Tumor-derived exosomes containing circ_0008039 promote the stemness of colorectal cancer cells by inhibiting ferroptosis

Abstract

This study was to investigate the impact of exosomes-derived circ_0008039 on ferroptosis and stemness in colorectal cancer cells, as well as its associated molecular mechanisms. The cell colony formation ability was evaluated using the MTT assay and colony formation assay, respectively. Additionally, the sphere formation assay was employed to examine the cells’ ability to aggregate into spheres. The targeting relationship between circ_0008039, miR-302e, and SLC7A11 was verified through dual luciferase assay. Furthermore, RNA immunoprecipitation (RIP) analysis of circ_0008039 and miR-302e was conducted. Western blotting was utilized to detect the protein expression of exosome surface antigens (TSG101 and CD63), stem cell markers (CD133, Nanog, and SOX-2), as well as SLC7A11 in both exosomes and cell lysates. The levels of glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS), and Fe²⁺ were determined using corresponding kits. Circ_0008039 exhibited significant downregulation in cancer tissues and cells. Furthermore, exosome-derived circ_0008039 played a crucial role in promoting cell viability, clone formation, and resistance to ferroptosis by inhibiting MDA, ROS, and Fe²⁺ levels. Additionally, circ_0008039 acted as a miR-302e sponge to regulate SLC7A11 expression. In colorectal cancer (CRC) tissues, miR-302e was negatively correlated with SLC7A11 expression while circ_0008039 was negatively correlated with miR-302e expression. Mechanistic validation demonstrated that circ_0008039 regulated CRC cell proliferation, stemness maintenance and ferroptosis through the modulation of the miR-302e/SLC7A11 axis. The Circ_0008039/miR-302e/SLC7A11 axis plays a pivotal role in facilitating the proliferation and maintenance of stemness in CRC cells, while enhancing resistance to ferroptosis.

Introduction

Colorectal cancer (CRC) is currently the third most common cancer and the second leading cause of cancer death in the world.⁽¹⁾ In 2020, there were 1.9 million new cases of CRC and 935,000 related deaths.⁽²⁾ Currently, China is undergoing a cancer transition, with an increasing burden of gastrointestinal cancer. The economic burden associated with CRC has been increasing in recent years. Studies have shown that the medical expenses related to CRC have increased rapidly, and the per capita direct medical expenses of patients with CRC in China exceed the per capita GDP of the year, which causes a heavy disease burden.^(3,4) Current studies estimate that 3.2 million new cases will occur globally by 2040, an increase of 63% from the nearly 2 million estimated in 2020.⁽⁵⁾ However, because population structure was not considered, this study may underestimate the risk of CRC in China. China is one of the countries with the fastest aging growth in the world. By 2040, due to the extension of life expectancy and the decline of fertility rate, the proportion of people over 60 years old in China is expected to reach 28%. Due to the large population base, the number of people over 60 years old in China is expected to reach 402 million by 2040.⁽⁶⁾ The incidence of CRC increases exponentially with age. Consequently, an in-depth investigation into the pathogenesis of CRC and the development of effective prevention and treatment strategies are critical challenges that require immediate attention.

Ferroptosis, an iron-dependent and lipid peroxide-driven form of cell death, has demonstrated significant potential in CRC research.⁽⁷⁾ Studies have revealed that CRC cell growth is intricately linked to multiple gene mutations, which modulate the sensitivity of CRC cells to ferroptosis through the regulation of metabolic pathways. By inhibiting key enzymes such as GPX4 or SLC7A11, ferroptosis can be effectively induced in CRC cells leading to inhibition of tumor growth.⁽⁸⁾ Previous studies have demonstrated that miR-509-5p facilitates ferroptosis in CRC cells through the targeted regulation of SLC7A11, thereby influencing tumor progression.⁽⁹⁾ Furthermore, the application prospects of ferroptosis inducers are extensive in CRC treatment as they can be used not only as monotherapy but also in combination with chemotherapy drugs and immune checkpoint inhibitors to elicit synergistic anticancer effects.⁽¹⁰⁾ However, challenges still remain regarding specificity, mechanistic understanding, and clinical translation.⁽¹¹⁾ In the future, as our understanding of the mechanisms underlying ferroptosis deepens and technological advancements continue, it is anticipated that ferroptosis will play a pivotal role in the treatment of CRC.

