LncRNA FTX promotes colorectal cancer radioresistance through disturbing redox balance and inhibiting ferroptosis via miR-625-5p/SCL7A11 axis

Abstract

BACKGROUND

Radiotherapy is widely employed in colorectal cancer (CRC) treatment, but the occurrence of radioresistance severely limits the clinical benefit to patients and significantly contributes to treatment failure and recurrent metastasis.

AIM

To explore the role and underlying mechanism of the lncRNA FTX in radiotherapy resistance in CRC.

METHODS

LncRNA FTX expression in colorectal parent cells (HT29 and HCT116) and radioresistant cells (HT29R and HCT116R) was determined by real-time quantitative PCR, and the viability of HT29R-shFTX and HCT116R-shFTX cells under ionizing radiation was evaluated using the cell counting kit-8 assay and colony formation experiment. The levels of glutathione and reactive oxygen species in cells after irradiation were determined, and the association between ferroptosis and lncRNA FTX expression in cancer cells was tested. A dual-luciferase assay was used to validate gene interactions. A xenotransplantation mouse model was established to explore the effects of FTX on the CRC tumor radiosensitivity in vivo.

RESULTS

FTX was upregulated in radioresistant CRC cells, and FTX knockdown inhibited cell survival and increased cell ferroptotic death in response to ionizing radiation. Moreover, lncRNA FTX restricted the SLC7A11 expression by sponging with miR-625-5p, and inhibition of the lncRNA FTX or SLC7A11 significantly increased cellular oxidant levels and DNA damage to ionizing radiation in cancer cells. However, SLC7A11 overexpression reversed the effects of decreased FTX levels on ferroptosis and high oxidation levels in cancer cells exposed to ionizing radiation.

CONCLUSION

Inhibition of the lncRNA FTX/miR-625-5p/SLC7A11 axis can induce ferroptosis and disturb intracellular redox balance, further sensitizing CRC cells to ionizing radiation, suggesting its potential as a therapeutic target for improving CRC response to radiation therapy.

Key Words: Colorectal cancer; LncRNA FTX; Solute carrier family 7 member 11; Ferroptosis; Redox; Radioresistance

Core Tip: Radiotherapy is widely employed in colorectal cancer (CRC) treatment, but the occurrence of radioresistance severely limits the clinical benefit to patients and significantly contributes to treatment failure and recurrent metastasis. In this study, our results demonstrated that inhibition of the lncRNA FTX/miR-625-5p/SLC7A11 axis can induce ferroptosis and disturb intracellular redox balance, further sensitizing CRC cells to ionizing radiation, suggesting its potential as a therapeutic target for improving CRC response to radiation therapy.

INTRODUCTION

Colorectal cancer (CRC) is among the most common malignant tumors of the gastrointestinal tract caused by environmental and multiple genetic factors[1], and it accounts for the third highest incidence[2] and the fourth highest mortality rate in the world, which is a severe threat to the global public health security[3]. Radiation therapy, as among the main treatment modalities for CRC, particularly rectal cancer, plays a vital role in reducing the clinical stage of tumors, preventing postoperative recurrence and metastasis, and alleviating the clinical symptoms of patients in advanced stages, making it an indispensable mainstay in the clinical treatment of CRC[4]. However, developing resistance to radiotherapy often leads to failure of tumor control and poor patient prognosis, which in turn leads to recurrence and metastasis. Therefore, exploring the molecular mechanisms of radioresistance and improving the effectiveness of radiotherapy is an urgent clinical problem that needs to be solved in CRC treatment.

Numerous studies have demonstrated that cancer cells undergo significant changes in gene expression after exposure to ionizing radiation and that the aberrant expression of some genes involved in cell cycle regulation and DNA damage repair is closely correlated with resistance to radiotherapy[5,6]. When DNA damage is induced by ionizing radiation, activated ataxia-telangiectasia mutated (ATM) protein is recruited to the vicinity of the damaged DNA, phosphorylates checkpoint kinase 2 (CHK and CDC25c, induces G2 phase block, and initiates repair)[7]. High levels of ATM expression correlate closely with cellular radiation resistance[8]. Caspase-activated DNase is also expressed in cancer cells upon radiation injury and actively induces G2 phase arrest, thereby promoting cancer cell survival in response to irradiation[9]. Moreover, the activation of some genes increases intracellular redox levels, and increasing the sensitivity of cancer cells to ferroptosis reduces their resistance to radiotherapy[10]. Radiotherapy can also act on the tumor microenvironment, causing chronic inflammation, fibrotic changes, local hypoxia, vascular injury, and a state of local immunosuppression[11], and is accompanied by many cytokine secretion disorders, which contribute to radiotherapy resistance.

