anti-miR-NC as the control. Small interference RNA targeting SMAD4 (si-SMAD4) was synthesized by Ribobio for SMAD4 knockdown, using si-NC as the negative control. These transfections were introduced into the experimental cells using Lipofectamine 3000 (Invitrogen).

The isolated cells were co-cultured with HCT116 and DLD1 cells for 24 h. Then, cells were collected for the subsequent functional assays.

Flow cytometry assay

Cell cycle distribution at different phases was monitored using a Cell Cycle Analysis Kit (Beyotime). The experimental cells were trypsinized and collected in 1 mL pre-cooled phosphate-buffered saline (PBS). Cells were next fixed in 70% ethanol and washed with PBS, followed by staining using propidium iodide (PI) staining buffer (containing RNase A) at 37 °C for 30 min in a dark room. Cell distribution at different phases was identified utilizing an Attune NxT Flow Cytometer (Invitrogen).

The apoptotic cells were investigated using an Annexin V-FITC Apoptosis Detection Kit (Beyotime). The experimental cells after treatment were washed with PBS, trypsinized and resuspended in 195 μL Annexin V-FITC binding buffer. Afterwards, cells were treated with 5 μL Annexin V-FITC and 10 μL PI, mixing gently and incubating for 15 min in the dark. The apoptotic cells were analyzed utilizing an Attune NxT Flow Cytometer (Invitrogen).

MTT assay

The experimental cells were seeded into a 96-well plate (2000 cells/well) and continuingly cultured for different time (0, 24, 48 and 72 h). MTT reagent (Beyotime) was added in each well at 4 h before the culture time points. Dimethylsulfoxide (DMSO) was subsequently added into each well to dissolve formazan. The absorbance at 570 nm was measured using a microplate reader (Thermo Fisher Scientific).

Irradiation

Cells were cultured in culture plates at 70% to 80% confluent and then exposed to X-rays from an irradiator (HWM D-2000; Siemens AG, Munich, Germany) at a dose of 0, 2, 4 and 6 Gy.

Colony formation assay

The experimental cells were plated into a 6-well plate (200 cells/well) and next left for 4 h at 37 °C incubator containing 5% CO2 prior to irradiation. Colonies were allowed to grow for 12 days and finally fixed with paraformaldehyde and stained with crystal violet (Beyotime). Five replicate dishes were counted for each treatment.

Dual-luciferase reporter assay

The relationship between circ_PTPRA and miR-671-5p was predicted by circular RNA interactome (https://circinteractome.nia.nih.gov/). The relationship between miR-671-5p and SMAD4 was predicted by miRDB (http://www.mirdb.org/).

The wild-type and mutant-type luciferase reporter plasmids of circ_PTPRA and SMAD4 3’ UTR were constructed by Sangon Biotech (Shanghai, China), including pmiRGLO-circ_PTPRA-WT (circ_PTPRA-WT), pmiRGLO-circ_PTPRA-MUT (circ_PTPRA-MUT), pmiRGLO-SMAD4-WT (SMAD4-WT) and pmiRGLO-SMAD4-MUT (SMAD4-MUT). HCT116 and DLD1 cells were cotransfected with circ_PTPRA-WT, circ_PTPRA-MUT, SMAD4-WT or SMAD4-MUT and miR-671-5p or miR-NC using Lipofectamine 3000 (Invitrogen). After 48 h, the luciferase activity was measured by using a Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA).

Animal study

The animal study was permitted by the Animal Use and Care Committee of Hunan Cancer Hospital. Immune compromised Balb/C mice (6–8 weeks old, female) were purchased from Shanghai SLAC lab. (Shanghai, China) and kept at a pathogen-free room. The mice were divided into two groups (n = 5 per group) and subcutaneously injected with HCT116 cells (2 × 106). One week later, exosomes isolated from circ_PTPRA or Vector-transfected HCT116 cells (Exo-circ_PTPRA or Exo-Vector) were injected into the tumor nudes every 5 days. During tumor growth, tumor volume was measured with calipers (0.5 × length × width2). At 32 day post-injection, all mice were sacrificed to remove tumor tissues. These tissues were weighted, photographed and then preserved at − 80 °C for further analyses.

Statistical analysis

Each experiment contained at least three repeats. The data were collected and processed using GraphPad Prism (Version 7.0; La Jolla, CA, USA). The difference between two groups was compared using Student’s t-test, and the difference among multiple groups was determined using analysis of variance (ANOVA). The data were finally exhibited as mean ± standard deviation (SD). It was considered to be statistically significant when P value less than 0.05.