Exosomes (exos) are small membrane vesicles involved in intercellular communication, harboring a diverse array of functional RNA molecules including microRNAs (miRNAs),⁽¹²⁾ long non-coding RNAs (lncRNAs),⁽¹³⁾ and circular RNAs (circRNAs). These RNA species have been implicated in the progression of cancer.⁽¹⁴⁾ For instance, in CRC, exosomal circ-PACRGL has been identified as an instigator of CRC proliferation and metastasis.⁽¹⁵⁾ In addition, circRNA circ-122 is considered to promote CRC chemotherapy resistance by regulating the glycolysis process of cells.⁽¹⁶⁾ Therefore, the investigation of exos-derived circRNA holds great potential in addressing CRC.

Circ_0008039, a recently identified circRNA, is derived from the transcript of PRKAR1B, a regulatory subunit of protein kinase A. Research findings suggest that circ_0008039 exhibits upregulated expression levels in breast cancer,^(17,18) thereby promoting tumor growth and metastasis. However, the biological function and regulatory mechanisms of circ_0008039 in CRC remain largely unexplored. Therefore, it is imperative to further investigate the role and potential mechanisms of circ_0008039 in CRC progression. In this study, we have successfully validated the robust expression of circ_0008039 in exos derived from the serum of CRC patients. However, limited evidence currently exists to establish a direct association between circ_0008039 in serum exos and CRC progression. This article primarily elucidates the impact of exosomal circRNAs on CRC advancement and delves into their potential underlying mechanisms.

Materials and Methods

Sample collection

From 2019.6 to 2021.3, blood samples of 25 patients with CRC, tumor tissues, adjacent tissues and blood samples of 10 individuals in good health were collected from the Affiliated Hospital of Guizhou Medical University. Recruit participants aged 18 to 70 years with CRC, who have not previously been diagnosed with any other malignancies or significant systemic disease. These healthy volunteers received physical examinations at the same time, and there were no cancers or systemic diseases. After the patient’s blood stood for half an hour, the serum was collected by centrifugation. Then, ExoQuick-TC kit (System Biosciences, Palo Alto, CA) was used to separate exos. The present study obtained ethical approval from the Ethics Committee of the Affiliated Hospital of Guizhou Medical University, and all participants provided their informed consent.

Separation and analysis of exos in CRC

The separation of exos was carried out according to the research method of Roman Kornilov.⁽¹⁹⁾ Exos were isolated from various cell cultures grown in 10 ‍cm culture plates using fresh DMEM supplemented with serum. Cell debris was removed by centrifugation at 120,000 g for 1 ‍h. After 48 ‍h, the culture medium was filtered through a 0.22 ‍μm filter (Millipore, Burlington, MA) and collected. Exos were subsequently separated by ultracentrifugation using an Optima MAX-XP ultracentrifuge (Beckman Coulter, Brea, CA). High-resolution images of the exos were obtained using a transmission electron microscope (HT7700; Hitachi, Tokyo, Japan) for detailed analysis.

PKH67 staining

The presence of exos (5 ‍μg) was quantified utilizing the KH67 fluorescent cell labeling kit (Sigma-Aldrich, St. Louis, MO). In the initial step, PKH67-labeled exos are from a 100 ‍ml culture medium. Following this, recipient cells were. To evaluate the uptake of exos by receptor cells, each well was treated with DMEM either containing or devoid of labeled exosome solution.

Western blot (WB) assay

The RIPA lysis buffer (Beyotime, Shanghai, China) was employed for protein extraction. Subsequently, 10% SDS-PAGE gel electrophoresis was performed to separate the proteins, which were then transferred onto a PVDF membrane. Antibodies from Abcam (Cambridge, UK) were utilized to immunoblot the membrane against TSG101, CD63, CD133, Nanog, SOX-2, SLC7A11, and GAPDH proteins. After incubation with HRP-labeled IgG antibodies, the bands were detected using an enhanced chemiluminescence kit provided by Millipore.

Cell culture

The HT-29 and SW480 cell lines, as well as NCM460 cells and 293T human embryonic kidney cells, were obtained from Procell in China. NCM460 cells were cultured in RPMI-1640 medium (KeyGene, Chengdu, China), while HT-29 and SW480 cells were cultured in DMEM medium at a temperature of 37°C with a CO₂ concentration of 5%. Both media contained 10% FBS (Gibco, Waltham, MA), and a double antibody at a concentration of 1% was purchased from MCE Company (Monmouth Junction, NJ).