Besides coding genes, non-coding RNAs, as critical epigenetic regulators in the cell, are involved in the progression of various malignant tumors[12]. It is also often aberrantly expressed in cancer cells after receiving ionizing radiation, thereby regulating the sensitivity of cancer cells to radiotherapy[13]. LncRNA FTX is a transcript consisting of 2300 bp transcribed by FTX, a conserved gene located at the X-inactivation center region of the human X-chromosome q13.2. LncRNA FTX is highly expressed in colorectal, hepatocellular, gastric, and lung cancers and promotes tumor proliferation growth and invasive metastasis[14]. However, its role in malignant tumor radiotherapy resistance remains unclear. In this study, lncRNA FTX regulated the expression of SLC7A11, a vital antioxidant protein in the ferroptosis pathway, through sponge adsorption of miR625-5p, which in turn affects the intracellular redox level and tumor sensitivity to radiotherapy after ionizing radiation. The inhibition of lncRNA FTX/miR625-5p/SLC7A11 would provide a new approach to overcome radiotherapy resistance and improve radiotherapy strategies for CRC by providing new ideas.

MATERIALS AND METHODS

Cell culture

Human CRC cell lines, HT29 and HCT116, were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in 1640 medium (Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, United States) in a 37 °C incubator with 5% CO₂. These cells were used until the 20^th generation. All cell lines identified by short tandem repeat sequences were mycoplasma-free.

Culture of radiotherapy-resistant cells

HT29 and HCT116 cells were cultured to achieve 90% confluence and subjected to 2 Gy irradiation. The cells were passaged once and then continuously irradiated repeatedly at a single dose of 2 Gy 25 times, and the surviving cells after irradiation with a total radiation dose of 50 Gy were the radioresistant cells (HT29R and HCT116R). Briefly, 6 MV high-energy X-ray irradiation, rack angle 180°, collimator angle 0°, the field size of 30 cm × 30 cm, the source skin distance of 100 cm, the dose rate of 303 cGy/minute, and the culture bottle (plate) under the pad of 1 cm thickness of tissue compensation film.

Cell transfection

The specific sh-RNAs to lncRNA FTX or SLC7A11 were synthesized by JinHe Tech (Guizhou, China), and non-specific shRNAs worked as negative control (NC). miR-625-5p inhibitor along with NC inhibitor were synthesized by JinHe Tech (Guizhou, China). After reaching 40%-50% confluence, cells were transfected with Lipofectamine 3000 (Invitrogen, United States).

Colony formation assays

Different numbers of the cells were inoculated according to different doses of radiation irradiation; cells with 5000, 10000, 20000, and 40000 were inoculated in 60 mm dishes. Then, cells were cultured in colonies after 14 days of incubation and rinsed with PBS, fixed in methanol for 15 minutes, stained with 0.1% crystal violet, and counted.

CCK-8 assay

The digested cells (5000 cells/well) were inoculated in 96-well plates and subjected to radiation irradiation (0, 2, 4, and 6 Gy) after 24 hours in the cell incubator, and 10 μL of CCK-8 reagent was added to each well after 72 hours. The cells were incubated for 2 hours. The absorbance of the cells was measured at 405 nm using an enzyme marker.

Real-time quantitative polymerase chain reaction

Total RNA was extracted using the TRIzol method (Invitrogen), according to the manufacturer's instructions, and cDNA was obtained by reverse transcription using the qPCR RT kit (Takara). The levels of lncRNA FTX and miR-625-5p in the cells were examined by real-time quantitative PCR (RT-qPCR) analysis using SYBR Green Master Mix (Takara). U6 was used as a control for miR-625-5p, and GAPDH was used as a control for FTX and SLC7A11. The data were analyzed using the 2^–ΔΔCt method. For the lncRNA FTX gene, the forward primer was 5′-CAGTTTGCCTCCCTCTTTC-3′, and the reverse primer was 5′-GAGGGACACCGCCATTTGAT-3′. For the GAPDH gene, the forward primer was 5′-CAGGGACACCGCCATTTGAT-3′, and the reverse primer was 5′- GAAGGCTGGGGCTCATTT-3′. For the SLC7A11 gene, the forward primer was 5′-GCAGTGGCTCACACCTGTAATCC-3′, and the reverse primer was 5′-GGCTGGTCTT GAACTCCTGATCTTG-3′.

Western blotting analysis

Total protein from CRC cells was extracted with RIPA lysate buffer (Solarbio, China) following the manufacturer's instructions. After measuring the protein concentration by BCA protein assay, 30 μg of total protein was loaded and separated by 10% SDS-PAGE for 2 hours, transferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore Corporation, MA, United States), and incubated with 5% blocking solution for 1 hour. The membranes were incubated overnight at 4 °C using primary antibodies SLC7A11 (1:3000), GPX4 (1:2000), and tubulin (1:40,000, all from Abcam), ACSL4 (1:3000), TFR1 (1:8000, all from Genetex), MRE11 (1:1000), Ku80 (1:3000, all from Proteintech). After that, the membranes were washed in Tris-HCl buffered salt solution thrice for 7 minutes each time, after which the membranes were incubated with the relative secondary antibody (Abcam) for 1 hour. The protein bands were exposed with an enhanced chemiluminescence kit (Millipore, St. Louis, MO, United States), following intensity analysis using the Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA, United States). The relative protein expression was calculated using tubulin as an internal reference.

