2.1 Cell lines, culture and transfection
Human esophageal cancer cell line TE-1, human esophageal cancer cell line ECA109, human embryonic kidney cell 293T were purchased from Beyotime, China. TE-1 cells were cultured in RPMI 1640(with 10% FBS,Gibco, Australia). ECA109, 293T were cultured in DMEM (with 10% FBS,Gibco, Australia). All cells were cultured in an incubator suitable for 37℃, 5%CO2 and humidity.
shRNA-C9orf139, miR-661 mimics and anti-miR-661 inhibitor were synthesized by the company (50OD, purified by HPLC) and self-diluted for use. The pcDNA3.1-HDAC11 was derived from a subclone of the chemically synthesized CDS region of pcDNA3.1. Cells were collected, counted and seeded into 6-well plates the day before transfection. Cell transfection was performed by using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer's protocol. DMEM with 10% FBS was added to each well at 6 h following transfection.
2.2 RNA extraction and RT-qPCR
Collect total RNA by using kit reagents and mass of total RNA was evaluated by Nanodrop 2000 spectrophotometer(Thermo Scientific, America). Reverse transcription of 1.0 µg total RNA to cDNA and qPCR was performed by using the SYBR Green MasterMinds Kit Testing on the testing system platform(Biosystems 7500;ABI;America).The gene expression data was performed by 2 − ΔΔCt method to analysis the relative quantity.
2.3 Cell Counting Kit‑8 (CCK‑8) assay
Cells were inoculated 4000 cells/well into 96-well plates (100ul volume); 24h after cell inoculation, 100ul of fresh RPMI1640 medium (containing 2%FBS) was replaced. Lipofilter was about 0.6ul, 24.4ul was mixed with RPMI1640 medium (excluding FBS), and reached at room temperature for 5min. The prepared siRNA was about 50pmol (2ul)(or 0.25ug PCDNA3.1 plasmid containing the GENE CDS region) and mixed with 23ul RPMI1640 medium (excluding FBS). The prepared 2 tubes of solution were mixed, gently mixed, reached at room temperature for 20min, and then added into cells; After 6 hours of culture, 150ul of fresh RPMI1640 medium (containing 10%FBS) was replaced for further culture. After 72h foster, 10ul CCK8 reagent was added for further incubation for 1-4h. The absorbance was measured at 450nm.
2.4 Clone formation
A 6-well plate was taken, and the target cells of exponential growth period were inoculated :1000 cells/well. NC control group (or Ctrl group) and experimental group were set, and the number of cells could be adjusted appropriately according to experimental needs. 24h after cell inoculation, fresh RPMI1640 medium 1500ul (containing 2%FBS) was replaced. The lipofilter was about 5.0ul, mixed with 250ul RPMI1640 medium (containing 2%FBS), and reached at room temperature for 5min. The prepared siRNA was about 50pmol(2ul) or 0.25ug plasmid, and mixed with 250ul RMI1640 medium (containing 2%FBS). The prepared 2 tubes of solution were mixed, gently mixed, then left to reach at room temperature for 20 minutes before being added to cells. After 6h of culturing, 2000ul of fresh RMI1640 medium (containing 10%FBS) was replaced. After 7–10 days of continuous foster, the number of clones was observed under a microscope and photographed and counted.
2.5 Transwell assay
A 24-well Transwell plate was taken (pretreated with 0.2ml matrix glue for 1h), and the target cells (containing 5%FBS) in exponential growth period were inoculated in the upper chamber :10000 cells /250ul/ well, then NC control group and experimental group were set, with three multiple Wells/groups. 4h after cell inoculation, 2.4ul lipofilter was taken, 47.6ul was mixed in RPMI1640 medium (containing 5%FBS), and reached at room temperature for 5min.The prepared siRNA or microRNA was about 50pmol(2ul) or 0.25ug pcDNA3.1-HDAC11 plasmid, and mixed with 46ul RMI1640 culture base (containing 5%FBS). Mixed the prepared 2 tubes of solution, mixed gently, stood at room temperature for 20min, added into the cells and mixed gently again. After 6-12h culturing, 300ul of fresh RPMI1640 medium (containing 5%FBS) was replaced, and 500ul RPMI1640 medium (containing 20%FBS) was added into the lower chamber to continue culture. After 48 hours of culture, the upper chamber was removed and the cells attached to the upper surface of the upper chamber were gently scraped with cotton swabs. The cells on the lower surface were treated and stained with crystal violet staining, and observed under a microscope then photographed.
