Downregulation of lncRNA SLC7A11-AS1 decreased the NRF2/SLC7A11 expression and inhibited the progression of colorectal cancer cells

Patients

A total of six pairs of fresh CRC tissues and normal adjacent tissues were collected from the Affiliated Hospital of Hebei University in 2021 and stored at −80 °C. All the patients who participated in the study had written informed consents, got a pathological diagnosis, and had no treatment before surgery. Three men and three women were involved in the study with an age range of 56–80 years and a mean age of 70 years.

Bioinformatics analysis

The pan-cancer analysis of the expression of SLC7A11-AS1 was performed by the ncFANs v2.0 website (http://ncfans.gene.ac/) (Zhang et al., 2021). The transcriptome data of COAD patients has been got accessed from The Cancer Genome Atlas (TCGA) dataset (https://portal.gdc.cancer.gov/) for further analysis. The prognostic performance of SLC7A11-AS1 in COAD was assessed by Gene Expression Profiling Interactive Analysis (GEPIA) website (Tang et al., 2017). The expression of SLC7A11-AS1 in the normal and COAD tissues was compared by the ggbetweenstats package with Welch’s t-test in R software (version 4.1.0). The Pearson correlation coefficient between the expression of SLC7A11-AS1 and the other genes was also calculated by the cor.test function of the R software and visualized by the Cytoscape software (version 3.7.2). R <−0.5 or R > 0.5 and p < 0.05 were considered to be relevant. The expression of genes that related to SLC7A11-AS1 in COAD patients was visualized by using the pheatmap package in R software. The mutational profiles of the related genes in COAD patients were identified by the maftool package in R software to create the mutation annotation format from TCGA COAD database (Mayakonda et al., 2018). Moreover, the Gene Ontology (GO) functional enrichment analysis of the related genes was carried out by the Metascape database (https://metascape.org/gp/index.html#/main/step1) (Zhou et al., 2019).

Cell culture and transfection

The human CRC cell line HCT-8 was donated by Dr. Nan Chen the associate professor of the School of Basic Medical Sciences of Hebei University. The normal colon epithelial cell FHC and CRC cell HCT-116 were provided by Dr. Yan Qin the associate professor of Affiliated Hospital of Hebei University. All the cells were cultured in RPMI-1640 medium (Wisent Biotechnology, Nanjing, China) with 10% FBS (Biological Industries, Beit Haemek, Israel), containing 100 U/mL penicillin and 100 mg/mL streptomycin (Solarbio, Beijing, China). All cells were incubated at 37 °C in a humidified atmosphere of 5% carbon dioxide. GenePharma (Shanghai, China) synthesized antisense oligonucleotides (ASO), the NRF2 overexpression vector was purchased from Obio Technology (Shanghai), both of which were transiently transfected into cells with Lipofectamine 3000 reagent (Invitrogen, CA, USA). as directed by the manufacturer. The sequences of the SLC7A11-AS1 ASO and negative control (NC) ASO were as follows: SLC7A11-AS1 ASO 5′- UGACUAGGTAGGAAGGGUGC −3′; NC ASO 5′- GCGUATTATAGCCGATTAAC -3′.

Wound healing assay

When the HCT-8 cells arrived at approximately 90% confluence, ASOs were transiently transfected into the cultured cells in 6-well plates (Jet Biofil, Guangzhou, China). 200-µL pipette tip was used to scrape the cell monolayer to create a wound gap after transfection for 24 h. The wounded monolayers were then cultured with RPMI-1640 medium containing 1% FBS and observed at 24 h and 48 h. The scratches were captured by microscope using FLUOCA FCSnap software (version 1.1.19627) and five fields were randomly selected for each scratch wound. The gap lengths were measured with Image J software (version Java 1.8.0_172).

Transwell migration and invasion

After the transfection of ASOs into CRC cells for 24 h, 1 × 10⁴ cells per well were seeded in the upper chamber of the tissue culture plate insert (Jet Biofil, Guangzhou, China) in 100 µl of serum-free medium. At the same time, 600 µl of complete medium was added to the lower chamber of the tissue culture plate insert. After being incubated for 24 h at 37 °C, cotton swabs were used to remove the cells remaining at the upper surface of the membrane, and the migrated cells on the lower surface of the membrane were fixed. 0.1% crystal violet solution was used to stain the migrated cells after fixing them with 4% paraformaldehyde. 4 × 10⁴ cells per well were seeded in the upper chamber coated by Matrigel (BD Biosciences) and incubated for 48 h, when doing the invasion assay with the same procedure. The cells that passed through the membrane were observed by microscope and captured by FLUOCA FCSnap software (version 1.1.19627).

