Long noncoding RNA LINC00239 inhibits ferroptosis in colorectal cancer by binding to Keap1 to stabilize Nrf2.

Ferroptosis, a novel regulated cell death induced by iron-dependent lipid peroxidation, plays an important role in tumor development and drug resistance. Long noncoding RNAs (lncRNAs) are associated with various types of cancer. However, the precise roles of many lncRNAs in tumorigenesis remain elusive. Here we explored the transcriptomic profiles of lncRNAs in primary CRC tissues and corresponding paired adjacent non-tumor tissues by RNA-seq and found that LINC00239 was significantly overexpressed in colorectal cancer tissues. Abnormally high expression of LINC00239 predicts poorer survival and prognosis in colorectal cancer patients. Concurrently, we elucidated the role of LINC00239 as a tumor-promoting factor in CRC through in vitro functional studies and in vivo tumor xenograft models. Importantly, overexpression of LINC00239 decreased the anti-tumor activity of erastin and RSL3 by inhibiting ferroptosis. Collectively, these data suggest that LINC00239 plays a novel and indispensable role in ferroptosis by nucleotides 1-315 of LINC00239 to interact with the Kelch domain (Nrf2-binding site) of Keap1, inhibiting Nrf2 ubiquitination and increasing Nrf2 protein stability. Considering the recurrence and chemoresistance constitute the leading cause of death in colorectal cancer (CRC), ferroptosis induction may be a promising therapeutic strategy for CRC patients with low LINC00239 expression.

Introduction

Despite tremendous improvements in detection and treatment, colorectal cancer (CRC) remains one of the most aggressive malignancies of the digestive system, with the third highest incidence and second highest mortality worldwide [1, 2]. Uncontrollable cell proliferation and continuous inhibition of cell death are common causes of poor prognosis in patients with CRC [3, 4]. Unfortunately, the molecular and genetic alterations in these processes remain elusive.

Cell death is strictly regulated by complex intracellular and extracellular signals, which are essential for various biological processes (including redox homeostasis imbalance, development, and disease) [5]. Ferroptosis is a new form of regulated cell death that involves the accumulation of iron-dependent lipid peroxides (lipid-ROS) and causes fatal cell damage [6]. Central to controlling redox homeostasis in carcinogenesis, NF-E2-related factor 2 (Nrf2) has attracted much attention due to its important role in mediating adaptation to oncogene-stimulated oxidative stress [7]. Nuclear translocation and constitutive activation of Nrf2 protect cancer cells from death and induce cell proliferation [8]. In particular, ferroptosis, a new type of regulated cell death (RCD) in the presence of iron-driven lipid peroxidation, has been implicated in Nrf2-mediated carcinogenesis [9, 10]. Mounting evidence indicates that ferroptosis exerts an antitumor effect on tumor progression [11-13], yet the biological and mechanistic details underlying this complex process remain unclear.

Long noncoding RNAs (lncRNAs) are a class of transcripts that lack protein-coding capacity and have lengths greater than 200 nucleotides [14, 15]. Tens of thousands of lncRNAs may be encoded in the human genome, but the precise roles of a large number of them remain elusive [16]. Recent studies have shown that lncRNAs are powerful and multifunctional cell regulators during tumorigenesis and development [17]. LncRNAs play important roles in the occurrence, metastasis, and drug resistance of colorectal cancer [18]. Depending on their subcellular localization, lncRNAs function in various forms [19]. Nuclear lncRNAs are involved in transcriptional regulation in cis and trans, regulation of chromosomal interactions, transcription factor trapping, chromatin circularization, gene methylation, transcription factor recruitment, and chromatin modification [20-22]. Cytoplasmic lncRNAs can regulate target protein levels by interacting with proteins, mRNAs, or micro-RNAs [23]. Accumulating evidence has revealed that lncRNAs are important regulators of oxidative stress during the development of tumors [24-26]. However, the more specific roles of lncRNAs in CRC under ferroptosis remain largely unknown.