2.6 Annexin V-FITC assay
Cells were inoculated during exponential growth period :100000 cells/well, NC control group and experimental group were set, and 3 multiple cells/group were set.24h after cell inoculation, 500ul of fresh RPMI1640 medium (containing 2%FBS) was replaced. 2.4ul lipofilter was taken, 47.6ul was mixed with RPMI1640 medium (containing 2%FBS), and stood at room temperature for 5min. The prepared siRNA was about 50pmol(2ul) or 0.25ug plasmid, and mixed with 46ul RPMI1640 medium (containing 2%FBS). Mix 2 tubes of solution, mix gently, let stand at room temperature for 20 minutes, then add into cells. After 6h culture, 500ul of fresh RPMI1640 medium (containing 10%FBS) was replaced. After 72h culture,300g,4, centrifugation for 5min. The cells were re-suspended by pre-cooled PBS and centrifuged at 300g,4, for 5min. Discard PBS and add 100ul 1*Binding buffer to resuscitate cells. Annexin V-FITC 5ul was added, mixed gently, dark, and reacted at room temperature for 10-15min. 400ul 1*Binding Buffer was added, mixed evenly and placed on ice for flow detection within 1h.
2.7 Dual luciferase activity detection
After 48h transfection, the cells were washed gently with PBS, followed by adding 200ul cell lysis solution to each well and incubating on ice for 5-10min to fully lysis the cells. According to the kit instructions (Shanghai YEASEN Biotechnology), luciferase substrate was prepared and kept on ice. 20ul cell lysate and 100ul firefly luciferase reaction solution was added into each well of the 96-well plate and then the plate was put into the plate tester to detect firefly luciferase activity. After the firefly luciferase activity was detected, 100ul of aquiferase reaction solution was added to each well and detect aquiferase activity.
2.8 Western blotting assay
After the experimental cells were washed twice with PBS solution, 100ul cell lysis buffer (containing 1% protease inhibitor 1%EDTA) was added, fully mixed, and reached at room temperature for 30sec. Centrifugation at 13000rpm for 5min. Took 20ul supernatant, added 5ul protein loading buffer, and mixed thoroughly. The samples were boiled in boiling water for 5min, and then some samples were taken. After electrophoresis, the membrane was transformed. Blocked PVDF membrane with blocking solution IKK antibody(ab32041) (1:500),IkB antibody(ab76429)(1:500), NFκB antibody(ab32360) (1 : 1,000), HDAC11 antibody (ab18973)(1 : 1,000), NCOR1 antibody(ab3482) (1 : 2,000),GAPDH antibody(ab8245)(1 : 3,000). Put them at room temperature for 1 h; added antibody diluted in blocking solution (goat anti rabbit IgG HRP (ab6721) (1: 3,000), and incubated them at 37℃ for 2 h and 4℃ overnight. Then, the membrane was washed and incubated with the goat anti rabbit IgG HRP antibody for 1h at 37℃. After washing the membrane, chemiluminescence imager was used for chemiluminescence.
2.9 Mouse Xenograft Assay
The effects of C9orf139 on tumorigenesis and growth in vivo were detected via mouse xenograft assay. We used twenty 5 to 6-week-old female NU-Foxn1nu nude mice (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) for the mouse xenograft assay. A total of 3×106 TE-1-NC cells or C9orf139-knockdown stable TE-1 cells were used to inject into the right or left oxter of female NU-Foxn1nu nude mice, respectively. Tumor size and weight were determined with calipers and balanced twice a week. The mice were executed and the tumors were removed after 53 days. The formula V = (W 2×L)/2 was used to calculate the tumor volume. V is the tumor volume, W is the tumor width, and L is the tumor length. Tumor size was presented as mean ± standard deviation (SD).
2.10 Protein-protein interactionnetwork
The PPI interaction networks between the DEGs were constructed by Search Tool for the Retrieval of Interacting Genes (STRING) database (http://string-db.org/). Firstly, the DEGs were typed into the database. Then, high-resolution bitmaps were displayed and downloaded from the webpage. Only these interactors with combined confidence score > = 0.4 were shown in the bitmap.
2.11 Statistical analysis
All experiments were repeated at least 3 times during the study period. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA). All data are expressed as mean ± standard deviation (SD) unless otherwise stated. The main statistical methods were tested and one-way ANOVA (p < 0.05 was considered significant in STATISTIC).