EIF4a3-regulated circRABL2B regulates cell stemness and drug sensitivity of lung cancer via YBX1-dependent downregulation of MUC5AC expression

Identification of mucin modulators is of remarkable significance to facilitate mucin-based antineoplastic therapy. However, little is known about circular RNAs (circRNAs) on regulating mucins. Dysregulated mucins and circRNAs were identified via high-throughput sequencing and their relationships with lung cancer survival were analyzed in tumor samples of 141 patients. The biological functions of circRABL2B were determined via gain- and loss-of-function experiments and exosome-packaged circRABL2B treatment in cells, patient-derived lung cancer organoids and nude mice. We identified that circRABL2B was negatively correlated with MUC5AC. Patients with low circRABL2B and high MUC5AC displayed the poorest survival (HR=2.00; 95% CI=1.12-3.57). Overexpressed circRABL2B significantly inhibited cell malignant phenotypes, while it knock-down exerted opposite effects. CircRABL2B interacted with YBX1 to inhibit MUC5AC, and subsequently suppressed integrin β4/pSrc/p53 signaling and impoverished cell stemness, and promoted erlotinib sensitivity. Exosome-packaged circRABL2B exerted significant anti-cancer actions in cells, patient-derived lung cancer organoids and nude mice. Meanwhile, circRABL2B in plasma exosomes could distinguish early-stage lung cancer patients from healthy controls. Finally, we found circRABL2B was downregulated at the transcriptional level, and EIF4a3 involved the formation of circRABL2B. In conclusion, our data suggest that circRABL2B counteracts lung cancer progression via MUC5AC/integrin β4/pSrc/p53 axis, which provides a rationale to enhance the efficacy of anti-MUCs treatment in lung cancer.

knockdown, short hairpin RNAs (shRNAs) targeting circRABL2B and circSULF2 were synthesized and inserted into the pLshRNA plasmid. For gene knockdown, siRNAs targeting MUC5AC, YBX1, RABL2B and EIF4a3 were purchased from Ribo TM Company (Guangzhou, China). To generate stable overexpression cells and knockdown cells, the transfection of plasmids was performed using Lenti-Pac HIV Expression Packaging Kit (Genecopoeia, Rockville, MD), and the harvestable virus particles were used to infect A549 and PC9 cells. To knockdown MUC5AC, YBX1, and EIF4a3, the transfection of mixed siRNAs was performed using the Lipofectamine 2000 Kit (Invitrogen).

In vitro cell behavior assays
To test cell proliferation, approximately 1 × 10 3 cells were seeded in 100 μl culture media in 96-well plates and cell viability was analyzed using Cell Counting Shanghai,China). To test cell oncogenicity, approximately 200 cells were seeded in 2 ml culture media in 6-well plates for 7-14 days. Then the cells were stained using Crystal Violet and counted. To test cell migration and invasion, approximately 2 × 10 5 cells were seeded in Corning Transwell insert chambers (Corning, New York) and BD BioCoat Matrigel invasion Chambers (BD Biosciences, Bergen, NJ), respectively. Cells migrated through the membrane were fixed, stained, and counted under a light microscope. To test the cell cycle, approximately 2 × 10 4 cells were incubated with 10 ul propidium iodide (PI) and submitted to flow cytometry (FCM) on FACScan (BD Biosciences). To test the cell apoptosis, approximately 1 × 10 5 cells were stained with 5 μl FITC-Annexin V and 10 μl 7AAD and then submitted to flow cytometry (FCM).

In vivo tumorigenesis assay
To test xenograft in vivo, 6-8-week-old female BALB/c nude mice were used.
Approximately 5 × 10 6 circRABL2B overexpressed A549 cells or control A549 cells, or circRABL2B knockdown PC9 cells or control PC9 cells were injected subcutaneously into the dorsal flank of mice. After 2-to-3 weeks, all mice were sacrificed and tumor volume was measured using a caliper as 1/2×length 2 ×width. Tumors were then surgically extracted and weighted.All experiments and procedures involving animals were conducted in accordance with guidelines approved by the Laboratory Animal Center of Guangzhou Medical University.