In this study, we confirmed that LINC00239 is a ferroptosis suppressor in CRC. LINC00239 promotes CRC proliferation by interacting with Kelch-like ECH-associated protein 1 (Keap1), causing instability of the Keap1/Nrf2 complex. Therefore, LINC00239 enhanced Nrf2 protein stability by suppressing its ubiquitination and promoted CRC development. Importantly, Nrf2 also promotes LINC00239 transcription in a positive feedback manner. LINC00239 inhibition in combination with ferroptosis induction might be a promising therapeutic strategy for CRC patients.

Materials and methods

Patients and follow-up

The study protocol was approved by the ethics committee of Air Force Military Medical University (Shaanxi, China). Written informed consent was obtained from all participants in this study. Cohort I included freshly sampled CRC tissues with healthy adjacent tissues collected between January 2005 and December 2007 from 174 adult patients who underwent surgery at Xijing Hospital of the Fourth Military Medical University (Xi’an, China). Cohort II included CRC tissue samples from 180 adult CRC patients at the Shanghai Outdo Biotech Co., Ltd (Shanghai, China). All patients were staged pathologically based on the American Joint Committee on Cancer (AJCC)/International Union against Cancer criteria. All the research was carried out in accordance with the provisions of the Declaration of Helsinki of 1975. None of the patients had received radiotherapy or chemotherapy prior to surgery.

Cell culture and treatments

Human CRC cell lines RKO, HCT116, CaCo2, SW480, SW620, and normal intestinal epithelial cells (FHC) were purchased from American Type Culture Collection (ATCC, USA). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum, penicillin, and streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in an atmosphere containing 5% CO2. The cell lines were tested for mycoplasma contamination before use to ensure that they were mycoplasma-free. All drugs were ordered from MedChemExpress unless otherwise indicated. All drug use was performed according to the manufacturer’s instructions. All small interfering RNAs (siRNAs) were purchased from TsingKe Technology (Beijing, China). Lipofectamine 2000 (Thermo Fisher Scientific, USA) was used to transfect siRNA (50 nM) into colorectal cells, while nonspecific siRNA (50 nM) was used as a negative control. The sequences of siRNAs are listed in Supplementary Table S1.

RNA isolation and qRT-PCR

For RNA-seq and qRT-PCR analysis, a MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan) was used to isolate RNA, and a one-step PrimeScript RT-PCR kit was used to reverse transcribe 1 µg of total RNA (TaKaRa, Japan). TB Green® Fast qPCR Mix (TaKaRa, Japan) was used for quantitative PCR with three repeated reactions with the primers listed in Supplementary Table S2. Using the ddCt method to compare with the 18 S level, the relative RNA expression level was calculated and normalized with respect to the control sample [27].

Plasmids and cloning

Gibson cloning was used for all vectors. For gene knockdown, we cloned the sgRNA in Supplementary Table S1 into the pLentiRNACRISPR-hU6-DR-RfxCas13 (Addgene no. 138147) vector [28]. For gene overexpression analysis, the human LINC00239, Nrf2, and Keap1 full-length open-reading frames were subcloned into plenti-CMV-Luc-Puro (Addgene no. 17477) [29]. All constructs were confirmed by Sanger sequencing. All primers used for molecular cloning and primer sequences are shown in Supplementary Table S2.

In vivo experiments

To clarify the role of LINC00239 in vivo, we used 4-week-old male BALB/c nude mice provided by the Experimental Animal Center of the Air Force Military Medical University. HCT116 or SW620 cells (1 × 107 cells) were injected subcutaneously into the right flanks of these mice to establish a CRC xenograft model. One week after the injection of cells, the volume of xenografts was continuously monitored (once a week). Four weeks later, the xenografts were removed, and the weights were measured. All experimental procedures were approved by the Animal Care and Use Committee of Shanghai Air Force Military Medical University.

Immunoblotting

The cells were collected by scraping and lysed for 15 min on ice in lysis buffer containing protease and phosphatase inhibitors. The cell lysate was centrifuged at 12,000 rpm and 4 °C for 15 min. SDS loading buffer was added to the supernatant, and then the sample was heated at 95 °C for 5 min before loading on the polyacrylamide gel. Western blotting was performed as previously described. The antibodies are listed in Supplementary Table S3.

RNA FISH

Single-molecule RNA FISH was performed as previously described [27]. The probe was designed by the online probe designer at https://www.biosearchtech.com/products/rna-fish/ and labeled with Cy3. The probe for LINC00239 is listed in Supplementary Table S1.