Measurement of glutathione levels

The Glutathione Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) was used to detect the glutathione (GSH) levels according to the product instructions. The GSH content was quantified using a microplate assay.

Detection of reactive oxygen species

DHE staining was used to detect the reactive oxygen species (ROS) in cells pre or post-irradiation according to the manufacturer’s instructions incubated with 20 mmol/L DHE dye solution under normal temperature and dark conditions for 1 hour. The red dye was used as a molecular probe and observed, under a fluorescence microscope.

Luciferase assay

We constructed the wild-type and mutant lncRNA FTX, which were respectively subcloned into pGL3 vectors (Promega, Madison, WI, United States), constructing plasmids for further transfection with indicated mimics or shRNAs for 48 hours. The cells were digested with restriction enzymes; the amplified PCR product was cloned into the polyclonal site of the reconstructed pGL3 expression vector. Finally, the luciferase activity was detected using the Dual-Luciferase^® Reporter assay kit (Promega, Madison, WI, United States).

Xenograft mouse model

Five-week-old male nude mice were obtained from Laboratory Animal Co. Ltd. (Chongqing Tengxin Company). They were maintained in the central laboratory in a pathogen-free animal facility at 24 °C with access to distilled food and water. Nude mice were subcutaneously injected with 0.1 mL CRC cells (HT29 R shNC and shFTX; 1 × 10⁶/cell per mouse) and divided into four groups (n = 5 per group). The spirit, diet, defecation, and activities of the mice were observed daily. The length diameter (a) and width diameter (b) were measured with calipers every two days starting on day 8 of injection. Mice were administered a radiation dose of 10 Gy. When the average volume of the tumor reached approximately 100 mm³, the tumor volume was measured every two days according to the following formula: Volume = 1/2 × length × width². The mice were observed for tumor growth and euthanized on day 30. The xenografts were then weighed and observed. All animal experiments were approved by the Animal Care and Use Committee of the First People's Hospital of Zunyi (No. 2024-2-723).

Immunohistochemistry

All tumor tissues were cut into 2-μm sections. The sections were incubated overnight at 4 °C with anti-ki67 antibody (1:200, Abcam, Cambridge, United Kingdom), SLC7A11 antibody (1:400, GeneTex), and 4HNE antibody (1:300, GeneTex). The secondary antibody was then incubated at room temperature for 30 minutes, and color developed with a DAB working solution. Immunohistochemical (IHC) images were acquired under a microscope.

Statistical analysis

All data analyses were performed using Statistical Package for the Social Sciences software (version 21.0; Armonk, NY, United States). Data are expressed as mean ± SD. Comparisons between two groups were performed using the independent samples t-test, and comparisons between multiple groups were performed using one-way analysis of variance, with Tukey's test selected for χ² and Dunnett's T3 method for analysis of χ². P < 0.05 indicated statistically significant differences.

RESULTS

Downregulation of lncRNA FTX increased radiosensitivity in CRC cells

We first evaluated the expression of lncRNA FTX in CRC tissues from the TCGA database. We found that its expression in colon and rectal cancer tissues was higher than that in adjacent tissues (Figure 1A), suggesting that lncRNA FTX functions as a cancer-promoting factor to participate in the progression of CRC. To explore the role of lncRNA FTX in CRC radiosensitivity, we irradiated HT29 and HCT116 parental cells at doses ranging from 0 to 6 Gy. RT-PCR assay revealed that the expression of lncRNA FTX in the cells increased with the irradiation dose (Figure 1B). Comparative analysis of lncRNA FTX expression in HT29, HCT116, and their corresponding radiotherapy-resistant cell lines (HT29R and HCT116R) revealed that its expression was significantly higher in parental cells than in radioresistant cells (Figure 1C).

Figure 1 LncRNA FTX is upregulated in colorectal cancer cells and associated with the radiosensitivity of colorectal cancer. A: lncRNA FTX expression in colon, rectal cancer, and para-cancerous tissues; B: Relative expression of lncRNA FTX in HT29 and HCT116 cells after irradiation at different doses; C: LncRNA FTX expression was significantly higher in colorectal cancer radiotherapy-resistant cells than in the corresponding parental cells; D: Knockdown efficiency of lncRNA FTX in HT29R and HCT116R cells was validated by real-time quantitative PCR; E: Knockdown of FTX expression in HT29R and HCT116R markedly increased the sensitivity of cancer cells to radiotherapy; F: Colony formation assay revealed that knockdown of FTX significantly increased the sensitivity of HT29R and HCT116R cells to radiotherapy; G: Cell viability was determined using CCK-8 assay after transfection of sh-FTX and sh-negative control in HT29R or HCT116R cells, which were irradiated by 4 Gy. All representative data are from three independent experiments, and the results are presented as deviation mean ± SD. Statistical analysis was conducted using the student's t-test. ^aP < 0.05 and ^bP < 0.01. NC: Negative control.

