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Exosomal lncRNA FOXD3-AS1 upregulates ELAVL1 expression and activates PI3K/Akt pathway to enhance lung cancer cell proliferation, invasion, and 5-fluorouracil resistance

Exosomal lncRNA FOXD3-AS1 upregulates ELAVL1 expression and activates PI3K/Akt pathway to enhance... Abstract Long non-coding RNA (lncRNA) FOXD3-AS1 expression is upregulated in lung cancer; however, its effect and mechanism on 5-fluorouracil (5-FU) resistance remain unclear. In this study, we determined the effects of FOXD3-AS1-enriched exosomes derived from lung cancer cells on the proliferation, invasion, and 5-FU resistance of lung cancer cells. Online bioinformatics database analysis showed that FOXD3-AS1 was upregulated in lung cancer progression. Real-time quantitative PCR results confirmed that FOXD3-AS1 expression was upregulated in lung cancer tissues and cell lines, and FOXD3-AS1 was greatly enriched in lung cancer cell-derived exosomes. ELAV-like RNA-binding protein 1 (ELAVL1) was identified as an RNA-binding protein of FOXD3-AS1. The lung cancer cell-derived exosomes promoted A549 cell proliferation and invasion and inhibited apoptosis caused by 5-FU, and transfection of si-FOXD3-AS1 or si-ELAVL1 in exosome-incubated A549 cells reversed these effects. Moreover, exosome-incubated A549 cells were co-transfected with si-FOXD3-AS1 and pcDNA-ELAVL1, showing the same cell proliferation, invasion, and 5-FU resistance as those of A549 cells treated with lung cancer cell-derived exosomes alone. Mechanistic studies identified that lung cancer cell-derived exosomes activated the PI3K/Akt pathway, and transfection of si-FOXD3-AS1 or treatment with the PI3K inhibitor LY294002 reversed the activation of the PI3K/Akt axis induced by exosomes. In conclusion, our study revealed that lung cancer cell-derived exosomal FOXD3-AS1 upregulated ELAVL1 expression and activated the PI3K/Akt pathway to promote lung cancer progression. Our findings provide a new strategy for lung cancer treatment. lung cancer, exosomes, lncRNA FOXD3-AS1, ELAVL1, 5-FU resistance, PI3K/Akt pathway Introduction Lung cancer is the respiratory malignancy with the highest morbidity and mortality worldwide, accounting for 18 million new cases annually and 13% of all cancers [1–3]. Similarly, the incidence and mortality of lung cancer in China are also increasing year by year, seriously endangering human health and life. Lung cancer is mainly divided into small cell lung cancer and non-small cell lung cancer, in which non-small cell lung cancer accounts for 80–85% of all lung cancer cases. Surgical treatment is the first and most important treatment for lung cancer, but most of the patients are diagnosed at the middle and advanced stage, the curative effect of surgical treatment is not ideal, and the 5-year survival rate is still <15% [4,5]. Therefore, the identification of prognostic markers and potential drug targets needs further study to promote prognosis and personalized treatment. Long non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules with transcript length ranging from 200 nt to 100 kb [6]. Recent studies have shown that lncRNAs are abnormally expressed in a variety of human cancers such as colon cancer [7], prostate cancer [8], breast cancer [9], and liver cancer [10], participating in important cellular and molecular activities such as epigenetic modification, transcriptional regulation, RNA editing, and protein translation and playing a key role in tumorigenesis, metastasis, and metabolism. Exosomes are membrane vesicles with a diameter of 30–150 nm that are actively secreted by living cells and contain proteins, lipids, and nucleic acids derived from maternal cells. The main function of exosomes is to maintain cell homeostasis and regulate cell function, especially information transmission between cells [11–13]. In recent years, increasing evidence has demonstrated the important role of exosomes in diseases. Tumor cell-derived exosomes promote cell proliferation, invasion, and drug resistance by delivering small molecules that have positive effects on tumor growth. lncRNAs play an important role in the information transmission of exosomes. In colorectal cancer, lncRNA CCAL promotes drug resistance of colorectal cancer cells due to the transfer of CCAL-carrying exosomes from cancer-associated fibroblasts to cancer cells, thereby inhibiting tumor cell apoptosis in vitro and in vivo and inducing chemoresistance [14]. Another study found that the expression of lncRNA ZFAS1 was increased in tumor cells, tumor tissues, serum, and serum exosomes of gastric cancer patients, and interference with ZFAS1 expression inhibited cell cycle progression, induced apoptosis, and inhibited epithelial–mesenchymal transition [15]. Moreover, studies have reported an increase of H19 in gefitinib-resistant lung cancer cells compared with that in drug-sensitive parental cells, and H19 encapsulated in exosomes was transferred to non-resistant cells, thereby inducing gefitinib resistance [16]. The upregulation of FOXD3-AS1 expression in lung cancer has been confirmed; however, its effect on 5-fluorouracil (5-FU) resistance and the possible regulatory mechanism of drug resistance remains unclear. In this study, we determined the effects of FOXD3-AS1-enriched exosomes derived from lung cancer cells on the proliferation, invasion, and 5-FU resistance of lung cancer cells, in order to provide new ideas for the prevention and reversal of drug resistance treatment strategies. Materials and Methods Samples The lung cancer tissues and adjacent tissues of 25 patients with lung cancer were collected surgically, and the average age of the patients was (53.2±3.1) years. All patients had complete clinical data (Table 1) and did not receive any form of anticancer treatment 3 months before surgery. The study protocol was approved by the Ethics Committee of Peking University Shenzhen Hospital, and written informed consent was obtained from each participant. Table 1. Clinical data of 25 lung cancer patients Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Open in new tab Table 1. Clinical data of 25 lung cancer patients Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Open in new tab Cell culture Human lung cancer cell lines A549, SK-MES-1, SPC-A1, H2170, and PC9, and human bronchial epithelial cells (HBECs) were purchased from the American Typical Culture Collection (Manassas, USA). Cells were incubated in medium containing 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St Louis, USA) in RPMI 1640 medium (Gibco, Rockville, USA) at 5% CO2 at 37°C. Small interfering RNAs against FOXD3-AS1 (si-FOXD3-AS1, 5ʹ-GCAAUAGGGACGCGCCAAU-3ʹ) and ELAVL1 (si-ELAVL1, 5ʹ-AAGAGGCAAUUACCAGUUUCA-3ʹ), and scramble (5ʹ-CGAGGGATGAGCCCGCGTAG-3ʹ) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Transfection was performed by using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. pcDNA3.1 and pcDNA-ELAVL1 were purchased from RiboBio (Guangzhou, China) and transfected into cells according to the instructions. Exosome extraction The supernatant of the cell culture medium was collected, and the cell components and dead cells were removed by low-speed centrifugation (300 g, 10 min, 2000 g, 10 min) at 4°C. The supernatant containing exosomes was retained, and the cell debris was removed by high-speed centrifugation (10,000 g, 70 min). The supernatant containing extracellular vesicles was retained, and the exosomes were precipitated by ultracentrifugation (100,000 g, 70 min). An appropriate amount of phosphate buffered saline (PBS) was taken to resuspend the extracellular vesicle precipitation and then ultracentrifuged again (100,000 g, 70 min) to eliminate contaminated proteins. The pellet was collected and aliquoted and stored at −80°C for future use. Cell counting kit-8 assay A549 cells grown in logarithmic phase were digested with 0.25% trypsin, the supernatant was discarded, and then the cell density was adjusted to 3×104 cells/ml by adding Dulbecco’s modified Eagle’s medium (DMEM; Gibco). Cell suspension was seeded at 3×104 cells/ml in 96-well plates, and 100 μl was added into each well, and placed in an incubator at 37°C and 5% CO2 for 4 h. Then, 10 μl of CCK-8 solution (Leigen Bio, Beijing, China) was add to each well and incubated for 4 h. The absorbance of each well at 450 nm was detected with a microplate reader (Molecular devices, Shanghai, China). Real-time quantitative PCR Total RNA was isolated from patient tissues or A549 cells by using TRIzol (Invitrogen). Single-stranded complementary DNA was synthesized using the PrimeScript Reagent Kit (Promega, Madison, USA). Real-time qPCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, USA). The reaction was run on the ABI7500 Real-time PCR system (Applied Biosystems). The RT-qPCR cycling conditions consisted of the following: 95°C for 3 min; then 35-cycle amplification for 20 s at 95°C, 30 s at 55°C, 15 s at 72°C; followed by 1 min at 72°C. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China) and listed in Table 2. The expression level was determined by using the 2−ΔΔCt method. GAPDH was used as an endogenous control. Table 2. The sequences of the primers used in this study Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Open in new tab Table 2. The sequences of the primers used in this study Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Open in new tab Western blot analysis Protein homogenates from cells or tissues were extracted. The protein content of each sample was determined using the BCA Protein Assay Kits (Thermo Scientific). Then, equal amounts of protein (15 μg per lane) were separated by 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, USA). The membranes were blocked in 5% (w/v) skimmed milk powder in TBST (Tris buffered saline containing 0.1% Tween) for 2 h at 25°C and then incubated with the following antibodies from Abcam: rabbit polyclonal anti-β-actin antibody (1:1000, ab8227), rabbit monoclonal anti-CD63 antibody (1:1500, ab217345), mouse monoclonal anti-CD81 antibody (1:1200, ab79559), rabbit monoclonal anti-TSG101 antibody (1:2500, ab125011), rabbit monoclonal anti-Alix antibody (1:2000, ab186492), rabbit monoclonal anti-ELAVL1 antibody (1:800, ab200342), rabbit monoclonal anti-PI3K antibody (1:1000, ab32089), rabbit polyclonal anti-Akt antibody (1:700, ab8805), and rabbit polyclonal anti-Akt (phospho T308) antibody (1:800, ab38449). Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000, ab6721; Abcam) or goat anti-mouse IgG (1:3000, ab6728; Abcam). Finally, the protein bands were visualized using ECL reagent (Amersham Biosciences, Piscataway, USA) and quantified using ImageJ software (National Institutes of Health, Bethesda, USA). β-Actin was used as the loading control. Transwell assay Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), and 1×105 cells (100 μl) was seeded in a chamber with Matrigel and on a control with polyethylene terephthalate membrane. After incubation for 24 h, the cells were fixed with anhydrous methanol for 20 min, then stained with 0.1% crystal violet (Sigma) for 20 min, dried and observed with an inverted microscope (Olympus, Tokyo, Japan), and counted to evaluate the invasion ability of the cells. Flow cytometry The cells were collected and washed twice with PBS. A total of 100 μl cell suspension (1×105 cells/ml) was transferred to a culture tube and then incubated with 5 μl of Annexin V-FITC and 5 μl propidium iodide (BD Biosciences) at room temperature for 20 min in the dark. Finally, 400 μl of binding buffer was added, and apoptotic cells were determined by flow cytometry on a BD flow cytometer (BD Biosciences, San Jose, USA). RNA immunoprecipitation assay RNA immunoprecipitation (RIP) assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore, Billerica, USA). Briefly, A549 cells were lysed by using the RIPA lysate buffer, and the supernatant was collected by centrifugation. Next, the supernatant was incubated with 1 μg corresponding antibodies: anti-ELAVL1 (ab200342, 1:100; Abcam) or negative control (NC) antibody (IgG) (ab10948, 1:100; Abcam), and then 50 μl of protein A/G-beads was added and incubated overnight at 4°C. After immunoprecipitation, the mixture was centrifuged to remove the supernatant. The protein A/G beads were then washed with 1 ml lysis buffer and repeated 3 times. Finally, beads were added with 15 μl of 2× SDS buffer solution and then boiled for 10 min. RNA protein complexes were obtained by using the anti-ELAVL1 antibody, and then RNA on the complexes was isolated and purified and analyzed by RT-qPCR. RNA pull-down assay The binding relationship between FOXD3-AS1 and ELAVL1 was identified by RNA pull-down assay. Briefly, biotinylated wild-type FOXD3-AS1 (FOXD3-AS1-WT), mutant-type FOXD3-AS1 (FOXD3-AS1-MUT), and NC were synthesized and transfected into A549 cells using Biotin RNA Labeling Mix (Roche), respectively. After transfection for 24 h, the cells were washed with PBS and incubated with lysis buffer on ice for 10 min. Then, the lysate was incubated with streptavidin magnetic beads (Invitrogen) at 4°C for 3 h. Then, the beads were washed extensively, and the RNA–protein complex was eluted. Western blot analysis was used to detect the retrieved proteins. Statistical analysis All statistical analyses were performed using the SPSS software (ver. 21.0; SPSS, Chicago, USA). The quantitative data derived from three independent experiments were expressed as the mean±SEM. Significance was determined by one-way analysis of variance or Student’s t-test. P<0.05 was considered of statistically significant difference. Results The expression of FOXD3-AS1 is upregulated in lung cancer tissues and cell lines The results of online bioinformatics database analysis (http://gepia.cancer-pku.cn/detail.php?gene) showed that FOXD3-AS1 was abnormally highly expressed in lung adenocarcinoma and lung squamous cell carcinoma progression (Fig. 1A). Tumor tissues and paracancerous tissues were collected from 25 patients with lung cancer, and RT-qPCR results indicated that the expression of FOXD3-AS1 in tumor tissues was significantly higher than that in paracancerous tissues (Fig. 1B). Furthermore, the expression of FOXD3-AS1 was upregulated in human lung cancer cell lines, including A549, SK-MES-1, SPC-A1, H2170, and PC9 compared with normal HBECs (Fig. 1C). Figure 1. Open in new tabDownload slide FOXD3-AS1 was upregulated in lung cancer tissues and lung cancer cells (A) Online bioinformatics analysis of FOXD3-AS1 expression level in lung cancer (http://gepia.cancer-pku.cn/detail.php?). (B) The lung cancer tissues and adjacent tissues were collected from patients (25 tumor tissues and 25 adjacent tissues). RT-qPCR was used to detect the relative expression of FOXD3-AS1. *P<0.01 compared with the adjacent group. (C) Relative expression of FOXD3-AS1 in lung cancer cell lines and human bronchial epithelial cells (HBECs) was detected by RT-qPCR. *P<0.01 compared with the HBEC group. Figure 1. Open in new tabDownload slide FOXD3-AS1 was upregulated in lung cancer tissues and lung cancer cells (A) Online bioinformatics analysis of FOXD3-AS1 expression level in lung cancer (http://gepia.cancer-pku.cn/detail.php?). (B) The lung cancer tissues and adjacent tissues were collected from patients (25 tumor tissues and 25 adjacent tissues). RT-qPCR was used to detect the relative expression of FOXD3-AS1. *P<0.01 compared with the adjacent group. (C) Relative expression of FOXD3-AS1 in lung cancer cell lines and human bronchial epithelial cells (HBECs) was detected by RT-qPCR. *P<0.01 compared with the HBEC group. Identification of exosomes from A549 and SPC-A1 cells A549 and SPC-A1 cell culture supernatant was collected, and exosomes were extracted by ultracentrifugation. As expected, no expression of exosome marker proteins, including CD63, CD81, TSG101, and Alix, was detected in the cell supernatant after centrifugation, whereas the abundance of exosome marker proteins was increased in the sediment after centrifugation (Fig. 2A–C). Next, qPCR results confirmed that FOXD3-AS1 was abundantly enriched in exosomes derived from A549 and SPC-A1 cells (Fig. 2D). Furthermore, the representative images of exosomes from A549 cells identified by transmission electron microscope (TEM) were shown in Fig. 2E, which indicated that the extracted exosomes had a characteristic morphology of round-shaped particles. Figure 2. Open in new tabDownload slide Isolation and identification of exosomes derived from A549 and SPC-A1 cells The cell culture supernatant of A549 and SPC-A1 cells was collected, and the derived exosomes were isolated by ultracentrifugation. (A–C) The expression levels of exosome marker proteins in the supernatants (centrifugation) and extracts of A549 and SPC-A1 cells were detected by western blot analysis. (D) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (E) Representative images of exosomes derived from A549 cells. β-Actin was used as an internal control. *P<0.01 compared with the supernatant group. Figure 2. Open in new tabDownload slide Isolation and identification of exosomes derived from A549 and SPC-A1 cells The cell culture supernatant of A549 and SPC-A1 cells was collected, and the derived exosomes were isolated by ultracentrifugation. (A–C) The expression levels of exosome marker proteins in the supernatants (centrifugation) and extracts of A549 and SPC-A1 cells were detected by western blot