Cell lines and cell culture
Three NSCLC cell lines, A549 and SPCA1(no EML4-ALK1 gene), H3122 (EML4-ALK1 positive cells), as well as HBE (human bronchial epithelial cells) were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences and had been passed the STR cell identification in 2020. All cell lines have been tested for mycoplasma contamination before the experiment. These cells were cultured in RPMI 1640 medium (Gibco-BRL, USA). The cell culture medium contained 10% fetal bovine serum (FBS), (Gibco-BRL, USA) and 1% penicillin/streptomycin (Gibco-BRL, USA). Cells were cultured in a humidified incubator containing 5% CO2, and at 37°C.
Plasmid construction and cell transfection
The pGPU6-F-CircEA1-GFP/Neo (Genepharma, Shanghai, China) was constructed as a means to knock down F-circEA1 by targeting the back-splicing junction of F-circEA1, using shNC was used as the negative control. The complete sequence of F-circEA1 was insert into vector pEX3-GCMV-MCS-Neo (Genepharma,Shanghai, China), and an empty vector with no F-circEA1 sequence, was used as the control plasmid. All plasmids were transfected with Lipofectamine 3000 (Life Technologies, USA), and RNA and proteins were collected 48 hours and 72 hours after transfection, respectively. The interference sequence and the full-length sequence of F-circEA1 are presented in Additional file 1.
RNA or DNA extraction, RNA sequencing and quantification
Total RNA was extracted from cells using Trizol reagent (Life Technologies, USA) and genomic DNA was extracted from H3122 cells using a genomic DNA mini preparation kit with spin columns (Beyotime, Shanghai, China). To digest the linear RNA, 5 µg of total RNA was incubated at 37°C for 25 min with 20 units of RNase R (Geneseed, Guangzhou, China) and then reverse transcribed into cDNA with a SMART MMLV Reverse Transcriptase kit (Takara, Dalian, China) according to the manufacturer’s instructions. The cDNA was then amplified using Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China) and the relevant primers by PCR. The products were electrophoresed on an agarose gel and then sequenced in Invitrogen (Shanghai, China). The relative expression levels of the genes were quantified by QRT-PCR using TB Green® Premix Ex Taq™ (Takara, Dalian, China) and relative expression levels were compared using the 2−ΔΔCt method. The primer sequences involved in the above assays are listed in Additional file 1.
Fluorescence in situ hybridization (FISH) of F-circEA1 in H3122 cells
Cy3 and DAPI were used to label the backsplice junction (BSJ) (Cy3-5ʹ CAACT+TCATTTGTTGTCA+TGTGTCT-3ʹ-Cy3) of F-circEA1 and cell nuclei respectively, to observe the position of F-circEA1 in the H3122 cells. FISH technology was performed according to instructions from the RNA FISH kit (Genepharma, Shanghai, China) and images were taken with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Cell proliferation and cell viability assays
For the cell proliferation assay, cells were seeded in 96-well plates at a concentration of 3×103 cells per well and cultured in complete medium (RPMI1640 + 10% FBS + 1% penicillin/streptomycin) at 37°C. Then 10 µL of CCK8 was added into each well at fixed time points for 2 hours and the absorbance was measured at 450 nm using a microplate reader (BioTek, Epoch, USA). For the cell viability assays, 5×103 cells per well were seeded and cultured in 96-well plates with complete medium at 37°C for 24 hours, then crizotinib(Sigma, Aldrich, USA) or DMSO (as control) (Sigma, Aldrich, USA)were added at different concentrations. After 48 hours, 10 µL of CCK8 was added into each well for 2 hours and the absorbance read at 450 nm using a microplate reader (BioTek, Epoch, USA).
Transwell migration and invasion assays
Transwell chambers (Corning, NY, USA) were used for the cell migration and invasion assays. For the invasion assay, the upper chambers were covered with an even layer of Matrigel (BD Biosciences CA, USA) diluted with RPMI-1640 (1:6). The cells were suspended in serum-free RPMI-1640 medium and inoculated uniformly into the upper chamber at a volume of 700 µL and RPMI-1640 medium containing 10% FBS was added to the lower chamber. After incubation at 37°C, cotton wool swabs were used to carefully remove the cells from the upper surface membrane. The cells on the lower surface of the chamber were fixed with methanol for 15 min and stained with 1% Crystal Violet (Beyotime, Shanghai, China) for 15 min. At least five different random fields were chosen to image and count the cells under the microscope.