To further verify the relationship between lncRNA FTX and radiotherapy resistance in CRC cells, we used shRNA to silence the expression of lncRNA FTX in HT29R and HCT116R cells (Figure 1D). CCK-8 and colony formation assays demonstrated that the proliferation of HT29R and HCT116R cells was significantly inhibited (Figure 1E and F). Irradiation of CRC cells with knockdown of lncRNA FTX using 4 Gy radiation revealed that downregulation of lncRNA FTX significantly increased the sensitivity of the cells to ionizing radiation (Figure 1G). These results implicate that lncRNA FTX is involved in the radiotherapy resistance of CRC, and downregulation of lncRNA FTX can significantly improve the sensitivity of CRC to radiotherapy.

Downregulation of lncRNA FTX increased ferroptosis sensitivity in CRC cells

Ferroptosis is a programmed cell death caused by iron accumulation and an increase in lipid peroxides[15], and induction of ferroptosis enhances tumor sensitivity to radiotherapy[10]. To verify whether ferroptosis plays a role in lncRNA FTX-mediated regulation of radiotherapy sensitivity in CRC cells, we first measured the expression of ferroptosis-related proteins after downregulation of FTX using Western blotting in HT29R and HCT116R cells. The expression of TFRC and ACSL4 was significantly elevated after FTX knockdown, while SLC7A11 and GPX4 was significantly decreased (Figure 2A). The GSH content in the resistant cells also decreased significantly after FTX knockdown (Figure 2B). Further observation of intracellular mitochondrial morphology using electron microscopy (Figure 2C) revealed that mitochondria in HT29R and HCT116R cells treated with shFTX were significantly reduced in size compared to the shNC group, with a solidified morphology and disappearance of mitochondrial cristae. The treatment of HT29R and HCT116R cells with the ferroptosis inducer RLS3 revealed that the downregulation of FTX combined with RLS3 significantly increased the sensitivity of resistant cells to ionizing radiation (Figure 2D). These results suggested that the inhibition of FTX expression significantly increased the susceptibility of CRC radiotherapy-resistant cells to ionizing radiation and ferroptosis.

Figure 2 Downregulation of lncRNA FTX increases ferroptosis sensitivity in colorectal cancer cells. A: Expression of ferroptosis-related proteins in HT29R, HCT116R cells after down-regulating lncRNA FTX; B: Glutathione content in HT29R and HCT116R cells after downregulating lncRNA FTX; C: Morphological changes in intracellular mitochondria in HT29R and HCT116R cells after knockdown of the lncRNA FTX were observed by electron microscopy; D: Cell viability was determined using CCK-8 assay after treatment by sh-FTX combined with RSL3 in HT29R and HCT116R cells. ^aP < 0.05 and ^bP < 0.01. NC: Negative control.

LncRNA FTX sequestered miR-625-5p to regulate intracellular redox and radiosensitivity of CRC cells

LncRNAs usually act as competitive endogenous RNA sponges with microRNAs, which in turn regulate the expression of downstream target genes[16,17]. We queried the starbase database and found that there was a base complementarity between miR-625-5p and lncRNA FTX (Figure 3A). Further analysis revealed that miR-625-5p expression was lower in colon and rectal cancer tissues than in normal tissues (Figure 3B). The expressions of miR-625-5p and lncRNA FTX were significantly negatively correlated with CRCs (R = -0.235, P < 0.01, Figure 3C). The results of RT-qPCR analysis in vitro cultured cancer cells revealed that miR-625-5p expression in radiation-resistant cells was significantly lower than in the corresponding parental cells (P < 0.05, Figure 3D). After downregulation of lncRNA FTX in HT29R and HCT116R cells, miR-625-5p expression was likewise significantly elevated (Figure 3E). The results of the dual-luciferase assay revealed (Figure 3F) that miR-625-5p mimic transfection could effectively inhibit the luciferase activity of pGLO-FTX-WT but had no effect on the luciferase activity of pGLO-FTX-MUT. Taken together, these results reveal that miR-625-5p is regulated by lncRNA FTX in CRC cells. To further validate the role of miR-625-5p in CRC radiosensitivity, we first decreased the expression of lncRNA FTX using shRNAs in HT29R and HCT116R cells, followed by transfection with an miR-625-5p inhibitor. The results of CCK8 and colony formation assays revealed that the knockdown of lncRNA FTX combined with a miR-625-5p inhibitor could partially reverse the tolerance of radiation-resistant cells to ionizing radiation (Figure 3G and H). Detection and analysis of intracellular GSH content revealed that inhibition of miR-625-5p effectively restored the effect of the downregulated lncRNA FTX on GSH in HT29R and HCT116R cells (Figure 3I). However, the number of DHE-stained positive cells, which was originally increased in HT29R-shFTX and HCT116R-shFTX cells, was reduced than the control group after inhibition of miR-625-5p, suggesting that miR-625-5p inhibitor could reduce the high oxidative level in cancer cells due to the decrease of lncRNA FTX (Figure 3J). Taken together, these results suggest that lncRNA FTX can regulate the redox level and radiotherapy resistance of CRC cells through miR-625-5p.