analysis. (D) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (E) Representative images of exosomes derived from A549 cells. β-Actin was used as an internal control. *P<0.01 compared with the supernatant group. FOXD3-AS1-enriched exosomes enhance proliferation, invasion, and 5-FU resistance in A549 cells Our previous results showed that FOXD3-AS1 was upregulated in lung cancer tissues and cells and enriched in A549 and SPC-A1 cell-derived exosomes. To further confirm the effect of FOXD3-AS1 on tumor cells, 100 μl of exosomes was added to A549 cells and incubated together. We found increased FOXD3-AS1 expression (Fig. 3A) and enhanced proliferation (Fig. 3B) and invasion (Fig. 3C) in A549 cells. Moreover, 5-FU-treated A549 cells incubated with exosomes exhibited decreased apoptosis (Fig. 3E,F) and increased cell viability (Fig. 3G). A549 cells were incubated with exosomes alone or transfected together with si-FOXD3-AS1, and we observed attenuated cell proliferation and invasion ability. Transfection with si-FOXD3-AS1 in exosome-incubated 5-FU-treated A549 cells reversed the inhibitory effect of exosomes on cell apoptosis, as well as the promoting effect on cell survival. Moreover, pcDNA-FOXD3-AS1 or si-FOXD3-AS1 was transfected into A549 cells, and we found that transfection with pcDNA-FOXD3-AS1 promoted FOXD3-AS1 expression (Supplementary Fig. S1A), promoted cell proliferation (Supplementary Fig. S1B) and invasion (Supplementary Fig. S1C,D), and inhibited cell apoptosis (Supplementary Fig. S1E,F), while transfection with si-FOXD3-AS1 inhibited FOXD3-AS1 expression, inhibited cell proliferation and invasion, and promoted cell apoptosis. Overexpression of FOXD3-AS1 enhanced the viability of 5-FU-treated A549 cells, and interference with FOXD3-AS1 accelerated the death of 5-FU-treated A549 cells (Supplementary Fig. S1G). Figure 3. Open in new tabDownload slide Effects of FOXD3-AS1-enriched exosomes on A549 cells The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-FOXD3-AS1. (A) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (B) Cell proliferation was analyzed by CCK-8 assay. (C,D) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-FOXD3-AS1. (E,F) Cell apoptosis was detected by flow cytometry. (G) CCK-8 assay was used to detect the cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA group. Figure 3. Open in new tabDownload slide Effects of FOXD3-AS1-enriched exosomes on A549 cells The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-FOXD3-AS1. (A) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (B) Cell proliferation was analyzed by CCK-8 assay. (C,D) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-FOXD3-AS1. (E,F) Cell apoptosis was detected by flow cytometry. (G) CCK-8 assay was used to detect the cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA group. ELAVL1 is an RNA-binding protein of FOXD3-AS1 Numerous studies have reported that ELAVL1, as an RNA-binding protein, affects the development of a variety of diseases by interacting with genes. Here, the results of online bioinformatics database analysis (http://starbase.sysu.edu.cn/) suggested that ELAVL1 may be an RNA-binding protein of FOXD3-AS1, and the potential ELAVL1 target motif in FOXD3-AS1 sequence was shown in Fig. 4A. Furthermore, RNA IP (Fig. 4B) and RNA pull-down (Fig. 4C) were used to validate the binding relationship between ELAVL1 and FOXD3-AS1. Moreover, we observed that ELAVL1 expression was upregulated in lung cancer tissues (Fig. 4D) and cell lines (Fig. 4E). Figure 4. Open in new tabDownload slide ELAVL1 was an RNA-binding protein of FOXD3-AS1 (A) Online bioinformatics databases (http://starbase.sysu.edu.cn/) were used to predict and screen out that ELAVL1 is an RNA-binding protein of FOXD3-AS1, and the binding motifs were shown. (B,C) RNA IP and RNA pulldown assay were used to validate the binding relationship between ELAVL1 and FOXD3-AS1. (D,F) RT-qPCR and western blot analysis were used to detect the relative mRNA and protein expression of ELAVL1 in lung cancer tissues and lung cancer cell lines. β-Actin was used as an internal control. *P<0.01 compared with the adjacent group or the HBEC group. Figure 4. Open in new tabDownload slide ELAVL1 was an RNA-binding protein of FOXD3-AS1 (A) Online bioinformatics databases (http://starbase.sysu.edu.cn/) were used to predict and screen out that ELAVL1 is an RNA-binding protein of FOXD3-AS1, and the binding motifs were shown. (B,C) RNA IP and RNA pulldown assay were used to validate the binding relationship between ELAVL1 and FOXD3-AS1. (D,F) RT-qPCR and western blot analysis were used to detect the relative mRNA and protein expression of ELAVL1 in lung cancer tissues and lung cancer cell lines. β-Actin was used as an internal control. *P<0.01 compared with the adjacent group or the HBEC group. Knockdown of ELAVL1 inhibits proliferation, invasion, and 5-FU resistance of exosome-incubated A549 cells To further explore the effect of ELAVL1 in exosome-incubated A549 cells, 100 μl of exosomes was added into cells and incubated together. Western blot analysis results indicated that ELAVL1 protein expression was increased in A549 cells incubated with exosomes, and transfection with si-ELAVL1 downregulated the ELAVL1 protein expression level (Fig. 5A,B). Furthermore,we observed that exosomes promoted A549 cell proliferation (Fig. 5C) and invasion (Fig. 5D,F), while transfection with si-ELAVL1 reversed the promoting effect of exosomes on A549 cell proliferation and invasion. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-ELAVL1. We found that exosomes inhibited apoptosis and promoted cell survival in 5-FU-treated A549 cells. Knockdown of ELAVL1 reversed the inhibitory effect of exosomes on the apoptosis of 5-FU-treated A549 cells (Fig. 5E,G), as well as the promoting effect on the survival of 5-FU-treated A549 cells (Fig. 5H). Figure 5. Open in new tabDownload slide Effect of ELAVL1 knockdown on A549 cells incubated with FOXD3-AS1-enriched exosomes The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-ELAVL1. (A,B) Western blot analysis was used to detect the protein expression of ELAVL1. (C) Cell proliferation was analyzed by CCK-8 assay. (D,F) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-ELAVL1. (E,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as an internal reference. *P<0.01 compared with the control group or the EXO+NC siRNA group. Figure 5. Open in new tabDownload slide Effect of ELAVL1 knockdown on A549 cells incubated with FOXD3-AS1-enriched exosomes The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-ELAVL1. (A,B) Western blot analysis was used to detect the protein expression of ELAVL1. (C) Cell proliferation was analyzed by CCK-8 assay. (D,F) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-ELAVL1. (E,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as an internal reference. *P<0.01 compared with the control group or the EXO+NC siRNA group. Overexpression of ELAVL1 reverses the inhibitory effect of si-FOXD3-AS1 on A549 cell proliferation, invasion, and 5-FU resistance Exosome-incubated A549 cells were transfected with si-FOXD3-AS1 alone or together with pcDNA-ELAVL1, and we found that transfection with si-FOXD3-AS1 alone inhibited the proliferation (Fig. 6A) and invasion (Fig. 6B,C) of exosome-incubated A549 cells, and overexpression of ELAVL1 reversed the inhibitory effect of si-FOXD3-AS1 on exosome-incubated A549 cell proliferation and invasion. Next, exosome-incubated 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or together with pcDNA-ELAVL1. We found that transfection of si-FOXD3-AS1 alone promoted exosome-incubated A549 cell apoptosis (Fig. 6D,E) and inhibited the survival of exosome-incubated A549 cell (Fig. 6F), and overexpression of ELAVL1 reversed the promoting effect of si-FOXD3-AS1 on apoptosis of exosome-incubated A549 cells, as well as the inhibitory effect on the survival of exosome-incubated A549 cells. Figure 6. Open in new tabDownload slide The effect of si-FOXD3-AS1 was reversed by pcDNA-ELAVL1 si-FOXD3-AS1 was transfected into exosome-incubated A549 cells alone or together with pcDNA-ELAVL1. (A) Cell proliferation was analyzed by CCK-8 assay. (B,C) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or together with si-ELAVL1 in the presence of EXO. (D,E) Cell apoptosis was detected by flow cytometry. (F) CCK-8 assay was used to detect cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA+pcDNA3.1 group or the EXO+si-FOXD3-AS1 group. Figure 6. Open in new tabDownload slide The effect of si-FOXD3-AS1 was reversed by pcDNA-ELAVL1 si-FOXD3-AS1 was transfected into exosome-incubated A549 cells alone or together with pcDNA-ELAVL1. (A) Cell proliferation was analyzed by CCK-8 