Cell counting kit-8 assay

Cell viability was detected by Cell Counting Kit-8 (CCK8, Dojindo, Japan) assay in accordance with the manufacturer’s protocols. After the transfection of ASOs into CRC cells for 24 h, CRC cells were seeded at a density of 5 × 10³ perwell with 100 µl complete medium in 96-well plates (Jet Biofil, Guangzhou, China). After cultured for 24, 48, and 72 h, CCK-8 reagent with a volume of 10 µl was added to each well and then incubated for 2 h. The absorbance was measured at 450 nm using the microplate reader (Bio-Rad, CA, USA). The ability of cell proliferation was represented by the optical density (OD).

Colony formation assay

After the transfection of ASOs for 24 h, CRC cells were inoculated into the 6-well plates at a density of 1 × 10³ cells per well in a complete medium and cultured for 15 days to allow the formation of colonies. Afterwards, the plates were washed in cold PBS and fixed at room temperature in 4% polyformaldehyde. 0.1% crystal violet solution was used to stain the colonies for 30 min at room temperature. The colonies were counted by Image J software (version Java 1.8.0_172). Colony formation rate was calculated by the number of each SLC7A11-AS1 ASO/the number of NC ASO ×100%.

Western blot

Total proteins were extracted from tissues or HCT-8 cells with RIPA buffer (Biomed, Beijing, China) containing one mmol/L PMSF (Solarbio, Beijing, China). The protein concentration was detected using a BCA protein assay reagent kit (Biomed, Beijing, China). Separations of proteins were carried out using 12% polyacrylamide gel electrophoresis, and then the proteins were transferred to nitrocellulose filter membranes. After blocking in 5% skim milk for 1 h, the anti-NRF2 antibody (1:1000, GeneTex, CA, USA), anti-SLC7A11 antibody (1:1000; Zen-Bioscience, Chengdu, China), and anti- β actin antibody (1:3000; Proteintech, Wuhan, China) were used as the primary antibodies in this study for overnight incubation at 4 °C. Afterwards, the membranes were incubated at room temperature for 1 h with the corresponding secondary antibodies. Image J software (version Java 1.8.0_172) was used to analyze the protein bands after they were visualized using enhanced chemiluminescence (ECL; Biomed, Beijing, China).

Extraction of RNA and quantitative reverse transcription-PCR (RT-qPCR)

Total RNA was extracted from HCT-8 cells or tissues using TRNzol Universal reagent (TIANGEN, Beijing, China). cDNA was synthesized using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Shiga, Japan). RT-qPCR was subsequently conducted by using UltraSYBR One Step RT-qPCR Kit (CWBIO, Jiangsu, China) on Roche LightCyler 96. Normalization of each gene was done against 18S ribosomal RNA (18S) or GAPDH at transcript levels. The comparative cycle threshold (Ct) method (2^−ΔΔCt) was applied to calculate relative expression levels of genes. The sequence of the primers used in the RT-qPCR was synthesized by Sangon (Shanghai, China) and shown in Table 1.

Reactive oxygen species quantitation and visualization

HCT-8 cells were detached by trypsin (Solarbio, Beijing, China), seeded in a 96-well culture plate (Wohong Biotechnology, Shanghai, China) and cultured for 24 h, which has been transfected with ASOs for 24 h. Dihydroethidium, DHE (Beyotime, Shanghai, China) was diluted 1:1000 in serum-free medium to the final concentration of 10 µM and used to replace the culture medium in a 96-well culture plate to detect the ROS level. Using a fluorescence microplate reader (Thermo Fisher, Waltham, MA, USA), the fluorescence intensity was examined at wavelengths of 485 nm for excitation and 590 nm for emission.

After being transfected with ASOs for 24 h, HCT-8 cells were collected and seeded in 24-well plates (Jet Biofil, Guangzhou, China). After 24 h, the cell culture medium was removed and replaced by 500 µl diluted DHE for 20 min incubation. Cells were then visualized by fluorescence microscopy (Leica DMi8, Wetzlar, Germany) with Leica Application Suite X software (version 3.1.1.15751).

Cytoplasmic and nuclear RNA and protein extraction

Separation of cytoplasmic and nuclear RNA and protein were performed by using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) plus TRNzol Universal reagent or RIPA buffer. HCT-8 cells were harvested with trypsin-EDTA and centrifugation at 500 g for 3 min. The supernatant was removed and discarded as much as possible. 200 µl cytoplasmic extraction reagent A (for 2 × 10⁶ cells) (with RNase inhibitors for RNA isolation) was used to resuspend the precipitate. After incubation for 20 min on ice, 10 µl cytoplasmic extraction reagent B was added to the tube and centrifuged at 12,000 g for 10 min, 4 °C. The supernatant was moved to a new tube and the TRNzol Universal reagent was used to extract the cytoplasmic RNA or RIPA buffer for protein extraction. The precipitate was also exacted with TRNzol Universal reagent to harvest the nuclear RNA or RIPA buffer for protein extraction. We assessed the quality and quantity of the RNA using the NanoDrop (Thermo Fisher, Waltham, MA, USA) at 260 and 280 nm wavelengths.