Results

The expression of circ_PTPRA was decreased in serumal exosomes from CRC patients and CRC cell lines

The circRNA sequencing profile (GEO accession: GSE149200) showed that circ_PTPRA was one of the downregulated circRNAs in serum exosomes from CRC patients (Fig. 1A). Utilizing clinical serum samples, we isolated exosomes. The representative lipid bilayer structure of exosomes was observed by TEM (Fig. 1B), and exosomes markers, including HSP70, TSG101 and CD63, were strikingly increased in isolated exosomes (Fig. 1C), suggesting that serumal exosomes were successfully isolated. In addition, we noticed that circ_PTPRA expression was notably lower in serumal exosomes from CRC patients (CRC-Exo) than that from healthy volunteers (HV-Exo) (Fig. 1D). Circ_PTPRA expression was also notably decreased in HCT116 and DLD1 cells compared with that in NCM460 cells (Fig. 1E). To verify the existence of this circRNA, we used RNase R to treat the isolated RNA samples from HCT116 and DLD1 cells and then examined the expression of circ_PTPRA and PTPRA. The data presented that the expression of circ_PTPRA was unaffected by RNase R, while the expression of PTPRA mRNA was notably impaired (Fig. 1F and G). In short, circ_PTPRA was downregulated in serumal exosomes from CRC patients and CRC cell lines.

Fig. 1.

Fig. 1

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Circ_PTPRA was downregulated in serumal exosomes from CRC patients and CRC cell lines. A The expression of several circRNAs in CRC tissues and normal tissues from a circRNA profile (GEO accession: GSE149200). B TEM showed the morphology of serum-derived exosomes. C The levels of exosomes-specific markers (HSP70, TSG101 and CD63) were examined by western blot. D The expression of circ_PTPRA in serum-derived exosomes from CRC patients and healthy volunteers was detected by qPCR. E The expression of circ_PTPRA in NCM460, HCT116 and DLD1 cells was detected by qPCR. F and G The stability of circ_PTPRA and linear PTPRA was examined using RNase R. *P < 0.05; Exo exosomes, HV-Exo serum-derived exosomes from healthy volunteers, CRC-Exo serum-derived exosomes from colorectal cancer patients

Exosomal circ_PTPRA inhibited cell growth and promoted radiosensitivity in HCT116 and DLD1 cells

We next investigated the function of exosomal circ_PTPRA. HCT116 cells were transfected with circ_PTPRA overexpression plasmid and empty vector (control), and exosomes were isolated from these cells. The data showed that circ_PTPRA expression was strikingly increased in exosomes from cells transfected with circ_PTPRA (Exo-circ_PTPRA) compared with that in exosomes from cells transfected with vector (Exo-Vector) (Fig. 2A). Then, HCT116 and DLD1 cells were incubated with exosomes, including Exo-circ_PTPRA and Exo-Vector, respectively (Fig. 2B). After incubation, the expression of circ_PTPRA was significantly enhanced in HCT116 and DLD1 cells (Fig. 2C). In functional assays, we found that cell cycle arrest (at G0/G1 phase) was produced in HCT116 and DLD1 cells harboring Exo-circ_PTPRA compared to Exo-Vector (Fig. 2D and E). MTT assay introduced that HCT116 and DLD1 cells with Exo-circ_PTPRA harbored decreased proliferation capacity compared to cells with Exo-Vector (Fig. 2F and G), which was validated by the expression of proliferation-related protein, Ki67. The data showed that the protein level of Ki67 was notably declined in HCT116 and DLD1 cells containing Exo-circ_PTPRA but not Exo-Vector (Fig. 2H). Moreover, we performed colony formation assay and flow cytometry assay to determine the role of exosomal circ_PTPRA on radiosensitivity. As shown in Fig. 2I and J, HCT116 and DLD1 cells harboring Exo-circ_PTPRA led to decreased cell survival fraction after exposing to different doses of radiation (0, 2, 4 and 6 Gy). As shown in Fig. 2K and L, the apoptotic rate was strikingly enhanced in HCT116 and DLD1 cells incubated with Exo-circ_PTPRA after 0 or 6 Gy-radiation, suggesting that exosomal circ_PTPRA induced radiosensitivity of CRC cells. Additionally, the protein level of Bcl-2 was declined, while the protein level of Bax was enhanced in HCT116 and DLD1 cells after Exo-circ_PTPRA incubation compared to Exo-Vector incubation (Fig. 2M and N). Overall, these data indicated that exosomal circ_PTPRA inhibited CRC cell malignant growth and promoted radiosensitivity.