Figure 3 LncRNA FTX sequester miR-625-5p to regulate intracellular redox and radiosensitivity of colorectal cancer cells. A: The potential binding site between lncRNA FTX and miR-625-5p predicted by Starbase; B: Expression of miR-625-5p in colon and rectal cancer was higher than that in paracancerous tissues; C: Pearson's correlation analysis was performed to determine the correlation between lncRNA FTX and miR-625-5p expression; D: Relative expression of miR-625-5p in HT29R and HCT116R cells; E: Relative expression of miR-625-5p in HT29R-shFTX and HCT116R-shFTX cell lines; F: The targeting relationship between lncRNA FTX and miR-625-5p was confirmed by dual-luciferase reporter assay; G: Transfection of miR-625-5p inhibitor reversed the inhibitory effect of FTX depletion on HT29R and HCT116R cells; H: Colony formation assay confirmed that miR-625-5p inhibitor reversed the survival effect of FTX depletion on HT29R and HCT116R cells; I: MiR-625-5p inhibitor reversed the glutathione content in HT29R and HCT116R cells caused by deletion of FTX; J: Immunofluorescence analyses suggested that miR-625-5p inhibitor altered ROS levels in HT29R-shFTX and HCT116R-shFTX cells. ^aP < 0.05 and ^bP < 0.01. NC: Negative control.

SLC7A11 was a direct target of lncRNA FTX/miR-625-5p signaling axis

From the results obtained above, lncRNA FTX/miR-625-5p regulated the progress of cellular redox and ferroptosis. To further explore the downstream target of miR-625-5p, the website Targetscan was utilized to find a complementary binding site between SLC7A11 and miR625-5p (Figure 4A), Spearman correlation analysis revealed a positive correlation between SLC7A11 and lncRNA FTX (r = 0.21, P < 0.01, Figure 4B). SLC7A11 was highly expressed in colon and rectal cancer tissues (P < 0.01, Figure 4C). RT-qPCR analysis of intracellular SLC7A11 mRNA levels revealed that its expression was significantly higher in resistant cells than in the corresponding parental cells (Figure 4D). Moreover, SLC7A11 mRNA expression was significantly decreased in HT29R and HCT116R cells after FTX downregulation (Figure 4E). The results of the dual luciferase assay further confirmed that overexpression of miR-625-5p mimics in HT29R and HCT116R cells decreased the luciferase activity of wild-type SLC7A11 but had no effect on mutant SLC7A11 (P < 0.01, Figure 4F). The results of Western blotting experiments demonstrated that overexpression of miR625-5p inhibitor caused the originally decreased SLC7A11 to rise again after knocking down FTX in radiotherapy-resistant cells (Figure 4G). These results imply that SLC7A11 is a downstream target of miR625-5p that is regulated by the lncRNA FTX/miR-625-5p.

Figure 4 SLC7A11 is a direct target of lncRNA FTX/miR-625-5p signaling axis. A: The potential binding site between SLC7A11 and miR-625-5p predicted by Starbase; B: Pearson's correlation analysis was performed to determine the correlation between SLC7A11 and lncRNA FTX expression; C: SLC7A11 expression in colon cancer, rectal cancer, and paracancerous tissues; D: Relative expression of SLC7A11 in HT29R and HCT116R cells; E: Relative expression of SLC7A11 in HT29R-shFTX and HCT116R-shFTX cell lines; F: The targeting relationship between miR-625-5p and SLC7A11 was confirmed by dual-luciferase reporter assay in HT29R and HCT116R cells; G: Expression levels of SLC7A11 in HT29R-shFTX and HCT116R-shFTX cells with or without transfection of miR625-5p were determined by Western blotting. ^aP < 0.05 and ^bP < 0.01. NC: Negative control.