Immunofluorescence (IF) staining and RNA fluorescence in situ hybridization (FISH)
To detect MUC5AC in vitro, A549 or PC9 cells were fixed, permeabilized, blocked and incubated at 4°C overnight with MUC5AC antibody. Then fixed cells were rinsed in Dulbecco's Phosphate Buffered Saline (DPBS; Thermo Fisher Scientific), which was followed by a reaction with AlexaFluor 594-conjugated Goat Anti-Rabbit IgG (Abcam) for 1 h and DAPI (Invitrogen) for 10 min.
To determine co-localization of circRABL2B with YBX1 or SFPQ, A549 or PC9 cells were fixed, permeabilized, blocked and incubated at 4°C overnight with YBX1 or SFPQ antibody. Subsequently, fixed cells were rinsed in DPBS and incubated at room temperature with AlexaFluor 488-conjugated Goat Anti-Rabbit IgG. Then cells were washed with DPBS three times, which was followed by RNA-FISH using Ribo TM FISH Kit (Guangzhou, China) according to the operating manual. Cells were fixed, permeabilized, and prehybridized for 30 min. Then hybridization was carried out with circRABL2B probes that were designed and synthesized by Ribo TM Company at 37°C in the dark overnight. After being washed in wash buffer at 42°C and DPBS, cells were incubated with DAPI for 10 min. Imaging was performed on an LSM980 confocal laser scanning biological microscope (ZEISS, Jena, Germany). Antibody information is listed in Table S3.

RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP)
RNA pull-down assay was performed using BersinBio TM RNA pull-down Kit (BersinBio, Guangzhou, China) according to the manufacturer's instructions. Briefly, 2 × 10 7 cells were sonicated in 1.7 ml RIP buffer, supplemented with a 17 µl protease inhibitor. After centrifugation, the cell supernatant was incubated with agarose beads and then incubated with Streptavidin magnetic beads-treated biotinylated DNA oligo probes against circRABL2B or LacZ control, supplemented with auxiliary reagents for 2 h at room temperature. The beads were washed with ice-cold NT2 buffer five times and eluted with Protein elution buffer. The retrieved proteins were used for mass spectrometry or WB analysis. Mass spectrometry was finished by a commercial corporation (BersinBio). Sense, antisense of full length circRABL2B and truncated circRABL2B with deletion of predicted YBX1-binding sequences (nucleotides 118-134), and sense, antisense of 5'-end MUC5AC transcript (nucleotides 1-1950), and truncated 5'-end MUC5AC transcript with deletion of predicted YBX1-binding sequences (nucleotides 225-784), were cloned into a pMD18-T vectors (BersinBio) and were biotinylated with a Pierce RNA 3'-End Desthiobiotinylation Kit (Thermo Fisher Scientific, Waltham, MA).
RIP was performed using BersinBio TM RNA Immunoprecipitation Kit (BersinBio) according to the manufacturer's instructions. Briefly, cells were lysed using RNA lysis buffer and then incubated with the RIP washing buffer containing the magnetic beads conjugated with antibodies of indicated proteins or negative control IgG at 4°C, overnight. The beads were washed six times. The immunoprecipitated RNAs were extracted using phenolchloroform-isoamylol and were used for RT-qPCR.
ChIP was performed using a high-sensitivity ChIP kit (Abcam). Briefly, tissue cultures were fixed, quenched and lysed. Then the chromatin DNA was sheared to fragments. The samples were incubated with a 0.8 μg primary antibody against EIF4a3, or non-immune IgG and mixed with ChIP reaction buffer overnight at 4°C. After washing, DNA fragments were eluted and used for qRT-PCR. Fold enrichment was calculated as a ratio of the ChIP sample amplification efficiency to non-immune IgG, as suggested by the manufacturer. Primer information is listed in Table S2.

Exosome isolation, identification
Tissue exosomes were isolated from the xenografts of nude mice following standard centrifugation steps. The samples were treated by ExoQuick-TC (System Biosciences, Palo Alto, CA) at 4°C and then were centrifuged at 1500 g for 30 min. The pellet was re-suspended in PBS and further centrifuged at 20,000 g for 2 h. The pellet was diluted in PBS.
Transmission electron microscopy was performed to identify exosomes.