Fig. 2.

Fig. 2

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Exsomal circ_PTPRA inhibited CRC cell proliferation and promoted CRC cell radiosensitivity. A The expression of circ_PTPRA in Exo-circ_PTPRA and Exo-Vector was detected by qPCR. B CRC cells were co-cultured with exosomes. C The expression of circ_PTPRA in HCT116 and DLD1 cells after co-culture with exosomes. D and E The role of Exo-circ_PTPRA and Exo-Vector on cell cycle progression was determined by flow cytometry assay. F and G The role of Exo-circ_PTPRA and Exo-Vector on cell proliferation was determined by MTT assay. H The level of Ki67 was detected by western blot. I and J Colony formation assay was performed to determine the role of Exo-circ_PTPRA and Exo-Vector on radiosensitivity. K and L Flow cytometry assay was conducted to determine the role of Exo-circ_PTPRA and Exo-Vector on cell apoptosis after radiation. M and N The levels of Bcl-2 and Bax were detected by western blot to observe cell apoptosis. *P < 0.05

MiR-671-5p was targeted by circ_PTPRA

Bioinformatics tools exhibited several putative target miRNAs of circ_PTPRA, and we identified the targets to investigate the potential mechanism of circ_PTPRA action. As displayed in Fig. 3A, there was a special binding site between circ_PTPRA and miR-671-5p. Subsequently, the wild-type and mutant-type of circ_PTPRA luciferase reporter plasmids were constructed, and the data showed that miR-671-5p and circ_PTPRA-WT cotransfection remarkably reduced the luciferase activity in HCT116 and DLD1 cells, while miR-671-5p and circ_PTPRA-MUT cotransfection hardly changed the luciferase activity (Fig. 3B and C), suggesting that circ_PTPRA bound to miR-671-5p. Besides, the expression of miR-671-5p was strikingly lessened in HCT116 and DLD1 cells with circ_PTPRA overexpression (Fig. 3D), suggesting that circ_PTPRA inhibited the expression of miR-671-5p. In addition, we discovered that miR-671-5p expression was elevated in HCT116 and DLD1 cells relative to NCM460 cells (Fig. 3E).

Fig. 3.

Fig. 3

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MiR-671-5p was a target of circ_PTPRA. A Through the analysis of circular RNA interactome, circ_PTPRA harbored a special binding site with miR-671-5p. B and C The interaction between circ_PTPRA and miR-671-5p was verified by dual-luciferase reporter assay. D The expression of miR-671-5p in HCT116 and DLD1 cells after circ_PTPRA overexpression was detected by qPCR. E The expression of miR-671-5p in NCM460, HCT116 and DLD1 cells was detected by qPCR. *P < 0.05

Circ_PTPRA overexpression inhibited CRC cell growth and enhanced radiosensitivity by binding to miR-671-5p

To determine whether circ_PTPRA played functions partly by mediating miR-671-5p, HCT116 and DLD1 cells were transfected with circ_PTPRA or circ_PTPRA + miR-671-5p, with Vector or circ_PTPRA + miR-NC as the control. The data from qPCR showed that the expression of miR-671-5p was decreased in HCT116 and DLD1 cells transfected with circ_PTPRA but substantially recovered in cells transfected with circ_PTPRA + miR-671-5p (Fig. 4A). In function, circ_PTPRA overexpression notably induced cell cycle arrest, while miR-671-5p reintroduction relieved circ_PTPRA-induced cell cycle arrest (Fig. 4B and C). Besides, circ_PTPRA overexpression significantly suppressed HCT116 and DLD1 cell proliferation, while miR-671-5p reintroduction promoted cell proliferation (Fig. 4D and E). The protein level of Ki67 was strikingly lessened in HCT116 and DLD1 cells transfected with circ_PTPRA but recovered in cells transfected with circ_PTPRA + miR-671-5p (Fig. 4F). Moreover, circ_PTPRA overexpression enhanced radiosensitivity and further inhibited cell survival fraction, while miR-671-5p restoration increased HCT116 and DLD1 cell survival fraction (Fig. 4G and H). Not surprisingly, circ_PTPRA overexpression further promoted radiation-induced cell apoptosis, while miR-671-5p reintroduction ameliorated the number of apoptotic cells (Fig. 4I and J). After radiation, the protein level of Bcl-2 inhibited in circ_PTPRA-transfected HCT116 and DLD1 cells was recovered by miR-671-5p restoration, while the protein level of Bax was opposite to the level of Bcl-2 (Fig. 4K and L), which verified the results of flow cytometry apoptosis detection. Overall, circ_PTPRA overexpression inhibited CRC cell growth and enhanced radiosensitivity by depleting the expression of miR-671-5p.