Real-time quantitative polymerase chain reaction (qRT-PCR)

The RNA was extracted using TRIzol (Beyotime) and reverse transcribed into cDNA using the cDNA Kit (Sigma-Aldrich). The expression levels of circ_0008039, miR-302e, and SLC7A11 were quantified by real-time PCR kit (Taraka, Kusatsu, Japan), following the 2^−ΔΔCT method with GAPDH and U6 as reference genes. The primers used are shown in Supplemental Table 1*.

MTT assay

The Aldrich assay was employed to evaluate cell viability. Cells were seeded in 96-well plates and incubated for various time intervals: 0, 24, 48, 72, and 96 ‍h. At each designated time point, 20 ‍μl of MTT reagent was added to each well. After a 4-h incubation period, solubilizing solution was introduced to facilitate dissolution. The optical density (OD) at a wavelength of 570 ‍nm was measured using a microplate reader as an indicator of cell viability.

The logarithmically growing cells were harvested and subsequently, the cell suspension was seeded into a 12-well plate at a density of 400 cells per well and then incubated statically with 5% CO₂ at 37°C for two weeks. After PBS washing, the cells were fixed with 4% paraformaldehyde (Beyotime) for 15 ‍min and subsequently stained again using crystal violet solution (Beyotime) for microscopic observation and enumeration of clone numbers.

Sphere formation assay

The transfected cells were treated with trypsin and converted into a suspension. This cell suspension, containing 1 × 10⁴ cells per well, was then introduced into a 6-well ultra-low attachment plate (Corning, Corning, NY) and incubated at 37°C for a duration of 10 days. After the incubation period an examination was conducted to capture images of the quantity well.

Oxidation index detection

The levels of MDA and GSH in CRC cells were quantified using biochemical assay kits (Beyotime) following the manufacturer’s instructions. Each sample was analyzed in triplicate.

Determination of lipid reactive oxygen species (ROS)

Before collecting cells for 20 ‍min, the final concentration was 10 ‍μM by adding DCFH-DA (Invitrogen, Waltham, MA). Finally, lipid ROS positive cells were evaluated by BD FACSCanto II flow cytometry (BD Biosciences, Franklin Lakes, NJ).

Determination of Fe²⁺ content

CRC cells were inoculated with an initial density of 2 × 10⁵/well and cultured at 24°C and 37% CO₂ for 5 ‍h. The Fe²⁺ content was detected by using the Fe²⁺ content detection kit (Suolaibao, Beijing, China).

Dual luciferase reporter assay

The pmirGLO vector was employed for cloning and amplification of the wild-type and mutant sequences of circ_0082096 or SLC7A11 3'UTR. Subsequently, the constructed WT/MUT reporter vector was transfected into 293T cells, followed by luciferase activity analysis using Beyotime in the presence of either miR-302e mimic or miR-NC.

RNA binding protein immunoprecipitation (RIP) experiment

The detection of RNA levels was performed using the RIP Kit (Millipore) following the research methodology outlined by Li et al.⁽²⁰⁾ Magnetic beads conjugated with anti-Ago2 or anti-IgG were employed to incubate cell lysates, and QPCR was utilized for quantifying RNA levels.

Statistical analysis

The data were presented as mean ± SD, and statistical analysis was performed using GraphPad Prism 7.0 software. Comparative analysis between the two groups was conducted using the student’s t test, while comparison between multiple groups was done using ANOVA. A significance level of p<0.05 indicates a statistically significant difference.

Results

Circ_0008039 was up-regulated in CRC patients and CRC cells

In comparison to normal serum samples, the levels of TSG101 and CD63 proteins, which serve as exosomal markers, were significantly elevated in the serum of patients with CRC (Fig. 1A). Compared to healthy volunteers, a significant elevation in the levels of circ_0008039 was observed in serum-derived exos from CRC patients (Fig. 1B). Furthermore, circ_0008039 expression levels were found to be significantly higher in CRC cells (HT-29 and SW480) compared to NCM460 cells (Fig. 1C).

Fig. 1.