Analysis of the cell cycle and apoptosis by flow cytometry
Cells were collected and fixed with 70% ethanol and placed in a refrigerator at 4°C for 7-8 hours, then stained with propidium iodide (PI) solution for half an hour and then the different cell cycle stages were analyzed within one hour by flow cytometry (Mloflox xdp, Beckman, San Jose, CA, USA). To determine cell apoptosis, cells were stained with Annexin V-PE and 7-AAD according to the protocol within the Annexin V-PE/7-AAD apoptosis detection Kit (Vazyme, Nanjing, China) and then the ratio of apoptotic to healthy cells was measured after one hour.
Western blot analysis
Cell lysates were prepared with RIPA (Beyotime, Shanghai, China) buffer to isolate total cellular protein. The proteins were then resolved by SDS-PAGE using a 10% gel, and transferred wet to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% BSA and the following primary antibody applied overnight at 4°C: ALK (D5F3R,1:2,000), p-ALK (1:1,000), PI3K (1:1,000), p-PI3K (1:1,000), AKT (1:1,000), p-AKT (1:2,000), mTOR (1:1,000), p-MTOR (1:1,000), JAK3 (1:1,000), p-JAK3 (1:1,000), STAT3 (1:1,000), p-STAT3 (1:2,000), MEK1/2 (1:1,000), p-MEK1/2 (1:1,000), ERK1/2 (1:1,000), p-ERK1/2 (1:1,000), GAPDH (1:1,000) ( all from Cell Signaling Technology, Beverly, MA,USA). The next day, blots were incubated with secondary anti-rabbit (1:3000, Cell Signaling Technology, Beverly, MA,USA) at room temperature for 1 hour. Finally, protein bands were detected with Immobilob™ Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA), using a fully automated chemiluminescence imaging analysis system (Tanon 5200, Shanghai, China).
Establishment of stable cell lines and Nude mice xenografts
Lentiviral interference vector LV3-H1-F-CircEA1-GFP & Puro, and the control
lentivirus were constructed by Genepharma (Shanghai, China). After infecting H3122 cells, the transfectants were isolated using puromycin selection to form stable cell lines. Next 1×106 cells and 30 µL Matrigel (BD Biosciences CA, USA) were diluted with PBS to 100 µL, and then inoculated into the armpit of BALB/c nude mice Subcutaneously (5 weeks old, Nanjing Qinglong Mountain Animal Breeding Farm, China), and maintained in a pathogen-free environment. The body weight and tumor volume of the mice were measured every 3 days after tumor formation. Finally, tumorous tissues were collected after 25 days.
The transplanted tumor tissue was made into paraffin sections and incubated with anti-ALK (D5F3R 1: 250) primary antibody (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C, and incubated with secondary anti-rabbit for one hour at 37°C, then soaked in HRP-labeled antibody/streptavidin solution for 10 min and stained with diaminobenzidine (DAB). Target protein levels were evaluated based on their intensities within positive cells. Finally, five fields of view were analyzed for each slide, using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany), at a magnification of 400×. The total score was obtained by multiplying the dye intensity and the percentage of the number of cells. The dye intensity score: 0 points (negative), 1 point (light yellow), 2 points (brown yellow), 3 points (dark brown). Percentage of cells: 0 points (negative), 1 point (1-25% positive cells), 2 points (26-50% positive cells), 3 points (51-75% positive cells), 4 points (76-100 % Positive cells).
Statistical analyses were performed using GraphPad Prism 8.0 and differences between groups were mainly compared using Student's t test. Difference with p value <0.05 is statistically significant.The gray value of WB strips value was measured by Image J. The dates are reported as the mean ± standard deviation (SD) by IBM SPSS Statistics 25.