Fig. 4.

Fig. 4

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Circ_PTPRA inhibited CRC cell proliferation and promoted CRC cell radiosensitivity by suppressing miR-671-5p. HCT116 and DLD1 cells were transfected with circ_PTPRA, Vector, circ_PTPRA + miR-671-5p or circ_PTPRA + miR-NC. A The expression of miR-671-5p in these cells was detected by qPCR. B and C Cell cycle distribution at different phases was determined by flow cytometry assay. D and E Cell proliferation was determined by MTT assay. F The level of Ki67 in these cells was measured by western blot. G and H Colony formation assay was performed to assess radiosensitivity. I and J Cell apoptosis after radiation was checked by flow cytometry assay. K and L The levels of Bcl-2 and Bax in these cells were detected by western blot. *P < 0.05

MiR-671-5p bound to SMAD4 3’UTR, and circ_PTPRA functioned as miR-671-5p sponge to increase SMAD4 expression

To determine whether miR-671-5p regulated gene expression by binding to the 3’UTR of target genes, we identified the targets of miR-671-5p. Bioinformatics tool predicted that miR-671-5p bound to SMAD4 3′UTR through a special binding site (Fig. 5A). This prediction was next verified by dual-luciferase reporter assay. The data showed that the luciferase activity was strikingly declined in HCT116 and DLD1 cells after miR-671-5p and SMAD4-WT cotransfection but not miR-671-5p and SMAD4-MUT cotransfection (Fig. 5B and C). In miR-671-5p-transfected HCT116 and DLD1 cells, miR-671-5p expression was largely promoted (Fig. 5D), while SMAD4 expression was notably decreased (Fig. 5E and F). In anti-miR-671-5p-transfected HCT116 and DLD1 cell, miR-671-5p expression was largely lessened (Fig. 5D), while SMAD4 expression was notably strengthened (Fig. 5E and F). Moreover, the expression of SMAD4 was significantly lower in HCT116 and DLD1 cells compared with that in NCM460 cells (Fig. 5G and H). Interestingly, we discovered that the expression of SMAD4 was strikingly reinforced in HCT116 and DLD1 cells transfected with circ_PTPRA compared to Vector, while the expression of SMAD4 was largely declined in cells transfected with circ_PTPRA + miR-671-5p compared to circ_PTPRA + miR-NC (Fig. 5I and J). The data hinted that circ_PTPRA overexpression enriched the level of SMAD4 via competitively combining with miR-671-5p.

Fig. 5.

Fig. 5

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Circ_PTPRA functioned as miR-671-5p sponge to increase the expression of SMAD4. A The relationship between miR-671-5p and SMAD4 was predicted by bioinformatics tool miRDB. B and C The relationship between miR-671-5p and SMAD4 was verified by dual-luciferase reporter assay. D The expression of miR-671-5p in HCT116 and DLD1 cells after miR-671-5p or anti-miR-671-5p transfection was detected by qPCR. E and F The expression of SMAD4 in HCT116 and DLD1 cells after miR-671-5p or anti-miR-671-5p transfection was detected by qPCR and western blot. G and H The expression of SMAD4 in NCM460, HCT116 and DLD1 cells was detected by qPCR and western blot. I and J The expression of SMAD4 in HCT116 and DLD1 cells transfected with circ_PTPRA, Vector, circ_PTPRA + miR-671-5p or circ_PTPRA + miR-NC was detected by qPCR and western blot. *P < 0.05

MiR-671-5p inhibition blocked CRC cell growth and promoted radiosensitivity by increasing the level of SMAD4