Expression of circ 0008039 in tumor tissues and exosomes. (A) Detection of exosome marker expression. (B) The expression of circ_0008039 in serum exosomes of CRC patients. (C) Detection of the expression of circ_0008039 in CRC cell line. (D) Detection of exosome marker expression. (E) The expression of circ_0008039 in exosomes of each group was determined; (F) PKH67 fluorescence labeling to determine the exosome uptake level of HT-29 cells; *p<0.05.

Study on the characteristics of exos

As part of our investigation into the impact of circ_0008039 derived from exos on CRC cells, we proceeded to isolate exos from HT-29 cells for subsequent analysis. Moreover, both the exo-vector group and exo-circ group exhibited significantly augmented levels of TSG101 and CD63, well-established markers for exos. These findings indicate successful isolation of exos from cells (Fig. 1D).

The expression level of circ_0008039 was quantified in isolated exos. The results revealed a significantly higher abundance of circ_0008039 in the exo-circ group compared to the exo-vector group (Fig. 1E). To investigate the interaction between extracted exos and HT-29 cells, we employed PKH67 labeling to monitor the intracellular distribution of fluorescent exos. Fluorescence analysis revealed fluorescence signals in both the exo-vector group and exo-circ group, suggesting that HT-29 cells potentially internalize exos via endocytosis. Importantly, no PKH67 signal was observed in the NC group. These findings indicate a potential active role of HT-29 cells in mediating exosome uptake (Fig. 1F).

Exo-circ_0008039 promotes CRC cell proliferation and cell stemness

In addition, we investigated the impact of circ_0008039 derived from exos on the cell growth. The exo-cic group exhibited significantly enhanced cell viability, increased formation of clones, and improved ability of stem cells to aggregate into spheres compared to the exo group (Fig. 2A–D). Western blot analysis revealed a significant upregulation in the expression of stem cell surface markers (CD133, Nanog, and SOX-2) in cells treated with exo-cic compared to those treated with exo-vector (Fig. 2E).

Fig. 2.

Eccrine circ_0008039 promotes CRC cell proliferation and cell stemness. (A) Detecting cell viability. (B) Detecting the number of cell clones. (C, D) Sphere formation assay was used to detect the effect of each treatment group on the stemness of tumor cells. (E) Detecting the expression of the surface markers of the stem cells; *p<0.05.

Exo-circ_0008039 promotes CRC cell proliferation and cell stemness by mediating ferroptosis

The MTT assay demonstrated a significant decrease in the viability of CRC cells subsequent to Erastin treatment. However, exo-cic exhibited a mitigating effect on the inhibitory impact of Erastin on cell viability. (Fig. 3A). The GSH level was significantly decreased after Erastin treatment, while exo-circ could partially increase the GSH level (Fig. 3B). In addition, Erastin treatment increased the levels of MDA, ROS, and Fe²⁺ in CRC cells, while exo-cic could partially reduce the increase of MDA, ROS, and Fe²⁺ induced by Erastin (Fig. 3C–E). In comparison to the NC group, the Erastin-treated group demonstrated a notable decrease in clone formation efficiency along with compromised ability of stem cells to aggregate and reduced expression levels of surface markers linked to stemness (CD133, Nanog, and SOX-2). Nevertheless, it was noted that exo-circulation exerted a mitigatory influence on these aforementioned effects (Fig. 3F–H).

Fig. 3.

Eccrine circ_0008039 promotes CRC cell proliferation and cell stemness by mediating ferroptosis. (A) Detecting cell viability. (B–E) Detect the levels of GSH, MDA, ROS, and Fe²⁺ respectively. (F) Detecting the cell clone number; (G) Sphere formation assay was used to detect the effect of each treatment group on the stemness of tumor cells. (F) Detecting the expression of the surface markers of the stem cells; *p<0.05.

Circ_0008039 as the molecular sponge of miR-302e

To investigate the potential interaction between circ_0008039 and miRNA, we employed the StarBase website for predicting the putative target miRNA of circ_0008039 and SLC7A11. The computational analysis unveiled a significant binding affinity existing between circ_0008039 and miR-302e (Fig. 4A). Subsequently, a dual luciferase assay was performed to validate the interaction between circ_0008039 and miR-302e. Our results demonstrated that co-transfection of miR-302e mimetic significantly attenuated the luciferase activity of circ_0008039-WT, while exhibiting no impact on the luciferase activity of circ_0008039-MUT (Fig. 4B). Furthermore, the RIP experiment findings demonstrate a significant upregulation in the recruitment level of circ_0008039 within the Ago2 group compared to the group (Fig. 4C). Furthermore, we conducted comprehensive expression level analysis in both clinical samples and cell lines. Our findings unveiled a significant downregulation of miR-302e expression within the CRC (Fig. 4D). Furthermore, a significant inverse correlation was observed between the expression levels of circ_0008039 and miR-302e in colorectal tissues (Fig. 4E). The exo-cir group exhibited a significant downregulation in the expression level of miR-302e compared the exo-vector group (Fig. 4F).