Immunofluorescence staining

For Keap1 and Nrf2 immunofluorescence staining assays, cells were fixed with 2% PFA at room temperature for 15 min. Then, the cells were permeated with 0.5% Triton X-100 for 15 min on ice and washed three times with PBS. The cells were then subjected to a blocking step and incubated with anti-Keap1 or anti-Nrf2 antibody at 4 °C overnight, followed by incubation with a fluorescent secondary antibody. The nuclei were counterstained with DAPI, and images were obtained by a laser confocal microscope [30]. The antibodies are listed in Supplementary Table S3.

Co-immunoprecipitation (co-IP)

Co-IP was performed as described before [31]. In short, the input and immunoprecipitation samples were analyzed by western blotting using various antibodies at the specified dilution: Keap1 antibody, Nrf2 antibody, and normal rabbit IgG. The antibodies are listed in Supplementary Table S3.

Chromatin immunoprecipitation

The procedure was performed as previously described [32]. In short, the cells were cross-linked with 1% formaldehyde for 10 min at room temperature and neutralized by adding glycine to a final concentration of 0.125 M for 5 min. After washing twice with cold PBS, the cells were collected and suspended in cold lysis buffer (10 mM Tris-Cl, pH 8.0, 85 mM KCl, 0.5% NP40, 5 mM EDTA, 0.25% Triton, and protease inhibitor). After 15 min of incubation on ice, the nuclei were harvested, resuspended in cold lysis buffer, and sonicated to obtain 200–500 base pair DNA fragments. Magnetic beads coated with specific antibody or IgG control were added to the lysis buffer and incubated overnight. The next day, the beads were washed 7 times with washing buffer (50 mM HEPES, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% sodium deoxycholate), then washed once with TE buffer, after which the protein–DNA complex was eluted. After reverse cross-linking at 55 °C overnight, DNA was extracted and analyzed by qRT-PCR. All primers are listed in Supplementary Table S2.

Immunohistochemistry (IHC)

In the immunohistochemical analysis of our own cohort, samples were independently assessed by two pathologists blinded to the clinical characteristics of the patients on the basis of staining intensity and region of protein expression in the samples. The percentage of positive cells was scored as 0 (<10%), 1 (10–40%), 2 (40–70%), or 3 (>70%), and the immunostaining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The final immune response score was calculated as staining intensity score × percentage of positive cells. A score of 0-6 indicates that the expression of the target is considered “LINC00239 (−)”, while a score of 7–9 is considered “LINC00239 ( + )”.

Data analyses and statistics

Relative RNA levels were normalized to 18 S RNA levels. All statistical analyses were performed using the GraphPad Prism 8.0 software package and SPSS 22.0 statistical software package (Abbott Laboratories, USA) for Windows. Data are presented as the means ± SD of at least three independent experiments. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.

Table 1

Correlation between LINC00239 expression and clinicopathological characteristics of CRCs in two independent cohorts of human CRC tissues.

Clinicopathological variables	Cohort I (n = 174)		P value	Cohort II (n = 180)		P value
	Tumor LINC00239 expression			Tumor LINC00239 expression
	Negative (n = 70)	Positive (n = 104)		Negative (n = 80)	Positive (n = 100)
Age (years)
>60	38	58	0.846996	47	58	0.919218
≤60	32	46		33	42
Sex
Female	40	56	0.668081	35	45	0.866815
Male	30	48		45	55
Tumor location
Right colon	20	36	0.659117	20	30	0.755572
Left colon	27	39		33	39
Rectum	23	29		27	31
Tumor size
<5 cm	62	12	6.8684E-24	69	7	1.0527E-26
≥5 cm	8	92		11	93
Tumor invasion
T1	26	9	7.6288E-10	29	3	1.2432E-13
T2	28	21		31	17
T3	12	30		15	37
T4	4	44		5	43
Lymph node metastasis
Absent	40	54	0.498114	42	58	0.460574
Present	30	50		38	42
AJCC stage
Stage I	30	5	1.1328E-18	27	4	1.4835E-12
Stage II	28	8		31	14
Stage III	9	54		16	45
Stage IV	3	37		6	37