We next carried out experiments to verify that miR-671-5p functioned partly by inhibiting SMAD4 expression. HCT116 and DLD1 cells were transfected with anti-miR-671-5p or anti-miR-671-5p + si-SMAD4, with anti-miR-NC or anti-miR-671-5p + si-NC as the control. The expression of SMAD4 was markedly increased in HCT116 and DLD1 cells transfected with anti-miR-671-5p but largely impaired in cells transfected with anti-miR-671-5p + si-SMAD4 (Fig. 6A and B). In function, we found miR-671-5p inhibition notably induced cell cycle arrest, which was relieved by SMAD4 knockdown (Fig. 6C and D). Also, miR-671-5p inhibition inhibited HCT116 and DLD1 cell proliferation, while SMAD4 knockdown recovered the inhibitory cell proliferation (Fig. 6E and F). The protein level of Ki67 was decreased in HCT116 and DLD1 cells transfected with anti-miR-671-5p but restored in cells transfected with anti-miR-671-5p + si-SMAD4 (Fig. 6G). Moreover, anti-miR-671-5p transfection further diminished radiation-inhibited HCT116 and DLD1 cell survival fraction, while anti-miR-671-5p + si-SMAD4 transfection partly increased cell survival fraction (Fig. 6H and I). Undoubtedly, anti-miR-671-5p transfection further increased radiation-induced HCT116 and DLD1 cell apoptosis, while anti-miR-671-5p + si-SMAD4 transfection partly blocked cell apoptosis (Fig. 6J and K), which was validated by the protein levels of Bcl-2 and Bax. The data showed that the level of Bcl-2 was strikingly decreased by anti-miR-671-5p transfection but recovered by anti-miR-671-5p + si-SMAD4 transfection in HCT116 and DLD1 cells after radiation treatment, while the level of Bax was opposite to the level of Bcl-2 in these cells (Fig. 6L and M). Overall, the data suggested that miR-671-5p inhibition blocked CRC cell growth and promoted radiosensitivity by increasing the level of SMAD4.

Fig. 6.

Fig. 6

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MiR-671-5p inhibition suppressed CRC cell proliferation and enhanced CRC cell radiosensitivity. HCT116 and DLD1 cells were introduced with anti-miR-671-5p, anti-miR-NC, anti-miR-671-5p + si-SMAD4 or anti-miR-671-5p + si-NC. A and B The expression of SMAD4 in these cells was investigated by qPCR and western blot. C and D Cell cycle distribution at different phases in these cells was investigated by flow cytometry assay. E and F Cell proliferation in these cells was assessed by MTT assay. G The level of Ki67 in these cells was measured by western blot. H and I Colony formation ability of HCT116 and DLD1 cells after radiation was assessed by colony formation assay. J and K Cell apoptotic rate after radiation was investigated by flow cytometry assay. L and M The levels of Bcl-2 and Bax in these cells were investigated by western blot. *P < 0.05

Exosomal circ_PTPRA blocked tumor growth in Xenograft models

We further investigated the role of exosomal circ_PTPRA in vivo. In brief, HCT116 cells were injected into nude mice. After one week, tumor nodes were injected with Exo-circ_PTPRA or Exo-Vector. As result, Exo-circ_PTPRA injection significantly inhibited tumor volume and tumor weight, leading to decreased tumor size (Fig. 7A and B). After removing these tumor tissues, we detected that the expression of circ_PTPRA was markedly promoted in tumor tissues from the Exo-circ_PTPRA group (Fig. 7C), while the expression of miR-671-5p was strikingly declined (Fig. 7D). Certainly, the expression of SMAD4 was consistent with circ_PTPRA expression, showing an increased level in tumor tissues from the Exo-circ_PTPRA group (Fig. 7E and F). The data suggested that exosomal circ_PTPRA could inhibit CRC tumor growth by mediating the expression of circ_PTPRA, miR-671-5p and SMAD4.

Fig. 7.

Fig. 7

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Exosomal circ_PTPRA inhibited tumor growth in vivo. A Tumor volume was measured to observe tumor growth. B Tumor weight and tumor size were determined. C The expression of circ_PTPRA in treated tumor tissues was detected by qPCR. D The expression of miR-671-5p in treated tumor tissues was detected by qPCR. E and F The expression of SMAD4 in treated tumor tissues was detected by qPCR and western blot. *P < 0.05

Discussion

This study mainly introduced that the expression of circ_PTPRA was aberrantly decreased in serum-derived exosomes from CRC patients and CRC cells. Exosomes-transferred circ_PTPRA induced CRC cell cycle arrest, inhibited cell proliferation and further enhanced radiosensitivity. In depth, we found that circ_PTPRA functioned as miR-671-5p sponge to compete with SMAD4 for miR-671-5p binding site. The data supporting this were that miR-671-5p reintroduction reversed the effects of circ_PTPRA, and SMAD4 knockdown reversed the effects of miR-671-5p deficiency. These results provided a new mechanism for antagonizing radioresistance and tumor aggravation in CRC.