SLC7A11 overexpression reversed the effect of lncRNA FTX on cancer cells in sensitivity to ferroptosis and radiotherapy

SLC7A11 is a light chain subunit of cystine/glutamate reverse transporter (xCT), a key protein in the ferroptosis antioxidant pathway that forms a functional dimer with SLC3A2, which is responsible for the transport of cystine from extracellular to intracellular compartments for further synthesis of GSH and the activation of GPX4, which scavenges cytosolic ROS and lipid oxides to prevent ferroptosis in cells[18]. To further validate the role of SLC7A11 in the radioresistance of CRC cells, we first downregulated the expression of SLC7A11 using shRNA and found that the sensitivity of HT29R and HCT116R cells to ionizing radiation was significantly increased (Figure 5A). Meanwhile, the content of GSH in HT29R and HCT116R cells after shSLC7A11 was significantly lower than that in the shNC group (Figure 5B). In HT29R and HCT116R cells with knocked down lncRNA FTX, the results of CCK8 and colony formation assays revealed that SLC7A11 overexpression could help the resistant cells restore their tolerance to radiation (Figure 5C and D). Western blotting results also demonstrated that in HT29R-shFTX and HCT116R-shFTX cells, oeSLC7A11 caused the elevated expression of TFRC to decrease again while upregulating the expression of GPX4 (Figure 5E). Furthermore, GSH detection results revealed (Figure 5F) that its content in HT29R and HCT116R cells after shFTX alone was 18.63 ± 3.05 and 50.52 ± 0.98, respectively. However, it increased to 39.88 ± 0.64 and 67.20 ± 1.89 after oeSLC7A11, suggesting that SLC7A11 overexpression significantly reversed the downregulated effect of lncRNA FTX on GSH in resistance cells. In Figure 5G, SLC7A11 overexpression similarly resulted in a significant reduction in the number of DHE-stained positive cells in the HT29R-shFTX and HCT116R-shFTX groups, suggesting that oe-SLC7A11 attenuated the oxidative levels of the two cell lines. These results demonstrate that SLC7A11, as a downstream target molecule of lncRNA FTX, is vital for modulating radiotherapy sensitivity and redox levels of intestinal cancer-resistant cells by lncRNA FTX/miR625-5p.

Figure 5 SLC7A11 overexpression reverses the effects of radioresistant cells knockdown lncRNA FTX in sensitivity of ferroptosis and radiotherapy. A: Downregulation of SLC7A11 promoted radio-sensitivity in HT29R and HCT116R cells; B: Downregulation of SLC7A11 reduced glutathione (GSH) content in HT29R and HCT116R cells; C: CCK-8 assay revealed that SLC7A11 overexpression increased the tolerance of HT29R-shFTX and HCT116R-shFTX cells to ionizing radiation; D: Colony formation assay confirmed the promoting effect of overexpression of SLC7A11 on the growth of HT29R-shFTX and HCT116R-shFTXcells; E: Western blotting analyses determined the expression of ferroptosis-related proteins in HT29R-shFTX and HCT116R-shFTX cells after SLC7A11 over expression; F: Reversal effect GSH content in HT29R-shFTX, HCT116R-shFTX cells after overexpression of SLC7A11; G: Immunofluorescence analyses revealed overexpression of SLC7A11 and upregulated ROS levels in HT29R-shFTX and HCT116R-shFTX cells. ^aP < 0.05 and ^bP < 0.01. NC: Negative control.

SLC7A11 overexpression promoted the repair of damaged DNA induced by ionizing radiation in CRC cells

Intracellular DNA damage repair is a stress response in cancer cells after radiotherapy and is among the main molecular mechanisms by which radiotherapy resistance occurs[19]. To confirm whether SLC7A11 is involved in DNA damage repair, we detected the expression of γH2AX in HT29R and HCT116R cells using immunofluorescence staining. We found that FTX knockdown significantly increased intracellular γH2AX expression at 12 hours, whereas oeSLC7A11 significantly decreased its expression in HT29R-shFTX and HCT116R-shFTX cells (Figure 6A). Western blotting results demonstrated that shFTX caused a significant decrease in the expression of MER11 and Ku80 in HT29R and HCT116R cells, and oeSLC7A11 significantly restored the expression of MER11 in these cells, whereas Ku80 Levels were non-significant (Figure 6B), suggesting that SLC7A11 overexpression attenuates the DNA damage caused by shFTX and promotes DNA repair through homologous recombination.

Figure 6 Overexpression of SLC7A11 promotes the repair of damaged DNA induced by ionizing radiation in colorectal cancer cells. A: Immunofluorescence assay detected γH2AX expression in HT29R-shFTX and HCT116R-shFTX cells after overexpression of SLC7A11 at 1 or 12 hours; B: Western blotting analyses determined the expression of mismatch repair proteins in HT29R-shFTX and HCT116R-shFTX cells after SLC7A11 overexpression. ^aP < 0.05 and ^bP < 0.01. NC: Negative control.