Statistical analysis

A minimum of three independent experiments were conducted for each experiment. The student’s t-test was performed using GraphPad Prism Version 7 for the analysis between two groups (San Diego, CA, USA). Analysis of variance (ANOVA) was used for the comparison of more than two groups. The data were expressed as the mean ±SEM. p < 0.05 was considered statistically significant.

SLC7A11-AS1 was highly expressed in CRC and associated with a poor prognosis

After analysis by the ncFANS (version 2.0) website and the TCGA database, we found that the expression of SLC7A11-AS1 was significantly elevated in various types of cancers including CRC (Figs. 1A and 1B). SLC7A11-AS1 was also significantly overexpressed in our CRC tissues compared to normal tissues (Fig. 1C and Table S1). Moreover, high SLC7A11-AS1 level was related to lower overall survival (Fig. 1D).

Silencing the SLC7A11-AS1 expression suppressed the proliferation of CRC cells

The expression of SLC7A11-AS1 in HCT-8 and HCT-116 cells was significantly higher than the normal FHC cell (Fig. 2A and Table S1). Then, we confirmed that SLC7A11-AS1 was mainly distributed in the cell nucleus by separating the cytoplasmic and nuclear total RNA in HCT-8 cell (Fig. 2B and Table S1). Therefore, we knocked down the expression of SLC7A11-AS1 by transfecting the SLC7A11-AS1 ASO into CRC cells (Fig. 2C and Table S1). We found that silencing SLC7A11-AS1 expression significantly attenuated the HCT-8 and HCT-116 cell proliferation measured by the CCK-8 assay and the colony formation experiment (Figs. 2D, 2E and Table S3).

SLC7A11-AS1 knockdown inhibited the migration of CRC cells

We also evaluated the effect of SLC7A11-AS1 knockdown on cell migration by performing the wound healing assay. We found that the migration of HCT-8 cells was suppressed by low expression of SLC7A11-AS1 at 24 h and 48 h after SLC7A11-AS1 ASO transfection (Fig. S1 and Table S3). We further confirmed our results by transwell assay, which exhibited that silencing SLC7A11-AS1 expression hindered the ability of HCT-8 and the HCT-116 cells migration (Fig. 3A and Table S3). A similar inhibitory effect was also observed in the invasion array of HCT-8 and the HCT-116 cells after knocking down the expression of SLC7A11-AS1 (Fig. 3B and Table S3).

The SLC7A11-AS1 expression positively correlated with the expression of SLC7A11 in CRC

To identify the target genes of SLC7A11-AS1 in CRC, we performed the correlation analysis using the TCGA COAD transcriptome dataset. The results showed that the expression of SLC7A11-AS1 was positively related to SLC7A11, POLQ, ASPM, CENPF, KIF14, ZGRF1, CENPE, MSI2, ANKRD36C, and negatively associated with B3GNT8, PHGR1 (Fig. 4A). We also detected the expression relationship between SLC7A11-AS1 and the mRNA level of SLC7A11 in our clinical tissues. The positive relation was confirmed in our clinical tissues, as shown in Fig. 4B (R = 0.8059). Enrichment analysis indicated that these genes were mostly enriched in the GO terms of metaphase plate congression (containing CENPE, CENPF, KIF14, ASPM, and MSI2) and brain development (including CENPF, KIF14, SLC7A11, ASPM) (Fig. 4C). The tumor mutation burden (TMB) of these genes was also analyzed, and ASPM, CENPF, and POLQ had higher mutation rates than the other genes (Fig. 4D). The expression profile of these related genes was shown by the heatmap in Fig. 4E, and we found that SLC7A11 was significantly upregulated in the TCGA COAD dataset in accordance with the expression profile of SLC7A11-AS1 (Fig. 4E).

NRF2 over expression could rescue the SLC7A11 expression and ROS level after SLC7A11-AS1 knocked down

To further investigate the relationship between SLC7A11-AS1 and SLC7A11, we silenced the lncRNA SLC7A11-AS1 by its’ ASO. We found that SLC7A11-AS1 knockdown decreased the expression of SLC7A11 in HCT-8 cells (Fig. 5A and Fig. S2). Since SLC7A11-AS1 could stabilize NRF2 in pancreatic cancer (Yang et al., 2020) and SLC7A11 is also a common target of NRF2 (Fan et al., 2017), we further analyzed the effect of SLC7A11-AS1 on the NRF2 expression. We found silencing SLC7A11-AS1 could decrease the nuclear level of NRF2 and increase the ROS level (Figs. 5A, 5B and Table S3). However, this effect could be relieved by NRF2 overexpression (Figs. 5B, 5C and Table S3). Meanwhile, we found that there were upregulated SLC7A11 and NRF2 expression levels in the clinical CRC tissues with overexpressed SLC7A11-AS1 (Fig. 5D). Therefore, a high SLC7A11-AS1 level might help to clear the extra ROS by upregulation of the NRF2/SLC7A11 antioxidant system.