assay. (B,C) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or together with si-ELAVL1 in the presence of EXO. (D,E) Cell apoptosis was detected by flow cytometry. (F) CCK-8 assay was used to detect cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA+pcDNA3.1 group or the EXO+si-FOXD3-AS1 group. FOXD3-AS1-enriched exosomes promote A549 cell proliferation, invasion, and 5-FU resistance by activating the PI3K/Akt pathway A549 cells were incubated with exosome alone or transfected together with si-FOXD3-AS1. Western blot analysis results suggested that exosomes upregulated the phosphorylation levels of PI3K and Akt in A549 cells, and transfection with si-FOXD3-AS1 reversed the promoting effects of exosomes on the phosphorylation of PI3K and Akt, which were similar to the effects of LY294002, a PI3K inhibitor (Fig. 7A,B). Moreover, FOXD3-AS1-enriched exosomes promoted A549 cell proliferation and invasion, and transfection with si-FOXD3-AS1 reversed the promoting effects of exosomes on cell proliferation (Fig. 7C) and invasion (Fig. 7D,F), which were similar to the effects of LY294002. The 5-FU-treated A549 cells were incubated with exosome alone or transfected together with si-FOXD3-AS1, and we found that exosomes inhibited apoptosis and promoted cell survival in 5-FU-treated A549 cells, whereas si-FOXD3-AS1 or LY294002 abolished the inhibitory effect of exosomes on apoptosis (Fig. 7E,G) and the promotion (Fig. 7H) of cell survival. Figure 7. Open in new tabDownload slide FOXD3-AS1-enriched exosomes activated the PI3K/Akt axis in A549 cells (A,B) A549 cells incubated with exosomes were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002, and the phosphorylation levels of PI3K and AKT were detected by western blot analysis. (C) Cell proliferation was analyzed by CCK-8 assay. (D,E) The invasion of A549 cells was detected by Transwell invasion assay. Exosome-incubated 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002. (F,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as the loading control. *P<0.01 compared with the control group or the EXO+NC siRNA group. Figure 7. Open in new tabDownload slide FOXD3-AS1-enriched exosomes activated the PI3K/Akt axis in A549 cells (A,B) A549 cells incubated with exosomes were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002, and the phosphorylation levels of PI3K and AKT were detected by western blot analysis. (C) Cell proliferation was analyzed by CCK-8 assay. (D,E) The invasion of A549 cells was detected by Transwell invasion assay. Exosome-incubated 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002. (F,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as the loading control. *P<0.01 compared with the control group or the EXO+NC siRNA group. Discussion Numerous studies have reported that lncRNAs participate in the occurrence and development of many diseases, such as leukemia, lymphoma, gastric cancer, and lung cancer. In recent years, FOXD3-AS1 has received much attention. The expression of FOXD3-AS1 was significantly increased in Müller glial cells, mimicking retinal infection with Toxoplasma gondii infection, suggesting its potential role in regulating the immune response of retinal Müller cells [17]. In the study of myocardial ischemia–reperfusion injury, FOXD3-AS1 expression was increased in H9C2 cells treated with OGD/R, and overexpression of FOXD3-AS1 significantly upregulated LC3-II, Beclin1, and ATG5 expression; promoted the secretion of proinflammatory factors in cells; and enhanced oxidative stress, while these changes in cell behavior were reversed after treatment with autophagy inhibitors. These findings suggest that FOXD3-AS1 aggravates myocardial hypoxia–reperfusion injury by promoting autophagy [18]. Furthermore, FOXD3-AS1 has been reported as a competing endogenous RNA to regulate the progression of colon adenocarcinoma [19], neuroblastoma [20], melanoma [21], thyroid cancer [22], liver cancer [23], and other tumors. In this study, we found that FOXD3-AS1 expression was upregulated in lung cancer tissues and cell lines and was largely enriched in A549 and SPC-A1 cell-derived exosomes, suggesting that FOXD3-AS1-enriched exosomes may have a potential regulatory role in lung cancer progression. As a nano-scale membrane structure, exosomes are mainly responsible for carrying various contents and are widely involved in the biological process of tumors through mechanisms such as plasma membrane fusion, endocytosis, and binding to cell surface receptors. As one of the molecular basis of tumor invasion and metastasis, exosomes are of great significance for early diagnosis and target therapy of lung cancer. It has been found that exosomes derived from highly metastatic lung cancer cells and serum of patients with advanced lung cancer induce vimentin expression, induce epithelial–mesenchymal transition of HBECs, and induce migration, invasion, and proliferation of non-metastatic cancer cells [24]. Wang et al. [25] found that exosomes derived from metastatic SCLC cells had a greater impact on tumor cell migration and invasion than those derived from early non-small cell lung cancer (NSCLC) cells. Especially under hypoxic conditions, the contents of transforming growth factor-beta and interleukin-10, which are closely related to tumor cell migration and invasion, were increased in the exosomes of metastatic small cell lung cancer cells. As the important substance for intercellular information exchange, exosomes transmit relevant signaling molecules to target cells by means of autocrine or long-distance dissemination, resulting in a series of biological effects. Therefore, exosome-targeted tumor therapy approaches may have broad prospects. The PI3K protein family is a dimer composed of the regulatory subunit p85 and the catalytic subunit p110, which is widely involved in regulating cell phenotypes such as proliferation, differentiation, apoptosis, and migration. It has been reported that abnormal activation of PI3K signal is involved in the development of afatinib resistance, while shikonin inhibits the proliferation and induces apoptosis of afatinib-resistant NSCLC cells by activating the apoptotic signaling pathway and negatively regulating the PI3K/Akt signaling pathway, delaying the further development of lung cancer [26]. Overexpression of Fer-1-like family member 4 in lung cancer cell lines A549 and 95D inhibited colony formation, cell proliferation, and migration, resulting in decreased PI3K/Akt expression in cells, while activation of PI3K/Akt signaling using small molecule inhibitors of phosphatase and tensin homolog reversed the inhibitory effect of Fer-1-like family member 4 on cell proliferation and metastasis [27]. Similarly, lncRNA FOXO1 expression was downregulated in lung cancer tissues or cells, and knockdown of FOXO1 promoted A549 cell viability, colony formation, and invasion by activating the PI3K/Akt pathway, accelerating tumor metastasis and recurrence [28]. In this study, we found that FOXD3-AS1-enriched exosomes activated the PI3K/Akt signaling system by interacting with ELAVL1 to promote lung cancer cell proliferation, invasion, and 5-FU resistance. In conclusion, therapeutic strategies targeting exosomes may bring new therapeutic hope for patients with lung cancer. Supplementary Data Supplementary data is available at Acta Biochimica et Biophysica Sinica online. Funding This work was supported by the grant from the National Natural Science Foundation of China (No. 81672273). Conflict of Interest The authors declare that they have no conflict of interest. References 1. Shi JF , Wang L, Wu N, Li JL, Hui ZG, Liu SM, Yang BY, et al. Clinical characteristics and medical service utilization of lung cancer in China, 2005–2014: overall design and results from a multicenter retrospective epidemiologic survey . Lung Cancer (Amsterdam, Netherlands) 2019 , 128 : 91 – 100 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Warner ET , Lathan CS. Race and sex differences in patient provider communication and awareness of lung cancer screening in the health information National Trends Survey, 2013-2017 . Prev Med 2019 , 124 : 84 – 90 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Ho JC , Leung CC. 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Ding X , Wang Q, Tong L, Si X, Sun Y. Long non-coding RNA FOXO1 inhibits lung cancer cell growth through down-regulating PI3K/AKT signaling pathway . Iran J Basic Med Sci 2019 , 22 : 491 – 498 . Google Scholar PubMed OpenURL Placeholder Text WorldCat © The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Biochimica et Biophysica Sinica Oxford University Press

Exosomal lncRNA FOXD3-AS1 upregulates ELAVL1 expression and activates PI3K/Akt pathway to enhance lung cancer cell proliferation, invasion, and 5-fluorouracil resistance