In vivo experiments confirmed that inhibition of lncRNA FTX increased the radiosensitivity of CRC

We used an animal model to further verify the effect of lncRNA FTX on the sensitivity of radiotherapy for CRC (Figure 7A). Results suggest that the tumor volume 1004.96 ± 129.53 mm³vs 1491.23 ± 127.35 mm³ and tumor weight 0.76 ± 0.15 g vs 1.24 ± 0.15 g of the nude mice in shFTX group alone were lower than those in shNC control group (both P < 0.05; Figure 7B and C). However, after being irradiated by 10 Gy, tumors in the shNC and shFTX groups of mice were significantly suppressed, the volume in the shNC group decreased from 1491.23 ± 127.35 mm³ before radiotherapy to 1160.19 ± 152.16 mm³ after radiotherapy, and that in the shFTX group decreased from 1004.96 ± 129.53 mm³ before radiotherapy to 614.79 ± 104.24 mm³ after radiotherapy (both P < 0.05). The same trend was observed in the tumor weight of the two groups: 1.24 ± 0.15 g vs 0.86 ± 0.20 g, 0.76 ± 0.15 vs 0.32 ± 0.08 g. However, the shFTX combined radiotherapy group mice had the lowest tumor volume or weight, suggesting that downregulation of lncRNA FTX could likewise significantly reduce the tolerance of living tumors to ionizing radiation and increase their radiosensitivity. IHC analysis demonstrated that the expression of Ki-67 and SLC7A11 decreased in the shFTX and shNC combined IR groups, with the most obvious decrease observed in the sh-FTX combined IR group. However, the expression of 4-HNE revealed the opposite trend; its expression was the highest in the shFTX combined IR group (Figure 7D). These results suggest that the downregulation of lncRNA FTX inhibits tumor growth and that shFTX combined with radiotherapy can significantly increase the radiosensitivity of tumor xenografts in nude mice.

Figure 7 Inhibition of lncRNA FTX increases the radiosensitivity of colorectal cancer in vivo. A: Representative images of the tumors after receiving or not by 10 Gy irradiation, formed by HT29R-sh-FTX or control cells subcutaneous vaccination; B: Tumor volume in xenograft mice of each group at the indicated times; C: Tumor weights of xenograft mice from each group; D: Representative immunohistochemical staining images of Ki-67, SLC7A11, and 4-HNE in the subcutaneous tumor xenograft tissues. Scale bar: 50 μm. Data are expressed mean ± SD. ^aP < 0.05 and ^bP < 0.01. NC: Negative control; IR: Ionizing radiation.

DISCUSSION

Radio resistance is a major impediment to radiotherapy failure in patients with CRC[20]. Overcoming radiotherapy resistance is particularly important for improving the prognosis of patients with cancer. LncRNA FTX is widely involved in cancer development and progression as a tumor oncogenic factor[14]. It is highly expressed in various malignancies, including pancreatic cancer[21], non-small cell lung cancer[22], and osteosarcoma[23], and serves as an indicator of poor patient prognosis[24,25]. Although FTX represses the progression of non-alcoholic fatty liver disease to hepatocellular carcinoma by regulating the M1/M2 polarization of Kupffer cells[26], it promotes aerobic glycolysis and tumor progression through the PPARγ pathway in hepatocellular carcinoma[27]. LncRNA FTX could upregulate SIVA1 to promote gastric cancer cell growth and tumor progression through binding to miR-215-3p[28]. As a pro-oncogenic factor, FTX can stimulate CRC progression by activating multiple downstream target genes[29-31]; however, its role in radiotherapy resistance has not been reported. In this study, we revealed for the first time the role of lncRNA FTX in CRC radiotherapy resistance. We observed that lncRNA FTX can regulate the sensitivity of CRC cells to radiotherapy and enhance the ability of cancer cells to resist antioxidative damage after ionizing radiation through the miR-625-5p/SLC7A11 signaling axis, which contributes to tumor resistance to radiotherapy.

It is currently accepted that lncRNAs mainly act as intracellular epigenetic regulators and play essential roles in regulating target genes by competitively binding to different miRNAs and regulating the transcription of mRNAs[32,33]. Studies have illustrated that different lncRNAs are not the same in resistance to radiotherapy in malignancy. Liu et al[34] reported that the lncRNA CASC19, which is highly expressed in radiotherapy-resistant nasopharyngeal carcinoma cells and enhances radiotherapy resistance in nasopharyngeal carcinoma by modulating the miR-340-3p/FKBP5 axis. In CRC, lncRNA SP100-AS1 could sponge adsorb miR-622 and stabilize ATG3 to induce tolerance to ionizing radiation in CRC cells[35]. The bioinformatics analysis found that lncTUG1 could bind to miR-144-3p and regulating the MET/EGFR/AKT axis, improved the radiotherapy resistance of ESCC[36]. However, lncRNA GAS5 expression was downregulated in irradiated breast cancer cells, and GAS5 overexpression resulted in increased G2/M arrest and unrepaired DNA damage, which sensitizes breast cancer cells to ionizing radiation[37]. Our results demonstrated that lncRNA FTX in the parental cells of CRC increased with increasing irradiation, while lncRNA FTX was significantly upregulated in HT29R and HCT116R resistance cells than in parental cells. FTX downregulation markedly increased CRC cell susceptibility to ionizing radiation and RSL3, which indicated that ionizing radiation induced the lncRNA FTX aberrant expression in CRC cells while decreasing lncRNA FTX significantly increased the tolerance of CRC cells to radiotherapy or drugs.