Fig. 4.

Circ_0008039 as a molecular sponge of miR-302e. (A) Prediction of the binding site between circ_0008039 and SLC7A11 by the Starbase database. (B, C) To verify the targeted binding relationship between circ_0008039 and miR-302e. (D) The expression of miR-302e in the tissues of CRC patients. (E) The correlation between the expression of circ_0008039 and the expression of miR-302e. (F) Detecting the expression of miR-302e in each group; *p<0.05.

MiR-302e targeted regulation of SLC7A11

According to the Starbase online database, it has been observed that miR-302e could regulate SLC7A11. The target sequence was illustrated in Fig. 5A. The double luciferase assay revealed that transfection of miR-302e mimetic significantly attenuates luciferase activity in SLC7A11-WT cells, while exhibiting no significant impact on luciferase activity in SLC7A11-MUT cells. (Fig. 5B). The subsequent analysis revealed a significant upregulation of SLC7A11 expression in CRC tissue compared to adjacent tissues (Fig. 5C). The expression of miR-302e showed a negative correlation with SLC7A11 (Fig. 5D). Additionally, the overexpression of miR-302e resulted in a significant reduction in protein and mRNA levels of SLC7A11 (Fig. 5E and F). Furthermore, cells treated with exo-circ significantly higher expression levels of SLC7A11 compared to those treated with the exo-vector group (Fig. 5G).

Fig. 5.

MiR-302e targeted regulation of SLC7A11. (A) Prediction of the interaction site between miR-302e and SLC7A11 using the Starbase database. (B) To validate the specific binding association of miR-302e and SLC7A11. (C) The expression of SLC7A11 in the tissues of CRC patients. (D) The correlation between the expression of SLC7A11 and miR-302e. (E–G) Detect the expression of SLC7A11 in each group; *p<0.05.

Eccrine circ_0008039 enhances the proliferation and stemness of CRC cells via the miR-302e/SLC7A11 axis, while suppressing ferroptosis

Currently, it remains unclear whether the effects of circ_0008039 derived from desiccation and resistance to ferroptosis are mediated through the miR-302e/SLC7A11 pathway. To further investigate this matter, we treated CRC cells with exo-circ_0008039 [exo-circ-mimic (miR-mimic)]. Compared to the NC group, there was a significant upregulation in SLC7A11 expression in the exo-circ group but a notable downregulation in the miR-mimic group. Moreover, SLC7A11 expression in the exo-circ + miR-mimic group was significantly lower than that observed in the miR-mimic group but considerably higher than that seen in control (Fig. 6A). Furthermore, the exo-circ group exhibited significant enhancements in cell viability (Fig. 6B), clone formation capacity (Fig. 6C), stem cell aggregation ability (Fig. 6D), and expression of stem cell to the NC group (Fig. 6E). Conversely, these factors were markedly suppressed in the miR-mimic group. The exo-circ group demonstrated higher levels of cell viability, clone formation capacity, stem cell aggregation ability, and expression of stem cell surface markers than the miR-mimic group. In addition, GSH level in exo-circ group increased significantly, while MDA, ROS, and Fe²⁺ levels decreased significantly, while the above indexes in exo-circ + miR-mimic group partially returned to the control level (Fig. 7A–D).

Fig. 6.

Eccrine circ_0008039 desiccation of CRC cells via modulation of the miR-302e/SLC7A11 pathway. (A) Detecting the expression of SLC7A11. (B) cell viability. (C) Detecting the cell clone number. (D) Sphere formation assay was used to detect the effect of each treatment group on the stemness of tumor cells. (E) Detecting the expression of the surface markers of the stem cells; *p<0.05.

Fig. 7.

Eccrine circ_0008039 enhances ferroptosis resistance through the miR-302e/SLC7A11 axis. (A–D) Detect the levels of GSH, MDA, ROS, and Fe²⁺ respectively; *p<0.05.