Acta Biochimica et Biophysica Sinica , Volume Advance Article – Oct 4, 2021

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Oxford University Press
Copyright
Copyright © 2021 Institute of Biochemistry and Cell Biology, SIBS, CAS
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1672-9145
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1745-7270
DOI
10.1093/abbs/gmab129
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Abstract

Abstract Long non-coding RNA (lncRNA) FOXD3-AS1 expression is upregulated in lung cancer; however, its effect and mechanism on 5-fluorouracil (5-FU) resistance remain unclear. In this study, we determined the effects of FOXD3-AS1-enriched exosomes derived from lung cancer cells on the proliferation, invasion, and 5-FU resistance of lung cancer cells. Online bioinformatics database analysis showed that FOXD3-AS1 was upregulated in lung cancer progression. Real-time quantitative PCR results confirmed that FOXD3-AS1 expression was upregulated in lung cancer tissues and cell lines, and FOXD3-AS1 was greatly enriched in lung cancer cell-derived exosomes. ELAV-like RNA-binding protein 1 (ELAVL1) was identified as an RNA-binding protein of FOXD3-AS1. The lung cancer cell-derived exosomes promoted A549 cell proliferation and invasion and inhibited apoptosis caused by 5-FU, and transfection of si-FOXD3-AS1 or si-ELAVL1 in exosome-incubated A549 cells reversed these effects. Moreover, exosome-incubated A549 cells were co-transfected with si-FOXD3-AS1 and pcDNA-ELAVL1, showing the same cell proliferation, invasion, and 5-FU resistance as those of A549 cells treated with lung cancer cell-derived exosomes alone. Mechanistic studies identified that lung cancer cell-derived exosomes activated the PI3K/Akt pathway, and transfection of si-FOXD3-AS1 or treatment with the PI3K inhibitor LY294002 reversed the activation of the PI3K/Akt axis induced by exosomes. In conclusion, our study revealed that lung cancer cell-derived exosomal FOXD3-AS1 upregulated ELAVL1 expression and activated the PI3K/Akt pathway to promote lung cancer progression. Our findings provide a new strategy for lung cancer treatment. lung cancer, exosomes, lncRNA FOXD3-AS1, ELAVL1, 5-FU resistance, PI3K/Akt pathway Introduction Lung cancer is the respiratory malignancy with the highest morbidity and mortality worldwide, accounting for 18 million new cases annually and 13% of all cancers [1–3]. Similarly, the incidence and mortality of lung cancer in China are also increasing year by year, seriously endangering human health and life. Lung cancer is mainly divided into small cell lung cancer and non-small cell lung cancer, in which non-small cell lung cancer accounts for 80–85% of all lung cancer cases. Surgical treatment is the first and most important treatment for lung cancer, but most of the patients are diagnosed at the middle and advanced stage, the curative effect of surgical treatment is not ideal, and the 5-year survival rate is still <15% [4,5]. Therefore, the identification of prognostic markers and potential drug targets needs further study to promote prognosis and personalized treatment. Long non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules with transcript length ranging from 200 nt to 100 kb [6]. Recent studies have shown that lncRNAs are abnormally expressed in a variety of human cancers such as colon cancer [7], prostate cancer [8], breast cancer [9], and liver cancer [10], participating in important cellular and molecular activities such as epigenetic modification, transcriptional regulation, RNA editing, and protein translation and playing a key role in tumorigenesis, metastasis, and metabolism. Exosomes are membrane vesicles with a diameter of 30–150 nm that are actively secreted by living cells and contain proteins, lipids, and nucleic acids derived from maternal cells. The main function of exosomes is to maintain cell homeostasis and regulate cell function, especially information transmission between cells [11–13]. In recent years, increasing evidence has demonstrated the important role of exosomes in diseases. Tumor cell-derived exosomes promote cell proliferation, invasion, and drug resistance by delivering small molecules that have positive effects on tumor growth. lncRNAs play an important role in the information transmission of exosomes. In colorectal cancer, lncRNA CCAL promotes drug resistance of colorectal cancer cells due to the transfer of CCAL-carrying exosomes from cancer-associated fibroblasts to cancer cells, thereby inhibiting tumor cell apoptosis in vitro and in vivo and inducing chemoresistance [14]. Another study found that the expression of lncRNA ZFAS1 was increased in tumor cells, tumor tissues, serum, and serum exosomes of gastric cancer patients, and interference with ZFAS1 expression inhibited cell cycle progression, induced apoptosis, and inhibited epithelial–mesenchymal transition [15]. Moreover, studies have reported an increase of H19 in gefitinib-resistant lung cancer cells compared with that in drug-sensitive parental cells, and H19 encapsulated in exosomes was transferred to non-resistant cells, thereby inducing gefitinib resistance [16]. The upregulation of FOXD3-AS1 expression in lung cancer has been confirmed; however, its effect on 5-fluorouracil (5-FU) resistance and the possible regulatory mechanism of drug resistance remains unclear. In this study, we determined the effects of FOXD3-AS1-enriched exosomes derived from lung cancer cells on the proliferation, invasion, and 5-FU resistance of lung cancer cells, in order to provide new ideas for the prevention and reversal of drug resistance treatment strategies. Materials and Methods Samples The lung cancer tissues and adjacent tissues of 25 patients with lung cancer were collected surgically, and the average age of the patients was (53.2±3.1) years. All patients had complete clinical data (Table 1) and did not receive any form of anticancer treatment 3 months before surgery. The study protocol was approved by the Ethics Committee of Peking University Shenzhen Hospital, and written informed consent was obtained from each participant. Table 1. Clinical data of 25 lung cancer patients Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Open in new tab Table 1. Clinical data of 25 lung cancer patients Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Parameters . Squamous cell carcinoma . Small cell carcinoma . Adenocarcinoma . Gender Female 2 3 5 Male 3 5 7 Age <50 3 4 8 ≥50 2 4 4 Clinical stage I–II 1 5 6 III–IV 4 3 6 Lymph node metastasis No 5 6 8 Yes 0 2 4 Open in new tab Cell culture Human lung cancer cell lines A549, SK-MES-1, SPC-A1, H2170, and PC9, and human bronchial epithelial cells (HBECs) were purchased from the American Typical Culture Collection (Manassas, USA). Cells were incubated in medium containing 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St Louis, USA) in RPMI 1640 medium (Gibco, Rockville, USA) at 5% CO2 at 37°C. Small interfering RNAs against FOXD3-AS1 (si-FOXD3-AS1, 5ʹ-GCAAUAGGGACGCGCCAAU-3ʹ) and ELAVL1 (si-ELAVL1, 5ʹ-AAGAGGCAAUUACCAGUUUCA-3ʹ), and scramble (5ʹ-CGAGGGATGAGCCCGCGTAG-3ʹ) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Transfection was performed by using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. pcDNA3.1 and pcDNA-ELAVL1 were purchased from RiboBio (Guangzhou, China) and transfected into cells according to the instructions. Exosome extraction The supernatant of the cell culture medium was collected, and the cell components and dead cells were removed by low-speed centrifugation (300 g, 10 min, 2000 g, 10 min) at 4°C. The supernatant containing exosomes was retained, and the cell debris was removed by high-speed centrifugation (10,000 g, 70 min). The supernatant containing extracellular vesicles was retained, and the exosomes were precipitated by ultracentrifugation (100,000 g, 70 min). An appropriate amount of phosphate buffered saline (PBS) was taken to resuspend the extracellular vesicle precipitation and then ultracentrifuged again (100,000 g, 70 min) to eliminate contaminated proteins. The pellet was collected and aliquoted and stored at −80°C for future use. Cell counting kit-8 assay A549 cells grown in logarithmic phase were digested with 0.25% trypsin, the supernatant was discarded, and then the cell density was adjusted to 3×104 cells/ml by adding Dulbecco’s modified Eagle’s medium (DMEM; Gibco). Cell suspension was seeded at 3×104 cells/ml in 96-well plates, and 100 μl was added into each well, and placed in an incubator at 37°C and 5% CO2 for 4 h. Then, 10 μl of CCK-8 solution (Leigen Bio, Beijing, China) was add to each well and incubated for 4 h. The absorbance of each well at 450 nm was detected with a microplate reader (Molecular devices, Shanghai, China). Real-time quantitative PCR Total RNA was isolated from patient tissues or A549 cells by using TRIzol (Invitrogen). Single-stranded complementary DNA was synthesized using the PrimeScript Reagent Kit (Promega, Madison, USA). Real-time qPCR was conducted by using SYBR Premix Ex TaqTM Kit (Applied Biosystems, Foster City, USA). The reaction was run on the ABI7500 Real-time PCR system (Applied Biosystems). The RT-qPCR cycling conditions consisted of the following: 95°C for 3 min; then 35-cycle amplification for 20 s at 95°C, 30 s at 55°C, 15 s at 72°C; followed by 1 min at 72°C. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China) and listed in Table 2. The expression level was determined by using the 2−ΔΔCt method. GAPDH was used as an endogenous control. Table 2. The sequences of the primers used in this study Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Open in new tab Table 2. The sequences of the primers used in this study Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Gene . Forward (5ʹ→3ʹ) . Reverse (5ʹ→3ʹ) . U6 CTGCTTCGCACGACGA CTTCACGGGACCGTTGCGT GAPDH AGATCCCACAGTCCATCA GCCTGCGGCGACGCCTTTG FOXD3-AS1 CCTTCGGCTCACAGCTC GCTTGGCTCGGACTTGAT ELAVL1 CGCAGAGATTCAGGTTCTCC CCAAACCCTTTGCACTTGTT Open in new tab Western blot analysis Protein homogenates from cells or tissues were extracted. The protein content of each sample was determined using the BCA Protein Assay Kits (Thermo Scientific). Then, equal amounts of protein (15 μg per lane) were separated by 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, USA). The membranes were blocked in 5% (w/v) skimmed milk powder in TBST (Tris buffered saline containing 0.1% Tween) for 2 h at 25°C and then incubated with the following antibodies from Abcam: rabbit polyclonal anti-β-actin antibody (1:1000, ab8227), rabbit monoclonal anti-CD63 antibody (1:1500, ab217345), mouse monoclonal anti-CD81 antibody (1:1200, ab79559), rabbit monoclonal anti-TSG101 antibody (1:2500, ab125011), rabbit monoclonal anti-Alix antibody (1:2000, ab186492), rabbit monoclonal anti-ELAVL1 antibody (1:800, ab200342), rabbit monoclonal anti-PI3K antibody (1:1000, ab32089), rabbit polyclonal anti-Akt antibody (1:700, ab8805), and rabbit polyclonal anti-Akt (phospho T308) antibody (1:800, ab38449). Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000, ab6721; Abcam) or goat anti-mouse IgG (1:3000, ab6728; Abcam). Finally, the protein bands were visualized using ECL reagent (Amersham Biosciences, Piscataway, USA) and quantified using ImageJ software (National Institutes of Health, Bethesda, USA). β-Actin was used as the loading control. Transwell assay Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), and 1×105 cells (100 μl) was seeded in a chamber with Matrigel and on a control with polyethylene terephthalate membrane. After incubation for 24 h, the cells were fixed with anhydrous methanol for 20 min, then stained with 0.1% crystal violet (Sigma) for 20 min, dried and observed with an inverted microscope (Olympus, Tokyo, Japan), and counted to evaluate the invasion ability of the cells. Flow cytometry The cells were collected and washed twice with PBS. A total of 100 μl cell suspension (1×105 cells/ml) was transferred to a culture tube and then incubated with 5 μl of Annexin V-FITC and 5 μl propidium iodide (BD Biosciences) at room temperature for 20 min in the dark. Finally, 400 μl of binding buffer was added, and apoptotic cells were determined by flow cytometry on a BD flow cytometer (BD Biosciences, San Jose, USA). RNA immunoprecipitation assay RNA immunoprecipitation (RIP) assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore, Billerica, USA). Briefly, A549 cells were lysed by using the RIPA lysate buffer, and the supernatant was collected by centrifugation. Next, the supernatant was incubated with 1 μg corresponding antibodies: anti-ELAVL1 (ab200342, 1:100; Abcam) or negative control (NC) antibody (IgG) (ab10948, 1:100; Abcam), and then 50 μl of protein A/G-beads was added and incubated overnight at 4°C. After immunoprecipitation, the mixture was centrifuged to remove the supernatant. The protein A/G beads were then washed with 1 ml lysis buffer and repeated 3 times. Finally, beads were added with 15 μl of 2× SDS buffer solution and then boiled for 10 min. RNA protein complexes were obtained by using the anti-ELAVL1 antibody, and then RNA on the complexes was isolated and purified and analyzed by RT-qPCR. RNA pull-down assay The binding relationship between FOXD3-AS1 and ELAVL1 was identified by RNA pull-down assay. Briefly, biotinylated wild-type FOXD3-AS1 (FOXD3-AS1-WT), mutant-type FOXD3-AS1 (FOXD3-AS1-MUT), and NC were synthesized and transfected into A549 cells using Biotin RNA Labeling Mix (Roche), respectively. After transfection for 24 h, the cells were washed with PBS and incubated with lysis buffer on ice for 10 min. Then, the lysate was incubated with streptavidin magnetic beads (Invitrogen) at 4°C for 3 h. Then, the beads were washed extensively, and the RNA–protein complex was eluted. Western blot analysis was used to detect the retrieved proteins. Statistical analysis All statistical analyses were performed using the SPSS software (ver. 21.0; SPSS, Chicago, USA). The quantitative data derived from three independent experiments were expressed as the mean±SEM. Significance was determined by one-way analysis of variance or Student’s t-test. P<0.05 was considered of statistically significant difference. Results The expression of FOXD3-AS1 is upregulated in lung cancer tissues and cell lines The results of online bioinformatics database analysis (http://gepia.cancer-pku.cn/detail.php?gene) showed that FOXD3-AS1 was abnormally highly expressed in lung adenocarcinoma and lung squamous cell carcinoma progression (Fig. 1A). Tumor tissues and paracancerous tissues were collected from 25 patients with lung cancer, and RT-qPCR results indicated that the expression of FOXD3-AS1 in tumor tissues was significantly higher than that in paracancerous tissues (Fig. 1B). Furthermore, the expression of FOXD3-AS1 was upregulated in human lung cancer cell lines, including A549, SK-MES-1, SPC-A1, H2170, and PC9 compared with normal HBECs (Fig. 1C). Figure 1. Open in new tabDownload slide FOXD3-AS1 was upregulated in lung cancer tissues and lung cancer cells (A) Online bioinformatics analysis of FOXD3-AS1 expression level in lung cancer (http://gepia.cancer-pku.cn/detail.php?). (B) The lung cancer tissues and adjacent tissues were collected from patients (25 tumor tissues and 25 adjacent tissues). RT-qPCR was used to detect the relative expression of FOXD3-AS1. *P<0.01 compared with the adjacent group. (C) Relative expression of FOXD3-AS1 in lung cancer cell lines and human bronchial epithelial cells (HBECs) was detected by RT-qPCR. *P<0.01 compared with the HBEC group. Figure 1. Open in new tabDownload slide FOXD3-AS1 was upregulated in lung cancer tissues and lung cancer cells (A) Online bioinformatics analysis of FOXD3-AS1 expression level in lung cancer (http://gepia.cancer-pku.cn/detail.php?). (B) The lung cancer tissues and adjacent tissues were collected from patients (25 tumor tissues and 25 adjacent tissues). RT-qPCR was used to detect the relative expression of FOXD3-AS1. *P<0.01 compared with the adjacent group. (C) Relative expression of FOXD3-AS1 in lung cancer cell lines and human bronchial epithelial cells (HBECs) was detected by RT-qPCR. *P<0.01 compared with the HBEC group. Identification of exosomes from A549 and SPC-A1 cells A549 and SPC-A1 cell culture supernatant was collected, and exosomes were extracted by ultracentrifugation. As expected, no expression of exosome marker proteins, including CD63, CD81, TSG101, and Alix, was detected in the cell supernatant after centrifugation, whereas the abundance of exosome marker proteins was increased in the sediment after centrifugation (Fig. 2A–C). Next, qPCR results confirmed that FOXD3-AS1 was abundantly enriched in exosomes derived from A549 and SPC-A1 cells (Fig. 2D). Furthermore, the representative images of exosomes from A549 cells identified by transmission electron microscope (TEM) were shown in Fig. 2E, which indicated that the extracted exosomes had a characteristic morphology of round-shaped particles. Figure 2. Open in new tabDownload slide Isolation and identification of exosomes derived from A549 and SPC-A1 cells The cell culture supernatant of A549 and SPC-A1 cells was collected, and the derived exosomes were isolated by ultracentrifugation. (A–C) The expression levels of exosome marker proteins in the supernatants (centrifugation) and extracts of A549 and SPC-A1 cells were