Exosomes serve as information exchange carriers, which mediate the transformation of proteins, lipids, mRNA, circRNAs, miRNAs and others, thus regulating the stability and function of intracellular environment (Kim et al. 2017). Exosomes are increasingly recognized as forceful regulators in cancers, and exosomes are accordingly becoming the focus of research as biomarkers, diagnostic/prognostic indicators and therapeutic tools (Kim et al. 2017). Exosomes-transferred circRNAs have been reported to play vital roles in CRC development. For example, exosomal circPACRGL notably promoted CRC cell proliferation, migration and invasion by mediating the miR-142-3p/ miR-506-3p-TGF-β1 networks (Shang et al. 2020). Herein, we reported that circ_PTPRA could be transferred by exosomes, and exosomes-transferred circ_PTPRA not only blocked CRC growth but also strengthened CRC cell radiosensitivity. Circ_PTPRA was previously shown to be downregulated in bladder cancer tissues by RNA sequencing (Li et al. 2017), and a study addressed that circ_PTPRA acted as a tumor suppressor to inhibit bladder cancer growth (He et al. 2019). Besides, circ_PTPRA was also reported to be downregulated in non-small cell lung cancer (NSCLC) tissues, and circ_PTPRA repressed epithelial-mesenchymal transitioning (EMT) in vitro and blocked tumor metastasis in vivo (Wei et al. 2019). The present documents indicated that circ_PTPRA was a tumor suppressor in various cancers. Our data also showed that circ_PTPRA was resistant to RNase R and stably expressed in CRC cells, hinting that exosomes-packaged circ_PTPRA might be used as a stable biomarker for CRC diagnosis and therapy.

Further analysis demonstrated that circ_PTPRA acted as sponge of miR-671-5p to inhibit miR-671-5p expression and function. MiR-671-5p was an oncogenic role in CRC by the findings from a previous study (Jin et al. 2019). This study recorded that miR-671-5p high expression was associated with poor prognosis in colon cancer, and miR-671-5p overexpression largely promoted colon cancer cell proliferation, migration and invasion (Jin et al. 2019). The similar role of miR-671-5p was also demonstrated in glioblastoma. MiR-671-5p was overexpressed in glioblastoma biopsies, and miR-671-5p overexpression restrained apoptosis but accelerated proliferation of glioblastoma cells (Barbagallo et al. 2016; Li and Diao 2019). Consistent with these results, we found that miR-671-5p restoration reversed the effects of circ_PTPRA, thus recovering CRC cell proliferation and weakening CRC cell radiosensitivity. Besides, miR-671-5p deficiency inhibited CRC cell growth and increased radiosensitivity. Contradictorily, miR-671-5p, as a tumor suppressor, blocked cell malignant behaviors in gastric cancer, esophageal squamous cell carcinoma and so on (Qiu et al. 2018; Li et al. 2019), indicating that the function of miR-671-5p was diverse in different type of cancers. The role of miR-671-5p in CRC needed further exploring.

Moreover, we identified that miR-671-5p suppressed SMAD4 expression by binding to SMAD4 3’UTR. It was shown that loss of SMAD4 from colorectal tumors was associated with decreased survival time, and cell migration and invasion were accelerated in SMAD4-negative CRC cells (Voorneveld et al. 2014). Besides, loss of SMAD4 facilitated metastasis of CRC to liver (Itatani et al. 2013). MiR-130a, miR-301a and miR-454 shared the same binding site with SMAD4 3’UTR, and all of them played oncogenic effects to promote CRC cell proliferation, invasion and tumorigenesis in vivo by degrading the expression of SMAD4 (Liu et al. 2013). Consistent with these findings, our data showed that SMAD4 expression was decreased in CRC cells, and SMAD4 knockdown abolished the role of miR-671-5p deficiency and then restored cell proliferation capacity and radioresistance, which enriched the function of SMAD4 in CRC progression and radiosensitivity.

In conclusion, our study strongly supported that exosomal circ_PTPRA blocked CRC malignant development and enhanced CRC cell radiosensitivity, which was accomplished partly by modulating the miR-671-5p-SMAD4 network. Exosomal circ_PTPRA is a promising biomarker in CRC diagnosis, treatment and prognosis.

Acknowledgements

None.

Funding

There is no funding.

Data availability

Data analyzed for this study will be available on a reasonable request.

Declarations

Conflict of interest

The authors declare that they have no financial conflicts of interest.

Ethical approval

All the procedures involving human subjects were compliant with the ethical guideline of the Declaration of Helsinki and were allowed by the Ethics Committee of Hunan Cancer Hospital.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

Data analyzed for this study will be available on a reasonable request.

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