Discussion

Previous studies have consistently demonstrated the presence of circRNA in exos, which can be reliably detected in both blood and urine samples.⁽²¹⁾ An increasing amount of evidence suggests that exocrine circRNAs have a significant impact on the proliferation of tumors,⁽²²⁾ immune escape,⁽²³⁾ angiogenesis,⁽²⁴⁾ metastasis,⁽²⁵⁾ and chemotherapy resistance⁽²⁶⁾ of CRC. In the present investigation, circ_0008039 exhibited up-regulation in both CRC patients and CRC cells. Exos-derived circ_0008039 demonstrated the ability to enhance cell viability, clone formation, and stemness while conferring resistance against ferroptosis-induced cell death. Dou D and others also revealed that circ_0008039 was highly expressed in breast cancer tissues, and circ_0008039 promoted the proliferation, migration, invasion, and glycolysis of breast cancer cells by regulating the miR-140-3p/SKA2 axis.⁽²⁷⁾ In a word, circ_0008039 may promote cancer progression.

Iron-induced mortality is characterized by the absence of apoptosis and programmed cell death, which occurs subsequent to the deactivation of GPX4 and XC. The latter involves SLC7A11, a transport mechanism that facilitates the reverse movement of cystine and glutamic acid, followed by iron-dependent lipid oxidation.^(28,29) The reduction of GPX4 is facilitated by GSH, which serves as a pivotal intracellular antioxidant that effectively safeguards against the accumulation of ROS. The inhibition system of XC can efficiently impede GSH metabolism and induce iron-mediated cell death.^(29,30) The present study demonstrates a significant decrease in intracellular GSH levels following Erastin treatment in HT-29 cells, accompanied by elevated levels of MDA, ROS, and Fe²⁺. Overexpression of exosomal circ_0008039 partially attenuates the Erastin-induced reduction in GSH levels in NCM460 cells and mitigates the increases in MDA, ROS, and Fe²⁺ content. Therefore, this study uncovers a novel mechanism by which exosomal circ_0008039 promotes the proliferation and survival of CRC cells through suppression of ferroptosis.

Recently conducted studies have demonstrated the pivotal role of miR-302e in various tumor types, including glioma and CRC.^(31–34) In this investigation, we discovered that circ_0008039 targets SLC7A11 and actively participates in regulating ferroptosis levels in CRC. To the best of our knowledge, this is the first report documenting these findings. Our data unequivocally demonstrate that circ_0008039 significantly influences ferroptosis levels through miR-302e, thereby further elucidating the mechanistic impact of circRNA in CRC.

The in vitro assays conducted in this study revealed that circ_0008039, derived from exos, enhances the proliferation of CRC cells through the miR-302e/SLC7A11 axis. Moreover, it also confers resistance to ferroptosis. These findings suggest that exos-derived circ_0008039 holds promise as a valuable therapeutic target for patients with CRC. However, further in vivo validation is required to confirm its efficacy.

Overall, this research provides novel insights into the mechanisms underlying CRC progression and has significant implications for diagnosis and the identification of new therapeutic targets.

Author Contributions

MS and XLiao made contribution to the conception and design; SW and XY analyzed and interpreted data; XLiu and MS drafted the article; LM and XLiao revisied it critically for important intellectual content; All authors approved the final version to be published.

Funding

Science and Technology Planning Project of Guizhou Province (Basic of Guizhou Science and Technology Cooperation-ZK) [2023] Key Project [038]; The Doctoral Startup Fund of the Affiliated Hospital of Guizhou Medical University [2022], gyfybsky-2022-15.

Availability of Data and Materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Ethics Approval

The present study obtained ethical approval from the Affiliated Hospital of Guizhou Medical University Ethics Committee, and all participants provided their informed consent.

Consent to Participate

Informed consent was obtained from all individual participants included in the study.

Abbreviations

circRNAs

circular RNAs

CRC

colorectal cancer

Exos

exosomes

GSH

glutathione

lncRNAs

long non-coding RNAs

MDA

malondialdehyde

miRNAs

microRNAs

qRT-PCR

real-time quantitative polymerase chain reaction

RIP

RNA binding protein immunoprecipitation

ROS

reactive oxygen species

WB

Western blot

Conflict of Interest

No potential conflicts of interest were disclosed.