detected by western blot analysis. (D) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (E) Representative images of exosomes derived from A549 cells. β-Actin was used as an internal control. *P<0.01 compared with the supernatant group. Figure 2. Open in new tabDownload slide Isolation and identification of exosomes derived from A549 and SPC-A1 cells The cell culture supernatant of A549 and SPC-A1 cells was collected, and the derived exosomes were isolated by ultracentrifugation. (A–C) The expression levels of exosome marker proteins in the supernatants (centrifugation) and extracts of A549 and SPC-A1 cells were detected by western blot analysis. (D) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (E) Representative images of exosomes derived from A549 cells. β-Actin was used as an internal control. *P<0.01 compared with the supernatant group. FOXD3-AS1-enriched exosomes enhance proliferation, invasion, and 5-FU resistance in A549 cells Our previous results showed that FOXD3-AS1 was upregulated in lung cancer tissues and cells and enriched in A549 and SPC-A1 cell-derived exosomes. To further confirm the effect of FOXD3-AS1 on tumor cells, 100 μl of exosomes was added to A549 cells and incubated together. We found increased FOXD3-AS1 expression (Fig. 3A) and enhanced proliferation (Fig. 3B) and invasion (Fig. 3C) in A549 cells. Moreover, 5-FU-treated A549 cells incubated with exosomes exhibited decreased apoptosis (Fig. 3E,F) and increased cell viability (Fig. 3G). A549 cells were incubated with exosomes alone or transfected together with si-FOXD3-AS1, and we observed attenuated cell proliferation and invasion ability. Transfection with si-FOXD3-AS1 in exosome-incubated 5-FU-treated A549 cells reversed the inhibitory effect of exosomes on cell apoptosis, as well as the promoting effect on cell survival. Moreover, pcDNA-FOXD3-AS1 or si-FOXD3-AS1 was transfected into A549 cells, and we found that transfection with pcDNA-FOXD3-AS1 promoted FOXD3-AS1 expression (Supplementary Fig. S1A), promoted cell proliferation (Supplementary Fig. S1B) and invasion (Supplementary Fig. S1C,D), and inhibited cell apoptosis (Supplementary Fig. S1E,F), while transfection with si-FOXD3-AS1 inhibited FOXD3-AS1 expression, inhibited cell proliferation and invasion, and promoted cell apoptosis. Overexpression of FOXD3-AS1 enhanced the viability of 5-FU-treated A549 cells, and interference with FOXD3-AS1 accelerated the death of 5-FU-treated A549 cells (Supplementary Fig. S1G). Figure 3. Open in new tabDownload slide Effects of FOXD3-AS1-enriched exosomes on A549 cells The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-FOXD3-AS1. (A) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (B) Cell proliferation was analyzed by CCK-8 assay. (C,D) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-FOXD3-AS1. (E,F) Cell apoptosis was detected by flow cytometry. (G) CCK-8 assay was used to detect the cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA group. Figure 3. Open in new tabDownload slide Effects of FOXD3-AS1-enriched exosomes on A549 cells The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-FOXD3-AS1. (A) RT-qPCR was used to detect the relative expression of FOXD3-AS1. (B) Cell proliferation was analyzed by CCK-8 assay. (C,D) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-FOXD3-AS1. (E,F) Cell apoptosis was detected by flow cytometry. (G) CCK-8 assay was used to detect the cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA group. ELAVL1 is an RNA-binding protein of FOXD3-AS1 Numerous studies have reported that ELAVL1, as an RNA-binding protein, affects the development of a variety of diseases by interacting with genes. Here, the results of online bioinformatics database analysis (http://starbase.sysu.edu.cn/) suggested that ELAVL1 may be an RNA-binding protein of FOXD3-AS1, and the potential ELAVL1 target motif in FOXD3-AS1 sequence was shown in Fig. 4A. Furthermore, RNA IP (Fig. 4B) and RNA pull-down (Fig. 4C) were used to validate the binding relationship between ELAVL1 and FOXD3-AS1. Moreover, we observed that ELAVL1 expression was upregulated in lung cancer tissues (Fig. 4D) and cell lines (Fig. 4E). Figure 4. Open in new tabDownload slide ELAVL1 was an RNA-binding protein of FOXD3-AS1 (A) Online bioinformatics databases (http://starbase.sysu.edu.cn/) were used to predict and screen out that ELAVL1 is an RNA-binding protein of FOXD3-AS1, and the binding motifs were shown. (B,C) RNA IP and RNA pulldown assay were used to validate the binding relationship between ELAVL1 and FOXD3-AS1. (D,F) RT-qPCR and western blot analysis were used to detect the relative mRNA and protein expression of ELAVL1 in lung cancer tissues and lung cancer cell lines. β-Actin was used as an internal control. *P<0.01 compared with the adjacent group or the HBEC group. Figure 4. Open in new tabDownload slide ELAVL1 was an RNA-binding protein of FOXD3-AS1 (A) Online bioinformatics databases (http://starbase.sysu.edu.cn/) were used to predict and screen out that ELAVL1 is an RNA-binding protein of FOXD3-AS1, and the binding motifs were shown. (B,C) RNA IP and RNA pulldown assay were used to validate the binding relationship between ELAVL1 and FOXD3-AS1. (D,F) RT-qPCR and western blot analysis were used to detect the relative mRNA and protein expression of ELAVL1 in lung cancer tissues and lung cancer cell lines. β-Actin was used as an internal control. *P<0.01 compared with the adjacent group or the HBEC group. Knockdown of ELAVL1 inhibits proliferation, invasion, and 5-FU resistance of exosome-incubated A549 cells To further explore the effect of ELAVL1 in exosome-incubated A549 cells, 100 μl of exosomes was added into cells and incubated together. Western blot analysis results indicated that ELAVL1 protein expression was increased in A549 cells incubated with exosomes, and transfection with si-ELAVL1 downregulated the ELAVL1 protein expression level (Fig. 5A,B). Furthermore,we observed that exosomes promoted A549 cell proliferation (Fig. 5C) and invasion (Fig. 5D,F), while transfection with si-ELAVL1 reversed the promoting effect of exosomes on A549 cell proliferation and invasion. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-ELAVL1. We found that exosomes inhibited apoptosis and promoted cell survival in 5-FU-treated A549 cells. Knockdown of ELAVL1 reversed the inhibitory effect of exosomes on the apoptosis of 5-FU-treated A549 cells (Fig. 5E,G), as well as the promoting effect on the survival of 5-FU-treated A549 cells (Fig. 5H). Figure 5. Open in new tabDownload slide Effect of ELAVL1 knockdown on A549 cells incubated with FOXD3-AS1-enriched exosomes The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-ELAVL1. (A,B) Western blot analysis was used to detect the protein expression of ELAVL1. (C) Cell proliferation was analyzed by CCK-8 assay. (D,F) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-ELAVL1. (E,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as an internal reference. *P<0.01 compared with the control group or the EXO+NC siRNA group. Figure 5. Open in new tabDownload slide Effect of ELAVL1 knockdown on A549 cells incubated with FOXD3-AS1-enriched exosomes The A549 cells were incubated with 100 μl of exosomes alone or transfected together with si-ELAVL1. (A,B) Western blot analysis was used to detect the protein expression of ELAVL1. (C) Cell proliferation was analyzed by CCK-8 assay. (D,F) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were incubated with exosomes alone or transfected together with si-ELAVL1. (E,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as an internal reference. *P<0.01 compared with the control group or the EXO+NC siRNA group. Overexpression of ELAVL1 reverses the inhibitory effect of si-FOXD3-AS1 on A549 cell proliferation, invasion, and 5-FU resistance Exosome-incubated A549 cells were transfected with si-FOXD3-AS1 alone or together with pcDNA-ELAVL1, and we found that transfection with si-FOXD3-AS1 alone inhibited the proliferation (Fig. 6A) and invasion (Fig. 6B,C) of exosome-incubated A549 cells, and overexpression of ELAVL1 reversed the inhibitory effect of si-FOXD3-AS1 on exosome-incubated A549 cell proliferation and invasion. Next, exosome-incubated 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or together with pcDNA-ELAVL1. We found that transfection of si-FOXD3-AS1 alone promoted exosome-incubated A549 cell apoptosis (Fig. 6D,E) and inhibited the survival of exosome-incubated A549 cell (Fig. 6F), and overexpression of ELAVL1 reversed the promoting effect of si-FOXD3-AS1 on apoptosis of exosome-incubated A549 cells, as well as the inhibitory effect on the survival of exosome-incubated A549 cells. Figure 6. Open in new tabDownload