Our study revealed that lncRNA FTX knockdown in HT29R and HCT116R resistance cells resulted in significant upregulation of miR-625-5p and downregulation of the downstream molecule SLC7A11. Moreover, the miR-625-5p inhibitor could rescue the effect of FTX down regulation SLC7A11. The dual luciferase assay results demonstrated the binding between lncRNA FTX and miR-625-5p, revealing that the lncRNA FTX/miR-625-5p regulated downstream molecule SLC7A11 plays a vital role in mediating FTX to promote radiation tolerance in CRC. MiR-625-5p is a downstream molecule that is bound to and negatively regulated by lncRNA FTX, which may be among the reasons for the low expression of miR-625-5p in CRC tissues or cells. As an essential tumor suppressor factor, miR-625-5p also demonstrated low expression in tumor tissues or cells, such as cervical[38] and gastric cancers[39], and was negatively associated with PKM2 and glycolysis levels in malignant melanoma cells[40]. Deregulation of miR-625-5p on downstream target genes leads to tumor progression[41,42]. Our results indicate that miR-625-5p regulates the expression of SLC7A11 in CRC cells by binding to SLC7A11. As among the main functional subunits of xCT, SLC7A11 can bind to SLC3A2 to form a functional transporter to transfer cystine into the cell for further GSH generation. miR-625-5p regulation of SLC7A11 suggests that besides governing the proliferation and growth of the cells, it is also linked to the amino acid metabolism and redox levels in CRC cells.

SLC7A11/GSH/GPX4, as among the most important antioxidant pathways for ferroptosis, can effectively scavenge the large amount of ROS and lipid peroxides caused by cellular iron overload and maintain intracellular redox homeostasis and internal environment stability[43]. SLC7A11 overexpression counteracted the growth inhibition and radiosensitivity of CRC cells caused by the knockdown of lncRNA FTX. However, intracellular oxidative levels were efficiently reversed, suggesting that upregulation of SLC7A11 and an increase in intracellular GSH levels by lncRNA FTX are the main reasons for its mediation of radiotherapy resistance. Indeed, besides causing DNA strand breaks, ionizing radiation induces the formation of large amounts of ROS, which in turn causes severe oxidative damage to molecules such as lipids, proteins, and nucleic acids[44]. The maintenance of intracellular redox homeostasis is the basis of molecular signal transduction and cellular activities[45]. SLC7A11 overexpression and its mediated activation of intracellular signaling pathways; and the subsequent increase in GSH reductants appear to be essential for the rapid neutralization and counteracting of oxidative damage for post-irradiation cell survival[43,46].

Numerous studies have revealed that cancer cells are susceptible to ferroptosis due to oxidative damage after irradiation[47], and targeting ferroptosis is considered a potential strategy for tumor radio sensitization therapy[10]. Our results exhibit that ferroptosis resistance due to the lncRNA FTX/miR-625-5p/SLC7A11 axis in radioresistant CRC cells is critical for inducing radiotherapy tolerance in CRC. Notably, DNA damage due to ionizing radiation allows cancer cells to undergo necrosis and apoptosis[48], and increased cell necrosis or apoptosis also improves the efficacy of radiotherapy for tumors[49,50]. However, lncRNA FTX and its downstream SLC7A11 are involved in regulating radiation-induced programmed cell death (necrosis or apoptosis), which requires further studies for confirmation. Our work reveals that the regulation of intracellular redox levels by the lncRNA FTX/miR-625-5p/SLC7A11 pathway helps cancer cells achieve adaptive survival under radiation conditions and facilitates the tolerance of ferroptosis and repair of DNA damage in CRC cells, which contributes to the transformation of CRC cells into radiotherapy-resistant cells (Figure 8).

Figure 8 Molecular mechanism of lncRNA FTX in colorectal cancer radio resistance. LncRNA FTX/miR-625-5p regulated downstream molecule SLC7A11 pathway helps cancer cells facilitates the tolerance of ferroptosis and repair of DNA damage in colorectal cancer cells, which contributes to the transformation of colorectal cancer cells into radiotherapy-resistant cells. CRC: Colorectal cancer.

CONCLUSION

Our findings confirmed the role of the lncRNA FTX/miR-625-5p/SLC7A11 pathway in regulating redox levels and radiotherapy resistance in CRC cells, and targeted inhibition of the lncRNA FTX will provide new ideas for strategies to overcome radiotherapy resistance and improve CRC sensitivity to radiotherapy.

ACKNOWLEDGEMENTS

The author thanks Scientific Research Center, The Third Affiliated Hospital of Zunyi Medical University (The First People’s Hospital of Zunyi), Mrs. Xiao-Qian Li, Mrs. Cheng-Min Deng and Dr Meng Ye for their valuable help.