slide The effect of si-FOXD3-AS1 was reversed by pcDNA-ELAVL1 si-FOXD3-AS1 was transfected into exosome-incubated A549 cells alone or together with pcDNA-ELAVL1. (A) Cell proliferation was analyzed by CCK-8 assay. (B,C) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or together with si-ELAVL1 in the presence of EXO. (D,E) Cell apoptosis was detected by flow cytometry. (F) CCK-8 assay was used to detect cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA+pcDNA3.1 group or the EXO+si-FOXD3-AS1 group. Figure 6. Open in new tabDownload slide The effect of si-FOXD3-AS1 was reversed by pcDNA-ELAVL1 si-FOXD3-AS1 was transfected into exosome-incubated A549 cells alone or together with pcDNA-ELAVL1. (A) Cell proliferation was analyzed by CCK-8 assay. (B,C) The invasion of A549 cells was detected by Transwell invasion assay. The 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or together with si-ELAVL1 in the presence of EXO. (D,E) Cell apoptosis was detected by flow cytometry. (F) CCK-8 assay was used to detect cell viability. *P<0.01 compared with the control group or the EXO+NC siRNA+pcDNA3.1 group or the EXO+si-FOXD3-AS1 group. FOXD3-AS1-enriched exosomes promote A549 cell proliferation, invasion, and 5-FU resistance by activating the PI3K/Akt pathway A549 cells were incubated with exosome alone or transfected together with si-FOXD3-AS1. Western blot analysis results suggested that exosomes upregulated the phosphorylation levels of PI3K and Akt in A549 cells, and transfection with si-FOXD3-AS1 reversed the promoting effects of exosomes on the phosphorylation of PI3K and Akt, which were similar to the effects of LY294002, a PI3K inhibitor (Fig. 7A,B). Moreover, FOXD3-AS1-enriched exosomes promoted A549 cell proliferation and invasion, and transfection with si-FOXD3-AS1 reversed the promoting effects of exosomes on cell proliferation (Fig. 7C) and invasion (Fig. 7D,F), which were similar to the effects of LY294002. The 5-FU-treated A549 cells were incubated with exosome alone or transfected together with si-FOXD3-AS1, and we found that exosomes inhibited apoptosis and promoted cell survival in 5-FU-treated A549 cells, whereas si-FOXD3-AS1 or LY294002 abolished the inhibitory effect of exosomes on apoptosis (Fig. 7E,G) and the promotion (Fig. 7H) of cell survival. Figure 7. Open in new tabDownload slide FOXD3-AS1-enriched exosomes activated the PI3K/Akt axis in A549 cells (A,B) A549 cells incubated with exosomes were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002, and the phosphorylation levels of PI3K and AKT were detected by western blot analysis. (C) Cell proliferation was analyzed by CCK-8 assay. (D,E) The invasion of A549 cells was detected by Transwell invasion assay. Exosome-incubated 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002. (F,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as the loading control. *P<0.01 compared with the control group or the EXO+NC siRNA group. Figure 7. Open in new tabDownload slide FOXD3-AS1-enriched exosomes activated the PI3K/Akt axis in A549 cells (A,B) A549 cells incubated with exosomes were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002, and the phosphorylation levels of PI3K and AKT were detected by western blot analysis. (C) Cell proliferation was analyzed by CCK-8 assay. (D,E) The invasion of A549 cells was detected by Transwell invasion assay. Exosome-incubated 5-FU-treated A549 cells were transfected with si-FOXD3-AS1 alone or treated with PI3K inhibitor LY294002. (F,G) Cell apoptosis was detected by flow cytometry. (H) CCK-8 assay was used to detect cell viability. β-Actin was used as the loading control. *P<0.01 compared with the control group or the EXO+NC siRNA group. Discussion Numerous studies have reported that lncRNAs participate in the occurrence and development of many diseases, such as leukemia, lymphoma, gastric cancer, and lung cancer. In recent years, FOXD3-AS1 has received much attention. The expression of FOXD3-AS1 was significantly increased in Müller glial cells, mimicking retinal infection with Toxoplasma gondii infection, suggesting its potential role in regulating the immune response of retinal Müller cells [17]. In the study of myocardial ischemia–reperfusion injury, FOXD3-AS1 expression was increased in H9C2 cells treated with OGD/R, and overexpression of FOXD3-AS1 significantly upregulated LC3-II, Beclin1, and ATG5 expression; promoted the secretion of proinflammatory factors in cells; and enhanced oxidative stress, while these changes in cell behavior were reversed after treatment with autophagy inhibitors. These findings suggest that FOXD3-AS1 aggravates myocardial hypoxia–reperfusion injury by promoting autophagy [18]. Furthermore, FOXD3-AS1 has been reported as a competing endogenous RNA to regulate the progression of colon adenocarcinoma [19], neuroblastoma [20], melanoma [21], thyroid cancer [22], liver cancer [23], and other tumors. In this study, we found that FOXD3-AS1 expression was upregulated in lung cancer tissues and cell lines and was largely enriched in A549 and SPC-A1 cell-derived exosomes, suggesting that FOXD3-AS1-enriched exosomes may have a potential regulatory role in lung cancer progression. As a nano-scale membrane structure, exosomes are mainly responsible for carrying various contents and are widely involved in the biological process of tumors through mechanisms such as plasma membrane fusion, endocytosis, and binding to cell surface receptors. As one of the molecular basis of tumor invasion and metastasis, exosomes are of great significance for early diagnosis and target therapy of lung cancer. It has been found that exosomes derived from highly metastatic lung cancer cells and serum of patients with advanced lung cancer induce vimentin expression, induce epithelial–mesenchymal transition of HBECs, and induce migration, invasion, and proliferation of non-metastatic cancer cells [24]. Wang et al. [25] found that exosomes derived from metastatic SCLC cells had a greater impact on tumor cell migration and invasion than those derived from early non-small cell lung cancer (NSCLC) cells. Especially under hypoxic conditions, the contents of transforming growth factor-beta and interleukin-10, which are closely related to tumor cell migration and invasion, were increased in the exosomes of metastatic small cell lung cancer cells. As the important substance for intercellular information exchange, exosomes transmit relevant signaling molecules to target cells by means of autocrine or long-distance dissemination, resulting in a series of biological effects. Therefore, exosome-targeted tumor therapy approaches may have broad prospects. The PI3K protein family is a dimer composed of the regulatory subunit p85 and the catalytic subunit p110, which is widely involved in regulating cell phenotypes such as proliferation, differentiation, apoptosis, and migration. It has been reported that abnormal activation of PI3K signal is involved in the development of afatinib resistance, while shikonin inhibits the proliferation and induces apoptosis of afatinib-resistant NSCLC cells by activating the apoptotic signaling pathway and negatively regulating the PI3K/Akt signaling pathway, delaying the further development of lung cancer [26]. Overexpression of Fer-1-like family member 4 in lung cancer cell lines A549 and 95D inhibited colony formation, cell proliferation, and migration, resulting in decreased PI3K/Akt expression in cells, while activation of PI3K/Akt signaling using small molecule inhibitors of phosphatase and tensin homolog reversed the inhibitory effect of Fer-1-like family member 4 on cell proliferation and metastasis [27]. Similarly, lncRNA FOXO1 expression was downregulated in lung cancer tissues or cells, and knockdown of FOXO1 promoted A549 cell viability, colony formation, and invasion by activating the PI3K/Akt pathway, accelerating tumor metastasis and recurrence [28]. In this study, we found that FOXD3-AS1-enriched exosomes activated the PI3K/Akt signaling system by interacting with ELAVL1 to promote lung cancer cell proliferation, invasion, and 5-FU resistance. In conclusion, therapeutic strategies targeting exosomes may bring new therapeutic hope for patients with lung cancer. 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Ding X , Wang Q, Tong L, Si X, Sun Y. Long non-coding RNA FOXO1 inhibits lung cancer cell growth through down-regulating PI3K/AKT signaling pathway . Iran J Basic Med Sci 2019 , 22 : 491 – 498 . Google Scholar PubMed OpenURL Placeholder Text WorldCat © The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Acta Biochimica et Biophysica SinicaOxford University Press

Published: Oct 4, 2021

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